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Identification and characterization of a unique subpopulation of double negative splenic T cells which… Ingram, Andrea 1995

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IDENTIFICATION AND CHARACTERIZATION OF A UNIQUE SUBPOPULATION OF DOUBLE NEGATIVE SPLENIC T CELLS WHICH EXPRESS THE ap T CELL RECEPTOR BY ANDREA INGRAM B.Sc, University of Waterloo A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) THE UNIVERSITY OF BRITISH COLUMBIA THE UNIVERSITY OF BRITISH COLUMBIA APRIL 28, 1995 (c) ANDREA INGRAM, 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of rPqyAfr/r^> ^ The University of British Columbia Vancouver, Canada Date / j /99<r DE-6 (2/88) 11 ABSTRACT A unique class of T lymphocytes has been identified in the spleen of normal mice. This splenic T cell population, which expresses the ap TCR associated with the CD3 complex yet lacks the CD4 and CD8 coreceptor molecules, constitutes approximately 0.1-0.3% of total nucleated cells and 17-22% of CD4" CD8" HSA" Mac-1' cells in the normal murine spleen. Phenotypic analysis of splenic TCR a p + CD4" CD8" HSA" Mac-1- (DNapT) cells demonstrated the expression of Lyt-1, Pgp-1, ICAM-1 and the transferrin receptor whereas minimal to no B220 or YE1/19 expression was observed. Amongst splenic DNapT cells there is a higher frequency of Vpg usage in comparison with mature SP T cells. Similar to TCR a p + DN thymocytes, splenic DNa.pT cells proliferate in the presence of IL-7 and this response is enhanced by the addition of IL-1. CD4 + and CD8 + SP. T cells, in contrast do not respond to IL-7 alone or in combination with IL-1. Splenic DNapT cells are considered to be mature based on the expression of the heat stable antigen. Furthermore, this splenic T cell subpopulation expresses a functional T cell receptor as suggested by their responsiveness to crosslinking of the TCR associated CD3 complex which is comparable to that of mature SP T cells. In contrast, splenic TCR y5 + DN T cells were nonresponsive to anti-CD3 crosslinking despite the expression of the TCR/CD3 complex. The addition of IL-1, however, appeared to restore the responsiveness of TCR y5 + DN towards the activation of the TCR/CD3 complex. The functional maturity of the splenic DNapT cell population was confirmed by their ability to express cytokine specific mRNA. DNapT cells constitutively express IFNy message whereas IL-4 mRNA was induced by crosslinking of the TCR associated CD3 complex. SP T cells, in contrast, express messages for IFNy and IL-4 as well as IL-2 only in response to anti-CD3 crosslinking. The above results support the hypothesis that splenic DNapT cells represent a unique class of mature T cells which are related to the phenotypically similar cells found in the thymus yet are distinct from the classical type of mature T cell which expresses the SP phenotype. Ill TABLE OF CONTENTS Page CHAPTER I: CHAPTER II: CHAPTER III: ABSTRACT ii LIST OF TABLES iv LIST OF FIGURES v LIST OF ABBREVIATIONS vi INTRODUCTION 1.1 The T cell antigen receptor complex 1 1.2 T cell differentiation 7 1.3 T cell activation 24 1.4 T cell function 30 1.5 Thesis Objectives 37 1.6 References 39 MATERIALS AND METHODS 2.1 Animals 65 2.2 Antibodies 65 2.3 Isolation of T cell populations 65 2.4 Phenotypic analysis 67 2.5 Proliferation assay 68 2.6 Lymphokine mRNA expression 68 2.7 References 74 3.1 3.2 3.3 Identification of Splenic DNapT cells Characterization of Splenic DNapT cells References 75 75 92 CHAPTER IV: CONCLUSIONS AND DISCUSSION References 93 113 iv LIST OF TABLES Page TABLE I Cytokine stimulation of spleen cell subpopulations 89 TABLE II Activation of spleen cell subpopulations by anti-CD3 crosslinking 90 TABLE III Activation of DNapT cells by PMA and ionomycin 91 LIST OF FIGURES Page FIGURE 1 Schematic overview of the thymic differentiation pathway 38 FIGURE 2 Procedure for the isolation of spleen cell subpopulations 72 FIGURE 3 PCR cytokine oligonucleotide primers. 73 FIGURE 4 Identification of DNapT cells in the spleen of normal mice 80 FIGURE 5 Phenotypic analysis of splenic DN cells 81 FIGURE 6 Phenotypic analysis of CD3" DN spleen cells 82 FIGURE 7 Phenotypic analysis of splenic DNapT cells 83 FIGURE 8 Determination of Vpg usage by splenic DNapT cells 84 FIGURE 9 Confirmation that nylon wool purified cells are indeed SP T cells 85 FIGURE 10 Presence of DNapT cells in the TCR y8" DN spleen cell population 86 FIGURE 11 Lymphokine mRNA expression 87 FIGURE 12 Confirmation of the specificity of the IFNy message by southern 88 hybridization vi LIST OF ABBREVIATIONS aa amino acid AIDS acquired immune deficiency syndrome APC antigen presenting cell bp base pair BSA bovine serum albumin C constant region Ca+2 calcium ion CD cluster of differentiation CD25 Interleukin-2 receptor a chain IL-2Ra CD44 Pgp-1 CDR complementarity determining region cDNA complementary DNA CHO Chinese hamster ovary cells C 0 2 carbon dioxide ConA concanavalin A CTL cytotoxic T lymphocyte D diversity region DAG diacyl glycerol DN double negative DNapT CD4" CD8- HSA" Mac-1" TCR a p + T cell DP double positive EBV Epstein Barr Virus EDTA ethylenediamine tetraacetic acid FACS flourescence activated cell sorter FCS fetal calf serum FITC fluorescein isothiocyanate FTOC fetal thymic organ culture GVHD graft-versus-host disease GVL graft-versus-leukemia HEPES N-2-hydroxyethylpiperazine HTV human immunodeficiency virus HLA human leukocyte antigen HSA heat stable antigen ICAM intracellular adhesion molecule IFN. interferon Ig immunoglobulin IL interleukin IP3 inositol 1,4,5-triphosphate kb kilobase kd kilodalton LFA lymphocyte function-associated antigen MAb monoclonal antibody MAP-2K microtubule-associated protein-2-kinase 2ME 2-mercaptoethanol MHC major histocompatibility complex VU MLC mixed leukocyte culture MLR mixed leukocyte reaction Mis minor lymphocyte stimulatory antigen MLTC mixed lymphocyte tumour cell culture MNC mononuclear cell MRL-lpr/lpr autoimmune lymphoproliferative murine mRNA messenger RNA NaOH sodium hydroxide NK natural killer PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PHA phytohemagglutinin PI phosphatidylinositol PIP 2 phosphatidyl inositol 4,5-biphosphate PKC protein kinase C PLC phospholipase C PMA phorbol myristate acetate PTK protein tyrosine kinase RAG recombinase activation genes RBC red blood cell SA-PE streptavidin-phycoerythrin SCA stem cell antigen SCID severe combined immunodeficient SDS sodium dodecyl sulphate SEB staphylococcal enterotoxin B SEM standard error of mean SLE systemic lupus erythematosus SP single positive SSC saline sodium citrate buffer SSPE saline sodium phosphate EDTA buffer TBE Tris borate EDTA buffer T C cytotoxic T cell TCR T cell receptor TE Tris EDTA TGF tumour growth factor T H helper T cell TN triple negative TNF tumour necrosis factor Tris tris(hydroxymethyl)aminomethane tRNA transfer RNA T S suppressor T cell VLA very late antigen 1 C H A P T E R I INTRODUCTION Thymus-derived lymphocytes, otherwise referred to as T cells, are critical components of the immune system. T lymphocytes express an antigen receptor complex which is capable of recognizing foreign antigens when presented in association with the products of the self major histocompatibility complex. As a consequence of this recognition, T cells are activated to perform their effector functions, whether it be as regulator T cells which control the development and activation of effector T and B cells through the delivery of either helper or suppressor signals, or as effector T cells capable of destroying infected or abnormal cells. Thus T cells contribute to the ability of the immune system to provide a protection against foreign pathogens. 1.1 THE T CELL ANTIGEN RECEPTOR COMPLEX The T cell antigen receptor complex consists of two alternate forms of the TCR, namely the ap and y5 heterodimers associated with the CD3 complex and either CD4 or CD 8 coreceptors. 1.1.1 T Cell Receptor 1.1.1 A) Identification of the T Cell Receptor T cell receptor (TCR) ap heterodimers were initially identified on human and mouse T cells with the use of monoclonal antibody (MAb) specific to individual cell lines (Allison et al. 1982; Haskins et al. 1983; Meuer et al. 1983b). Such clonotypic antibodies could block the interaction of T cells with their targets (Haskins et al. 1983; Lancki et al. 1983; Samelson et al. 1983; Staerz et al. 1984) as well as activate T cell clones (Kappler et al. 1983; Kaye et al. 1983) and were therefore used to biochemically characterized the antigen receptors. Subsequently, complementary DNAs (cDNAs) were isolated which were strikingly similar to those encoding immunoglobulins (Igs), rearranged specifically in T cells but not B cells, encoded the a and p chains identified serologically (Hedrick et al. 1984; Chien et al. 1984; Saito et al. 1984b) and transferred both antigen and major histocompatibility complex (MHC) specificity from one T cell to another in gene transfer studies (Dembic et al. 1986; Saito et al. 1987b). During the search for TCR a, the y chain of the second heterodimer, y5, was isolated (Saito et al. 1984a). A subsequent serological study demonstrated y chain expression together with CD3 and a fourth polypeptide 5 (Brenner et 2 al. 1986) whose isolation was facilitated by its location within the TCR a locus (Lindstein et al. 1987; Chien era/. 1987). 1.1.1 B^ ) Structure of the T Cell Receptor Human TCR a (43-49 kd) and p (38-44 kd) chains are heterogeneously charged glycoproteins with TCR a containing N-linked oligosaccharides of the complex type and TCR P containing both high mannose and complex N-linked glycan side chains attached to polypeptide backbones of 32 and 34 kilodalton (kd) respectively. The murine TCR ap heterodimer consists of 40 to 50 kd TCR a and P glycoproteins containing N linked oligosaccharides attached to polypeptide backbones of 28 and 32 kd respectively (Allison and Lanier, 1987; Toyonaga and Mak, 1987). The TCR y5 heterodimer, composed of 55 kd (TCR y) and 40 kd (TCR 8) proteins, was co-precipitated with anti-CD3 MAb from human CD4" CD8" CD3 + T cell clones with natural killer (NK) activity but lacking reactivity with the MAb WT31 specific for a common epitope on human TCR ap chains (Brenner et al. 1987). The murine TCR y protein is a 35 kd glycoprotein with a polypeptide backbone of 32 kd which is disulfide linked to a 45 kd TCR 5 chain with a 31 kd polypeptide backbone (Chien et al. 1987; Pardoll et al. 1987b). The primary amino acid (aa) sequences deduced from four TCR cDNA clones (Toyonaga and Mak, 1987) reveal an Ig-like organization with variable (V) and constant (C) regions linked by a short joining (J) segment. An additional diversity (D) segment has been identified on the TCR p and y proteins only. V(D)J recombination is thought to be mediated by a recombinase encoded by the two genes RAG-1 and RAG-2 (Lieber et al. 1987; Schatz et al. 1989) whose expression correlates with those stages of thymocyte development associated with such recombination events, namely TCR" and TCR + DP thymocytes (Turka et al. 1991). Although the number of hypervariable regions within the TCR sequences is controversial, diversity is evident in regions equivalent to the classic Ig hypervariable or complementarity determining regions (CDRs) which form principle points of contact with antigen. TCR regions equivalent to CDR1 and CDR2 are encoded within the V gene whereas the conjunction of V and J forms the CDR3 equivalent region. The latter region is thought to be responsible for antigen contact whereas the remainder of the TCR heterodimer interacts with the MHC molecule (Davis and Bjorkman, 1988; Chothia et. al. 1988; Claverie et al. 1989). TCR constant segments, varying in length from 138-179 aa, are divided into four functional regions, the first of which includes a cysteine residue thought to link two p sheets in the characteristic Ig fold (Davis and Bjorkman, 1988). An additional cysteine residue located in a separate exon may be involved in the interchain sulphydral bond. Distinct hydrophobic segments have been found in all four TCR chains which represent membrane spanning regions containing a \ 3 highly conserved lysine residue thought to interact with conserved negatively charged residues (aspartate or glutamate) in the transmembrane region of four of the CD3 polypeptides. Beyond the transmembrane regions, short cytoplasmic tails of varying length (5-12 aa) have been identified. 1.1.1 C) Generation of T Cell Receptor Diversity Similar to B cells, T cells use several mechanisms for the generation of diversity in the antigen receptor including use of several gene segments (V,D,J) for variable region formation, random combinatorial joining of different germline gene segments, addition of N nucleotides not encoded in the genome to the junction between joined segments and use of D gene segment in all 3 translation reading frames. However, TCR genes lack three important features of Ig expression and diversity (Davis, 1990). First of all, unlike the differential splicing which allows either a membrane or secretory form of Ig to be produced depending on the stage of response, no secretory form of TCR has been identified reflecting the fact that T cells are engaged solely in cell-cell interactions. Furthermore, somatic hypermutation evident in Ig is not permissible in T cell receptors as it would be counterproductive to thymic selection, possibly promoting autoimmunity. In addition, isotype (heavy chain class) switching which enables an antibody VJJVL combination to be expressed as a fusion protein with constant regions of different functional and localizational properties, has not been observed for the TCR and is thought to be unnecessary in the absence of secretory forms. Although TCR genes lack the above mechanisms and employ fewer V gene segments than do Ig, they are still considered to be more sophisticated than Ig in generating diversity particularily at the V-J interface equivalent to the CDR3 region of Igs (Davis and Bjorkman, 1988). Several unique TCR gene characteristics contribute to the diversity observed in this region including: 1) N region diversification observed for all four TCR polypeptides (Elliot et al. 1988) but only in heavy chain genes of Ig; 2) large numbers of TCR J region gene segments in a and P genes as opposed to the four J regions identified in mouse Ig heavy and K chain loci; 3) flexibility of the position of 3' joining points of several Vets, Vys, V5s; 4) the ability to use both D regions in the adult 5 chain; 5) three different positions for N region diversification and 6) the ability to translate D region sequences in all three reading frames, although rare in Ig heavy chains, is coruiiion in TCR p and 5 chains. The above features contribute to the highly diverse CDR equivalent region on the TCR which is considered to be the site of antigen contact (Davis and Bjorkman, 1988). 1.1.1 D) Association of TCR-CD3 complex with a Cytoplasmic Tyrosine Kinase A member of the sre family of protein tyrosine kinases (PTK) referred to as p59^n has been shown to be associated with the TCR-CD3 complex based on the detection of low levels of the 4 protein tyrosine kinase (PTK) in TCR immunoprecipitates (Samelson et al. 1990). Two forms of fyn, the products of mutually exclusive splicing of alternative seventh exons, are expressed in either neuronal or hemopoietic cells (Cooke and Perlmutter, 1989). The expression of fyn late in thymocyte development correlates with the ability to respond to TCR mediated signals (Cooke et al. 1991). Studies in which the overexpression offyn in a T cell hybridoma cell line (Davidson et al 1992) or in transgenic mice (Cooke et al. 1991) enhanced TCR sisnal transduction have suggested a possible role for this PTK in T cell activation but not development. 1.1.2 CD3 Complex 1.1.2 A) Identification of the CD3 Complex The CD3 antigen was initially identified by immune precipitation with the MAb OKT3 as a 20 kd glycoprotein present on the surface of all human T lymphocytes (van Agthoven et al. 1981). Subsequent studies demonstrated CD3 expression as a complex of the major 20 kd glycoprotein (CD3-5), 20 kd non-glycoprotein (CD3-e) and a 25-28 kd glycoprotein (CD3-y) (Borst et al. 1983; Borst et al. 1984). Studies with an anti-TCR MAb have identified a murine TCR/CD3 complex consisting of five CD3-like proteins including 2 N-glycosylated proteins of 21 and 28 kd (CD3-y and CD3-5 respectively), one 25 kd glycoprotein which may contain O-linked oligosaccharides (CD3-e), 34 kd non-glycosylated homodimer not previously found in human cells by co-precipitation with anti-TCR or anti-CD3 MAbs (CD3-Q and an additional 21 kd non-glycosylated murine protein (CD3-r|) often disulphide linked to CD3 C, (Samelson et al. 1985a; Oettgen et al. 1986; Berkhout et al. 1987). 1.1.2 B) Structure of the CD3 Complex With respect to the CD3 complex, y and 8 are glycoproteins whose core protein sizes are approximately 15 kd. Following glycosylation, the apparent molecular weight of y is 21 kd in humans and 25 kd in mouse, whereas 8 is 25-28 kd in humans and 21 kd in mouse. In contrast, the 8 chain is non-glycosylated. Each with an N terminal extracellular domain, transmembrane segment and cytoplasmic domain, these three CD3 components are highly homologous, closely linked within 50-300 kb on human chromosome 1 lq23 and mouse chromosome 9 (Tunnacliffe et al. 1987) and are considered to belong to the Ig gene superfamily (Gold et al. 1987). In contrast to the TCR, a significant percentage of the primary structure of these CD3 components is located in the cytoplasmic domain and is thought to facilitate the role of CD3 in signal transduction. The C, chain, on the other hand, is a 16 kd non-glycosylated protein with no sequence or structurai 5 homology to either of the above three CD3 components or the TCR. Encoded by a gene found on human and mouse chromosome 1 (Baniyash et al. 1989), the C, chain has a very short (6-9 aa) extracellular domain with the vast majority of the protein (113 aa) represented in the cytoplasmic segment. Although the majority of the C, chain exists as a 32 kd homodimer, 5-20% is disulfide linked to a highly homologous 22 kd n, protein forming a 38 kd heterodimer (Frank et al. 1990). The n chain is thought to be derived by alternate splicing of the C, transcript (Jin et al. 1990) and lacks one of six possible tyrosine phosphorylation sites as well as a putative nucleotide binding site identified in CD3-c^  (Weissman et al. 1988). Interestingly, C, expression is not restricted to T cells as it is also expressed on NK cells in association with CD 16, the ligand binding subunit of FcyRIII (Anderson et al. 1989; Lanier, e. al. 1989). 1.1.2C) Association of CD3 with the TCR The T cell receptor complex consists of the disulfide-linked TCR noncovalently associated with the CD3 y,5 and s chains and the disulfide linked homo- or heterodimer containing the C, chain. Evidence for such an association between the CD3 complex and the TCP heterodimers has been provided by numerous studies. First of all, MAb against TCR has been shown to co-precipitate CD3 and vice versa. Incubation of T cells with TCR or CD3 specific MAb leads to the co-modulation of both TCR and CD3 due to the apparent uptake of TCR-CD3 into multivesicular bodies. (Meuer et al. 1983; Zanders et al. 1983). Futhermore, restoration of cell surface TCR-CD3 complex expression has been observed following gene transfer of either TCR a or p cDNA (Ohashiera/. 1985; Saito et al. 1987a). Studies of somatic cell mutants have shown that surface expression of the TCR complex is dependant upon the expression of the TCR a and p chains (Weiss and Stobo, 1984c; Ohashi et al. 1987; Saito et al. 1987a) as well as the C, chains (Sussman et al. 1988; Weissman et al. 1989). Recently the requirement for the CD3 chains has been demonstrated by a reconstitution study of TCR in CHO cells (Carson et al. 1991). TCR-CD3-c^ associations are thought to be mediated through hydrophobic transmembrane domains based on studies demonstrating the varying ability of different detergents in preserving the integrity of the TCR complex during solubilization (Weiss et al. 1986; Koning et al. 1990). Lysine and arginine residues within the transmembrane domain of the four TCR chains are thought to form salt bridges with the glutamic and aspartic acids located within the transmembrane regions of the CD3-y and CD3-8, s and C, chains respectively. Site-directed mutagenesis Lus identified residues lysine 126 and arginine 121 of the TCR a chain (John et al. 1989; Blumberg et al. 1990) and 6 lysine 290 of the TCR p chain (Morely et al. 1988) as playing an essential role in the proper assembly and expression of the TCR complex. 1.1.3 CD4andCD8 CD4 and CD8 glycoproteins are nonpolymorphic members of the Ig gene superfamily expressed on two mutually exclusive subsets of mature T cells. Although most helper T cells (Tj-r) express CD4 whereas cytotoxic T cells (JQ) and suppressor T cells (Tg) cells express CD8, this correlation is not as strict as initially thought. Anti-CD8 MAb have been shown to inhibit CD8 + cytotoxic and CD8 + helper T cells specific for class I MHC antigen (Swain, 1981). Furthermore, CD4 expression has been demonstrated on certain cytolytic T cells specific for antigen presented in association with class II MHC proteins (Krensky et al. 1982a; Krensky et al. 1982b). Therefore CD4 and CD8 expression is more closely correlated with MHC specificity of the T cell than with function. 1.1.3 A) Structure of CD4 The human and murine CD4 molecule, also referred to as T4, Leu-3 or L3T4, is a 55 kd single chain glycoprotein with four extracellular domains of which the most distal bears a striking homology to the Ig light chain variable genes, a stretch of hydrophobic transmembrane residues and a highly conserved 40 aa cytoplasmic tail (Maddon et al. 1985; Liftman and Gettner, 1987). Although considered to be a T cell specific marker, CD4 glycoprotein expression has been observed on monocytes, macrophages, Langerhan cells and brain cells. 1.1.3 B) Structure of CD8 Unlike CD4 which exists as a 55 kd monomer in both human and murine T cells, CD8 expression differs between the two species. In humans, the 34 kd CD8 molecule forms disulfide-linked homodimers and homomultimers on peripheral T cells (Snow and Terhorst, 1983) yet on thymocytes, in addition to homodimers, the CD8 polypeptide forms complexes with the 45 kd CD1 (T6) glycoprotein (Snow et al. 1985). In contrast, the murine CD8 homologue, both in the thymus and on peripheral T cells, consists of two chains, the 38 kd CD8 a (Tyt-2) chain or its corresponding 34 kd product of alternative splicing of the cytoplasmic tail, and the 30 kd P (Lyt-3) glycoprotein which are expressed as disulfide-linked ap or ap' heterodimers (Reilly et. al. 1980; Ledbetter et al. 1981; Ledbetter and Seamen, 1982). Whereas the 38 kd a chain of murine CD8 is thought to be the equivalent of the human CD8 molecule, the existence of a human Lyt-3 7 homologue has yet to be demonstrated. The human CD8 protein and the corresponding murine a chain of Lyt-2 both contain an amino terminal hydrophobic signal sequence followed by an Ig-like domain, a short extracellular hinge region, a hydrophobic transmembrane segment and a highly charged cytoplasmic domain (Littman et al. 1985; Sukhatme et al. 1985; Nakauchi et al. 1985). 1.1.3 O Association of CD4 and CD8 with a Cytoplasmic Tyrosine Kinase Early studies suggested that CD4 and CD 8 might have a signal transduction function in addition to ligand binding capacity and it has been subsequently shown that the cytoplasmic domains of these two glycoproteins are indeed associated with the cytoplasmic tyrosine kinase p56 ^  (Marth et al. 1985; Rudd et al. 1988; Veillette et al. 1988). Identified initially as a tyrosine kinase overexpressed in malignant lymphocytes, Ick belongs to the src family of tyrosine kinases. In addition to an amino terminal region facilitating binding to cysteine residues in the cytoplasmic tails of the CD4 and CD8 coreceptor molecules, Ick also contains a site for myristylation to allow association with the inner leaflet of the plasma membrane. Similar to other members of the src family kinases, Ick also expresses a conserved carboxy terminus which includes two regulatory tyrosine residues, amino acid residues 505 and 394, which when phosphorylated inhibit and stimulate kinase activity respectively (Veillett et al. 1991). 1.2 T CELL DIFFERENTIATION 1.2.1 Thymocyte Ontogeny Epithelial components of the murine embryonic microenvironment arise from the ectoderm and endoderm of the third pharyngeal cleft and pouch respectively at 10-11 days in ontogeny (Owen and Jenkinson, 1984) and subsequently organize into cortical and medullary stroma. By the 11th day of gestation, the thymus is first colonized by hemopoietic cells (Moore and Owen, 1967) which lack TCR, CD4 and CD8 expression (Bluestone et al. 1987) and represent the earliest intrathymic developmental stage. Southern blot analysis has shown that the majority of all TCR loci of these murine thymocytes remain in germline configuration up to day 13-14 when progressive rearrangement occurs. Although full length y (V-J-C) and 8 (V-D-J-C) mRNA are detected on day 14 of gestation, the majority of p messenger RNA (mRNA) at this time exists as 1 kb transcripts of incomplete D-J rearrangements with significant levels of full length 1.3 kilobases (kb) (V-D-J-C) P mRNA not observed until day 16 (Samelson et al. 1985). Full length a mRNA (V-J-C), on the other hand does not appear until.day 17. 8 With respect to surface expression, CD3 is first detected on day 14-15 in association with TCR y5 (Pardoll et al. 1987). TCR ap expression follows 2-3 days later as cells bearing this receptor increase in proportion until the time of birth on day 20 when they represent the major thymocyte population. Initial accessory molecule expression occurs on day 15-16 of gestation with the transient appearance of CD3" CD4" CD8 + thymocytes (Ceredig et al. 1983; Kisielow et al. 1984). By day 17 however, essentially all accessory molecule-bearing thymocytes co-express both CD4 and CD8 and progessively increase in number until they represent 75-80% of the total thymocyte population shortly after birth. Although initially CD3", by day 18 approximately 5% of CD4 + CD8 + cells have begun to express low levels of TCR ap. The first CD4 + CD8" thymocytes begin to appear by day 17-18, followed shortly thereafter by an increase in the number of CD4" CD8 + cells. In contrast to the double positive thymocytes, both subsets of single positive cells express high levels of TCR ap. Studies of human thymocyte ontogeny have revealed a sequence of TCR expression similar to that observed in the murine model. While approximately 10% of thymocytes from 9 1/2 week fetuses express TCR y5, not until 10 weeks of gestation do TCR ap expressing thymocytes appear and progressively increase in proportion concomitant with a decrease in number of TCR y5+ thymocytes (Haynes et al. 1988). 1.2.2 Classical T Cell Differentiation Pathway From murine ontogeny studies, a basic outline of the stages of thymocyte development has evolved (Figure 1). More specifically, hemopoietic CD3" CD4" CD8" (triple negative) progenitors rearrange TCR genes with subsequent surface expression of TCR ap and associated accessory molecules giving rise to TCR a p + CD4 + CD8 + (double positive) thymocytes. Through positive and negative selection processes, these double positive thymocytes give rise to CD4 + CD8" and CD4" CD8 + single positive cells which emigrate from the thymus to become functional T cells. Finer phenotypic analyses have further subdivided the above stages into additional steps providing an increasingly more detailed pathway for T cell differentation. 1.2.2 A) Hemopoietic Stem Cell The initial stage of T cell development involves the seeding of the thymus by multipotent stem cells derived from fetal liver and adult bone marrow. Such precursor cells, capable of reconstituting B 9 lymphoid, myeloid and erythroid lineages in addition to T lymphoid cells, have been purified from adult murine bone marrow (Sprangrude et al. 1988; Sprangrude and Scollay, 1990) and have been shown to express low levels of Thy-1 yet lack markers characteristic of other hemopoietic lineages. These multipotent stem cells also express stem cell antigen-1 (Sca-1), CD44 (Pgp-1), heat stable antigen (HSA) and MHC class I. 1.2.2 B) CD4 l p CD8- CD3" thymocytes Although the earliest intrathymic precursors were once thought to be contained within a small population of thymocytes expressing neither CD4 or CD8 accessory molecules (CD4" CD8", double negative thymocytes) (Fowlkes et al. 1985), even the most immature cells of this heterogenous group have already partially rearranged TCR y and p genes (Pearse et al. 1989). Recently, a minor population of lymphoid cells with both TCR and IgH genes in the germline state has been identified in the murine thymus (Wu et al. 1991). Referred to as "low CD4 precursors based on the expression of moderate levels of CD4, this novel population is thought to represent a newly defined stage in T cell differentiation linking the bone marrow derived stem cells which continuously colonize the thymus to the CD4" CD8" precursors engaged in TCR rearrangement. Although phenotypically similar to bone marrow hemopoietic stem cells which are also Thy-1+ HSA + Pgp-1 + + H-2k + + Sca-1+, the low CD4 precursors appear to be more mature in several respects including a higher proportion in the S+G2+M phases of cell cycle, more rapid progression to mature T cells following intrathymic transfer and the expression of Sca-2 (stem cell antigen-2) characteristically absent on purified bone marrow stem cells. Furthermore, '.."alike bone marrow stem cells, low CD4 precursors are not multipotent as suggested by the inability to form either myeloid or erythroid colonies in the spleen or in agar culture. Instead low CD4 precursors appear to be restricted to lymphoid lineage development although not exclusively to T cells. When transferred intravenously, this intrathymic precursor population is capable of producing both Thy-1 + T cell as well as B220 + B cell progeny in the bone marrow, spleen and lymph nodes. However, this low CD4 precursor population does display a preference for thymic seeding, giving rise to thymocytes of both TCR ap and y8 lineages as well as thymic dendritic cells (Ardavin et al. 1993) in the irradiated hosts. In contrast to the CD4" CD8" population, low CD4 precursors are considered to be less mature with respect to reconstitution behavior and TCR gene rearrangement status. Therefore, low CD4 precursors are considered to be the earliest T-lineage precursor cells in the adult murine thymus. 10 1.2.2 C) Triple Negative Thymocytes Following thymic seeding, low CD4 precursors cease expression of CD4 to enter the CD3" CD4" CD8' triple negative stage of development, characterized by extensive expansion and differentiation as cells begin to rearrange and express TCR genes in association with the accessory molecules CD4 and CD8. These triple negative (TN) thymocytes belong to a heterogenous group which can be further subdivided into subpopulations based on the expression of other surface markers. Progression through the developmental stages of this population is marked by the aquisition of high level Thy-1 and HSA expression, a reduction in CD44 (Pgp-1) levels and a transient expression of the low affinity IL-2 receptor a chain (CD25). Progressive increases in TCR Y and P gene rearrangement further signify maturation along this pathway. The earliest cells in this pathway, which are CD44 + CD25", have the slowest repopulation kinetics in irradiated thymi but demonstrate the greatest potential for expansion (Scollay et al. 1988). Few cells of this phenotype are in the active phases of the cell cycle. Although limited rearrangement of the TCR p genes has occured by this stage, no TCR a mRNA is detectable (Pearse et al. 1989). From the CD44 + CD25" phenotype, these cell progress to a CD25 + stage which can be further subdivided based on the expression of CD44. CD44 + CD25 + thymocytes are considered to be less mature than CD44" CD25 + cells in several respects (Godfrey et al. 1993). CD44+ CD25 + cells are larger based on forward scatter, and are Thy-1+ HSA + and c-kit^ whereas CD44" CD25 + thymocytes are significantly smaller and express higher levels of Thy-1 and HSA and are c-kitl0. As both Thy-1 and HSA levels characteristically increase with TN maturation and c-kit expression is down regulated during the CD25 + TN stage, the changes in expression levels of these markers suggest a more immature phenotype for the CD44 + CD25 + thymocytes. Furthermore, CD44 + CD25 + thymocytes have TCR p genes in the germline state whereas the CD44" CD25 + counterparts are rearranged at the TCR p chain gene locus. This recent finding is, however, in contradiction with an earlier study (Pearse et al. 1989) which demonstrated partial TCR p gene rearrangement in the least mature TN subpopulation, CD44 + CD25" thymocytes. The final subpopulation in the TN developmental pathway consists of CD44" CD25" thymocytes which demonstrate very rapid but short lived thymic reconstitution potential in irradiated recipients (Scollay et al. 1988). When transplanted intrathymically into non irradiated hosts, CD44" CD25" thymocytes give rise to CD4 + CD8 + cortical cells within one day and to mature single positive cells following an additional 2-4 days (Petrie et al. 1990a). Following one day culture in simple medium, these cells spontaneously and rapidly acquire high levels of CD4 and CD8 (Wilson et al. 1989) accompanied by CD3 expression at a significantly slower rate (Nikolic-Zujic and Moore, 1989) and are therefore considered to be preprogramed to become CD3l° CD4 + CD8 + cortical 11 thymocytes. Thymocytes of the CD44" CD25" phenotype express completely rearranged TCR P genes and high levels of mature TCR P mRNA and are the only cell type in the TN population to express TCR a mRNA (Pearse et al. 1989). Although traditionally considered to be members of the TN population, mRNA and corresponding low levels of surface CD4 and CD8 expression have been observed for this CD44" CD25" population, therefore suggesting that the accessory molecule expression begins concurrent with the down regulation of IL-2Rcc (Petrie et. al. 1990b) such that CD44" CD25" thymocytes, actually represent a transition stage between TN and CD3 1 0 CD4+ CD8 + cells. On their way to becoming CD4 + CD8+ C D3 1 q cells, preprogramed CD44' CD25" thymocytes are thought to pass through a transient single positive (SP) stage- either as CD3" CD4" CD8+ or CD3" CD4 + CD8" (Matsumoto et al. 1989; Hugo et al. 1990) cells. Similar to their immediate precursors, CD3" CD4" CD8 + and CD3" CD4+ CD8" SP cells express low levels of CD4 and CD8 respectively (Nikolic-Zujic and Moore, 1989; Petrie et al. 1990b; Hugo et al. 1991). Evidence that both SP subsets represent similar stages of differentiation include similar phenotypes (MacDonald et al. 1988a; Shortman et al. 1989; Yagita et al. 1989; Hugo et al. 1991) and timing of appearance during in vivo and in vitro T cell ontogeny and during thymic reconstitution following cortisone treatment and irradiation (Hugo et al. 1990; Hugo et al. 1991; Matsumoto et al. 1989; Arase et al. 1990). 1.2.2 D) Double Positive Thymocytes Approximately 80-85% of adult thymocytes are defined by coexpression of the accessory molecules, CD4 and CD8, and as such are the predominant cell type in the thymic cortex (Bluestone et al. 1987). Of this double positive (DP) population, 20% are large and medium sized dividing cells, whilst the remaining 80%, the typical small cortical thymocytes, are the noncycling products of this division. Interestingly, the majority of these DP cortical thymocytes are believed to die in the thymus as a result of a stringent thymic selection process involving positive selection for appropriate interaction with the self-MHC proteins of the antigen presenting system, and negative selection against reactivity with a variety of self antigens. However, a small proportion (approximately 3%) of DP thymocytes are not dead end cells but differentiate further to become mature SP medullary cells. Support for the direct precursor relationship of these DP thymocytes to mature SP cells is based on the observation of SP progeny following intrathymic transfer of CD4 + CD8 + TCR!0 blasts (Guidos et al. 1989). In addition, intrathymic expression of the self antigens Mls a and I-E, recognized by CD4 + SP T cells bearing TCR Vpg and V p i ^ respectively, eliminate not only CD4 + but also CD8+ SP cells (Kappler et al. 1987, MacDonald et al. 1988). Futhermore, the CD8 + TCR Vp5+ and Vpi7+ cells may be rescued by CD4 blocking monoclonal 12 antibodies during thymic development (Fowlkes et al. 1988, MacDonald et al. 1988a) therefore suggesting that all T cells must pass through a CD4 + CD8 + stage. The DP population is thought to represent a stage in T cell development when TCR a.p is first expressed in association with the CD3 complex (Shortman et al. 1990). Low level expression of surface TCR-CD3 has indeed been observed on approximately 50% of DP thymocytes (Havran et al. 1987, Crispe et al. 1987), the majority of which are rapidly dividing blast cells (Shortman et al. 1991). Furthermore, a small proportion of DP thymocytes have been shown to express high levels of TCR-CD3 (Bluestone et a/. 1987, Havran et al. 1987, Blue et a/. 1987) and represent the nondividing small cortical thymocytes (Shortman et al. 1991). It is from this nondividing DP population that mature SP thymocytes are thought to differentiate based on kinetic data (Penit, 1986, Edgerton et al. 1990). The intermediate phenotype (Thy-l1" HSA1" CD44" CD5^i MEL-i4int/hi LFA-l 1" and H-2Klo/hi) (Hugo et al. 1991) and timing of appearance during in vivo and in vitro embryonic development and thymic reconstitution following cortisone treatment (Hugo et al. 1991a) provide further evidence that CD4 + CD8 + CD3hi thymocytes represent a transitional stage between the CD4 + CD8 + CD3^° thymocytes and mature SP medullary cells. The importance of the TCR ap during this stage of thymocyte development has been demonstrated in numerous studies. First of all, the expression of the TCR p chain genes is thought to play a key role in the progression of TN thymocytes to the DP stage based on studies of severe combined immunodeficient (SCID) mice whose T cell development is arrested at the TN stage. Crossing of SCID mice with those mice whose T cells express TCR consisting of the P chain only facilitates the progression of some TN thymocytes to the DP stage (Kishi et al. 1991; Groettrup et al. 1992). Furthermore, the arrest in thymocyte development at the TN stage observed in mice in which the recombinase activation genes RAG-1 and RAG-2 have been inactivated (Mombaerts et al. 1992b; Shinkai et al. 1992) has been reversed by the crossing of RAG-1 knockout mice with mice expressing the transgenic TCR P chain (Mombaerts et al. 1992a). Functional deletion of the TCR a chain, on the other hand, arrests thymocyte development at the DP stage (Philpott et al. 1992; Mombaerts et al. 1992a). Unlike the TCR P chain, termination of die TCR a chain gene rearrangements that occurs during the DP stage is not a consequence of the expression of productive a chain gene rearrangements on the other chromosome, but rather due to the termination of RAG-1 and RAG-2 expression following the engagement of the TCR in DP thymocytes during positive selection (Borgulya et al. 1992; Brandle et al. 1992). TCR ap also plays a key role in the thymic selection process which occurs during the DP stage of development. More specifically, appropriate interaction of TCR with self MHC expressed primarily on thymic epithelial cells facilitates the rescue of DP thymocyte from programmed cell death through a 13 process of positive selection whereas reactivity of TCR against self antigens expressed on bone marrow derived cells leads to clonal deletion via negative selection or tolerance. Positive Selection Post thymic T cell repertoire is thought to arise from a subset of DP thymocytes with TCR binding affinity for the particular MHC molecules expressed in the thymus via a process referred to as positive selection. Those DP thymocytes which express TCR with affinity for either class I or II self MHC on thymic epithelial cells, in the absence of nominal antigen, are rescued from preprogramed cell death to become either CD8 + or CD4 + SP cells respectively. Evidence in support of positive selection has been provided by studies of transgenic animals which have shown that the specificity of the TCR along with the particular MHC expressed by the animal both determine the fate of developing thymocytes. While T cell development is skewed towards the production of CD8 + SP cells in transgenic animals that express both a clors I MHC-specific TCR and the appropriate class I MHC (Teh et al. 1988; Sha et al. 1988b), CD4 + SP cells selectively develop in transgenic animals expressing the class II MHC TCR and appropriate class II MHC (Berg, et al. 1989; Kaye et al. 1989; Murphy et al. 1990). Furthermore, in transgenic mice expressing MHC molecules not recognized by the transgene-encoded TCR, iymocyte maturation from the DP stage to SP cells is blocked (Scott et a/. 1989; Sha et al. 1988a; Berg et al. 1989; Kaye et al. 1992). TCR-MHC interactions, however, are not the only requirement for successful positive selection. The importance of CD4/CD8-MHC interactions in positive selection have been demonstrated in studies where mice, treated from birth with MAb specific for either CD4 or CD8, failed to develop CD4 + or CD8 + SP thymocytes respectively (Zuniga-Pflucker et al. 1989, Ramsdell and Fowlkes, 1989, Zuniga-Pflucker et al, 1990). Evidence for thymic epithelium as the source of MHC mediated positive selection has been provided by a study in which dGuo-treated fetal thymuses from A mice were grafted into thymectomized A X B ¥\ recipients (Lo and Sprent, 1986). Although the thymuses were recolonized with bone marrow derived cells expressing both A-and B-type MHC molecules, the T cells developing in these chimeric thymuses displayed preferential restriction of A-type MHC antigens. Furthermore, a recenv study has shown that intrathymic injection of a cloned thymic epithelium cell line expressing class I MHC induces developing thymocytes to recognize those MHC antigens expressed on the thymic epithelium cell line as self-MHC (Vukmanovic et al. 1992). Two models have been proposed for the positive selection and commitment to the CD4 or CD8 lineage (Robey et al. 1990). The first model, referred to as the "instruction" model, suggests that TCR specificity dictates CD4 or CD8 expression via an instructive signal generated when 14 thymocytes engage self MHC. In this model, positive selection occurs during the double positive stage prior to commitment to either CD4 or CD8 lineages. During positive selection, a DP thymocyte whose TCR is specific for self class I MHC would, for example, receive a signal directing its differentiation to the CD8 lineage. Commitment to the CD4 lineage, on the other hand, would be directed by a different signal generated during selection by class II MHC. Consistent with this model are studies demonstrating the failure of the expression of CD8 coreceptors, encoded by a transgene expressed in all T cells, to rescue CD4 + SP cells with MHC class I restricted receptors from cell death (Borgulya et al. 1991). In further support is a recent study demonstrating that when placed in culture, small CD4 + CD8 + TCR m thymocytes can differentiate further into CD4 + CD8" or CD4" CD8 + thymocytes in the absence of thymic stromal cells indicating that this DP thymocyte population has already recieved signals allowing maturation to SP cells (Swat et al. 1992). The instructive model assumes that two different intracellular signals exist for those DP thymocytes being positively selected on class I versus class II MHC, although the nature of these instuctive signals is elusive. It is possible that the CD4 and CD8 molecules themselves are responsible for the differential signalling. Differences in the properties of p56 l c k when bound to CD4 as opposed to CD8, including the degree of autophosphorylation in vitro and dissociation following protein kinase C (PKC) activation, have indeed been observed (Hurley et al. 1989). Furthermore, a recent study demonstrating higher kinase specific activities of p56 l c k as well as p59fyn in DP thymocytes undergoing positive selection versus nonselecting thymocytes provides evidence for the role of tyrosine phosphorylation in the positive selection process (Carrea et al. 1992). An alternate model, referred to as the "selection" model, suggests that the commitment to CD4 or CD8 lineages is stochastic and precedes positive selection. Therefore positive selection occurs during the single positive stage and requires the coengagement of TCR and CD4 or CD8. Those SP thymocytes with a mismatch between CD4 or CD 8 expression and TCR specificity are unable to be positively selected and thus die within the thymus. Unlike the first model, the intracellular event for both MHC class I and II positive selection are similar in the selection model. Negative Selection Negative selection was initially proposed to provide an explanation as to how the developing immune system learns to distinguish self from non-self. The initial evidence that tolerance to self-antigens occurs during intrathymic development was provided by the demonstration that T cells maturing in fetal thymic organ culture (FTOC) are significantly more reactive against allogenic stimulator cells than against syngenic stimulator cells (Robinson and Owen, 1977). It was 15 subsequently shown that developing T cells are tolerant to self-antigens only in the context of thymically expressed MHC antigens (Dos Reis and Shevach, 1983; Groves and Singer, 1983; Rammensee and Bevan, 1984; Matzinger et al. 1984). Negative selection is thought to involve deletion rather than suppression or functional inactivation of self reactive clones based on studies demonstrating the absence of peripheral T cells expressing a particular Vp TCR product in mouse strains expressing the corresponding stimulating antigen. The C57BR mouse strain, for example, expresses the class II MHC molecule, I-E, and possesses a functional V p i 7 a + gene yet has virtually no peripheral T cells or SP thymocytes, of either CD4 + CD8" or CD4" CD8 + phenotype, expressing V p i 7 a + TCR (Kappler et al. 1987). V p i 7 a + DP thymocytes, however, are numerous in the C57BR strain suggesting that negative selection occurs during the DP ^ .tage of development (Kappler et al. 1987). DP thymocytes have indeed been shown to be rosetted around thymic stromal cells thought to be involved in negative selection (Kyewski et al. 1987). Furthermore, injection of anti-CD4 MAb into mice following irradiation and bone marrow reconstitution inhibited the deletion of CD4" CD8 + cells expressing V p i 7 a , confirming that deletion occurred at the DP stage and required the interaction of CD4 with MHC class II (Fowlkes et al. 1988). Similar results have been obtained for Vpg \ and Vpg products which confer anti-Mlsa reactivity (Kappler et al. 1988; MacDonald et al. 1988). Evidence for the deletion of class I restricted TCR has been provided by studies of transgenic mice expressing both a and p TCR chains from a CTL clone reactive with male H-Y antigen + H-2D^ (Kisielow et al. 1988). Significantly diminished numbers of SP thymocytes were observed in the transgenic male H-2^ mice than in their female littermates. Interestingly, those T cells which escaped the deletion process in the male mice expressed normal levels of the transgenic receptor but expressed low levels of the CD8 molecule. In a second transgenic model, T cells expressing a receptor reactive against the class I molecule were significantly diminished in number in L"+ mice (Sha et al. 1988a). Evidence that negative selection by clonal deletion is primarily mediated by bone marrow derived cells residing in the thymus has been provided by numerous models including radiation chimeras (Ready et al. 1984; von Boehmer and Schubiger, 1984, Widera et al. 1987; van Ewijk et al. 1988), dGuo-treated thymic transplantations (Jenkinson et al. 1985), frog (Flajnik et al. 1985) and chick (Ohki et al. 1987) chimeras. These bone marrow derived cells include dendritic cells expressing both class I and II MHC antigens which are located in the corticomedullary and medullary regions of the thymus, as well as class I MHC expressing macrophages residing primarily in the medulla (Barclay and Mayrhofer, 1981; Jenkinson et al. 1981). Other thymic cells of hemopoietic origin may also participate in negative selection including B cells whose expression of self-antigens is thought to induce both clonal deletion and anergy (Inaba et al. 1991; Mazda et al. 1991; Iwabuchi et al. 1992). Thymic epithelial cells have also been implicated in negative selection. Recent 16 studies have demonstrated that the expression of foreign antigens on thymic epithelial cells in TCR-transgenic mice induce functional inactivation of the corresponding antigen specific T cells (Carlowefo/. 1992; Schonrich et al. 1992; Husbands et al. 1992; Hoffinui. et al. 1992). Clonal deletion of negatively selected DP thymocytes is thought to involve a form of programmed cell death referred to as apoptosis in which membrane blebbing is accompanied by nuclear and cytoplasmic shrinking and DNA fragmentation. Recent studies have been focused on elucidating the signals and genes involved in this apoptotic process. Although crosslinking of the surface protein Fas has been shown to transduce an apoptotic signal (Itoh et al. 1991), the Fas gene alone is not responsible for thymic negative selection since deletion of self reactive T cells is observed in lymphoproliferative (Ipr) mice despite a mutation in the Fas gene (Sidman et al. 1992). Furthermore, the apoptosis-blocking Bcl-2 gene alone is also not sufficient to control negative selection as suggested by studies of transgenic mouse lines expressing high Bcl-2 protein levels in the T lineage (Sentman et al. 1991; Strasser etal. 1991; Siegel et al. 1992). A question remains as to how DP thymocytes distinguish between positive and negative selection signals since both processes involve the interaction of TCR and CD4/CD8 accessory molecules with self MHC. The first model proposed to explain this paradox was based on receptor affinity (Sprent et al. 1988). This affinity model suggests that it is the qualitative differences in the avidity of the interaction between DP thymocytes and thymic stroma or bone marrow derived cells that determine whether positive or negative selection occurs. More specifically, if the avidity, which takes into consideration the density and affinity of the TCR-MHC interactions as well as those of the CD4 and CD8 accessory molecules and adhesion molecules, is low, positive selection occurs whereas high avidity leads to negative selection. Recent studies demonstrating that the fate of thymocytes expressing a particular transgene was altered from positive to negative selection following the introduction of a CD8 transgene (Lee et al. 1992; Robey et al. 1992) provide support for this model. An alternate model (Marrack et al. 1988; Marrack and Kappler, 1988) suggests that thymic selection is "compartmentalized" as a result of the different signals delivered by differential peptide, costimulatory molecule or adhesion molecule expression on thymic epithelial cells as opposed to bone marrow derived dendritic cells. More specifically, recognition of self peptides derived from epithelial cell proteins facilitates positive selection whereas recognition of self proteins derived from dendritic cell proteins is required for negative selection. This model, however, fails to explain why proteins would not be released from the epithelial cells to be taken up by the dendritic cells leading to the induction of tolerance to epithelial peptides as has been previously shown for minor histocompatibility antigens (von Boehmer and Hafen, 1986) and Mis (Pullen et al. 1988). Additional studies demonstrating negative selection by thymic epithelial cells 17 (Carlow et al. 1992; Schonrich et al. 1992; Husbands et al. 1992; Hoffman et al. 1992) and positive selection by thymic APCs under certain circumstances (Bix and Raulet, 1992) provide further evidence against this compartmentalization theory. A third model has been proposed (Finkel et al. 1991) based on the observed differential responses of DP thymocytes to ligation of the TCR. Those DP thymocytes susceptible to negative selection respond to ligation of TCR ap as well as CD3-E with mobilization of calcium (Ca+2) which signals cell death. Those DP thymocytes resistant to tolerance, on the other hand, do not respond to TCH ligation with Ca+2 mobilization perhaps as a result of the uncoupling of the TCR to CD3 as suggested by the absence of C, homodimers in the receptor complex. 1.2.2 E) Single positive thymocytes DP thymocytes that progress beyond the thymic selection stage have continued upregulation of TCR-CD3 accompanied by loss of either CD4 or CD8 depending on whether class I or class II MHC restriction occured during selection. The final maturation process of these CD4 + CD8" or CD4" CD8 + SP thymocytes, which now reside in the medulla and constitute 10-15% of the adult thymus, is gradual and involves the loss of HSA and the acquisition of Qa-2 (Fowlkes and Pardoll, 1989). Further maturation occurs including altered signal transduction through the TCR-CD3 complex as well as the acquisition of antigen responsiveness and cortisone resistance. 1.2.2 F) Single positive T cells Following the acquisition of functional maturity, SP CD4 + CD8" and CD4" CD8 + thymocytes are ready to leave the thymus to colonize peripheral lymphoid tissues including the lymph nodes and spleen as helper and cytotoxic/suppressor T cells respectively. Following activation by the presentation of foreign antigen in the context of the class II MHC, CD4 + SP helper T cells respond by the secretion of soluble mediators which, for example, provide positive signals, to assist in the differentiation of B cells into antibody secreting plasma cells. CD8 + SP T cells, in contrast, assume cytotoxic activity resulting in the destruction of cells presenting antigen in the context of class I MHC by direct cell contact. 1.2.3 Alternate T Cell Differentiation Pathways Although the majority of peripheral T cells express the TCR aP-CD3 complex along with either CD4 or CD8 co-receptors, a distinct T cell population has been identified in a number of peripheral organs which express CD3 associated with either TCR ap or yS heterodimers yet lack 18 both CD4 and CD8. TCR yS + DN T cells have been identified in such peripheral tissues as lymph nodes, spleen (Cron et al. 1988), skin (Stingl et al. 1987) and intestinal epithelia. DN T cells expressing the TCR ap have been recently identified in normal human peripheral blood (Shivakumar et al. 1989; Quaratino et al. 1991), as well as murine bone marrow (Sykes, 1990; Levitsky et al. 1991), lymph nodes (Guidos et al. 1989a), spleen (Levitsky et al. 1991; Prud'homme et al. 1991) and liver (Seki et al. 1991; Ohteki et al. 1992). Although thymic equivalents of these two peripheral CD3 + DN T cell populations have been identified (Lew et al. 1986; Nakanishi et al. 1987; Fowlkes et al. 1987) , neither TCR a p + or y8 + DN thymocytes are thought to follow the conventional T cell differentiation pathway that gives rise to mature SP T lymphocytes. 1.2.3 A) TCR a.p+ DN Lineage TCR a p + double negative (DN) thymocytes constitute 2-30% of the thymic CD4" CD8" subpopulation depending on the mouse strain studied and display a CD3 + HSA" CD5 + CD25" CD44" B220" Qa-2+ NK1.1+ phenotype (Fowlkes et al. 1987; Howe and MacDonald, 1988; Wilson et al. 1988a,b; Levitsky et al. 1991). This thymic subpopulation is thought to be composed of late stage differentiated cells based on studies demonstrating the lack of HSA expression, the relatively late appearance in FTOC and during ontogeny, occuring between birth and day 5 of life in the mouse (Fowlkes et al. 1987) and reaching plateau levels between 6 to 10 weeks of age (Takahama et al. 1991) as well as lack of precursor activity following intrathymic transfer (Crispe et al. 1987). In addition, TCR a p + DN thymocytes express predominantly the products of a single Vp gene family, namely Vpg, in a frequency twofold higher than in SP T cells (Fowlkes et al. 1987; Papiernik and Pontoux, 1990; Takahama et al. 1991). It was therefore suggested that TCR a p + DN thymocytes may be derived from DP thymocytes which, due to potential self reactivity, have escaped clonal deletion by the downregulation of surface expression of their CD4 and CD8 determinants. Evidence in support of this hypothesis was provided by a study of TCR transgenic mice in which the majority of thymocytes and T cells in male mice expressing the transgenic anti-male TCR were of the DN phenotype and expressed high levels of the transgenic TCR but were nonetheless tolerant to male self antigen (Teh et al. 1989). A comparison of TCR + HSA" DN thymocytes expressing Vpg i in Mls-l a and Mls-1^ mice demonstrated similar proportions of this thymic subpopulation in the two strains (Egerton and Scollay, 1990). Vpg i + mature T cells, in contrast, undergo clonal deletion in Mls-1?- mice suggesting an apparent escape of the Vpg \ + DN thymocytes from this selection process. The observed reactivity of Vpg \ + with Mls-l a in association with not only class II MHC but also class I MHC suggested that the TCR Vpg i + DN thymocytes may be derived from thymic DP 19 cells which had downregulated both CD4 and CD8 expression following interaction with both class I and II MHC. Furthermore, the demonstration of demethylation of DNA sequences 5' to the CD8a gene implied that TCR a f i + DN thymocytes are derived from a cell population that had at one time expressed the CD8ct gene locus (Wu et al. 1990; Takahama et al. 1991). Cytosine residues of developmentally regulated genes are often demethylated at the time of first expression and progressive demethylation of several CCGG sites has been observed as T cells develop from immature TN through the DP stage to peripheral SP T lymphocytes (Carbone et al. 1988). However, the demethylation pattern observed was distinct from that of thymic or peripheral CD8 + T cells, suggesting that TCR ap+ DN thymocytes are not derived from SP thymocytes or peripheral T cells that have returned to the thymus and downregulated CD8 expression. Instead the CD8a demethylation pattern was considered as further evidence in support of a DP origin for the TCR ap+ DN thymocyte population. Recent evidence however suggests that TCR ap+ DN thymocytes may not be derived from thymic DP cells but represent a distinct T cell lineage which has branched off from the classical T cell differentiation pathway prior to the DP stage. First of all, in conflict with the suggestion that TCR ap+ DN thymocytes were derived from the down regulation of CD4 and CD8 molecules on potentially self reactive DP thymocytes was the demonstration that the frequency of TCR ap+ DN thymocytes expressing various anti-self TCR including either Mls-l a reactive Vpg and Vpg \ or I-E reactive Vpy and Vp^7a were not increased in those strains expressing their putative self antigen but were either unaffected or significantly reduced (Takahama et al. 1991). Although Vpg overexpression appeared to be independent of the developmental appearance of the basic thymocyte repertoire, TCR ap+ Vpg+ DN thymocytes were also susceptible to deletion by the introduction of the super-antigen staphylococcal enterotoxin B (SEB) into the neonatal differentiation environment. Secondly, following intrathymic transfer of DP thymocytes, no detectable levels of CD3 + DN cells were obtained although SP progeny were observed (Guidos et al. 1989b). With respect to the association of CD8a demethylation with a potential DP thymic origin for TCR ap+ DN thymocytes, it has been recently shown that the proportion of the thymic subpopulation referred to as TN based on FACS analysis actually express low levels of CD8a (Nikolic-Zugic et al. 1989). Furthermore, CD8a methylation has indeed been observed in a portion of the TN thymocytes and may correspond to the CD8^° subset (Wu et al. 1990). Support for this hypothesis has been provided by the study of CD4"^° CD8l° thymocytes which were isolated by anti-CD8a MAb coated plates but were identified by FACs analysis as lacking both CD4 and CD8 (Nikolic-Zugic and Moore, 1989). Although in short term culture, the majority of CD4"^° CD8^° thymocytes spontaneously differentiated into thymic DP cells, a significant number of these cells maintained their DN phenotype and expressed CD3. Therefore demethylated CD8a genes observed in TCR 20 a p + DN thymocytes may be reminiscent of a TN phenotype during a previous step in development. Recent studies have provided support for the TN origin of the TCR a.p+ DN thymocytes (Vissinga et al. 1991; Suda and Zlotnik, 1991; Suda and Zlotnik, 1993). Those TN thymocytes which express CD25 were shown to contain precursor cells for all TCR + subsets found within the thymus including thymic CD3 + DN cells as well as DP and SP thymocytes. When CD25 + TN thymocytes were cocultured in IL-7, a proportion of this thymic population acquired the expression of CD3. Of these CD3 + DN thymocytes, the majority of those which expressed the TCR ap were HSA" CD44 + except for approximately 10% which were HSA" CD44"/10. TCR y 5 + DN thymocytes were also included in the CD3 + DN population and consisted of three major subsets: HSA + CD44", HSA" CD44" and HSA" CD44+ cells. When HSA+ CD44" CD3 + DN thymocytes were cocultured with interleukin-7 (IL-7), a reduction in HSA expression was observed for both TCR a p + and y 5 + subsets whereas stimulation with IL-7 and anti-CD3 gave rise to the HSA" CD44 + phenotype. When CD3 + DN thymocytes were subdivided based on the expression of either of the two types of TCR and subsequently stimulated with IL-7 +/- anti-CD3, the majority of TCR a p + HSA" CD44" DN cells acquired CD44 with IL-7 alone whereas TCR y 8 + HSA" CD44" DN thymocytes required the addition of anti-CD3 to the culture conditions suggesting that the former may have already acquired a TCR mediated signal and were committed to become CD44 + prior to isolation. This preprogrammed differentiation was further supported by the detection of both complete and incomplete TCR p mRNA in the CD25 + TN population. CD3 + DN thymocytes obtained from IL-7 stimulated cultures of CD25 + TN cells were also shown to undergo differentiation similar to that of freshly isolated CD3 + HSA + CD44" DN thymocytes when cocultured with IL-7 and anti-CD3. Therefore it has been proposed that CD25 + TN thymocytes represent a stage in T cell development when those cells destined to become CD3 + DN branch off from the classical differentiation pathway to acquire either TCR ap or y5 and differentiate from HSA + CD44" via HSA" CD44" to the HSA" CD44+ phenotype. It has also been shown that positive selection of TCR a p + DN thymocytes may occur during the transition from HSA + to HSA" phenotype based on Vpg 2 overexpression in the latter population only. As mentioned earlier, peripheral equivalents of thymic TCR a p + DN cells, referred to as DNapT cells, have been identified in a number of tissues including the bone marrow, peripheral blood, lymph nodes, liver and spleen. Whether these peripheral DNapT cells are derived from the phenotypically similar thymocyte population or are a result of an extrat'*ymic differentiation pathway is the subject of current research. Evidence of a thymic origin for at least some of these peripheral DNapT cells has been provided by the detection of a minor population of Thy-1+ CD3 + DN T cells in the lymph nodes and peripheral blood of the irradiated mice following the intrathymic injection of either adult or fetal CD4" CD8" thymic precursors (Guidos et al. 1989). 21 Similar CD5 + HSA" phenotype as well as similar frequencies of Vpg usage were demonstrated between the peripheral DNapT cells of the manipulated mice and their putative TCR ap+ DN thymocytes precursors. Furthermore, although a significant proportion of DNapT cells identified in the bone marrow and spleen of normal mice have been shown to express the NK1 surface antigen, there is a virtual absence of NK1 + TCR ap+ cells in either of the two tissues in C57B10 nude mice (Sykes, 1990; Levitsky et al. 1991). However, following fetal thymic engraftment into nude mice, reconstitution of the peripheral NK1 + TCR ap+ populations occured therefore providing support for the thymic origin of the bone marrow and splenic NK1 + DNapT cells which are also distinct from the thymic independent NK cells. Not all peripheral DNapT cells are thought to be of a thymic origin but rather are the products of an extrathymic differentiation pathway. A significantly higher proportion of bone marrow T cells express the TCR ap+ DN phenotype than in either the thymus or spleen suggesting that the bone marrow is a potential site for extrathymic differentiation (Sykes, 1990; Palathumpat et al. 1992b). Support for the potential extrathymic origin of the bone marrow DNapT cells was provided by the observation of the apparent in vitro conversion of TCR aP" CD4" CD8~ bone marrow cells to the TCR ap+ DN phenotype (Palathumpat et. al. 1992b). Although TCR rearrangement, as detected by PCR analysis of genomic DNA amplified with Vp7 and Jp2-Cp2 primers, was not observed in freshly isolated TCR aP" DN marrow cells, P chain gene rearrangement associated with the conversion of this marrow subset to the TCR ap+ phenotype was observed following 48 hr incubation in tissue culture medium suggesting an apparent maturation of bone marrow T cells in the absence of a thymic microenvironment. In a second study, IL-3 was shown to induce the generation of DNapT cells with predominant Vp2 gene usage from CD4 +/CD8 + depleted bone marrow of both normal and nude BALB/c mice (Kubota et al. 1992). The absence of negative selection of normal bone marrow DNapT cells expressing the self reactive Vp5 receptors in BALB/c mice was also considered to be consistent with the putative lack of dependence of this bone marrow subpopulation on the presence of a mature thymus (Palathumpat et al. 1992b). Studies in which the deletion of T cells expressing self reactive VP receptors did not occur in either nude mice or neonatally thymectomized mice (Hodes et al. 1989; Jones et al. 1990) have implicated a requirement for the development of a mature thymic microenvironment in this selection process. If the hypothesis suggesting an extrathymic origin for bone marrow DNapT cells were true then one would expect the proportion of DNapT cells in the bone marrow in athymic nude mice to be similar to that in euthymic mice. Two studies have demonstrated that the proportion of NK1.1+ TCR ap+ T cells actually decrease in the bone marrow of nude mice in comparison to normal mice (Sykes, 1990; Levitsky et al. 1991). The majority of bone marrow NK1.1+ TCR ap+ cells, however, have been shown to express CD4 and CD8 and therefore the 22 reduction of this population in nude mice can not necessarily be extrapolated to the DN subset. A subsequent study has demonstrated, however, that although the percentage of typical TCRap + CD4+ and CD8+ SP T cells was markedly reduced in the spleens of nude mice, the proportion of DNapT cells in the bone marrow of this athymic murine strain was indeed similar to that of normal adult mice (Palathumpat et. al. 1992b). Similarily, the percentage of splenic DNapT cells are also not reduced in nude mice unlike the mature SP T cells of this tissue. In addition, the spleens of neonatal thymectomized mice also demonstrate a similar percentage of DNapT cells and a significantly reduced number of typical SP T cells in comparison to the normal adult spleen. Furthermore, during the early recovery period following total lymphoid in puliation, the ratio of splenic DNapT cells to typical SP T cells was significantly elevated. These results suggest that DNapT cells may be thymic independent and may be present in the spleen prior to the emigration of typical T cells from the thymus. Whether these splenic DNaPT cells are derived from the phenotypically similar bone marrow cells or have undergone differentiation within the spleen itself has yet to be determined. A recent study has shown that production of DNapT cells could be induced in the spleens of both normal and nude mice by the lymphokine IL-3 (Kubota et al. 1992) The liver has been proposed as an alternate site for extrathymic differentiation of DNapT cells. While a single peak pattern of bright TCR ap and Vpg expression was observed in intestinal epithelia as well as mononuclear cells (MNC) of the lymph nodes, spleen and blood of normal mice, a two peak pattern, more specifically intermediate and bright expression of both TCR ap and Vp was detected in the liver (Seki et al. 1991). Further characterization of such intermediate hepatic cells revealed that although a large proportion of Vpg + T cell with intermediate TCR also expressed either CD4 or CD8, a significant percentage of Vpg + DN cells with an intermediate TCR were also present. Although the intermediate TCR ap cells constitute a small proportion of cells in the liver of young mice (6-8 wks), this population has been shown to significantly expand as a consequence of aging with DNapT cells becoming a predominant phenotype in the older mice (60 wks) (Ohteki et al. 1992). At older ages when the thymus is characteristically involuted, the extrathymic pathway was in its most activated state and the apparent intermediate TCR progeny of this pathway began to appear in the periphery, first in the peripheral blood and then eventually in the spleen and lymph nodes. Confirmation that the liver was a site of extrathymic differentiation was provided by the demonstration of only intermediate TCR cells including DNapT cells in the liver of congenitally athymic nude mice. Furthermore, an age dependent increase in the proportion of this hepatic population was also observed in the nude mice. 23 1.2.3 B) TCR Y8 + DN Lineage A second population of CD3 + DN T cells which express the alternate TCR y8 heterodimers has been identified in numerous tissues including the thymus, lymph nodes, spleen, blood, skin, female reproductive organs, tongue, lung and liver. Although both express the CD3 + DN phenotype, TCR y8 + DN T cells and DNapT cells are two distinct T cell lineages which arise from a common precursor cell. Divergence of these two lineages is thought to occur relatively late in intrathymic development just prior to CD4 and CD8 expression (Petrie et al. 1992). Recent studies have shown that following incubation with IL-7, CD25 + TN thymocytes acquire CD3 expression associated with either the ap or yS TCR heterodimers (Vissinga et al. 1991; Suda and Zlotnik, 1991). IL-7 was also associated with the loss of HSA expression whereas the addition of anti-CD3 MAb induced a CD44 + phenotype (Suda and Zlotnik, 1993). Whether DN thymocytes commit to the y5 or aP lineage depends on the activation of a y chain silencer which acts to silence in-frame as well as out-of-frame rearranged y genes leading to the development of TCR ap+ T cells (Heilig and Tonegawa, 1986; Bonneville et al. 1989). Following expression of the yS heterodimers, TCR y5 + DN thymocytes are thought migrate to peripheral organs without the acquisition of either CD4 or CD8 accessory molecules. TCR yS + T cells can be divided into several subpopulations based on differences in appearance m ontogeny, thymic dependence, V gene usage, junctional variability and tissue localization. Those TCR yS + T cells which disseminate in epithelia, more specifically, the V5 subpopulation in the epidermis of skin (Asarnow et al. 1988) as well as the V6 subset in the mucosal surfaces of the tongue and, vagina and uterus (Itohara et al. 1990) are both generated from precursors in the fetal thymus (Havran and Allison, 1988; Ito et al. 1989). Shortly after birth, the majority of thymic y8 cells express the Vy4 and Vyl genes (Bluestone et al. 1991). Whereas the V4 subset circulate through the blood as well as lymph nodes and spleen, V y l + cells reside in skin and intestinal epithelia in addition to the spleen. A minority of TCR y8 + T cells arising from the postnatal thymus express either Vy2 or Vy7 genes. However, not all TCR y8 + T cells are thymic dependent. One TCR y8 + subset, thought to arise in the intestinal epithelia shortly after birth (Bandeira et al. 1991), has been characterized by predominant Vyl and Vy7 usage, multiple Vy chain expression and high junctional diversity. Furthermore, similar to thymic independent aP T cells, the majority of cells from this yS subset express CD8a homodimers but lack Thy-1 (Guy-Grand et al. 1991). Support for the thymic independence of the V7 T cells has been provided by the observation of this y8 subset in the intestines of athymic nude mice as well as lethally irradiated mice reconstituted with syngeneic bone marrow (Bandeira et. al. 1991; Guy-Grand et al. 1991). It has been 24 suggested that the order of appearance in ontogeny may be due to preprogrammed rearrangement of the specific Vy and Vg gene segments (Raulet et al. 1991). 1.3 T CELL ACTIVATION Interaction of the TCR with degraded fragments of antigen presented in the context of MHC is considered to be the central event in the initiation and propagation of immune responses toward foreign antigens. Crystallographic analysis of several class I MHC molecules has revealed N-terminal domains consisting of two long parallel a helices supported on a platform of p strands creating a groove in which the site of antigen binding is contained (Bjorkman et al. 1987; Madden et al. 1991). An extra density representing an antigen fragment has indeed been detected within this groove. Although a high resolution structure of the class II MHC molecule has not yet been determined, similarities to the class I MHC structure are expected based on sequence comparisons (Brown et al. 1988). The peptide within the MHC groove is thought to interact with the CDR3 loops of the TCR whereas as the flanking a helices of the MHC molecule coinact TCR CDR1 and CDR2 loops (Davis and Bjorkman, 1988). Also important for T cell recognition and activation is the interaction of MHC on APC with CD4 and CD8 accessory molecules. Initial evidence for the participation of CD4 and CD8 in T cell function was provided by studies demonstrating the inhibition of TCR mediated responses to class II or class I restricted antigens by antibodies directed against either CD4 (Nakayama et al. 1979; Dialynas et al. 1983) or CD8 (Reinherz et al. 1981; Hollander, 1982 ). Therefore it was proposed that CD4 and CD8 were involved in the augmentation of T cell recognition by binding to monomorphic determinants on class II and class I MHC molecules respectively. The subsequent identification of a lymphocyte specific tyrosine kinase noncovalently associated with the cytoplasmic tails of CD4 and CD8 as well as the demonstration of an increase in tyrosine phosphorylated substrates in mature T cells following CD4 or CD8 crosslinking (Veillette et al. 1989; Luo and Sefton, 1990) suggested a possible role for these accessory molecules in the TCR signal transduction pathway (Veillette et al. 1988; Barber et al. 1989; Turner et al. 1990). As a consequence of occupancy of the T cell receptor by antigens presented in the context of MHC, two signal transduction pathways, namely protein tyrosine kinase and phospholipase C (PLC) pathways, are activated giving rise to a cascade of intracellular biochemical changes including protein phosphorylations, cytoplasmic alkalinization and fluxes in ions and cyclic nucleotides. Furthermore TCR signal transduction activates a regulated cascade of sequential gene activation events, including the induction of expression of IL-2 and its corresponding receptor which ultimately lead to the proliferation and aquisition of effector functions by the T cells. 25 1.3.1 TCR Signal Transduction Pathway Two signal transduction pathways have been associated with TCR stimulation by antigens, mitogen lectins or anti-TCR and anti-CD3 MAb, namely the activation of the protein tyrosine kinase and phospholipase C pathways. 1.3.1 A) Protein Tyrosine Kinase Pathway The first signalling event thought to occur following activation of the TCR involves an increase in the tyrosine phosphorylation of several proteins (June et al. 1990a). With the use of anti-phosphotyrosine MAb to immunoblot lysates of stimulated cells, an increase in tyrosine phosphorylation activity was observed within 5 sec. following TCR activation and prior to the detection of increased PLC activity. Furthermore, tyrosine phosphorylation appears to be required for the activation of the PLC pathway based on studies demonstrating the inhibition of PLC activation and subsequent events following cellular stimulation by tyrosine kinase inhibitors (Mustelin era/. 1990; June et al. 1990b). Although the TCR lacks intrinsic PTK function, interactions with several cytoplasmic PTKs have been documented. The two src family PTKs fyn and Ick have, for example, been implicated in this inital signalling event. TCR does indeed coimmunoprecipitate with fyn although the stoichiometry of this association appears to be low (Samelson et al. 1990). Furthermore, overexpression of fyn in transgenic mice (Cooke et al. 1991) renders T cells hyper-responsive to TCR stimulation whereas a hyporesponse was observed for those mice expressing the kinase defective form suggesting therefore a possible role for fyn in TCR mediated signal transduction. The second src family candidate, Ick, is directly associated with the cytoplasmic tail of the CD4 and CD8 coreceptors (Veillette et al. 1988; Barber et al. 1989; Turner et al. 1990). Similar to fyn, hypersensitivity to TCR stimulation has also been observed following the overexpression of the activated form of Ick in a T cell hybridoma (Abraham et al. 1991). Furthermore, a defect in signalling through the TCR in T cell mutants which lack Ick expression can be restored by reexpression of the PTK (Strauss and Weiss, 1992; Karnitz et al. 1992). As members of the src family PTKs, both fyn and Ick share a src homology (SH)-2 domain capable of binding phosphotyrosine residues (Koch et al. 1991) and a carboxy-terminal tyrosine residue, which when dephosphorylated is activated (Cantley et al. 1991). CD45 is thought to be the phosphatase responsible for the activation of both Ick and fyn as suggested by the in vitro activation of these PTKs by CD45 (Mustelin et. al. 1989; Mustelin et al. 1992) and the increased phosphorylation of 26 Ick on tyrosine 505 in CD45" mutants (Ostergarrd et al. 1989). Recently, a third possible candidate for PTK activity in TCR signal transduction has been identified. Found not only in T cells but also NK cells, ZAP-70 has been shown to associate with the TCR subsequent to receptor stimulation (Chan et al. 1992). An association of ZAP-70 with either fyn or Ick has been suggested by the apparent requirement of the expression of these two src family PTKs for the interaction of ZAP-70 with C, in cotransfection studies with COS cells. An additional PTK implicated in TCR signalling is the IL-2 inducible T cell kinase Itk (Siliciano et al. 1992). Unlike the src family PTKs, ZAP-70 and Itk lack the negative regulatory carboxy terminal tyrosine residues and the inner membrane anchormg-amino-terminal myristoylation sites (Desiderio, 1993). A number of substrates for tyrosine phosphorylation following TCR stimulation have been identified. Phosphorylation of the TCR C\ chain, for example, has been observed following TCR stimulation (Samelson et al. 1986). Murine and human C\ chains include either 6 or 7 potential sites for tyrosine phosphorylation respectively and immunoprecipitation analyses have identified several forms of phospho-^, suggesting a complex pattern of phosphorylation (Klausner et al. 1987; Irving, and Weiss, 1991). The association of CD4 with TCR is thought to be a requirement for effective tyrosine phosphorylation of the C\ chains possibly due to the delivery of the PTK Ick to the receptor (Dianzani et al. 1992). Other phosphoproteins which have been identified include vav (Margolis et. al. 1992), valosin containing protein (Egerton et al. 1992a), ezrin (Egerton et al. 1992b) and CD5 (Davies et al. 1992). The ability of PTK inhibitors to block TCR induced PLC activity has provided an additional substrate for tyrosine phosphorylation. TCR stimulation induces tyrosine phosphorylation of PLC-yl which can be blocked by PTK inhibitors (Weiss et al. 1991). 1.3.1 B) Phospholipase C Pathway Activation of the phosphatidylinositol (PI) pathway was the first TCR-regulated signal transduction event to be identified (Imboden and Stobo, 1985). Activation of PLC by tyrosine phosphorylation following TCR stimulation leads to the hydrolysis of the phosphodiester bond of the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2) (Rhee et al. 1989). The end products of this reaction, inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) function as second messengers facilitating the mobilization of cytoplasmic free calcium [Ca +^]j and activation of protein kinase C (PKC) (Berridge and Irvine, 1989). Activation of this PI pathway is thought to contribute to the production of IL-2 based on studies demonstrating the ability of phorbal ester and calcium ionophore reagents, which activate PKC and increase [Ca +^]j respectively, to induce IL-2 production and T cell proliferation (Weiss et al. 1984b; Truneh et al. 1985) while agents which 27 block PKC activation and [Ca + 2]j increase inhibit production of this lymphokine (Weiss et al. 1984a; Nel et al. 1987). Furthermore, elements within the regulatory region of the IL-2 gene have been identified as being responsive to activation of PKC and increases in [Ca + 2]i (Crabtree, 1989). Although the mechanisms for the transduction of the initial biochemical signals, namely PKC activation and increased [Ca + 2]i, to the nucleus remain to be elucidated, a number of potential intermediate targets have been identified. Activation of the ras family of G proteins occurs within minutes of TCR stimulation as well as in the presence of phorbal esters suggesting that an apparent dependence on PKC activation (Downward et al. 1992). Inhibition of ras -mediated GTP hydrolysis is thought to be involved in the TCR activation of this G protein. Recent studies have suggested a role for ras in the regulation of TCR induced transcription of the IL-2 gene. While IL-2 promoter activity was enhanced by the expression of an activated form of ras, this activity was significantly inhibited by the dominant negative form of ras, Ha-rasN17 (Rayter et al. 1992). Additional downstream events dependent on PKC activation include the stimulation of a number of serine/lhreonine protein kinases. Phosphorylation on serme/lhreonine residues and subsequent increase in activity has been observed for both the c-raf proto-oncogene as well as the ribosomal protein S6 kinase in response to TCR crosslinking or treatment with phorbal esters (Siegel et al. 1990; Calvo et al. 1992). Furthermore microtubule-associated protein-2-kinase (MAP-2K) is activated within minutes of TCR stimulation and is dependent on both a serine/threonine kinase such as PKC as well as a tyrosine kinase such as Ick (Nel et al. 1990). It has been proposed that raf can activate the MAP kinases which in turn stimulate the 90 kDa S6 kinase (Howe et al. 1992). Therefore these three kinases may function via a single pathway which may be involved in lymphokine gene expression since studies have demonstrated that MAP kinases accumulate in HeLa cell nuclei following serum stimulation and are capable of phosphorylating a number of transcription factors (Chen et al. 1992). Inositol 1,4,5-triphosphate mediated increase in [Ca + 2]i is thought to be involved in lymphokine gene expression via the activation of calmodulin regulated enzymes. A calcium/calmodulin dependent serine/threonine phosphatase, calcineurin, has been identified as a result of an investigation into the action of the immunosuppressant drugs cyclosporin A and FK506. Both the overexpression of calcineurin or the expression of the activated form have been shown to enhance IL-2 gene inducibility (O'Keefe et al. 1992; Clipstone and Crabtree, 1992). Therefore the increase in [Ca + 2]j following activation of the PI pathway may be involved in the stimulation of calcineurin phosphatase activity, the substrate for which may be the cytosolic component of the transcription factor, NF-AT, which regulates the expression of the IL-2 gene (Flanagan et al. 1992). 28 1.3.1 C) IL-2 Gene Activation Following the activation of the TCR signalling pathway, T cells proliferate and differentiate to effector functions. The critical event involved in this response is the production of the T cell growth inducing cytokine, IL-2. Activation of IL-2 gene expression is thought to involve a sequential series of transcriptional events based on the requirement for protein synthesis and the delay in the initiation of transcription. Induction of IL-2 promoter activity by immediate-early genes is thought to be one such event. The induction, within rninutes of TCR activated signal transduction, of transcription of several immediate-early genes involved in the regulation of T cell proliferation and lymphokine expression including c-fos and c-myc has indeed been observed. It is the products of such immediate-early genes which are considered to facilitate the production and activity of factors required for lymphokine transcription. Post-translational modifications involving serine/threonine kinases and phosphatases may also regulate the activity of the transcription factors required for IL-2 gene expression. One transcription factor associated with IL-2 expression, AP-1, is composed of the c-fos and c-jun protein family and is subject to regulation by phosphorylation (Hunter and Karin, 1992). In addition, promoter activation by NF-AT, AP-3 and NF-KB may be blocked by treatment with cyclosporin A and FK506 which inactivate the serine/threonine phophatase, calcineurin (Schreiber and Crabtree, 1992). Expression of the IL-2 gene produces a 15.5 kDa glycoprotein consisting of 133 aa organized in anti-parallel a helices (Bazan, 1992) which recognizes three different forms of the IL-2 receptor with differing affinities. Three critical IL-2 receptor subunits have been identified, namely IL-2Ra, IL-2RP and IL-2Ry. Identified by the anti-Tac MAb, IL-2Ra is a 55 kDa glycosylated protein (Leonard et al. 1984). A second 70/75 kDa IL-2 binding protein, referred to as IL-2RP was identified by crosslinking with radiolabelled IL-2 (Tsudo et al. 1986). Recently, a third IL-2 receptor subunit, the 64 kDa y chain has been identified (Takeshita et al. 1992). When expressed alone, neither IL-2Ra nor IL-2RP demonstrate significant binding to IL-2. In contrast, intermediate binding affinity has been observed when both IL-2RP and IL-2Ry are coexpressed, such as on large granular lymphocytes. Cells which express all three subunits as a noncovalently associated complex, including activated T cells, demonstrate high affinity binding. Two alternative signalling pathways have been associated with the occupation of the IL-2 receptor (Shibuya et al. 1992). The first pathway involves the induction of the c-myc gene via an unknown mechanism and enables T cells to enter the S phase of cell cycle. In the alternate pathway, c-fos and c-jun induction occurs following activation of cytoplasmic protein tyrosine kinases. Following the production of IL-2, T cells are induced to proliferate as well as differentiate to effector function. More specifically, IL-2 29 augments the production of cytokines involved in T cell functions including IL-4 and IFNy. Furthermore, IL-2 facilitates the differentiation of CD8 + SP T cells into cytotoxic effector cells. 1.3.2 Accessory Receptor Signalling Pathways Although TCR signal transduction plays a key role in the activation of several transcription factors involved in lymphokine gene expression, TCR mediated signals on their own are not sufficient to induce lymphokine production and in fact often induce anergy. An increasing number of accessory receptors are now thought to also be involved in T cell activation. One possible accessory receptor providing assistance to the TCR during T cell activation is the 44 kDa glycoprotein member of the Ig supergene family, CD28 (June et al. 1990c). Approximately 50% of CD8 + T cells and virtually all CD4 + T cells express this costimulatory molecule. CD28 is thought to play a significant role in T cell activation although the signals delivered by this accessory molecule appear to be distinct from those provided by the TCR itself. Activation of CD28 has been shown to enhance proliferation as well as IL-2 production by anti-TCR or mitogen stimulated T cells in the absence of enhanced phospholipid hydrolysis. Furthermore, this CD28-induced expression of IL-2 is resistent to cyclosporin A suppression. CD28 crosslinking does however induce tyrosine phosphorylation activity (Vandenberghe et al. 1992). In addition to the upregulation of IL-2, CD28 activation has been shown to increase mRNA levels and secretion of other lymphokines including IL-3, IFNy, TNF-ct, GM-CSF and lymphotoxin (Thompson et al. 1989). Therefore the primary role of CD28 appears to be the regulation of the amount of lymphokine produced by T cells via an increase in mRNA half-life. In the absence of CD28, mRNA is labile and subject to rapid degradation such that little if any is secreted. Activation of CD28 leads to the stabilization and enhanced translation of lymphokine mRNA. A cell surface ligand for CD28, referred to as B7/BB1 was identified initially on activated B cells and subsequently on activated monocytes and dendritic cells (Linsley et al. 1990, Freeman et al. 1989; Larsen et al. 1992). Cocculturing of B7-transfected Chinese hamster ovary cells (CHO) or B7-Ig fusion protein with suboptimally stimulated T cells leads to increased proliferation and expression of IL-2 mRNA (Linsley et al. 1991) Furthermore, addition of MAb specific for either B7 or CD28 inhibit T cell responses including alloantigen-induced T cell proliferation and cytotoxicity (Azuma etal. 1992). Additional accessory receptors implicated in T cell activation include lymphocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) which are required for the adhesion between T cells and their corresponding antigen presenting cell (APC). Several of 30 the adhesion molecules including CD2, CD44, LFA-1 and very late antigen-4 (VLA-4) are also capable of mediating transmembrane signalling events. MAb specific for HSA expressed on murine B cells has been shown to suppress the costimulatory activity of the APC although the identity of the ligand for HSA on T cells remains to be identified (Liu et al. 1992). 1.4 T CELL FUNCTION Although both CD4 + and CD8 + SP T cells have been shown to perform key helper, suppressor and cytotoxic roles, there is increasing evidence to suggest that both subpopulations of CD3 + T DN cells, namely TCR a p + and y5 + DN T cells, also contribute to the immune response to foreign antigen. 1.4.1 CD4+ SP Helper T Cells Following the recognition of foreign antigen presented in the context of class II MHC, CD4 + SP T cells acquire the ability to regulate both B and T cell activity through the delivery of "helper" signals. The CD4 + SP population, however, is heterogeneous and can be further subdivided based on the pattern of cytokine secretion following receptor activation. Two major subsets of CD4 + SP helper T cells have been identified, namely Tj^l and Tjj2 which mediate distinct immune responses. Recently it has been shown that the cytokine environment is a major determinant of which type of TJJ cell is activated during an immune response. More specifically, interferon y (IFNy) drives naive CD4 + SP T cells towards a TJJI phenotype (Scott, 1991) whereas 11-4 plays a key role in the differentiation of the T^2 subset ( Seder et al. 1992). T j j l cells have been shown to produce IL-2 and IFNy whereas Tj-r2 cells secrete IL-4 and IL-5. Other lymphokines including GM-CSF and IL-3 are the products of both types of helper T cells. Of the lymphokine signals delivered by CD4+ SP T cells, IL-4, IL-5 and IFNy are known to have selective activities on the induction of specific Ig isotypes. While IL-4, selectively enhances IgGi and IgE responses to LPS (Coffman et al. 1986; Snapper and Paul, 1987), IL-5 and IFNy preferentially enhance polyclonal B cell IgA (Coffman et al. 1987) and IgG2a (Snapper and Paul, 1987) responses to the antigen respectively. Furthermore the ability of T j j l and TJJ2 cells to induce IgG2a and IgGi responses respectively in TNP specific B cells has been confirmed (Stevens et al. 1988). IL-2, in contrast has been shown to play a significant role in the activation and differentiation of mature cytotoxic T lymphocyte (CTL) effector cells from their precursors (Nabholz and MacDonald, 1983). 31 1.4.2 CD8+ SP Cytotoxic T Cells The effector function of CD8 + SP T cells involves the killing of target cells which bear antigen in the context of class I self MHC. Following recognition of antigen by the TCR, intracellular signals result in the induction of possibly several different cytotoxic mechanisms. Two major alternatives for CD8 + SP T cell effector function include target cell death triggered either by the loss of plasma membrane integrity and therefore ion imbalance due to perforin induced pore formation (Ortaldo et al. 1992) or by CTL mediated signalling of target cell receptors leading to cell disintegration (Berke, 1991). Recently, the Fas gene has been implicated in CTL mediated triggering of programmed target cell death (Rouvier et al. 1993). 1.4.3 SP Suppressor T cells Suppressor T cells (Tg) are heterogeneous group of T lymphocytes which are thought to include both conventional and modified Thl, Th2 and CTL subsets as well as T cells specialized for suppression. A number of mechanisms have been proposed for T cell mediated immunosuppression including the release of inhibitory lymphokines including tumour growth factor-P (TGF-p), IFNy and IL-10, secretion of soluble antigen-specific suppressor factors encoded at least in part by TCR a and/or p chain genes, cytolysis and delivery of a non-cytolytic negative signal via direct contact with the target cell (Murphy, 1993). 1.4.4 TCR ap +DNT Cells Based on the late appearance in ontogeny, the absence of precursor activity and the ability to respond to lectins and anti-TCR ap MAb (Ceredig et al. 1987), TCR ap+ DN T (DNapT) cells are considered to be a mature population of T cells. Although the role of DNapT cells in the immune response has yet to be definitively established, a number of possible functions have been proposed. One potential role for DNapT cells in the immune response is that of a suppressor function. Numerous studies have demonstrated that normal adult bone marrow cells are capable of suppressing the proliferative response towards mitogens of normal spleen cells as well as the mixed leukocyte reaction (MLR) regardless of the stimulator and responder strain combinations employed. Characterization of the bone marrow cells capable of mediating this suppressive 32 function has revealed a "null" lymphocyte function, more specifically Thy-1" Ig" Mac-1" (Weingenberg et al. 1984; Holda et al. 1986) and a similarity in phenotype and function to natural suppressor cells identified in the neonatal mice or adult mice exposed to total lymphoid irradiation or suffering from chronic graft versus host disease (GVHD) (Oseroff et al. 1984; Maier et al. 1986). When murine bone marrow cells were further fractionated by Percoll density gradients, those cells comprising the low density fraction were also found to be enriched for natural suppressor activity and were capable of inhibiting MLR (Benveniste et al. 1990; Palathumpat et al. 1992b). Furthermore, this low density fraction of murine bone marrow cells was also capable of inhibiting GVHD (Palathumpat et al. 1992a) Interestingly, the low density bone marrow fraction was found to be enriched in cells with the TCR a.p+ CD4" CD8" phenotype. When bone marrow DNapT cells were further purified by sorting, inhibition of MLR as well as GVHD was again observed. The ability to inhibit both MLR and GVHD has also been observed with cloned cell lines of the DNapT phenotype derived from the spleens of neonatal or adult mice recovering from either irradiation or bone marrow transplantation (Hertel-Wulff et al. 1984; Schwadron et al. 1985; Strober et al. 1987; Sykes et al. 1990a). Furthermore, suppression of acute GVHD was detected in the low density fraction of normal murine spleen cells and was correlated with the depletion of typical SP T cells and the enrichment of DNapT cells (Palathumpat et al. 1992a). Lethal GVHD suppression was also performed by DNapT cells obtained from the spleens of Mls-1° mice following immunization with Mls-l a cells (Bruley-Rosset et al. 1990). In further support of a suppressor like function for DNapT cells, consistent increases in splenic CD3 + CD4" CD8" cells have been demonstrated in animals receiving GVH-inducing inocula (Abraham et al. 1992). The percentage of this splenic subpopulation was further enhanced when the recipients of the allogeneic inocula were treated with IL-2. Early administration of IL-2 following bone marrow transplantation has indeed been shown to provide potent prophylaxis against GVHD mortality without preventing alloengraftment or reducing graft versus leukemia effects (Sykes et al. 1990b,c). A role for DNapT cells in cytotoxic response and allogeneic tumour rejection has also been suggested. CTL with the TCR a p + CD4" CD8" phenotype were generated in mixed lymphocyte tumour cell culture (MLTC) spleen cells from anti-CD8 MAb treated B6 mice (Mieno et al. 1991). It was suggested that when CD8 + cells were blocked by anti-CD8 MAb, CD4 + cells were capable of mediating tumour rejection by the induction of DNapT cells. Similarily, rejection of an I A + variant line of FBL-3 leukemia in a CD8 depleted syngeneic B6 mice was also mediated by DNapT cells as well as CD4 + CTL generated by the activation of CD4 + helper cells (Yoshimura et al. 1993). Furthermore, IL-2 dependent T cell lines of the DNapT phenotype with non-MHC-restricted cytolytic activity against tumour targets, syngeneic and allogeneic fibroblasts and 33 phytohemagglutinin (PHA) blasts from allogeneic donours have been developed from the peripheral blood of a patient with combined immunodeficiency and tissue injury resembling GVHD who had a massive expansion of this cell type (Brooks et al. 1990). When activated by mitogens or phorbol myristate acetate (PMA) and IL-1, these cell lines were capable of producing IFNy as well as tumour necrosis factor-a (TNF-a) and -p. A number of studies have also implicated DNapT cells with the development of autoimmune disease. A marked expansion of DNapT cells has been observed in the peripheral blood of patients with the autoimmune disease systemic lupus erythematosus (Shivakumar et al. 1989). Along with CD4 + SP T cells, DNapT cells are responsible for the induction of oligoclonal B cell production of highly cationic Ig anti-DNA autoantibodies. In addition, TCR ap+ CD4" CD8" B220+ Pgp-1+ cells of which a high percentage are Vpg+, have been shown to accumulate in the massively enlarged lymph nodes and spleen of the autoimmune lymphoproliferative MRL-lpr/lpr mice (Wofsy et al. 1984; Nemanzee et al. 1985). Strains which carry the mutant autosomal recessive gene, Ipr, spontaneously develop a severe and rapidly progressive autoimmune disease characterized by the production of a variety of autoantibodies, immune complex-mediated glomerulonephritis and vasculitis in addition to the massive lymphadenopathy (Andrews et al. 1978; Theofilopoulos and Dixon, 1981). The thymus is thought to play a role in the acceleration of this disorder based on studies which have shown that neonate! thymectomy reduces lymphadenopathy and the autoimmune syndrome (Steinberg et al. 1980) whereas a recurrence of the lymphadenopathy occurs following thymic grafting into the neonatal thymectomized Ipr mice irrespective of the thymic genotype (Theofilopoulos et al. 1981). It has been lecently shown that the liver is a major site for the proliferation of the abnormal ap-T cell population based on the demonstration by in vivo [^ H] tritiated mymidine injection of hepatic DNapT cell migration to the periphery with subsequent lymphadenopathy (Ohteki et al. 1990). Furthermore, the abnormal ap-T cells in Ipr mice are thought to be a counterpart of the normal T cells in the liver based on similarities with respect to the TCR ap+ CD4" CD8" Pgp-1+ phenotype, intermediate TCR expression and Vpg expression (Seki et al. 1991). In a subsequent study it was shown that the increased expansion in the liver of DNapT cells is a common feature of other autoimmune mouse strains including C3H/HeJ-gld/gld, BxSB, NOD, MRL-+/+ and NZB/W Fi mice (Masuda et al. 1991). Furthermore, the accumulation of this abnormal DNapT cell population was also age dependent. Additional evidence for a potential role of DNapT cells in the development of autoimmune disease was provided by a study in which the neonatal thymectomy of CBA/H mice resulted in a marked transient increase in CD3 + CD4" CD8' cells in the peritoneal cavity which selectively overexpressed the products of the TCR Vpii gene family (Andreu-Sanchez et al. 1991). Intraperitoneal inoculation of an IL-2 vaccinia virus construct which provides high titers of 34 human IL-2 in vivo, induced the conversion of this DN population to either CD4 + or CD8 + SP T cells and provoked autoimmune manifestations including anti-DNA antibodies, rheumatoid factors and interstitial nephritis. An apparent capacity for autoreactivity has also been demonstrated in splenic DNapT cells of normal mice (Prud'homme et al. 1991). Although DNapT cells were unresponsive in MLC, following activation with either concanavalin A (Con A) or anti-TCR ap antibodies this splenic subpopulation acquired CD4 expression and high reactivity in both syngeneic and allogeneic mixed leukocyte culture (MLC) which could be blocked by anti-CD4 antibodies. 1.4.5 TCRyS + T Cells The y8 T cell receptor has been shown to recognize a diverse selection of antigens. Such antigens include both classical and non-classical MHC molecules (Matis et al. 1987; Bonneville et al. 1989b), bacterial antigens including mycobacterial heat shock proteins (Born et. al. 1990b) and superantigens such as staphylococcal enterotoxins (Rust et al. 1990), and numerous self antigens including the P2-microglobulin-associated human CD1 molecules (Porcelli et al. 1989), cell surface immunoglobulin (Wright et al. 1989) and autologous heat shock proteins (Born et al. 1990a). The ability of TCR y 8 + T cells to display specificity for conventional MHC antigens has been suggested by the overlap in specificity thought to occur between TCR y8 and TCR ap cells as a result of the interspersal of V a and V 5 elements in the germline (Raulet, 1989) as well as by the detection of known V a elements which are rearranged to DJ5 to encode functional yS receptors (Elliot et al. 1988). TCR y 8 + T cells specific for both conventional class I (Bluestone et al. 1988; Rivas et al. 1989) and class II MHC molecules (Matis et al. 1989; Rellahan et al. 1991) have indeed been isolated. Not all TCR yS + T cells are thought to be restricted by conventional MHC molecules as suggested by an essentially normal development of TCR yS + T cells observed in either MHC class II or P2-microglobulin deficient mice (Correa et al. 1992; Bigby et al. 1993). Alternatively, TCR y 5 + T cells may display specificity for non-classical MHC antigens. Human TCR y 8 + cells isolated from peripheral blood have been shown, for example, to be specific for CD1 molecules (Porcelli et al. 1989). These non-MHC linked CD1 antigens demonstrate limited homology to class I MHC molecules and are expressed in noncovalent association with P2 microglobulin. Furthermore, Lam et al. (1990) have isolated human TCR y 8 + cell lines specific for autologous non-classical human leukocyte antigen (HLA)-like molecules serologically related to HLA B and C. Whether non-classical class I MHC molecules play a prominent role in antigen presentation to TCR y 8 + cells, however, remains to be determined. 35 TCR y5 + T cells are thought to play a key role in infectious diseases. T cells expressing the y8 heterodimers have been shown to accumulate in numerous tissues following bacterial infection including in the peritoneal cavity of mice infected with Listeria monocytogenes (Hiromatsu et al. 1992), in the draining lymph nodes of mycobacteria infected mice (Janis et al. 1989), and in the skin lesions of patients infected with the tuberculosis form of leprosy (Modlin et. al. 1989). Protozoan infections have also been associated with TCR y8 + T cell accumulation including that observed in the spleens of mice infected with Trypanosoma cruzi and Plasmodium chabaudi (Minoprio et al. 1989) as well as in the skin lesions of patients with cutaneous leischmaniasis (Falini et al. 1989). TCR y8 + T cell accumulation has also been demonstrated in viral infections including the lungs of mice infected intranasally with influenza virus (Eichelberger et al. 1991), and in the blood of human immunodeficiency virus (HIV) infected acquired immunodeficiency syndrome (AIDS) patients (Autran et. al. 1989; De Paoli et al. 1991) and during the acute phase of Ebstein Barr virus (EBV) infection (De Paoli et al. 1990). An involvement of TCR yS + T cells in immune surveillance against cancer has been suggested by the ability of this T cell subset to kill tumour cells following activation with IL-2. Subsets of TCR y8 + cells have also been shown to recognize EBV transformed B cells (Hacker et al. 1992) as well as superantigen expression on Burkitt lymphoma cells (Sturm et al. 1990). Furthermore, autologous tumour cells have been recognized by TCR y8 + cells obtained from patients suffering from Burkitt lymphoma as well as from acute lymphoblastic leukemia in complete remission (Bensussan et al. 1989). Class II MHC dependent lysis of autologous tumour cells has also been shown to be mediated by TCR yS + T cell lines derived from tumour infiltrating lymphocytes (Zocchiera/. 1990). TCR yS + T cells have also been implicated in a number of pathological immune responses. Although TCR y8 + cells of the VI subset represent a relatively minor percentage of circulating y8 cells in healthy individuals, significant numbers of these cells have been detected in the synovial fluid of patients with rheumatoid arthritis (Holoshitz et al. 1989). While V81 cells have been shown to accumulate both in intestinal lesions of patients with coeliac disease (Rust et al. 1992) as well as in the blood of a patient with the type 1 autoimmune polyglandular syndrome associated with aplastic anemia (Hara et al. 1990), an expansion of V82 cells has been observed in the blood of an atopic dermatitis patient (De Paoli et al. 1990) as well as in brain tissue from multiple sclerosis patients (Hvas et al. 1992). 36 TCR y8 + T cells have also been shown to accumulate in a number of immunodeficiency disorders including ectodermal dysplasia syndrome, bare lymphocyte syndrome, partial DiGeorge syndrome, common variable immunodeficiency (Seki et al. 1989), Wiskott Aldrich syndrome (Morio et al. 1990) and ataxia telangiectasia (Carbonari et al. 1990). 37 1.5 Thesis Objectives Although thymic maturation involves the aquisition of the TCR along with the CD4 and CD8 co-receptors, not all thymocytes are thought to follow this conventional T cell pathway. Indeed subpopulations of thymocytes which express either the a.p or y5 TCR heterodimers yet lack CD4 and CD8 have been identified and are thought to be equivalent to the phenotypically similar cells residing in a number of peripheral organs. Furthermore both TCR cq3+ and y8 + DN T cells are thought to be functionally mature and their accumulation in tissues have been correlated with pathological immune responses including several autoimmune disorders. The objective of this thesis was to characterize the TCR cq3+ DN (DNcq3T) cell population identified in the spleen of normal mice. Splenic DNapT cells were characterized with respect to their surface phenotype and preferential usage of Vp TCR genes. Furthermore, the ability of splenic DNapT cells to respond in the presence of cytokines, MAb which crosslink the TCR as reagents which bypass the TCR and activate intracellular events was also assessed. Finally the function of this splenic DNapT cell population was also studied specifically with respect to lymphokine production. The purpose of these studies was to provide support for the hypothesis that splenic DNapT cells represent a unique class of mature T cells which are related to the phenotypically similar cells found in the thymus and are distinct from single positive mature T cells. 38 Periphery a T C R y6+ CD4" CD8" Bone Marrow Thymus T C R - C D 4 L 0 C D 8 -CD44- CD25" o TCR" CD4" CDS' C D 4 4 + cKi t + TCRop + CD4-CDS- * % o TCR" C D 4 1 0 CD8" CD44+ CD25" 6 TCR' C D * " CD8" C D 4 4 + CD25" 0 TCR" CD4" CD8" CD44+ C D 2 5 + 1 - o — o — o — a T C R T 8 + C D 4 " C D 8 " T C R " C D 4 " C D 8 T C R o p * CD4' CDS' T C R a B + CD4" CD8' C D 4 4 + C D 2 5 " CDU-CD25 CD44" CD25" H S A + CD44+CD25" HSA" 1 o TCR" CD4- CD8" CD44" CD25" TCR" CD4" C D 8 1 0 CD44"CD25" Poalive md Negative Selection T C R op1 oph ia > 4 + C D 8 -T C R af)1" CD4" C D 8 + \Spleen b TCRaP* CD4" CD8" - e x T C R t i P + C D 4 + C D 8 - * % Periphery a T C R o P + C D 4 - a M + Figure 1 Schematic overview of the thymic differentiation pathway. 39 1.5 REFERENCES Abraham, N., Miceli, M.C., Parties, J.R. and Veillette, A. (1991) Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kiase p56lck. Nature 350:62. Abraham, V.S., Sachs, D.H. and Sykes, M. (1992) Mechanism of protection from graft-versus-host disease mortality by IL-2. III. Early reductions in donor T cell subsets and expansion of a CD3+ CD4" CD8" cell population. J. Immunol. 148:3746. Allison, J.P., Mclntyre, B.W., Bloch, D. (1982) Tumor-specific antigen of murine T-lymphoma defined with monoclonal antibody. J. Immunol. 129:2293. Allison, J.P. and Lanier, L.L. (1987) Structure, function and serology of the T-rell antigen receptor complex. Ann. Rev. Immunol. 5:503. Anderson, P., Caligiuri, M., Ritz, J. and Schlossman, S.F. (1989) CD3-negative natural killer cells express C\ TCR as part of a novel molecular complex. Nature 341:159. Andreu-Sanchez, J.L., Moreno de Alboran, I., Marcos, M.A.R., Sanchez Movilla, A., Martinez, A. and Kroemer, G. (1991) Interleukin-2 abrogates the nonresponding state of T cells expressing a forbidden T cell repertoire and induces autoimmune disease in neonatally thymectomized mice. J. Exp. Med. 173:1323. Andrews, B.S., Eisenberg, R.A., Theofilopoulos, A.N., Izui, S., Wilson, C.B., McConahey, P.J., Murphy, E.D., Roths, J.B. and Dixon, F.J. (1981) Etiopathogenesis of murine SLE. Immunol. Rev. 55:179. Arase, H., Fukushi, N., Hatakeyama, S., Ogasawara, K., Iwabuchi, K., Ivvabuchi, C, Negishi, I., Good, R.A. and Oroe, K. (1990) Sequential analysis of the thymocyte differentiation in fully allogeneic bone marrow chimera in mice. II Further characterization of the CD4 + or CD8 + sinple positive thymocytes. Immunobiol. 180:267. Ardavin, C, Wu, L., Li, C-L. and Shortman, K. (1993) Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 362:761. Asarnow, D.M., Kuziel, W.A., Bonyhadi, M., Tiegelaar, R.E., Tucker, P.W. and Allison, J.P. (1988) Limited diversity of y/5 antigen receptor genes of thy-l + dendritic epidermal cells. Cell 55:837. Autran, B., Triebel, F., Katlama, C, Rozenbaum, W., Hercend, T. and Debre, P. (1989) T-cell receptor y8 + lymphocyte subsets dduring rHV infection. Clin. Exp. Immunol. 75:206. Azuma, M., Cayabyab, M., Buck, D., Phillips, J.H. and Lanier, L.L. (1992) CD28 interaction with B7 costimulates primary allogeneic proliferative responses and cytotoxicity mediated by small resting T lymphocytes. J. Exp. Med. 175:353. 40 Bandeira, A., Itohara, S., Bonneville, M., Burlen-Defranoux, 0., Mota-Santos, T., Coutinho, A. and Tonegawa, S. (1991) Extrathymic origin of intestinal intraepithelial lymphocytes bearing T-cell antigen receptor y5. Proc. Natl. Acad. Sci. USA 88:43. Baniyash, M., Hsu, V.W., Seldin, M.F., Klausner, R.D. (1989) The isolation and characterization of the murine T cell antigen receptor £\ chain gene. J. Biol. Chem. 244:13252. Barber, E.K., Dasgupta, J.D., Schlossman, S.F., Trevillyan, J.M. and Rudd, C.E. (1989) The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56^) that phosphorylates the CD3 complex. Proc. Natl. Acad. Sci. USA 86:3277. Barclay, A.N. and Mayrhofer, G. (1981) Bone marrow origin of la-positive cells in the medulla of rat thymus. J. Exp. Med. 153:1666. Bazan, J.F. (1992) Unraveling the structure of 11-2. Science 257:410. Bensussan, A., Lagabrielle, J. and Degos, L. (1989) TCR y5 bearing lymohocyte clones with lymphokine-activated killer activity against autologous leukemic cells. Blood 73:2077. Benveniste, P., Chadwick, B.S., Miller, R.G. and Reimann, J. (1990) Characterization of cells with T cell markers in athymic nude mice bone marrow and of their in v/Yra-derived clonal progeny. Comparison with euthymic bone marrow. J. Immunol. 144:411. Berg, L.J., Pullen, A.M., Fazekas de St. Groth, B., Mathis, D., Benoist, C, and Davis, M.M. (1989) Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand. Cell 58:1035. Berke, G. (1991) Lymphocye-triggered internal target disintegration. Immunol. Today 12:396. Berkhout, B., Alarcon, B. and Terhorst, C. (1988) Transfection of genes encoding the T cell receptor-associated CD3 complex into COS cells results in assembly of the macromolecular structure. J. Biol. Chem. 263:8528. Berridge, M.J. and Irvine, R.D. (1989) Inositol phosphates and cell signalling. Nature 341:197. Bigby, M., Markowitz, J.S., Bleicher, P.A., Grusby, M.J., Simha, S., Siebrecht, M., Wagner, M., Nagler-Anderson, C. and Glimcher, L.H. (1993) Most y5 T cells develop normally in the absence of MHC class II molecules. J. Immunol. 151:4465. Bix, M. and Raulet, D. (1992) Ineffecient positive selection of T cells directed by hemopoietic cells. Nature 359:330. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L. and Wiley, D.C. (1987) Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329:506. Bluestone, J.A., Pardoll, D., Sharrow, S.O. and Fowlkes, B.J. (1987) Characterization of murine thymocytes with CD3-associated T-cell receptor structures. Nature 326:82. 41 Bluestone, J.A., Cron, R.Q., Cotterman, M., Houlden, B.A. and Matis, L.A. H988) Structure and specificity ofT cell receptor y/5 on major histocompatibility complex antigen-specific CD3 +, CD4" CD8" T lymphocytes. J. Exp. Med. 168:1899. Bluestone, J.A., Cron, R.Q., Barrett, T.A., Houlden, B., Sperling, A.I., Dent, A., Hedrick, S., Rellahan, B. and Matis L.A. (1991) Repertoire development and ligand specificity of murine TCR y5 cells. Immunol. Rev. 120:5. Blumberg, R.S., Alarcon, B., Sancho, J., McDermott, F.V., Lopez, P., Breitmeyer, J. and Terhorst, C. (1990) Assembly and function of the T cell receptor. J. Biol. Chem. 265:14036. Boehmer von, H. and Schubiger, K. (1984) Thymocytes appear to ignore class I major histocompatibility complex antigen expressed on thymus epithelial cells. Eur. J. Immunol. 14:1048. Boehmer von, H. and Hafen, K. (1986) Minor but not major histocompatibility antigens of thymus epithelium tolerize precursors of cytolytic T cells. Nature 320:626. Bonneville, M., Ishida, I., Mombaerts, P. Katsuki, M., Verbeek, S., Burns, A. and Tonegawa, S. (1989a) Blockade of ap T cell development by TCR yS transgenes. Nature 342:931. Bonneville, M. Ito, K., Krecko, E.G., Itohara, S., Kappes, D. and Tonegawa, S. (1989b) Recognition of a self major histocompatibility complex TL region product byy5 T cell receptors. Proc. Natl. Acad. Sci. USA 86:5928. Borgulya, P., Kishi, H., Muller, U. Kirberg, J. and von Beohmer, H. (1991) Development of the CD4 and CD8 lineage of T cells: instruction versus selection. EMBO J. 10:913. Borgulya, P. Kishi, H, Uematsu, Y. and von Boehmer, H. (1992) Exclusion and inclusion of a and p T cell receptor alleles. Cell 65:529. Born, W., Hall, L., Dallas, A., Boymel, J., Shinnick, T., Young, D., Brennen, P. and O'Brien, R. (1990) Recognition of a peptide antigen by heat shock-reactive y8 T lymphocytes. Science 249:67. Born, W., Happ, M.P., Dallas, A., Reardon, C, Kubo, R., Shinnick, T., Brennen, P.and O'Brien, R. (1990b) Recognition of heat shock proteins and y5 cell function. Immunol. Today. 11:40. Borst, J., Prendiville, M.A. and Terhorst, C. (1983) The T3 complex on human human thyrnus-derived lymphocytes contains two different subunits of 20 kDa. Eur. J. Immunol. 13:576. Borst, J., Colligan, J.E., Oettgen, H, Pessano, S., Malin, R. and Terhorst, C. (1984) The 5- and e-chains of the human T3/T-cell receptor complex are distinct polypeptides. Nature 312:455. Brandle, D., Muller, C. Rulicke, T. Hengartner, H. and Pircher, H. (1992) Engagement of the T-cell receptor during positive selection in the thymus down-regulates RAG-1 expression. Proc. Natl. Acad. Sci. 89:9529. 42 Brenner, M.B., Mclean, J., Dialynas, D.P., Strominger, J.L., Smith, J.A, Owen, F.L., Seidman, J.G., Ip, S., Rosen, F. and Krangel, M.S. (1986) Identification of a putative second T-cell receptor. Nature 322:145 Brenner, M.B., McLean, J., Schejt, K., Riberdy, J., Ang, S.L., Seidman, T G., Devlin, P. and Krangel, M.S. (1987) Two forms of the T-cell receptor y protein found on peripheral blood cytotoxic T lymphocytes. Nature 325:689. Brooks, E.G., Wirt, D.P., Goldblum, R.M., Vaidya, S., Asuncion, M.T., Patterson, J.C., Ware, C.F. and Klimpel, G.R. (1990) Double-negative (CD4" CD8") T cells with an a/p T cell receptor. Non-MHC-restricted cytolytic activity and lymphokine production. J. Immunol. 144:4507. Brown, J.H., Jardetzky, T., Saper, M.A., Samraoui, B., Bjorkman, P.J. and Wiley, D.C. (1988) A hypothetical model of the foreign antigen binding site of class II histocompatibility molecule. Nature 332:845. Bruley-Rosset, M., Miconnet, I., Canon, C. and Halle-Pannenko, O. (1990) Mls a generated suppressor cells. 1. Suppression is mediated by double-negative (CD3 + CD5 + CD4" CD8") a/p T cell receptor-bearing cells. J. Immunol. 145:4046. Calvo, V., Bierer, B.E. and Vik, T.A. (1992) T cell receptor activation of a ribosomal S6 kinase activity. Eur. J. Immunol. 22:457. Cantley, L.C., Auger, K.R., Carpenter, C, Duckworth, B., Grazini, A., Kapellwe, R. and Soltoff, S. (1991) Oncogenes and signal transduction. Cell 64:281. Carbonari, M., Cherchi, M., Paganelli, R., Geannini, G., Galli, E., Gaetano, C, Papetti, C. and Fiorilli, M. (1990) Relative increase of T cells expressing the gamma/delta rather than the alpha/beta receptor in ataxia-telangiectasia. New Engl. J. Med. 322:73. Carbone, A.M., Marrack, P. and Kappler, J.W. (1988) Demethylated CD8 gene in CD4+ T cells suggests that CD4 + cells develop from CD8 + precursors. Science 242:1174. Carlow, D.A., Teh, S-J. and Teh, H-S. (1992) Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium. J. Immunol. 148:2988. Carrera, A.C., Baker, C, Roberts, T.M. and Pardoll, D.M. (1992) Tyrosine kinase triggering in thymocytes undergoing positive selection. Eur. J. Immunol. 22:2289. Carson, G., Kuestner, R., Ahmed, A., Pettey, C. and Concino (1991) Six chains of the human T cell antigen receptor-CD3 complex are necessary and sufficient for processing the receptor heterodimer to the cell surface. J. Biol. Chem. 266:7883. Ceredig, R., Dialynas, D.P., Fitch, F.W. and MacDonald, H.R. (1983) Precursors of T cell growth factor producing cells in the thymus: ontogeny, frequency and quantitative recovery in a subpopulation of phenotypically mature thymocytes defined by the monoclonal antibody GK1.5. J. Exp. Med. 158:1654. 43 Ceredig, R., Lynch, F. and Newman, P. (1987) Phenotypic properties, mterleukin-2 production, and developmental origin of a "mature" subpopulation of Lyt-2" L3T4" mouse thymocytes. Proc. Natl. Acad. Sci. USA 84:8578. Chan, A.C., Iwashima, M., Turck, C.W. and Weiss, A. (1992) ZAP-70: A 70 kd protein-tyrosine kinase that associates with the TCR C, chain. Cell 71:649. Chen, R.H., Sarnecki, C. and Blenis, J. (1992) Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol. Cell. Biol. 12:915. Chien, Y., Becker, P.M., Lindsten, T., Okamura, M., Cohen, D.I. and Davis, M.M. (1984) A third type of murine T-cell receptor gene. Nature 312:31. Chien, Y., Iwashima, M., Kaplan, K.B., Elliot, J.F. and Davis, M.M. (1987) A new T-cell receptor gene located within the alph locus and expressed early in T-cell differentiation. Nature 327:677. Chothia, C, Boswell, P.R. and Lesk, A.M. (1988) The outline structure of the T-cell ap receptor. EMBO J. 7:3745. Claverie, J.M., Prochnickia-Chalufour, A. and Bougueleret, L. (1989) Implications of a Fab-like structure for the T cell receptor. Immunol. Today 10:10. Clipstone, N.A. and Crabtree, G.R. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. (1992) Nature 357:695. Coffman, R., Ohara, J., Bond, M.W., Carty, J., Zlotnik, A. and Paul, W.E. (1986) B cell stimulatory factor-a enhances the IgE response of lipopolysaccharide-activated B cells. J. Immunol. 136:4538. Coffman, R.L., Schrader, B., Carty, J., Mosmann, T.R. and Bond, M.W. (1987) A mouse T cell product that preferentially enhances IgA production. I. Biological characterization. J. Immunol. 139:3685. Cooke, M.P. and Perlmutter, R.M. (1989) Expression of a novel form of the fyn proto-oncogene in hemopoietic cells. New. Biol. 1:66. Cooke, MP., Abraham, K.M., Forbush, K.A. and Perlmutter, R.M. (1991) Regulation of T cell receptor signalling by a src family protein-tyrosine kinase ($59^™). Cell 65:281. Correa I., Bix, M., Liao, N-s., Zijlstra, M., Jaenisch, R. and Raulet, D. (1992) T cells bearing y5 T cell receptors develop normally in P2-microglobulin-mutant mice. Proc. Natl. Acad. Sci. USA 89:653. Crabtree, G. (1989) Contingent genetic regulatory events in T lymphocyte activation. Science 243:355. Crispe, I.N., Moore, M.W., Husmann, L.A., Smith, L., Bevan, M.J. and Shimonkevitz, R.P. (1987) Differentiation potential of subsets of CD4" CD8" thymocytes. Nature 329:336. 44 Cron, R.Q., Koning, F., Maloy, W.L., Pardoll, D., Coligan, J.E. and Bluestone, J.A. (1988) Peripheral murine CD3 + CD4- CD8- T lymphocytes expressed novel T cell receptor y5 structures. J. Immunol. 141:1074. Davidson, D., Chow, L.M., Fournel, M. and Veillette, A. (1992) Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p5w" . J. Exp. Med. 175:1483. Davies, A.A., Ley, S.C. and Crumpton, M.J. (1992) CD5 is phosphorylated on tyrosine after stimulation of the T-cell antigen receptor complex. Proc. Natl. Acad. Sci. USA 89:6368. Davis, M.M. (1990) T cell receptor gene diversity and selection. Annu. Rev. Biochem. 59:475. Davis, M.M. and Bjorkman, P.J. (1988) T-cell antigen receptor genes and T-cell recognition. Nature 334:395. Dembic, Z., Haas, W., Weiss, S., McCubrey, J., Kiefer, H., von Boehmer, H. and Steinmetz, M. (1986) Transfer of specificity by murine a and p T-cell receptor genes. Nature 320:232. De Paoli. P., Gennari, D., Martelli, P., Cavazerani, V., Comoretto, R. and Santini, G. (1990) y5 T-cell receptor-bearing lymphocytes during Epstein Barr Virus infection. J. Infect. Dis. 161:1013. De Paoli, P., Gennari, D., Martelli, P., Basaglia, G., Grovatto, M. and Battistin, S. (1991) A subset of lymphocytes is increased during HTV-1 infection. Clin. Exp. Immunol. 83:187. Desiderio, S. (1993) Becoming B cells. Nature 361:202. Dialynas, D.P., Wilde, D.B., Marrack, P., Pierres, A., Wall, K.A., Havran, W., Often, G., Loken, M.R., Pierres, M., Kappler, J. and Fitch, F.W. (1983) Characterization of the murine antigenic determinant designated L3T4a, recognized by monoclonal antibody GK1.5: expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHC antigen reactivity. Immunol. Rev. 74:29. Dianzani, U., Shaw, A., Al-Ramadi, B.K., Kubo, R.T. and Janeway, A.C. (1992) Physical association of CD4 with the T cell receptor. J. Immunol. 148:678. Dos Reis, G.A. and Shevach, E.M. (1983) Antigen-presenting cells from non responder strain 2 guinea pigs are fully competant to present bovine insulin P chain to responder strain 13 T cells.J. Exp. Med. 157:1287. Downward, J., Graves, J. and Cantrell, D. (1992) The regulation and function of p21 r a s in T cells. Immunol. Today 13:89. Egerton, M. and Scollay, R. (1990) Intrathymic selection of murine TCR ccp+ CD4" CD8" thymocytes. Intl. Immunol. 2:157. 45 Egerton, M., Ashe, O.R., Chen, D., Drucker, B.J., Burgess, W.H. nad Samelson, L.E. (1992a) VCP, the mamalian homolog of cdc-48, is tyrosine phosphorylated in response to T cell antigen receptor activation. EMBO J. 11:3533. Egerton, M., Burgess, W.H., Chen, D., Drucker, B.J., Bretscher, A. and Samelson, L.E. (1992b) Identification of ezrin as an 81 kDa tyrosine phoshorylated protein in T cells. J. Immunol. 149:1847. Eichelberger, M., Allan, W., Carding, S.R., Bottomly, K. and Doherty, P.C. (1991) Activation status of the CD4" 8" y5-T cells recovered from mice with influenza pneumonia. J. Immunol. 147:2069. Elliot, J.F., Rock, E.P., Pattern, P.A., Davis, M.M. and Chien, Y-H. (1988) The adult T cell receptor 8 chain is diverse and distinct from that of fetal thymocytes. Nature 311:627. Ewijk, van, W., Ron, Y., Monaco, J., Kappler, J., Marrack, P., Le Meur, M., Gerlinger, P., Durand, B., Benoist, C. and Mathis D. (1988) Compartmentalization of MHC class II gene expression in transgenic mice. Cell 53:357. Falini, B., Flenghi, L., Pileri, S., Pelicci, P., Fagioli, M., Martelli, M.F., Moretta, L. and Ciccone, E. (1989) Distribution of T cells bearing different forms of the T cell receptor y/8 in normal and pathological human tissues. J. Immunol. 143:2480. Finkel, T.H., Kubo, R.T. and Cambier, J.C. (1992) T-cell development and transmembrane signalling: changing biological responses through an unchanging receptor. Immunol. Today 12:79. Flajnik, M.F., DuPasquier, L. and Cohen, N. (1985) Immune responses of thymus/lymphosyte embryonic chimeras: studies on tolerance with major histocompatibility complex restriction in Xenopus. Eur. J. Immunol. 15:540. Flanagan, W.M., Corthesy, B., Bram, R.J. and Crabtree, G.R. (1992) Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352:803. Fowlkes, B.J., Edison, L., Mathieson, B.J. and Chused, T.M. (1985) Early T lymphocytes: differentiation in vivo of adult intrathymic precursor cells. J. Exp. Med. 162:802. Fowlkes, B.J., Kruisbeek, A.M., Ton-That, H, Weston, M.A., Coligan, J.E., Schwartz, R.H. and Pardoll, D.M. (1987) A novel population of T-cell receptor ap bearing thymocytes which predominantly express a single Vp gene family. Nature 329:251. Fowlkes, B.J., Schwartz, R.H. and Pardoll, D.M. (1988) Deletion of self-reactive thymocytes occurs at a CD4 +8 + precursor stage. Nature 334:620. Fowlkes, B.J. and Pardoll, D.M. (1989) Molecular and cellular events of T cell development. Adv. Immunol. 44:207. Frank, S.J., Samelson, L.E. and Klausner, R.D. (1990) The structure and signalling functions of the invariant T cell receptor components. Semin. Immunol. 2:89. 46 Freeman, G.J.Freedman, A.S., Segil, J.M. Lee, G., Whitman, J.F. and Nadler, L.M. (1989) B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells. J. Immunol. 143:2714. Godfrey, D.L, Kennedy, J., Suda, T. and Zlotnik, A. (1993) A developmental pathway involving four phenotypically and functionally distinct subsets of CD3- CD4- CD8~ triple negative adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol. 150:4244. Gold, D.P., Clevers, H., Alarcon, B., Dunlap, S., Novotny, J., Williams, A.F. and Terhorst, C. (1987) Evolutionary relationship between the T3 chains of the T-cell receptor complex and the immunoglobulin supergene family. Proc. Natl. Acad. Sci. USA 84:7649. Groettrup, M., Baron, A., Griffiths, G., Palacios, R. and von Boehmer, H. (1992) T cell receptor (TCR) P chain homodimers on the surface of immature but not mature a, y, 6, chain deficient T cell lines. EMBO J. 7:2735. Groves, E.S. and Singer, A. (1983) Role of the H-2 complex in the induction of T cell tolerance to self minor histocompatibility antigens. J. Exp. Med. 158:1483. Guidos, C.J., Weissman, LL. and Adkins, B. (1989a) Developmental potential of CD4"8_ thymocytes: Peripheral progeny include mature CD4_8~ T cells bearing ap T cell receptor. J. Immunol. 142:3773. Guidos, C.J., Weissman, LL. and Adkins, B. (1989b) Intrathymic maturation of murine T lymphocytes from CD8 + precursors. Proc. Natl. Acad. Sci. USA 86:7542. Guy-Grand, D., Cerf-Benussan, N., Malissen, B., Malassis-Seris, M., Briottet, C. and Vassalli, P. (1991) Two gut intraepithelial CD8 + lymphocyte populations with different T cell receptors: A role for gut epithelium in T cell differentiation. J. Exp. Med. 173:471. Hacker, G„ Kromer, S., Falk, M., Heeg, K., Wagner, H. and Pfeffer, K. (1992) The V51 positive subset of human yS T cells responds to ligands expressed by Epstein-Barr virus infected Burkitt lymphoma cells and transformed B-lymphocytes. Eur. J. Immunol. Hara, T., Mizuno, Y., Nagata, M., Okabe, Y., Tanikguchi, S., Harada, M., Niho, Y., Pshirni, K., Ohga, S., Yoshikai, Y,, Nomoto, K., Yumara, K., Kawa-Ha, K. and Ueda, K. (1990) Human y5 T cell receptor-positive cell mediated inhibition of erythropoiesis in vitro in a patient with type 1 autoimmune polyglandular syndrome and pure red blood cell aplasia. Blood 75:941. Haskins, K., Kubo, R., White, J., Pigeon, M., Kappler, J. and Marrack, P. (1983) The major histocompatibility complex-restricted antigen receptor on T cells I. Isolation with a monoclonal antibody. J. Exp. Med. 157:1149. Havran, W.L. and Allison, J.P. (1988) Developmentally ordered appearance of thymocytes expressing different T-cell antigen receptors. Nature 335:443. 47 Haynes, B.F., Singer, K.H., Denning, S.M. and Martin, M.E. (1988) Analysis of expression of CD2, CD3 and T cell receptor molecules during early human fetal thymic development. J. Immunol. 141:3776. Hedrick, S.M., Nielsen, E.A., Kavaler, J., Cohen, D.I. and Davis, M.M. (1984) Sequence relationships between putative T-cell receptor polypeptides and immunoglobulins. Nature 308:153. Heilig, J.S. and Tonegawa, S. (1986) Diversity of murine gamma genes and expression in fetal and adult T lymphocytes. Nature 322:836. Hertel-Wulff, B., Okada, S., Oseroff, A. and Strober, S. (1984) In vitro propagation and cloning of murine natural suppressor (NS) cells. J. Immunol. 133:2791. Hiromatsu, K., Yoshikai, Y., Matsuzaki, G., Oliga, S., Muramori, K., Matsumoto, K., Bluestone, J.A. and Nomoto, K. (1992) A protective role of y5 T cells in primary infection with Listeria monocytogenes in mice. J. Exp. Med. 175:49. Hodes, R.J., Sharrow, S.O. and Solomon, A. (1989) Failure of T cell receptor Vp negative selection in an athymic environment. Science 246:1041. Hoffman, M.W., Allison, J. and Miller, J.F.A.P. (1992) Tolerance induction by thymic medullary epithelium. Proc. Natl. Acad. Sci. 89:2526. Holda, J.H., Maier, T. and Claman, H.N. (1986) Natural suppressor activity in graft-versus-host spleen and normal bone marrow is augmented by IL-2 and Interferon-y. J. Immunol. 137:3538. Hollander, N. (1982) Effects of anti-Ly-2 antibodies on T cell function. Immunol. Rev. 68:43. Holoshitz, J., Koning, F., Coligan, J.E., De Bruyn, J. Strober, S. (1989) Isolation of CD4" CD8" mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 339:226. Howe, R.C. and MacDonald, H.R. (1988) Heterogeneity of immature (Lyt-27L3T4") thymocytes: Identification of four major phenotypically distinct subsets differing in cell cycle status and in vitro activation requirements. J. Immunol. 140:1047. Howe, L.R., Leevers, S.J., Gomez, N., Nakielny, S., Cohen, P. and Marshall, C.J. (1992) Activation of the MAP kinase pathway by the protein kinase raf. Cell 71:335. Hvas, J., Oksenberg, J., Fernando, R., Steinman, L. and Bernard, C C A . (1992) y5 T cell receptor repertoire in brain lesions of patients with multiple sclerosis. Hugo, P. Waanders, G.A., Scollay, R, Shortman, K. and Boyd, R.L. (1990) Ontogeny of a novel CD4 + CD8" CD3 - thymocyte subpopulation: a comparison with CD4" CD8 + CD3" thymocytes. Int. Immunol. 2:209. Hugo, P., Waanders, G.A., Scollay, R., Pertrie, H.T. and Boyd, R.L. (1991) Characterization of immature CD4 + CD8' CD3" thymocytes. Eur. J. Immunol. 21:835. 48 Hunter, T. and Karin, M. (1992) The regulation of transcription by phosphorylation. Cell 70:375. Hurley, T.R., Luo, K. and Sefton, B.M. (1989) Activators of protein kinase C induce dissociation of CD4, but not CD8, from p56lck. Science 245:407. Husbands, S.D., Schonrich, G., Arnold, B., Chandler, P.R., Simpson, E., Philpott, K.I., Tomlinson, P., O'Reilly, L. Cooke, A. and Mellor, A.L. (!992) Expression of major histocompatibility complex class I antigens at low levels in the thymus induces T cell tolerance via a non-deletional mechanism. Eur. J. Immunol. 22:2655. Imboden, J.B. and Stobo, J.D. (1985) Transmembrane signalling by the T cell antigen receptor: perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores. J. Exp. Med. 161:446. Inaba, M., Inaba, K., Hosono, M., Kumamoto, T., Ishida, T., Muramatsu S., Masuda, T. and Dcehara, S. (1991) Distinct mechanisms of neonatal tolerance induced by dendritic cells and thymic B cells. J. Exp. Med. 173:549. Irving, B. and Weiss, A. (1991) The cytoplasmic domain of the T cell receptor C, chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 64:891. Ito, K., Bonneville, M., Takagaki, Y., Nakanishi, N., Kanagawa, O., Krecko, E. and Tonegawa, S. (1989) Different y5 T-cell receptors are expressed on thymocytes at different stages of development. Proc. Natl. Acad. Sci. USA 86:631. Itohara, S., Farr, A.G., Lafaille, J.J., Bonneveille, M., Takagaki, Y., Haas, W. and Tonegawa, S. (1990) Homing of a y/5 thymocyte subset with homogenous T-cell receptors to mucosal epithelial. Nature 343:754. Iwabuchi, K., Nakayama, K-I, McCoy, R.I., Wang, F., Nishimura, T., Habu, S., Murphy, K.M. and Loh, D.Y. (1992) Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes. Proc. Natl. Acad. Sci. 89:9000. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S-L, Sameshima, M., Hase, A., Seto, Y. and Nagata, S. (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66:233. Janis, E.M., Kaufmann, S.H.E., Schwartz, R.H. and Pardoll, D.M. (1989) Activation of y5 T cells in primary immune response to Mycobacterium tuberculosis. Science 244:713. Jenkinson, E.J., Van Ewijk, W. and Owen, J.J.T. (1981) Major histocompatibility complex antigen expression on the epithelium of the developing thymus in normal and nude mice. J. Exp. Med. 153:280. Jenkinson, E.J., Jhittay, P., Kingston, R. and Owen, J.J.T. (1985) Studies of the role of the thymic environment in the induction of tolerance to MHC antigens. Transplantation 39:331. 49 Jin, Y-J., Clayton, L.K., Howard, F.D., Koyasu, S., Sieh, M., Steinbrich, R., Tarr, G.E. and Reinherz, E.L. (1990). Molecular cloning of the CD3C>related product in thymus-derived cells. Proc. Natl. Acad. Sci. USA 87:3319. John, S., Banting, G.S., Goodfellow, P.N. and Owen, M.J. (1989) Surface expression of the T cell receptor complex requires charged residues within a chain transmembrane region. Eur. J. Immunol. 19:3355. Jones, L.A., Chin, L.T., Merriam, G.R., Nelson, L.M. and Kruisbeek, A.M. (1990) Failure of clonal deletion in neonatally thymectomized mice: tolerance is preserved through clonal anergy. J. Exp. Med. 172:1277. June, C.H., Fletcher, M.C., Ledbetter, J.A. and Samelson, L.E. (1990a) Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation. J. Immunol. 144:1591. June, C.H., Fletcher, M.C., Ledbetter, J.A., Schieven, G.L., Siegel, J.N., Phillips, A.F. and Samelson, L.E. (1990b) Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction. Proc. Natl. Acad. Sci. USA 87:7722. June, C.H., Ledbetter, J.A., Linsley, P.S. and Thompson, C.B. (1990c) Role of the CD28 receptor in T-cell activation. Immunol. Today 11:211. Kappler, J. Kubo, R., Haskins, K., White, J. and Marrack, P. (1983) The mouse T cell receptor, comparison of MHC-restricted receptors on two T cell hybridomas. Cell 34:727. Kappler, J.W., Roehm, N.and Marrack, P. (1987) T cell tolerance by clonal elimination in the thymus. Cell 49:273. Kappler, J.W., Staerz, U., White, J. and Marrack, P.C. (1988) Self-tolerance eliminates T cells specific for Mis-modified products of the major histocompatibility complex. Nature 332:35. Karnitz, L., Sutor, S.L., Torigoe, T. Reed, J.C., Bell, M.P., McKean, D.J., Leibson, P.J. and Abraham, R.T. (1992) Affects of p56 l c k deficiency on the growth and cytolytic effector function of an interleukin 2 dependent cytotoxic T-cell line. Mol. Cell. Biol. 12:4521. Kaye, J., Porcelli, S., Tite, J., Jones, B. and Janeway, J. (1983) Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen presenting cells in the activation of T cells. J. Exp. Med. 158:836. Kaye, J., Hsu, M.-L., Sauron, M.E-., Jameson, S.C., Gascoigne, N.R.J., and Hedrick, S.M. (1989) Selective development of CD4 + T cells in transgenic mice expressing a class II MHC-restricted antigen receptor. Nature 341:746. Kaye, J., Vasquez, N.J., and Hedrick, S.M. (1992) Involvement of the same region of the T cell receptor in thymic selection and foreign peptide recognition. J. Immunol. 148:3342. 50 Kishi, H., Borgulya, P., Scott, B., Karjalainen, K., Traunecker, A., Kaufman, J. and von Boehmer, H. (1991) Surface expression of the p T cell receptor (TCR) chain in the absence of other TCR or CD3 proteins on immature T cells. EMBO J. 10:93. Kisielow, P., Leiserson, W. and von Boehmer, H. (1984) Differentiation of thymocytes in fetal organ culture: analysis of phenotypic changes accompnying the appearance of cytolytic and interleukin-2 producing cells. J. Immunol. 133:1117. Kisielow, P., Bluthmann, H. Staerz, U.D., Steinmetz, M. and von Boehmer, H. (1988) Tolerance in T-cell-receptor transgeneic mice involves deletion of nonmature CD4 +8 + thymocytes. Nature 333:742. Klausner, R.D., O'Shea, J.J., Luong, H., Ross, P., Bluestone, J.A. and Samelson, L.E. (1987) T cell receptor tyrosine phosphorylation: variable coupling for different activating ligands. J. Biol. Chem. 262:12654. Koch, C.A., Anderson, D., Moran, M.F., Ellis, C. and Pawson, T. (1991) SH2 qand SH3 domains: elements that control interactions of cytoplasmic signalling proteins. Science 252:668. Koning, F., Maloy, W.L. and Coligan, J.E. (1990) The implications of subunit interactions for the structure of the T cell receptor-CD3 complex. Eur. J. Immunol. 20:299. Krensky, A.M., Clayberger, C, Reiss, C.S., Strominger, J.L. and Burakoff, S.J. (1982a) Specificity of OKT4 + cytotoxic T lymphocyte clones. J. Immunol. 129:2001. Krensky, A.M., Reiss, C.S., Mier, J.W., Strominger, J.L. and Burakoff, S.J. (1982b) Long-term human cytolytic T-cell lines allospecific for HLA-DR6 antigen are OKT4+. Proc. Natl. Acad. Sci. USA 79:2365. Kubota, H., Okazaki, H., Onuma, M., Kano, S., Hattori, M. and Minato, N. (1992) CD3+ 4" 8' aP T cell population with biased T cell receptor V gene usage. J Immunol. 149:1143. Lam, V., De Mars, R., Chen, R.P., Hank, J.A., Kovats, S., Fisch, P. and Sondel, P.M. (1990) Human T cell receptor y8-expressing T cell lines recognize MHC-controlled elements on autologous EBV-LCL that are not HLA -A, -B, -C, -DR, -DQ, or DP. J. Immunol. 145:36. Lancki, D.W., Lorber, M.J., Loken, M.R. and Fitch, F.W. (1983) A clone-specific monoclonal antibody that inhibits cytolysis of a cytolytic T cell clone. J. Exp. Med. 157:921. Lanier, L.L., Yu, G. and Phillips, J.H. (1989) Co-association of CD3t; with a receptor (CD16) for IgG Fc on human natural killer cells. Nature 342:803. Larson, CP., Ritchie, S.C., Pearson, T.C, Linsley, P.S. and Lowry, R.P. (1992) Functional expression of the costimulatory molecule B7/BB1, on murine dendritic cell populations. J. Exp. Med. 176:1215. 51 Ledbetter, J.A., Seamen, W.E., Tsu, T.T. and Herzenberg, L.A. (1981) Lyt-2 and Lyt-3 antigens are on two different polypeptides subunits linked to disulfide bonds: Relationship of subunits to T cell cytolytic activity. J. Exp. Med. 153:1503. Ledbetter, J.A. and Seamen, W.E. (1982) The Lyt-2, Lyt-3 macromc'.ecules: Structural and functional studies. Immunol. Rev. 68:197. Lee, N.A., Loh, D.Y. and Lacy, E. (1992) CD8 surface levels alter the fate of a/p T cell receptor-expressing thymocytes in transgenic mice. J. Exp. Med. 175:1013. Leonard, W.J., Depper, J.M., Crabtree, G.R., Rudikoff, S., Pumphrey, J., Robb, R.J., Kronke, M., Svetlik, P.B., Peffer, N.J., Waldmann, T.A. and Greene, W.C. (1984) Molecular cloning and expression of cDNAs for the human interleukin-2 receptor. Nature 311:626. Levitsky, H.Y., Golumbek, P.T. and Pardoll, D.M. (1991) The fate of CD4"8- T cell receptor-aP+ thymocytes. J. Immunol. 146:1113. Lew, A.M., Pardoll, D.M., Maloy, W.L., Fowlkes, B.J., Kruisbeek, A., Cheng, S-F., Germain, R.N., Bluestone, J.A., Schwartz, R.H. and Coligan, J.E. (1986) Characterization of T-cell receptor gamma chain expression in a subset of murine thymocytes. Science 234:1401. Lieber, M.R.,Hesse, J.E., Mizuuchi, K. and Gellert, M. (1987) Developmental stage specificity of the lymphoid V(D)J recombination activity. Genes Dev. 1:751. Lindstein, T., Fowlkes, B.J., Samelson, L.E., Davis, M.M. and Chier, Y. (1987) Transient rearrangement of the T cell antigen receptor a locus in early thymocytes. J. Exp. Med. 166:761. Linsley, P.S., Clark, E.A. and Ledbetter, J.A. (1990) T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1. Proc. Natl. Acad. Sci. USA 87:5031. Linsley, P.S., Brady, W., Grosmaire, L. Affuffa, A., Damle, N.K. and Ledbetter, J.A. (1991) Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 173:721. Liftman, D.R., Thomas, Y., Maddon, P.J., Chess, L. and Axel, R. (1985) The isolation and sequencing of the gene encoding T8: a molecule defining functional classes of T lymphocytes. Cell 40:237. Liftman, D.R. and Gettner, S.N. (1987) Unusual intron in the immunoglobulin domain of the newly isolated murine CD4 (L3T4) gene. Nature 325:453. Liu, Y., Jones, B., Aruffo, A. Sullivan, K.M., Linsley, P.S. and Janeway, C.A.(1992) Heat-stable antigen is a costimulatory molecule for CD4 T cell growth. J. Exp. Med. 175:437. Lo, D. and Sprent, J. (1986) Identity of cells that imprint H-2-restricted T cell specificity in the thymus. Nature 319:672. 52 Luo, K. and Sefton, B.M. (1990) Crossliriking of T cell surface molecules CD4 and CD8 stimulates phosphorylation of the Ick tyrosine protein kinase at the autophosphorylation site. Mol. Cell. Biol. 10:5305. MacDonald, H.R., Budd, R.C. and Howe, R.C. (1988a) A CD3" subset of CD4'8+ thymocytes: a rapidly cycling intermediate in the generation of CD4 +8 + cells. Eur. J. Immunol. 18:519. MacDonald, H.R., Schneider, R., Lees, R.K., Howe, R.C, Acha-Orbea, H, Festenstein, H, Zinkernagel, R.M. and Hengartner, H. (1988b) T-cell receptor Vp use predicts reactivity and tolerance to Mls a- encoded antigens. Nature 332:40. Madden, D.R., Gorga, J.C., Strominger, J.C. and Wiley, D.C. (1991) The structure of HLA-B27 reveals nonamer self-peptides bound in an extended conformation. Nature 353:321. Maddon, P.J., Littman, D.R., Godfrey, M., Maddon, D.E., Chess, L. and Axel, R. (1985) The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell 42:93. Maier, T., Holda, J.H. and Claman, H.N. (1986) Natural suppressor (NS) cells: Members of the LGL regulatory family. Immunol. Today 7:312. Margolis, B., Hu, P., Katzav, S., Li, W., Oliver, J.M., Ullrich, A., Weiss, A. and Schlessinger, J.(1992) Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domains and transcription factor "motifs. Nature 356:71. Marrack, P. and Kappler, J. (1988) The T cell repertoire for antigen and MHC. Immunol. Today 9:308. Marrack, P., Lo, D., Brinster, R., Palmiter, R., Burkly, L., Flavell, R.H. and Kappler, J. (1988) The effect of the thymus enviroment on T cell development and tolerance. Cell 53:627. Marth, J.D., Peet, E.G., Krebs, E.G. and Perlmutter, R.M. (1985) A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell 43:393. Masuda, T., Ohteki, T., Abo, T., Seki, S., Nose, M., Nagura, H. and Kumagai, K. (1991) Expansion of the population of double negative CD4"8" Tap-cells in the liver is a common feature of autoimmune mice. J. Immunol. 147:2907. Matis, L.A., Cron, R.Q. and Bluestone, J.A. (1987) Major histocompatibility -complex linked specificity of y8 receptor-bearing T lymphocytes. Nature 330:262. Matis, L.A., Fry, A.M., Cron, R.Q., Cotterman, M.M., Dick, R.F. and Bluestone, J.A. (1989) Structure and specificity of a class II MHC alloreactive y5 T cell receptor. Science 245:746. Matsumoto, K., Yoshikai, Y., Matsuzaki, G., Asano, T. and Nomoto, K. (1989) A novel CD3" Jl l d + subset of CD4 + CD8" cells repopulating thymus in radiation bone marrow chimeras. Eur. J. Immunol. 19:1203. 53 Matzinger, P. Zamoyska, R. and Waldmann, H. (1984) Self tolerance is H-2-restricted. Nature 308:738. Mazda, 0., Watanabe, Y., Gyotoku, J-I. and Katsura, Y. (1991) Requirment of dendritic cells and B cells in the clonal deletion of Mis-reactive T cells in the thymus. J. Exp. Med. 173:539. Meuer, S.C., Acuto, 0., Hussey, R.E., Hodgdon, J.C., Fitzgerald, K.A., Schlossman, S.F. and Reinherz, E.L. (1983) Evidence for the T3-associated 90 K heterodimer as the T cell antigen receptor. Nature 303:808. Meuer, S.C., Fitzgerald, K.A., Hussey, R.E., Hodgdon, J.C., Schlossman, S.F. and Reinherz, E.I. (1983b) Clonotypic structures involved in antigen-specific human T cell function. J. Exp. Med. 157:705. Mieno, M., Suto, R., Obata, Y., Udono, H., Takahashi, T., Shiku, H. and Nakayama, E. (1991) CD4" CD8" T cell receptor ap T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumour rejection. J. Exp. Med. 174:193. Minoprio, P., Itohara, S., Heussere, C, Tonegawa, S., Coutinho, A. (1989) Immunobiology of murine T. cruzi infection: the predominance of parasite-nonspecific responses and the activation of TCR1 T cells. Immunol. Rev. 112:183. Modlin, R.L., Pirmez, C, Hofman, F.M., Torigian, V., Uyemura, K., Rea, T.H., Blooem, B.R. and Brenner, M.B. (1989) Lymphocytes bearing antigen-specific y5 T cell receptors accumulate in human infectious disease lesions. Nature 338:544. Mombaerts, P., Clarke, A.R., Rudnicki, M.A., Iacomini, J., Itohara, S., Lafaille, J.J., Wang, L., Ichikawa, Y., Jaenisch, R., Hooper, M.L. and Tonegawa, S. (1992a) Mutations is T-cell antigen receptor genes a and P block thymocyte development at different stages. Nature 360:255. Mombaerts, P., Iacomini, J., Johnson, R.S., Herrup, K., Tonegawa, S. and Papiaoannou, E. (1992b) RAG-1 -deficient mice have no mature B or T lymphocytes. Cell 68:869. Moore, M. and Owen, J.J.T. (1967) Experimental studies on the development of the thymus. J. Exp. Med. 126:715. Morely, B.J., Chin, K.N., Newton, M.E. and Weiss, A. (1988) The lysine residue in the membrane-spanning domain of the P chain is necessary for the cell surface expression of the T cell antigen receptor. J. Exp. Med. 168:1971. Morio, T., Nagasawa, M., Nonoyama, S., Okawa, H. and Yata, J-I. (1990) Phenotypic profile and functions of T cell receptor-y8-bearing cells from patients with primary immunodeficiency syndrome. J. Immunol. 144:1270. Murphy, K.M., Heimberger, A.B., and Loh, D.Y. (1990) Induction by antigen of intrathymic apoptosis of CD4 + CD8+ TCR 1 0 thymocytes in vivo. Science 250:1720. 54 Murphy, D.B. (1993) T cell mediate immunosuppression. Curr. Opinion Immunol. 5:411. Mustelin, T., Coggeshall, K.M. and Altaian, A. (1989) Rapid activation of the T cell tyrosine protein kinase p 5 6 ^ by the CD45 phosphotyrosine phosphatase. Proc. Natl. Acad. Sci. USA 86:6302. Mustelin, T., Coggeshall, K.M., Isakov, N. and Altman, A. (1990) T cell antigen receptor-mediated activation of phospholipase C requires tyrosine phosphorylation. Science 247:1584. Mustelin, T., Pessamorikawa, T., Autero, M., Gassmann, M., Anderson, L.C., Gahmberg, C.G. and Burn, P. (1992) Regulation of the p59 (fyn) protein tyrosine kinase by the CD45 phosphotyrosine phosphatase. Eur. J. Immunol. 22:1173. Nabholz, M. and MacDonald, H.R. (1983) Cytolytic T lymphocytes. Annu Rev. Immunol. 1:273. Nakauchi, H., Nolan, G.P., Hsu, C, Huana, H.S., Kavothas, P. and Herz.euberg, L.A. (1985) Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: molecular homology to its human counterpart, Leu-2/T8, and to immunoglobulin variable regions. Proc. Natl. Acad. Sci. USA 82:5126. Nakanishi, N., Maeda, K., Ito, K-I., Heller, M. and Tonegawa, S. (1987) Ty protein is expressed on murine fetal thymocytes as a disulfide-linked heterodimer. Nature 325:720. Nakayama, E., Shiku, H, Stockert, E., Oetgen, H.F. and Old, L.J. (1979) Cytotoxic T cells: Lyt phenotype and blocking of killing activity by Lyt antisera. Proc. Natl. Acad. Sci USA 76:1977. Nel, A.E., Schabort, I., Rheeder, A., Bouic, P. and Wooten, M.W. (1987) Inhibition of antibodies to CD3 surface antigen and phytohemagglutinin-mediated T cellular responses by inhibiting Ca + 2/ phospholipid-dependent protein kinase activity with the aid of l-5-isoquinolinylsulfonyl-2-methylpiperazine dihychloride. J. Immunol. 139:2230. Nel, A.E., Pollack, S., Landreth, G., Ledbetter, J.A., Hultin, L., Williams, K., Katz, R. and Akerley, B. (1990) CD3 -mediated activation of MAP-2 kinase can be modified by ligation of the CD4 receptor. Evidence for tyrosine phosphorylation during activation of this kinase. J. Immunol. 145:971. Nemanzee, D.A., Studer, S., Steinmetz, M., Dembie, Z. and Kiefer, M. (1985) The lymphoproliferating cells of MRL-lpr/lpr mice are a polyclonal population that bear the T lymphocyte receptor for antigen. Eur. J. Immunol. 15:760. Nikolic-Zugic, J. and Moore, M.W. (1989) T cell receptor expression on immature thymocytes with in vivo and in vitro precursor potential. Eur. J. Immunol. 19:1957. Nikolic-Zugic, J., Moore, M.W. and Bevan, M.J. (1989) Characterization of the subset of immature thymocytes which undergo rapid in vitro differentiation. Eur. J. Immunol. 19.649. Oettgen, H, Pettey, C, Maloy, W. and Terhorst, C. (1986) A T3-like protein complex associated with the antigen receptor on murine T cells. Nature 320:272. 55 Ohashi, P., Mak, T., Van den Elsen, P., Yanagi, Y., Yasunobu, Y., Caiman, A. Terhorst, C, Stobo, J. Weiss, A. (1985) Reconstitution of an active surface T3/T-cell antigen receptor by DNA transfer. Nature 316:606. Ohki, H., Martin, C, Corbel, C, Coltey, M. and LeDouarin, N.M. (1987) Tolerance induced by thymic epithelial grafts in birds. Science 237:1032. Ohteki, T., Seki, S., Abo, T. and Kumagai, K. (1990) Liver is a possible site for the proliferation of abnormal CD3 + 4" 8" double-negative lymphocytes in autoimmune MRL-lpr/lpr mice. J. Exp. Med. 172:7. Ohteki, T., Okuyama, R., Seki, S., Abo, T., Sugiure, K., Kusumi, A., Ohmori, T., Watanabe, H. and Kumagai, K. (1992) Age-dependent increase of extrathymic T cells in the liver and their appearance in the periphery of older mice. J. Immunol. 149:1562. OKeefe, S.J., Tamura, J., Kincaid, R.L., Tocci, M.J. and ONiel, E.A. (1992) FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature 357:692. Ortaldo, J.R., Winkler, Pickett, R.T., Nagashima, K., Yagita, H. and Okumura, K. (1992) Direct evidence for release of pore-forming protein during NK cellular lysis. J. Leukocyte Biol. 52:483. Oseroff, A., Okada, S. and Strober, S. (1984) Natural suppressor (NS) cells found in the spleen of neonatal mice and adult mice given total lymphoid irradiation (TLI) express the null surface phenotype. J. Immunol. 132:101. Owen, J.J.T. and Jenkinson, E.J. (1984) Early events in lympocyte genesis in the fetal thymus. Am. J. Anat. 170:301. Palathumpat, A., Dejbakhsh-Jones, S., Holm, B., Wang, H., Liang, O. and Strober, S. (1992) Studies of CD4" CD8" ap bone marrow T cells with supressor activity. J. Immunol. 148:373. Palathumpat, A., Dejbakhsh-Jones, S., Holm, B. and Strober, S. (1992a) Different subsets of T cells in the adult mouse bone marrow and spleen induce of suppress acute graft-versus-host disease. J. Immunol. 149:808. Palathumpat, A., Dejbakhsh-Jones, S., Holm, B., Wang, H., Liang, O. and Strober, S. (1992b) Studies of CD4" CD8" ap bone marrow T cells with suppressor activity. J. Immunol. 148:373. Papiernik, M. and Pontoux, (1990) In vivo and in vitro repertoire of CD3 + CD4" CD8" thymocytes. Intl. Immunol. 2:407. Pardoll, D.M., Fowlkes, B.J., Bluestone, J.A., Kruisbeek, A., Malory, W.L., Coligan, J.E. and Schwartz, R.H. (1987a) Differential expression of two distinct T-cell receptors during thymocyte development. Nature 326:79. Pardoll, D.M., Kruisbeek, A.M., Fowlkes, B.J., Coligan, J. and Scwartz, R.H. (1987b) FEBS J. 1:103. 56 Pearse, M., Wu, L., Egerton, M., Wilson, A., Shortman, K. and Scollay, R. (1989) A murine early thymocyte developmental sequence is marked by transient expression of the interleukin-2 receptor. Proc. Natl. Acad. Sci. 86:1614. Petrie, H.T., Hugo, P., Scollary, R. and Shortman, K. (1990a) Lineage relationships and developmental kinetics of immature thymocytes: CD3, CD4 and CD8 acquisition in vivo and in vitro. J. Exp. Med. 172:1583. Petrie, H.T., Pearse, M., Scollay, R. and Shortman, K. (1990b) Development of immature thymocytes: initiation of CD3, CD4 and CD8 acquisition parallels down-regulation of the interleukin 2 receptor a chain. Eur. J. Immunol. 20:2813. Petrie, H.T., Scollay, R. and Shortman, K. (1992) Commitment to T cell receptor-ap or -y8 lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes. Eur. J. Immunol. 22:2185. Philpott, K.I., Viney, J.L., Kay, G., Rastan, S., Gardiner, E.M., Chae, S., Hayday, A.C. and Owen, M.J. (1992) Lymphoid development in mice congenitally lacking T cell receptor ap-expressing cells. Science 256:1448. Porcelli, S., Brenner, M.B., Greenstein, J.L., Balk, S.P.,Terhorst, C. and Bleicher, P.A. (1989) Recognition of cluster of differentiation 1 antigens by CD4~ CD8" cytolytic T lymphocytes. Nature 341:447. Prud'homme, G.J., Bocarro, D.C. and Luke, E.C.H. (1991) Clonal deletion and autoreactivity in extrathymic CD4" CD8" (double negative) T cell receptor-a/p T cells. J. Immunol. 147:3314. Pullen A.M., Marrack, P. and Kappler, J.W. (1988) The T cell repertoire is heavily influenced by tolerance to polymorphic self-antigens. Nature 335:796. Quaratino, S., Murison, G., Knyba, R.E., Verhoff, A. and Londei, M. (1991) Human CD4" CD8" ctp + T cells express a functional T cell receptor and can be activated by superantigens. J. Immunol. 147:3319. Rammensee, H.G. and Bevan, M.J. (1984) Evidence from in vitro studies that tolerance to self antigens is MHC-restricted. Nature 308:741 Ramsdell, F. and Fowlkes, B.J. (1989) Engagement of CD4 and CD8 accessory molecules is required for T cell maturation. J. Immunol. 143:1467. Raulet, D.H. (1989) The structure, function and molecular genetics of the y8 T cell receptor. Annu. Rev. Immunol. 7:175. Raulet, D.H., Spencer, D.M. Hsiang, Y-H., Goldman, J.P., Bix, M., Liao, N-S., Zijlstra, M., Jaenisch, R. and Correa, I. (1991) Control of yS cell development. Immunol. Rev. 120:185. 57 Rayter, S.I., Woodrow, M., Lucas, S.C., Cantrell, D.A. and Downward, J. (1992) p21 r a s mediates control of IL-2 gene promoter function in T cell activation. EMBO J. 12:4549. Ready, A.R., Jenkinson, E.J., Kinston, R. and Owen, J.J.T. (1984) Successful transplantation across major histocompatibility barrier of deoxyguanosine-treated embryonic thymus expressing class II antigens. Nature 310:231. Reilly, E.B., Auditore-Hargreaves, K., Hammering, U. and Gottlieb, P.D. (1980) Lyt-2 and Lyt-3 alloantigens: precipitation with monoclonal and conventional antibodies on one- and two-dimensional polyacrylamide gels. J. Immunol. 125:2245. Reinherz, E.L., Hussey, R., Fitzgerald, K., Snow, P., Terhorst, C. and Schlossman, S.F. (1981) Antibody directed at a surface structure inhibits cytolytic but not suppressor function of human T lymphocytes. Nature 294:168. Rhee, S.G., Suh, P-G., Ryu, S-H. and Lee, S.Y. (1989) Studies of inositol phospholipid-specific phospholipase C. Science 244:546. Rivas, A., Koide, J., Cleary, M. and Engleman, E.G. (1989) Evidence for the involvement of the y5 T cell antigen in cytotoxicity mediated by human alloantigen-specific T cell clones. J. Immunol. 142:1840. Robey, E.A., Fowlkes, B.J. and Pardoll, D.M. (1990) Molecular mechanisms for lineage commitment in T cell development. Seminars in Immunology 2:25. Robey, E.A., Ramsdell, F., Kioussis, D., Sha, W., Loh, D., Axel, R. and Fowlkes. B.J. (1992) The level of CD8 expression can determine the outcome of thymic selection. Cell 69:1089. Robinson, J.H. and Owen, J.J.T. (1977) Generation of T-cell function in organ culture of foetal mouse thymus. Clin. Exp. Immunol. 27:322. Rouvier, E., Luciani, M.F. and Goldstein,P. (1003) Fas involvement in Ca+^-independent T-cell mediated cytotoxicity. J. Exp. Med. 177:195. Rudd, C.E., Trevillyan, J.M., Dasgupta, J.V., Wang, L.L. and Schlossman, S.F. (1988) The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc. Natl. Acad. Sci. USA 85:5190. Rust, C.J.J., Verreck, F., Victor, H. and Koning, F. (1990) Specific recognition of staphylococcal entertoxin A by human T cells bearing receptors with the VyQ region. Nature 346:572. Rust, C. Kooy, Y., Pena, S., Mearins, M.-L., Kluin, P. and Koning, F. (1992) Phenotypical and functional characterization of small intestinal TcRy5 + T cells in coeliac disease. Scand. J. Immunol. 35:459. Saito, H., Kranz, D.M., Takagaki, Y., Hayday, A.C., Eisen, H.N. and Tonegawa, S. (1984a) Complete primary structure of a heterodimeric T-cell receptor deduced from cDNA sequences. Nature 309:757. 58 Saito, H., Kranz, D.M., Takagaki, Y., Hayday, A.C., Eisen, H.N. (1984b) A third rearranged and expressed gene in a clone of cytotoxic T lymphocytes. Nature 312:36. Saito, T., Weiss, A., Gunter, K.C., Shevach, E.M. and Germain, R.N. (1987a) Cell surface T3 expression requires the presence of both y, a and p chains of the T cell receptor. J. Immunol. 139:625. Saito, T., Weiss, A., Miller, J., Norcross, M.A. and Germain, R.N. (1987b) Specific antigen-la activation of transfected human T cells expressing murine T: ap-human T3 receptor complexes. Nature 325:125. Samelson, L.E., Germain, R.N. and Schwartz, R.H. (1983) Monoclonal antibodies against the antigen receptor on cloned T-cell hybrid. Proc. Natl. Acad. Sci. USA 80:6972. Samelson, L.E., Harford, J.B. and Klausner, R.D. (1985) Identification of t l ; components of the murine T cell antigen receptor complex. Cell 43:223. Samelson, L.E., Lindsten, T., Fowlkes, B.J., van den Elsen, P., Terhorst, C, Davis, M.M., Germain, R.N. and Schwartz, R.H. (1985b) Expression of genes of the T-cell antigen receptor complex in precursor thymocytes. Nature 315:765. Samelson, L.E., Patel, M.D., Weissman, A.M., Harford, J.B. and Klausner, R.D. (1986) Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen receptor. Cell 46:1083. Samelson, L.E., Phillips, A.F., Luong, E.T. and Klausner, R.D. (1990) Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc. Natl. Acad. Sci. USA 87:4358. Schatz, D.G., Oettinger, M.A. and Baltimore, D. (1989) The V(D)J recombination activating gene, RAG-1. Cell 59:1035. Schonrich, G., Momburg, F., Hammerling, G.J. and Arnold, B. (1992) Anergy induced by thymic medullary epithelium. Eur. J. Immunol. 22:1687. Schreiber, S.L. and Crabtree, G.R. (1992) The mechanism of action of cyclosporin A and FK506. Immunol. Today 13:136. Schwadron, R.B., Gandour, D.M. and Strober, S. (1985) Cloned natural suppressor cell lines derived from the spleen of neonatal mice. J. Exp. Med. 162:297. Scollay, R., Wilson, A., D'Amico, A., Kelly, K., Egerton, M., Pearse, M., Wu, L. and Shortman, K. (1988) Developmental status and reconstitution potential of subpopulations of murine thymocytes. Immunol. Rev. 104:81. Scott, B., Bluthmann, H., Teh, H.S., and von Boehmer, H. (1989) The generation of mature T cells requires interaction of the a.p T-cell receptor with major histocompatibility antigens. Nature 338:591. 59 Scott, P. (1991) IFN-y modulates the early development of Thl and Yh2 responses in a murine model of cutaneous Leishmaniasis. J. Immunol. 147:3149. Seder, R.A., Paul, W.E., Davis, M.M. and Fazekas de St Groth, B. (1992) The presence of interleukin-4 during in vitro priming determines the lymphokine-producing potential of CD4 + T cells from T cell transgenic mice. J. Exp. Med. 176:1091. Seki, H., Nanno, M., Day, N.K., Good, R.A. and Platsoucas, CD. (1989) Disulfide-linked y5 T cell antigen receptors expressed on T cells derived from patients with primary immunodeficiency disorders. J. Clin. Invest. 84:1679. Seki, S., Abo, T., Ohteki, T., Sugiura, K. and Kumagai, K. (1991) Unusual cq3-T cells expanded in autoimmune Ipr mice are probably a counterpart of normal T cells in the liver. J. Immunol. 147:1214. Sentman, C.I., Shutter, J.R., Hockenbery, D., Kanagawa, O. and Korsmeyer, S.J. (1991) Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 67:889. Sha, W.C., Nelson, C.A., Newberry, R.D., Kranz, D.M., Russel, J.H., and Loh, D.Y. (1988a) Positive and negative selection of an antigen receptor on T cells in transgenic aiice. Nature 336:73. Sha, W.C., Nelson, C.A., Newberry, R.D., Kranz, D.M., Russel, J.H., and Loh, D.Y. (1988b) Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice. Nature 335:271. Shibuya, H., Yoneyama, M., Ninomiya-Tsuji, J., Matsumoto, K. and Taniguchi, T. (1992) IL-2 and EGF receptors stimulate the hemopoietic cell cycle via different signalling pathways and demonstration of a novel role for c-myc. Cell 70:57. Shinkai, Y., Rathbun, G., Lam, K-P. Oltz, E.M., Stewart, V., Mendelsohn, M., Charron, J., Dattam, Young, F., Stall, A.M. and Alt, F.W. (1992) &4G-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangements. Cell 68:855. Shivakumar, S., Tsokos, G.C. and Datta, S.K. (1989) T cell receptor a/p expressing double negative (CD47CD8") and CD4 + T helper cells in human augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis. J. Immunol. 143' 103. Shortman, K., Wilson, A., Egerton, M., Pearse, M. and Scollay, R. (1989) Immature CD4" CD8+ murine thymocytes. Cell. Immunol. 113:462. Sidman, C.L., Marshall, J.D. and von Boehmer, H. (1992) Transgenic T cell receptor interactions in the lymphoproliferative and autoimmune syndromes of Ipr and gld mutant mice. Eur. J. Immunol. 22:499. Siegel, J.N., Klausner, R.D., Rapp, U.R. and Samelson, L.E. (1990) T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protien kinase C-dependent pathway. J. Biol. Chem. 265:18472. 60 Siegel, R.M., Katsumata, M., Miyashita, T., Louie, D.C., Greene, M.I. and Reed, J.C. (1992) Inhibition of thymocyte apoptosis and negative antigenic selection in Bcl-2 transgenic mice. Proc. Natl. Acad. Sci. USA 89:7003. Siliciano, J.D., Morrow, T.A. and Desidero, S.V. (1992) Itk, a T-cell specific tyrosine kinase gene inducible by interleukin-2. Proc. Natl. Acad. Sci. USA 89:11194. Snapper, CM. and Paul, W.E. (1987) Interferon-y and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944. Snow, P.M. and Terhorst, C. (1983) The T antigen is a multimeric complex of two distinct subunits on human thymocytes but consists of homomultimeric forms on peripheral blood T lymphocytes. J. Biol. Chem. 258:14675. Snow, P.M., Van de Rijn, M. and Terhorst, C. (1985) Association between the human thymic differentiation antigens T6 and T8. Eur. J. Immunol. 15:529. Spangrude, G.J., Heimfeld, S. and Weissman, LL. (1988) Purification and characterization of mouse hemopoietic stem cells. Science 241:58. Spangrude, G.J. and Scollay, R. (1990) A simplified method for enrichment of mouse hematopoietic stem cells. Exp. Hematol. 18:920. Sprent, J., Lo, D., Gao, E.K. and Ron, Y. (1988) T cell selection in the thymus. Immunol. Rev. 101:173. Staerz, U.D., Pasternack, M.S., Klein, J.R., Benedetto, U.D. and Bevan, M.J. (1984) Monoclonal antibodies specific for a murine cytotoxic T-lymphocyte clone. Proc. Natl. Acad. Sci. USA 81:1799. Steinberg, A.D., Roths, J.B., Murphy, E.D., Steinberg, R.T. and Raveche, E.S. (1980) Effect of thymectomy or androgen administration upon the autoimmune disease of MRL/Mp-lpr/lpr mice. J. Immunol. 125:871. Stevens, T., Bossie, A., Sanders, V.M., Fernaandez-Botran, R., Coffman, R., Mossman, T. and Vitetta, E.S. (1988) Regulation of antibody isotype secretion by subsets of antigen specific helper T cells. Nature 334:255. Stingl, G., Koning, F., Yamada, H., Yokoyama, W.M., Tschachler, E., Bluestone, J.A., Steiner, G., Samelson, S., Lew, A.M., Coligan, J.E. and Shevach, E. (1987) Thy-1+ dendritic epidermal cells express T3 and T cell receptor gamma chain. Proc. Natl. Acad. Sci. USA 84:4586. Strasser, A., Harris, A.W. and Cory, S. (1991) Bcl-2 transgene inhibits T cell death and peerturbs thymic self-censorship. Cell 67:889. Straus, D. and Weiss, A. (1992) Genetic evidence for the involvement of the Ick tyrosine kinase in signal transduction through the T cell antigen receptor. Cell 70:585. 61 Strober, S., Palathumpat, V., Schwadron, R. and Hertel-Wulff. (1987) Cloned natural suppressor cells prevent lethal graft-versus-host disease. J. Immunol. 138:699. Sturm, E., Braakman, E., Fisch, P., Vreudgenhil, R.J., Sondel, P. and Bolhuis, R.L.H., (1990) Human Vy9-V82 T cell receptor-y5 lymphocytes show specificity to Daudi Burkitt's lymphoma cells. J. Immunol. 145:3202. Suda, T. and Zlotnik, A. (1991) IL-7 maintains the T cell precursor potential of CD3' CD4" CD8" thymocytes. J. Immunol. 146:3068. Suda, T. and Zlotnik, A. (1993) Origin, differentiation, and repertoire selection of CD3 + CD4" CD8" thymocytes bearing either ap or y8 T cell receptors. J. Immunol. 150:447. Sukhatme, V.P., Sizer,. K.C., Vollmer, A.C., Hunkapiller, T. and Parnes, J.R. (1985) The T cell differentiation antigen Leu-2/T8 is homologous to immunoglobulin and T cell receptor variable regions. Cell 40:591 Sussman, J.J., Bonifacino, J.S., Lippincott-Schwartz, J., Weissman, A.M., Saito, T., Klausner, R.D. and Ashwell, J.D. (1988) Failure to synthesize the T cell CD3-^ chain: structure and function of a partial T cell receptor complex. Cell 52:85. Swain, S.L. (1981) Significance of Lyt phenotypes: Lyt 2 antibodies block activities of T cells that recognize class I major histocompatibility complex antigens regardless of their function. Proc. Natl. Acad. Sci. USA 78:7101. Swat, W., Dessing, M., Baron, A., Kisielow, P. and von Boehmer, H. (1992) Phenotypic changes accompanying positive selection of CD4 + CD8 + thymocytes. Eur. J. Immunol. 22:2367. Sykes, M. (1990) Unusual T cell populations in adult murine bone marrow: prevelance of CD3 + CD4" CD8" and apTCR cells. J. Immunol. 145:3209. Sykes, M., Hoyles, K.A., Romick, M.L. and Sachs, D.H. (1990a) In vitro and in vivo analysis of bone marrow derived CD3 + CD4" CD8" NK1.1+ cell lines. Cell Immunol. 129:478. Sykes, M., Romick, M.L., Hoyles, K.A. and Sachs, D.H. (1990b) In vivo administration of interleukin-2 plus T cell-depleted syngeneic marrow prevents graft-versus-host mortality and permits alloengraftment. J. Exp. Med. 171:645. Sykes, M., Romick, M. and Sachs, D.H. (1990c) Interleukin-2 prevents graft-versus-host disease without diminishing the graft-versus-leukemia effect of allogeneic lymphocytes. Proc. Natl. Acad. Sci. USA 87:5633. Takahama, Y., Kosugi, A. and Singer, A. (1991) Phenotype, ontogeny, and repertoire of CD4" CD8" T cell receptor a p + thymocytes. Variable influence of self-antigens on T cell receptor VP usage. J. Immunol. 146:1134. Takeshita, T. Asao, H., Ontani, K., Ishii, N., Kumaki, S., Tanaka, N., Munakata, H., Nakamura, M. and Sugamura, (1992) Cloning of the y chain of the human IL-2 receptor. Science 257:379. 62 Teh, H.S., Kisielow, P., Scott, B., Kishi, H., Uetmatsu, Y., Bluthrnann, H., and von Boehmer, H. (1988) Thymic major histocompatibility complex antigens and the ap T-cell receptor determine the CD4/CD8 phenotype of T cells. Nature 335:229. Teh, H.S., Kishi, H., Scott, B. and von Boehemer, H. (1989) Detection of autospecific T cells in T cell receptor (TCR) transgenic mice spares cells with normal TCR levels and low levels of CD8 molecules. J. Exp. Med. 169:795. Theofilopoulos, A.N. and Dixon, F.J. (1981) Etiopathogenesis of murine SLE. Immunol. Rev. 55:179. Theofilopoulos, A.N., Balderas, R.S., Shawler, D.L., Lee, S. and Dixon, F.J. (1981) Influence of thymic genotype on the systemic lupus erythematosus-like disease and T cell proliferation of MRLMp-lpr/lpr mice. J. Exp. Med. 153:1405. Thompson, C.G., Lindsten, T., Ledbetter, J.A., Kunkel, S.L., Young, H.A., Emerson, S.G., Leiden, J.M. and June, C.H. (1989) CD28 activation pathway regulates the production of multiple T-cell derived lymphokines/cytokines. Proc. Natl. Acad. Sci. USA 86:1333. Toyonaga, B. and Mak, T. (1987) Genes of the T cell antigen receptor in liormal and malignant T cells. Ann. Rev. Immunol. 5:585. Truneh, A., Albert, F., Golstein, P. and Schmitt-Verhulst, A.M. (1985) Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester. Nature 313:318. Tsudo, M., Kozak, R.W., Goldman, C.K. and Waldmann, T.A. (1986) Demonstration of a non-Tac peptide that binds interleukin 2: A potential participant in a multichain interleukin 2 receptor complex. Proc. Natl. Acad. Sci. USA 83:9694. Tunnacliffe, A., Buluwela, L. and Rabbitts, T.H. (1987) Physical linkage of three CD3 genes on human chromosome 11. EMBO J. 6:2953. Turka, L.A., Schatz, D.G., Oettinger, M.A., Chun, J.J.M., Gorka, C, Lee, K., McCormack, W.T. and Thompson, C.B. (1991) Thymocyte expression of RAG-1 and RAG-2; termination by T cell receptor cross-linking. Science 253:778. Turner, J.M., Brodsky, M.H., Irving, B.A., Levin, S.D., Perlmutter, R.M. and Liftman, D.R. (1990) Interaction of the unique N-terminal region of tyrosine kinase p 5 6 ^ with cytoplasmic domains of CD4 and CD8 mediated by cysteine motifs. Cell 60:755. Van Agthoven, A., Terhorst, C, Reinherz, C. and Schlossman, S.F. (1981) Characterization of T cell surface glycoproteins T l and T3 present on all human peripheral T lymphocytes and functionally mature thymocytes. Eur. J. Immunol. 11:18. Vandeberghe, P., Freeman, G.J., Nadler, G. Fletche, M.C., Kamoun, M, Turka, L.A., Ledbetter, J.A., Thompson, C.B. and June, L.H. (1992) Antibody and B7/BB1-mediated ligation of the CD25 receptor induces tyrosine phosphorylation in human T cells. J. Exp. Med. 175:951. 63 Veillette, A., Bookman, M.A., Horak, E.M. and Bolen, J.B. (1988) The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56/c^. Cell 55:301. Veillette, A., Bolen, J.B. and Bookman, MA. (1989) Alteration in tyrosine phosphorylation induced by antibody-mediated cross-linking of the CD4 receptor of T lymphocytes. Mol. Cell. Biol. 9:4441. Veillette, A., Abraham, N., Caron, L. and Davidson, D. (1992) The lymphocyte-specific tyrosine protein kinase p56 Ick. Semin. Immunol. 3:143. Vukmanovic, S., Grandea, A.G., Faas, S.J., Knowles, B.B. and Bevan, M.J. (1992) Positive selection of T-lymphocytes induced by intra-thymic injection of thymic epithelial cell line. Nature 359:729. Weingensberg, M. Morecki, S., Weiss, L., Fuks, Z. and Slavin, S. (1984) Suppression of cell-mediated immune responses after total lymphoid irradiation (TLI). 1. Characterization of suppressor cells of mixed lymphocyte reaction. J. Immunol. 132:971. Weiss, A., Daley, J.F., Hodgdon, J.C. and Reinherz, E.L. (1984a) Calcium dependency of antigen-specific (T3-Ti) and alternative (Til) pathways of human T-cell activation. Proc. Natl. Acad. Sci. USA 81:6836. Weiss, A., Imboden, J., Schoback, D. and Stobo, J. (1984c) Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium. Proc. Natl. Acad. Sci. USA 81:4169. Weiss, A. and Sobo, J.D. (1984c) Requirement for the coexpression of T3 and the T cell antigen receptor on a malignant human T cell line. J. Exp. Med. 160:1284. Weiss, A., Newton, M. and Crommie, D. (1986) Expression of T3 in association with a molecule distinct from the T-cell antigen receptor heterodimer. Proc. Natl. Acad. Sci. USA 83:6998. Weiss, A., Koretzky, G., Schatzman, R. and Kadlecek, T. (1991) Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-yl. Proc. Natl. Acad. Sci. USA 88:5484. Weissman, A.M., Baniyash, M., Hou, D., Samelson, L.E., Burgess, W.H. and Klausner, R.D. (1998) Molecular cloning of the zeta chain of the T cell antigen receptor. Science 239:1018. Weissman, A.M., Frank, S.J., Orloff, D.G., Mercep, M. Ashwell, J.D. and Klausner, R.D. (1989) Role of the zeta chain in the expression of the T cell antigen receptor: genetic reconstitution studies. EMBO J. 8:3651. Widera, G., Burkly, L.C., Pinkert, C.A., Bottger, E.C., Cowing, C, Palmiter, R.D., Brinster, R.L and Favell, R.A. (1987) Transgenic mice selectively lacking MHC class II (I-E) antigen expression on B cells: an in vivo approach to investigate la gene function. Cell 51:175. 64 Wilson, A., D'Amico, A., Ewing, T., Scollay, R. and Shortman, K. (1988a) Subpopulations of early thymocytes: a cross-correlation flow cytometric analysis of adult mouse Ly-2" L3T4" (CD8~ CD4") thymocytes using eight different surface markers. J. Immunol. 140:1461. Wilson, A., Ewing, T., Owens, T., Scollay, R. and Shortman, R. (1988b) T cell antigen receptor expression by subsets of Ly-2- L3T4- (CD4" CD8") thymocytes. J. Immunol. 140:1470. Wilson, A., Petrie, H.T., Scollay, R. and Shortman, K. (1989) Acquisition of CD4 and CD8 during differentiation of early thymocytes in short term culture. Int. Immunol. 1:605. Wofsy, D., Hardy, R.R. and Seaman, W.E. (1984) The proliferating cells in autoimmune MRL/lpr mice lack L3T4, an antigen on "helper" T cells that is involved in the response to class II major histocompatibility antigens. J. Immunol. 132:2686. Wright, A., Lee, J.E., Link, M.P., Smith, S.D., Carroll, W. Levy, R., Clayberger, C. and Krensky, A.M. (1989) Cytotoxic T lymphocytes specific for self tumour immunoglobulin express T cell receptorS chain. J. Exp. Med. 169:1557. Wu, L., Pearse, M., Egerton, M., Petrie, H. and Scollay, R. (1990) CD4" CD8" thymocytes that express the T cell receptor may have previously expressed CD8. Int. Immunol. 2:51. Wu, L., Scollay, R., Egerton, M., Pearse, M., Spangrude, G.J. and Shortman, K. (1991) CD4 expressed on earliest T-lineage precursor cells in the adult murine thymus. Nature 349:71. Yagita, H, Asakawa, J.L, Tansyo, S., Nakamura, T., Habu, S. and Okomura, K. (1989) Expression and function of CD2 during murine thymocyte ontogeny. Eur. J. Immunol. 19:2211. Yoshimura, A., Shiku, H. and Nakayama, E. (1993) Rejection of an I A + variant line of FBL-3 leukemia by cytotoxic T lymphocytes with CD4 + and CD4" CD8" T cell ieceptor-ccP phenotypes generated in CD8-depleted C57BL/6 mice. J. Immunol. 150:4900. Zanders, E.D., Lamb, J.R., Feldman, M., Green, N. and Beverly, P.C.L. (1983) Tolerance of T-cell clones is associated with membrane antigen changes. Nature 303:625. Zochi, M.R., Ferrarini, M. and Rugarli, C. (1990) Selective lysis of the autologous tumour by 8TCS1 + y/S+ ramour-infiltrating lymphocytes from human lung carcinomas. Eur. J. Immunol. 20:2685. Zuniga-Pflucker, J.C., McCarthy, S.A., Weston, M., Longo, D.L., Singer, A.and Kruisbeek, A.M. (1989) Role of CD4 in thymocyte selection and maturation. J. Exp. Med. 169:2085. Zuniga-Pflucker, J.C., Jones, L.A., Longo, D.L. and Kriusbeek, A.M. (1990) CD8 is required during positive selection of CD4" CD8 + T cells. J. Exp. Med. 171:427. 65 C H A P T E R II MATERIALS AND METHODS 2.1 ANIMALS Inbred BALB/c mice were obtained from the Jackson Laboratories, Bar Harbor, ME. and maintained in the Animal Facility of the B.C. Cancer Research Center. 2.2 ANTIBODIES Hybridoma lines 53-6.72 (Ledbetter and Herzenberg, 1979) producing MAb to CD8, Ml/70 (Springer et al. 1979) producing MAb to the a unit of Mac-1 , Ml/69 (Springer et al. 1978) producing MAb to heat stable antigen, KJ16 producing MAb to Vpg \ and Vpg 2 of the TCR ap (Haskins et al. 1984), RG7/9.1 HLK (Springer et. al. 1982) producing MAb to rat K light chain were obtained from the American Type Culture Collection (Rockville, MD). Hybridoma lines GK1.5 (Dialynas et al. 1984) and 145-2C11 (Leo et al. 1987) were used to produce MAb to CD4 and CD3 respectively. Undiluted supernatants of overgrown cultures were used as the source of MAb. MAb RG7/9.1 and 145-2C11 were also purified and conjugated with fluorescien isothiocyanate (FITC) (Sigma Chemical Co., St. Louis, MO) or biotinylated (Sigma). Biotinylated anti-TCR aP and TCR y5 and FITC-conjugated anti-TCR y5 were obtained from Pharmingen (San Diego, CA). 2.3 ISOLATION OF T CELL POPULATIONS 2.3.1 DN splenic T cell subpopulations An outline of the procedure for the isolation of splenic T cell subpopulations is provided in Figure 2. Wire mesh was used to obtain single cell suspensions, in Hanks balanced salt solution supplemented with 5% heat inactivated fetal calf serum (FCS), of total splenic T cells from 5-6 week old BALB/c mice. Contaminating red blood cells (RBC) were lysed in 2X ammonium chloride solution (0.16 M Tris N H 4 C I pH 7.2) and washed in Hanks 5% FCS. CD4+ CD8+ HSA+ and Mac-1+ cells were depleted using a modified indirect panning method (Takei, 1988). Briefly, cells were incubated with a mixture of anti-CD4, anti-CD8, anti-HSA and anti-Mac-1 66 MAb (1 ml/spleen for each of the undiluted hybridoma supernatants) on ice for 45 min. The cells were then washed and incubated twice for 40 min. at 4°C on plastic plates precoated with purified mouse anti-rat I g K antibody, RG7/9.1 (0.1 mg/ml in 10 mM Tris pH 8.0) for 1 hr at room temperature followed by phosphate buffered saline (PBS) 5% FCS for 1 hr at 4°C. Nonadherent cells were restained on ice for 30 min. with CD4, CD8, HSA, and Mac-1 antibodies followed by FITC-conjugated RG7/9.1. Propidium iodide (1 pg/ml, 5 min. on ice) was added to stain dead cells. Positively stained cells were removed by sorting with a single beam flow cytometer (FACStar, Becton, Dickinson, Sunnyvale, CA) based on gating set by a negative control population of total nucleated spleen cells incubated with FITC-conjugated RG7/9.1 on ice for 30 min. For further purification of CD4" CD8" HSA" Mac-1" (DN) splenic T cell subpopulations, cells were stained for 30 min. on ice with either biotinylated TCR ap recognized by phycoerythrin-conjugated streptavidin (SA-PE) or FITC-conjugated TCR y5 and CD3 antibodies. TCR ap+ CD4" CD8" HSA" Mac-1" cells (DNapT cells) were isolated either by positively sorting for TCR ap+ DN cells or by negatively sorting for TCR y5" DN cells. TCR y 5 + CD4" CD8" HSA" Mac-1" cells, similarly, were isolated either by positively sorting for TCR y5 + DN cells or by negatively sorting for TCR aP" DN cells. In order to isolate TCR aP+/" DN, gating was determined from a negative control population of total nucleated spleen cells incubated with FITC-conjugated RG7/9.1 and SA-PE for 30 min. on ice. The negative control used for setting the gates in the isolation ofTCRy5 + / " DN and CD3 + /~ DN cells consisted of total nucleated spleen cells incubated with biotinylated RG7/9.1 for 30 min. on ice followed by SA-PE for 30 min. on ice. To reassess the purity following sorting, all cell populations were restained with the embodies used in the isolation procedure and analysed by flourescence activated cell sorter (FACS). 2.3.2 Mature splenic T cells Mature T cells were isolated from splenic cell suspension by passage over nylon wool column (Julius et al. 1973). Nylon wool was boiled for 10 min. in 25 mM eftylenediamine tetraacetic acid (EDTA) pH 8.0, washed twice with distilled H2O and dried overnight at 37°C in preparation for packing into the barrel of 6 ml plastic syringes (lg nylon wool per syringe) and subsequent autoclaving. The sterile packed syringe barrels were equilibrated overnight at 37°C with RPMI 1640 media 5% FCS. Nylon wool columns were loaded with 1 ml cell suspension in warm RMPI 1640 5% FCS from the spleens of 5-6 week old BALB/c mice (1 spleen/column). Following a 1 hi incubation at 37°C, the columns were washed slowly with RPMI 1640 5% FCS and the resulting effluent was collected for 45 min. The nonadherent cells were resuspended in 0.16 M Tris NH4CI pH 7.2 for depletion of contaminating RBCs. 67 2.3.3 DN thymocytes subpopulations The procedure for the isolation of DN thymocytes and their subpopulations was similar to that for splenic DN T cell subpopulations. 2.4 PHENOTYPIC ANALYSIS For phenotypic analysis, sorted populations were incubated with the specific antibody for 30 min. on ice. Propidium iodide was added prior to reanalysis to stain dead cells. 2.4.1 Splenic DN T cells In order to determine the proportion of splenic DN T cells which express either the ap yS TCR associated with the CD3 complex, sorted splenic CD4" CD8" HSA* Mac-1" (DN) T cells were stained with either biotinylated anti-TCR ap recognized by SA-PE or FITC-conjugated anti-TCR y8 or anti-CD3. 2.4.2 Splenic CD3" DN T cells In order to further characterize the phenotype of splenic CD3" DN T cells, sorted splenic CD3" CD4" CD8" HSA" Mac-1" T cells were stained with either of the MAb specific for Thy-1, Pgp-1, T220, Lyt-1, YE 1/19 (PCI), YE 1/22 (4F2), LFA-1, ICAM-1 and B220 recognized by FITC conjugated RG7/9.1. 2.4.3 Splenic DNapT cells In order to further characterize splenic DNapT cells, sorted splenic CD4" CD8" HSA" Mac-1" TCR ap T cells were stained with Pgp-1 recognized by FITC conjugated RG7/9.1 or with B220, YE1/19, YE1/9, ICAM-1 and Lyt-1 recognized by FITC conjugated goat anti-rat antibody. In order to determine whether splenic DNapT DNapT cells overexpress TCR Vpg genes, splenic DN cells as well as nylon wool purified mature splenic T cells were stained with either anti-TCR Vpg antibody KJ16 or anti-TCR ap MAb recognized by FITC conjugated RG7/9.1. 68 2.5 PROLIFERATION ASSAY In order to determine the cytokine concentrations which would give optimal stimulation, sorted DN thymocytes and spleen cells were assessed in triplicate for their proliferative response to varying concentrations of either IL-7 or IL-1 in wells previously coated for 1 hour at 4°C with 145-2C11 anti-CD3 antibody respectively. Optimal proliferative responses were obtained with 10 ng/ml IL-7 and 200 U/ml IL-1 in the presence of immobilized anti-CD3. Sorted subpopulations of DN splenic T cells and thymocytes as well as nylon wool non-adherent splenic T cells were stimulated in triplicate in round bottom microtiter wells in either 0.1 ml (if > 5.0 X 104 cells/well) or 0.05 ml (if < 5.0 X 104 cells/well) of RPMI 1640 medium containing 5% FCS and 50 uM 2-mercaptoethanol (2-ME) with either 10 ng/ml IL-7 (Genzyme, Boston, MA) or 200 U/ml IL-1 (Biogen, Geneva, Switzerland) or a combination of both. Cells were also cultured with or without 200 units/ml IL-1 in wells previously coated with 145-2C11 anti-CD3 antibody for 1 hr at 4°C. Furthermore, cells were stimulated with either 10 ng/ml PMA or 600 nM ionomycin or a combination of both. As a negative control, cells were incubated in media alone. Due to the rarity of the splenic DNapT cell population and therefore the minimal and often variable number of cells obtained from the purification procedure, the number of cells used per well often varied between experiments from 1.4 X 10 4to 1.0 X 105 cells per well in order to provide triplicate wells for ~.rh culture condition including negative and positive controls. All cultures were incubated for 72 hr at 37°C in a humidified atmosphere of 5% C 0 2 and subsequently pulsed with 50 pi ^H-mymidine (luCi) for 6 hr. Cells were then harvested and counted by a liquid scintillation counter. For each of the cell populations and stimulating conditions studied, the results given in Tables I, II and III are representative of at least three separate experiments. The standard error of means (SEM) given are calculated based on triplicate samples of the representative experiment. Standard error of means comparing the three experiments for each set of cell populations and stimulating conditions were not calculated as the number of cells/well were not consistent between experiments. 2.6 LYMPHOKINE mRNA EXPRESSION 2.6.1 Stimulation of splenic T cells Sorted splenic TCR a p + DN, TCR y5~ DN, CD3" DN and nylon column purified mature T cells were stimulated for 16 hr at 37°C in round bottom microtiter wells (1 X 10^ cells/well) in 100 pi of RPMI 1640 medium containing 5% FCS and 50 urn 2-ME with either 10 ng/ml IL-7 or 200 U/ml IL-1 or a combination of both. As mentioned previously, the concentrations of IL-7 and IL-1 69 used were those identified as providing optimal stimulation, as assessed by ^ H-mymidine incorporation, of thymic DN T cells and splenic DN T cells in wells previously coated with anti-CD3 antibodies respectively. Cells were also cultured in round bottom microtiter wells previously coated with 145-2C11 anti-CD3 antibody for 1 hr at 4°C. As a negative control, cells were incubated in media alone. All cultures were incubated for 16 hr at 37°C in a humidified atmosphere of 5% carbon dioxide (CO2). A minimum of three seperate experiments were performed in which the splenic subpopulations were stimulated under the conditions outlined above. 2.6.1 RNA extraction Total cellular RNA from sorted splenic DN subpopulations and nylon column purified mature T cells was isolated by the acid-phenol method (Chomczynski and Sacchi, 1987) either directly following purification or after 16 hr stimulation. Cells were washed 2 X in phosphate buffered saline (PBS) and the pellet was dissolved in 0.1 ml of solution D (4 M guanidine thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% Sarkosyl, 0.1 M 2-mercaptoethanol). Following this 10 pi of 1 mg/ml yeast transfer RNA (tRNA), 10 pi of 2 M sodium acetate pH 4.0, 100 pi water saturated phenol and 40 pi of chloroform:isoamyl alcohol (49:1) were added. The solution was thoroughly mixed and microfuged at 14,000 g for 10 min. at 4°C. The aqueous layer was mixed with 100 pi isopropanol, incubated at -20°C for 90 min and microfuged at 14,000 g for 15 min. at 4°C. The pellet was resuspended in 100 pi of solution D followed by 10 pi of 2 M sodium acetate pH 4.0, 100 pi of water saturated phenol, 40 pi chloroform:isoamyl alcohol (49:1). The solution was thoroughly mixed and microfuged at 14,000 g for 10 min. at 4°C. The aqueous layer was mixed with 100 pi isopropanol and incubated at -20°C for 90 min and microfuged at 14,000 g for 15 min at 4°C. The pellet was resuspended in 100 pi of distilled water, to which was added 10 pi of 3 M sodium acetate pH 6.6 and 220 pi ethanol. The solution was microfiiged for 10 min. at 4°C and the pellet was resuspended in 200 pi of 75% ethanol and centrifuged for 10 min. at 4°C. The supernatant was removed and the pellet was dried in a vacuum centrifuge. 2.6.2 cDNA synthesis Extracted RNA was converted to cDNA by reverse transcription using random hexamers as follows. The pellet obtained from RNA extraction was resuspended in 16.5 pi of distilled water to which was added 6 pi of 5 X buffer (250 mM tris HC1 pH 8.3, 300 mM potassium chloride, 15 mM magnesium chloride and 50 mM dithiothreitol), 3 pi of 1 mg/ml bovine serum albumin (BSA), 70 1 pi of 40 U/pl RNase, 1 pi of 100 ng/pl random hexamers, 1.5 pi dNTP (10 mM each of dATP, dGTP, dCTP and dTTP), and 1 pi of 200 U/pl Moloney Murine Leukemia Virus reverse transcriptase. The solution was incubated at 40°C for 1 hr. 2.6.3 Polymerase Chain Reaction Cytokine specific fragments in the cDNA samples were amplified by the polymerase chain reaction (PCR) as follows. To 5 pi of cDNA was added 26.75 pi of distilled water, J pi of 10 X reaction buffer (500 mM potassium chloride, 100 mM tris HC1 pH 8.3, 15 mM magnesium chloride, 0.1% gelatin), 8 pi dNTP (1.25 mM each of dATP, dGTP, dCTP, dTTP), 2.5 pi of 20 pM 5' oligonucleotide primer, 2.5 pi of 20 pM 3' oligonucleotide primer and 0.25 pi of 5 U/pl Taq polymerase. Specific 21-mer oligonucleotide primers for the cytokines IFNy, IL-2, IL-4 and IL-6 as well for the positive control, p actin, were used. Sequences for the oligonucleotide primers is included in Figure 3. Cytokine and actin specific DNA fragments were amplified by 30 cycles of PCR. Each cycle consisted of denaturation for 1 min. at 94°C, annealing for 2 min. at 50°C and extension for 3 min. at 72°C. 2.6.7 Agarose Gel Electrophoresis The amplified DNA (15 pi) was loaded onto 1% agarose mini-gels anu electrophoresed in tris borate EDTA (TBE) buffer (89 mM Tris, 89 mM borate, 25 mM EDTA pH 8.3) at 80 volts for 1.5 hr. Ethidium bromide was added to both the gel and the running buffer at a concentration of 500 ng/ml. Molecular weights were determined with a X Hindlll molecular weight standard. The gels were visualized and photographed under ultra violet light. Expected sizes of amplified fragments of p actin, IFNy, IL-2, IL-4 and IL-6 were 540, 460, 502, 399 and 638 base pairs (bp) respectively. 2.6.8 Southern Blotting The agarose gel containing the PCR amplified fragments was layered on top of a 3M Whatman filter paper saturated with 0.4 M sodium hydroxide (NaOH). The ends of the filter paper were in contact with the 0.4 M NaOH solution. A piece of Zeta Probe (Bio-rad Laboratories; Mississauga, Ontario), cut to the size of the gel, was presaturated with 0.4 M NaOH and placed on top of the gel. Two pieces of Whatman paper were layered on top of the membrane followed by approximately 10 cm of paper towels and then a glass plate and weight. The dry paper towels draw the 0.4 M NaOH solution upward by capillary action and provide a unidirectional transfer of 71 the DNA onto the membrane. The transfer was allowed to proceed overnight following which the membrane was rinsed in 6X saline sodium citrate buffer (SSC) (20 X SSC: 3 M NaCl, 0.15 M sodium citrate pH 7.0). 2.6.9 Prehybridization The membrane was incubated at 60°C for 2 hr in a prehybridization solution consisting of 3X saline sodium phosphate EDTA buffer (SSPE) (20 X SSPE: 3.6 M NaCl, 0.2 M NaH 2P0 4, 20 mM EDTA pH 7.4), 1% sodium dodecyl sulphate (SDS), 0.5% Blotto (Carnation Instant Skim Milk) and 0.5 mg/ml denatured salmon sperm DNA. 2.6.10 Oligolabelled Probe The IFNy cDNA (Clontech Laboratories, Palo Alto, CA) was radiolabelled by the following oligolabelling procedure (Feinberg and Vogelstein, 1983). Briefly, 20 ng of IFNy DNA was added to 200 ng of randomly generated hexanucleotides in a final volume of 14 pi. The solution was incubated at 95°C for 3 min. followed by 1 min. on ice. To this solution was added 2 pi of 10X HLB buffer (0.5 M HEPES pH 6.9, 0.1 M MgCl 2, 0.06 M 2-mercaptoethanol), 2 pi of a nucleotide mixture (dATP, dGTP, dTTP, 2.5 mM each), 5 pi [a-32P]-dCTP and 1 unit of Kienow polymerase (Pharmacia). The solution was then incubated at room temperature for 2 hr and the radiolabelled IFNy DNA was subsequently purified using a Nick Column. Briefly, the solution was added to a Nick column pre-equilibrated with 3 ml of tris EDTA (TE) (10 mM Tris pH 8.0, 1 mM EDTA). Two 400 pi aliquots of TE were passed through the column, the second of which was collected, boiled for 3 min. and then stored on ice. 2.6.11 Hybridization and Washing The membrane was removed from the prehybridization solution and transferred to the hybridization mixture consisting of 3X SSPE, 10% SDS, 0.5% Blotto and 0.5 mg/ml denatured salmon sperm DNA to which the oligolabelled probe was then added. The membrane was incubated overnight at 60°C and then washed in each of the following three solutions for 15 min. at room temperature; 2X SSC, 0.1% SDS; 0.5X SSC, 0.1% SDS; and 0.1X SSC, 0.1% SDS. The final wash of the membrane was in prewarmed 0.1 X SSC, 1% SDS at 55°C for 1 hr. The membrane was then dried at room temperature for 15 min. and exposed to Kodak XAR film at -70°C for 1 hr. 72 SPLEEN LYSE RBC STAIN CELLS WITH RAT-ANTI-MOUSE ANTIBODIES DIRECTED AGAINST CD4, CD8, HSA AND MAC-1 INDIRECT PANNING WITH MOUSE-ANTI-RAT lg ANTIBODY TIB169 STAIN NONADHERENT CELLS WITH ANTIBODIES DIRECTED AGAINST TCR ap (or TCR y5) SORT BY FLOW CYTOMETRY TCR ap+ (ORTCRy5+) CD4" CD8* T CELLS TCR ap" (OR TCR 76") CD4" CDS" T CELLS Figure 2 Procedure for the isolation of spleen cell subpopulations. Total spleen cells depleted of contaminating RBC were incubated with the rat-anti-mouse MAb specific for CD4, CD8, HSA and Mac-1. Positively stained cells were removed by two rounds of panning on mouse-anti-rat MAb coated plates. Nonadherent cells were restained with the above MAb and sorted by FACS. To further purify DNapT cells, panned cells were also stained with MAb towards the TCR ap prior to sorting into TCR a p + and aP" DN populations. This procedure was also used to purify DN cells expressing the alternate T C R y8 by including MAb towards this TCR in the staining step prior to sorting into T C R y 6 + and y8~ D N subpopulations. 73 a) P Actin 5' primer 3' primer 5'-GTG GGC CGC TCT AGG CAC CAA-3' 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3' b) IFNY 5' primer 3' primer 5'-GTG GGC CGC TCT AGG CAC CAA-3' 5'-CGA CTC CTT TTC CGC TTC CTG AG-3' c) IL-2 5' primer 3' primer 5'-ATG TAC AGC ATG CAG CTC GCA TC-3' 5'-GGC TTG TTG AGA TGA TGC TTT GAC A-3 d) IL-4 5' primer 3' primer 5'-ATG GGT CTC AAC CCC CAG CTA GT-3' 5'-GCT CTT TAG GCT TTC CAG GAA GTC-3' e) IL-6 5' primer 3' primer 5'-ATG AAG TTC CTC TCT GCA AGA GAC T-3 3'-CAC TAG GTT TGC CGA GTA GAT CTC-5' Figure 3 PCR cytokine oligonucleotide primers. Sequences for the 5' and 3' oligonucleotide primers used in the PCR amplification of a) p actin (Alonso et al. 1986), b) IFNy (Gray and Goeddel, 1983) c) IL-2 (Kashima et al. 1985), d) IL-4 (Otsuka et al. 1987) and e) IL-6. 74 2.7 R E F E R E N C E S Alonso, S., Minty, A., Bourlet, Y. and Buckingham, M.C. (1986) Comparison of three actin-coding sequences in the mouse: evolutionary relationships between the actin genes of warm-blooded vertebrates. J. Mol. Evol. 23:22. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156. Dialynas, D.P., Wilde, D.B., Marrack, P., Pierres, A., Wall, P., Havran, G., Otten, G., Pierres, M.R., and Fitch, F.W. (1984) Characterization of the murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK1.5: expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHC antigen reactivity. Immunol. Rev. 74:29. Gray, P.W. and Goeddel D.V. (1983) Cloning and expression of murin immune interferon cDNA. PNAS USA 80:5842. Haskins, K., Hannum, C, White, J., Roehm, N., Kubo, R., Kappler, J. and Marrack, P. (1984) The major histocompatibility complex-restricted antigen receptor on T cells. VI. An antibody to receptor allotype. J. Exp. Med. 160:452. Julius, M.H., Simpson, E. and Herzenberg, L.A. (1973) A rapid method for the isolation of functional thymus derived murine lymphocytes. Eur. J. Immunol. 3:645. Kashima, N., Nishi-Takgoka, C, Hamuro, J. and Taniguch, T. (1985) Unique structure of murine interleukin-2 as deduced from cloned cDNAs. Nature 313:402. Ledbetter, J.A. and Herzenberg, L.A. (1979) Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47:63. Leo, 0., Foo, M., Sachs, D.H., Samelson, L.E., and Bluestone, J.A. (1987) Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl. Acad. Sci. USA. 84:1374. Springer, T., Galfre, G., Secher, D.S. and Milstein, C. (1978) Monoclonal xenogeniec antibodies to murine cell surface antigens: Identification of novel leukocyte differentiation antigens. Eur. J. Immunol. 8:539. Otsuka, T., Villaret, D., Yokota, T., Takebe, Y., Lee, F., Arai, N. and Arai, K. (1987) Structural analysis of the mouse chromosomal gene encoding interleukin-4 which expresses B cell, T cell and mast cell stimulating activities. Nucl. Acids Res. 15:333. Springer, T., Galfre, G., Secher, D.S. and Milstein, C. (1979) A macrophage differentiation antigen identified by monoclonal antibody. Eur. J. Immunol. 9:301. Springer, T., Bhattacharva, A., Cardoza, J.T., and Sanchez-Madrid, F. (1982) Monoclonal antibodies specific to rat IgGi, IgG2a, Ig2B subclasses, and kappa chain monotypic and allotypic determinants. Hybridoma 1:257. Takei, F. (1988) IL-2 and IL-4 stimulate different subpopulations of double negative thymocytes. J. Immunol. 141:1114. 75 C H A P T E R III RESULTS 3.1 IDENTIFICATION OF SPLENIC DNaBT CELLS For the detection of splenic DNapT cells, total nucleated spleen cells from young adult BALB/c mice were stained with MAb to CD4, CD8, HSA and Mac-1 recognized by FITC-conjugated anti-rat Ig K (RG7/9.1) and biotinylated anti-TCR ap recognized by SA-PE. MAb against CD4 and CD8 were used to remove mature SP splenic T cells. HSA was a convenient marker for this study as it is widely distributed on murine leukocytes including immature thymocytes (Crispe and Bevan, 1987), most B cells (Bruce et al. 1981, Takei et al. 1981) and the majority of myeloid cells (Bruce et al. 1981), yet is absent on mature peripheral T cells (Crispe and Bevan, 1987) and thymic DNapT cells (Ceredig et al. 1987). Since initial FACS analysis revealed the presence of Mac-1+ cells in the splenic TCR a P + CD4" CD 8" HSA" population, anti-Mac-1+ antibody was included in subsequent studies. Based on two colour FACS analysis, DNapT cells constituted only 0.1-0.3% of total nucleated spleen cells (Fig. 4). Following two rounds of panning, DNapT cells represented approximately 1.4-2.5% of panned CD4" CD8" HSA" Mac-1" (DN) cells (Fig. 4). To further enrich for DNapT cells, panned CD4" CD8" HSA" Mac-1" cells were subsequently isolated by FACS sorting. Reanalysis of the isolated cells (Fig.5) suggested that of the 0.7-1.1% of total splenic cells which expressed the DN phenotype, 17-22% were TCR a P + whereas 21-25% were TCR yS+. Approximately 52-60% of DN spleen cells expressed the CD3 complex. The phenotype of the remaining CD3" DN was assessed (Fig. 6) in the hope of finding a distinct marker that could be used to deplete this population during the purification procedure. The remaining CD3" cells expressed significant levels of Pgp-1 (93.1%), T200 (99.8%) and LFA-1 (99.8%) whereas expression of ICAM-1, YE1/19 (PCI), YE1/22 (4F2), Lyt-1, B220 and surface Ig was minimal to absent. The CD3" DN population also consists two d i s t i l populations based on either the expression or lack of expression of Thy-1. 3.2 CHARACTERIZATION OF SPLENIC DNaBT CELLS 3.2.1 Phenotypic analysis of Splenic DNapT cells Following identification, splenic DNapT cells were then characterized with respect to their phenotype. As mentioned previously, splenic DNapT cells lack the expression of HSA, a marker 76 characteristically found on immature thymocytes. Furthermore, when DNapT were assessed with a number of MAb following isolation by panning and FACS sorting (Fig. 7), this splenic population was shown to express Lyt-1 (95.1%), Pgp-1 (90.9%), ICAM-1 (79.9%) and the transferin receptor (71.2%). Minimal to no expression of B220 (9.0%) and YE1/19 (plasma cell antigen) (18.0%) was observed. 3.2.2 Determination of Vpg Usage by Splenic DNapT Cells Thymic DNapT cells are known to express an unusual TCR repertoire with preferential usage of Vpg genes (Fowlkes et al. 1987; Papiernik and Pontoux, 1990; Takahama et al. 1991). In order to determine whether splenic DNapT share this characteristic Vpg skewing, CD4" CD8" HSA" Mac-1" spleen cells were analysed (Fig. 8), following purification by panning and FACS sorting, with MAb KJ16 which recognizes T cell receptors bearing either Vpg \ or Vpg 2 (Haskins et al. 1984). Approximately 11% of the DN spleen cells were KJ16 + whereas 23% were TCR ap +. Therefore the frequency of Vpg usage amongst splenic DNapT cells was approximately 48%. Vpg usage in DNapT cells was then compared to that in mature SP T cells isolated by a nylon wool column method (Julius et al. 1973). In order to confirm that nylon wool purified cells were indeed SP T cells, the phenotype of this population was assessed. As can be seen in Fig. 9, the majority (94.5%) of nylon wool purified spleen cells do indeed express either CD4 or CD8 and are Thy-l+(93.7%) HSA" and Mac-1" (61%). Although 61% of nylon wool purified spleen cells are HSA" Mac-1", approximately 39% of cells expressed low levels of either of these markers which may be accounted for in part by the presence of contaminating B cells. It has been previously shown that approximately 5-10% of nonadherent cells obtained following passage through a nylon column are indeed B cells (Julius et al. 1973). Furthermore a subpopulation of cells of the monocyte/macrophage lineage has been shown to express the CD4 molecule. In contrast to DNapT cells, approximately 21% of nylon wool purified spleen T cells were KJ16 + while 92% were TCR ap +. Therefore only 22% of the TCR a p + T cells expressing either CD4 or CD8 used members of this Vpg family. 3.2.3 Proliferative Response of Splenic DNapT cells to Cytokines Further characterization of this splenic DNapT cell population included an assessment of the response to antigen independent stimulation. Since CD4" CD8" thymocytes, including those which express the TCR ap have been shown to respond to IL-7 in the absence of additional co-mitogen (Sudae/o/. 1990, Vissinga et al. 1992), the effects of this cytokine on splenic DNapT cells were examined. Initially CD4" CD8" HSA" spleen cells, isolated by panning and sorting, were cultured 77 with purified recombinant IL-7 for 72 hr and the proliferative response was assessed by ^ H-thymidine incorporation. As indicated in Table I, CD4" CD8" HSA" spleen cells were directly responsive to IL-7 and this responsiveness is comparable to that observed for CD4" CD8" thymocytes. However, when this population was further purified by the renu.al of Mac-1+ cells, the response to IL-7 was significantly lower. The addition of IL-1, a known macrophage product, apparently restored this response. In order to confirm that the IL-7 responsive cells are indeed T cells and not other cells which may be present in the CD4" CD8" HSA" Mac-1" population such as pre-B cells, DN spleen cells were sorted into CD3 + and CD3" subpopulations and were studied. As shown in Table I, only those cells expressing the CD3 complex were responsive to IL-7, and this response was enhanced by the addition of IL-1. Since the CD3 complex is co-expressed on T cells with the T cell receptor, the CD3 + DN population was further subdivided into those DN cells expressing either the ap or y5 TCR. Both of these TCR expressing T cell populations were stimulated by IL-7 and this response was enhanced by IL-1. In order to determine whether the proliferative response was due to the activation of the TCR-CD3 complex by the antibodies used in the purification procedure, both populations were sorted negatively. DNapT cells, sorted negatively as TCR y5" DN, proliferated in the presence of IL-7 and this response was significantly enhanced by IL-1. Reanalysis of TCR y5" DN cells following sorting confirmed the presence of DNapT cells (25-33%) (Fig. 10). Similarly, minimal change in the proliferative response was observed when TCR y5+ DN cells were sorted negatively as TCR ap DN. Therefore the proliferative response observed for both TCR expressing populations was not influenced by the anti-TCR antibodies. Unlike DNapT cells, the proliferation of nylon wool purified splenic T cells in the presence of IL-7 was minimal and was not influenced by the addition of IL-1. 3.2.4 Stimulation of Splenic DNapT cells by anti-TCR crosslinking Splenic DNapT cells, which are HSA", are thought to be mature T cells since the loss of HSA expression is closely associated with the maturation of T lymphocytes (Crispe and Bevan, 1987). In order to assess the functional maturity of DNapT cells, the effects of TCR cross-linking on this splenic subpopulation were determined by ^ H-thymidine incorporation assay. Initially, splenic DN T cells were isolated by panning followed by FACS sorting and were incubated in wells pre-coated with MAb against CD3. As shown in Table II, splenic DN T cells proliferate vigorously in the presence of immobilized anti-CD3, a response comparable to that of nylon wool purified mature SP T cells. When DN spleen cells were further divided into CD3 + and CD3" subpopulations, only those DN cells expressing the TCR and therefore associated CD3 complex were responsive to anti-CD3 crosslinking. When the CD3 + DN population was further subdivided into those expressing either the ap or y8 TCR, an interesting difference was observed. DNapT cells, sorted either 78 positively as TCR ctp + DN or negatively as TCR y8" DN, vigorously proliferated in the presence of immobilized anti-CD3, a response comparable to that of nylon wool purified mature SP T cells. Although still capable of proliferating when cultured with immobilized anti-CD3 MAb, negatively sorted TCR y5" DN were significantly less responsive than their positively sorted counterparts. This may be due to the fewer number of DNapT cells within the TCR y8" DN population which, unlike the positively sorted subset, also contains CD3" DN cells which comprise approximately 67-75% of this negatively sorted population. In contrast, TCR yS + DN cells, sorted either positively as TCR y8 + DN or negatively as TCR aP" DN, were non-responsive to CD3 crosslinking. Interestingly, when this TCR y8 + subset is purified without the depletion of Mac-1+ contaminating cells, TCR y8 + CD4" CD8" HSA" cells appear to be capable of responding to crosslinking of the TCR associated CD3 complex. This restoration of responsiveness to anti-CD3 crosslinking is particularly evident when TCR y8 + cells, sorted negatively as TCR aP" CD4" CD8" HSA", are not depleted of Mac-1+ cells. Furthermore, the addition of IL-1 to TCR y8 + DN cells cultured with immobilized anti-CD3 appears to facilitate the response to TCR crosslinking thus suggesting a requirement for the presence of Mac-1+ cells such as monocytes and macrophages and their IL-1 product for the antigen dependent activation of TCR yS + DN subpopulation. The response of DNapT cells to anti-CD3 was also enhanced, by the addition of IL-1. Similar to their splenic counterparts, TCR a p + DN thymocytes also respond to anti-CD3, although not to the same extent, and this response is enhanced by IL-1. 3.2.5 Stimulation of Splenic DNaPT Cells by PMA and Ionomvcin Further analysis of the activation pathway (Table III) involved in the stimulation of DNapT cells revealed that this spleen cell population is responsive to PMA but not ionomycin alone. When cocultured in the presence of both PMA and ionomycin, DNapT cells vigorously proliferate, a response comparable to that observed in the presence of immobilized anti-CD3. In order to determine whether the antibodies used in the purification procedure influenced this response, DNapT cells were sorted negatively as TCR y8~ DN and assessed. As shown in Table 3, DNapT cells still respond to PMA and ionomycin but not PMA alone when sorted negatively as TCR y8" DN. The phorbal ester PMA and the calcium ionophore ionomycin bypass the TCR to directly influence PKC and [Ca + 2]j. 3.2.6 Cytokine mRNA expression by splenic DNapT cells Since splenic DNapT cells are thought to be functionally mature, the capacity for cytokine production in response to stimulation was assessed by PCR amplification of cytokine specific 79 messages (Fig. 11). Therefore RNA was extracted from purified spleen cell populations either directly following sorting or after 16 hr incubation in wells precoated with anti-CD3 or containing media, IL-7 and/or IL-1. The extracted RNA was converted to cDNA by reverse transcription and cytokine specific messages were amplified by 30 cycles of PCR. Interestingly, IFNy message was detected in freshly isolated DNapT cells prior to stimulation. Following ib hr incubation with either immobilized anti-CD3, or IL-7 alone or in combination with IL-1, IFNy message detected but at lower levels. In the absence of stimulatory factors, IFNy was not detected in cultured DNapT cells. Southern hybridization (Fig. 12) confirmed the specificity of the amplified sequence. The pattern of IFNy mRNA expression in TCR y5" DN cells (Fig. 11) was similar to that of the positively sorted DNapT cell population indicating that the anti-TCR antibodies used in the purification procedure had no influence on cytokine expression. Although only 30% of TCR y5" DN cells were TCR a p + (Fig. 10), the remaining 70% of cells which expressed neither TCR type were not responsible for the expression of the IFNy message as indicated by the absence of cytokine specific bands for the CD3" DN. In addition to IFNy message, splenic DNapT cells sorted either positively or negatively expressed IL-4 specific mRNA (Fig. 11) in response to anti-CD3. However, IL-4 message was not detected at Day 0 or following culture with IL-7 and/or IL-1 or with media alone. Furthermore, splenic DNapT cells expressed neither IL-2 or IL-6 messages under the conditions tested. Similar to DNapT cells, nylon wool purified mature SP T cells also express mRNA specific for IFNy and IL-4 in response to anti-CD3 crosslinking (Fig. 11). Unlike DNapT cells, however, significant IFNy mRNA was not detected in mature SP T cells following isolation but prior to stimulation, or after 16 hr incubation with immobilized anti-CD3 or IL-7 with or without IL-1. Although a very faint band corresponding to IFNy mRNA can be seen for freshly isolated SP T cells this may be attributed to the presence of a small proportion of activated T cells expressing IFNy mRNA within this normal splenic T cell population. Furthermore, a small proportion of DNapT cells contained within the nylon wool purified T cell population may have also contributed to the expression of IFNy messages detected by this sensitive assay. In addition, anti-CD3 stimulated mature SP T cells but not DNapT cells also express IL-2 message. 80 b CD4 CD8 HSA MAC-1 Figure 4 Identification of DNapT cells in the spleen of normal mice. Spleen cells obtained from 5-6 week old BALB/c mice following RBC depletion (a) and following a subsequent two rounds of panning to remove those cells stained with the rat anti-mouse MAb against CD4, CD8, HSA and Mac-1 using plastic plates precoated with anti-rat IgK (b) were stained with MAb to CD4, CD8, HSA and Mac-1 recognized by FITC-conjugated anti-rat IgK (RG7/9.1) and biotinylated anti-TCR ap followed by PE-conjugated streptavidin (SA-PE). Gates were set based on the negative control population of total nucleated spleen cells stained with both FITC-conjugated RG7/9.1 and SA-PE. DNapT cells constitute 0.1-0.3% of total nucleated spleen cells and 1.4-2.5% of panned splenic T cells based on the results of three experiments. « ... C - O I * 32 CD4 CD8 HSA Mac-1 o w S X 00 Q O ••t Q O CD3 Figure 5 Phenotypic analysis of splenic DN cells. Nucleated spleen cells were depleted of CD4+ CD8+ HSA+ Mac-1+ cells by an indirect panning method followed by FACS sorting. Following FACS sorting, isolated DN cell were restained with the above MAb recognized by FITC-conjugated mouse anti-rat Ig MAb RG7/9.1 to assess purity as well as with SA-PE. Sorted DN cells were also stained with either biotinylated anti-TCR ap or anti-TCR 7 8 plus SA-PE or FITC-conjugated anti-CD3 and analysed by FACS. Of the sorted DN spleen cells, 17-23% were TCR ap +, 21-25% were TCR y5+ and 52-60% were CD3+ based on the results of three experiments. 82 Figure 6 Phenotypic analysis of CD3" DN spleen cells. The purification procedure used to isolate CD3" DN spleen cells involved 2 rounds of indirect panning to remove those cells stained with rat anti-mouse MAb against CD4, CD8, HSA and Mac-1 using plastic plates precoated with anti-rat IgK MAb. Panned cells were then restained with MAb against CD4, CD8, HSA and Mac-1 recognized by FITC-conjugated anti-rat IgicMAb as well as with FITC-conjugated anti-CD3MAb and the resulting negatively stained cells (CD4" CD8" HSA" Mac-1" CD3" or CD3" DN) were then isolated by FACS. Purified CD3" DN spleen cells were then stained by a number of MAb including Pgp-1, YE1/19, Lyt-1, T200, YE1/22, B220, Thy-1, LFA-1 and ICAM-1 recognized by FITC-conjugated anti-rat IgK and analysed by FACS. Isolated CD3" DN cells were also restained with the same MAb used in the purification procedure (MAb against CD4, CD8, HSA, and Mac-1 recognized by mouse anti-rat Ig MAb RG7/9.1 and FITC-conjugated anti-CD3 MAb) in order to assess purity. In order to quantify positive staining, gates were set based on the negative control population of sorted cells stained with the FrrC-conjugated second antibody (superimposed). The percentage of DN cells staining positively for the above MAb are indicated in brackets as follows: Pgp-1"* (93.1%), YE1/19+ (3.8%), Lyt-1+ (18.7%), T200 (99.8%), YE1/22+ (27.7%), B220+ (24.9%), Thy-1+ (47.1%), LFA-1+ (99.8%) and ICAM-1 (61.7%). 83 Figure 7 Phenotypic analysis of splenic DNapT cells. The purification procedure used to isolate splenic DNapT cells involved 2 rounds of parining to remove those cells stained with the rat anti-mouse MAb against CD4, CD8, HSA and Mac-1 using plastic plates precoated with anti-rat IgK. Panned cells were then restained with MAb against CD4, CD8, HSA and Mac-1 recognized by FITC-conjugated anti-rat IgK as well as biotinylated anti-TCR ap recognized by SA-PE and those cells of the DNapT cell phenotype (TCR ap + CD4" CD8" HSA" Mac-1) were isolated by FACS. Purified splenic DNapT were stained by a number of MAb including B220, YE1/19, YE1/9, ICAM-1 and Pgp-1 recognized by FITC-conjugated anti-rat IGK. Isolated DNapT cells were also restained with the same MAb used in the purification procedure (MAb against CD4, CD8, HSA and Mac-1 recognized by mouse anti-rat Ig MAb and biotinylated anti-TCR ap recognized by SA-PE) in order to assess purity. In order to quantify positive staining, gates were set based on the negative control population of sorted cells stained with the FITC-conjugated second antibody (superimposed). The percentage of DNapT cells staining positively for the above MAb are indicated in brackets as follows: B220+ (9.0%), YE1/19+ (18.0%), YE1/9+ (71.2 %), ICAM-1+ (79.9 %), Lyt-1 (95.1 %) and Pgp-1+ (90.9 %). 84 Figure 8 Determination of Vpg usage by splenic DNapT cells. A) DN (CD4" CD8" HSA" Mac-1") spleen cells were isolated by two rounds of panning followed by sorting by FACS to deplete those cells stained by the rat anti-mouse MAb against CD4, CD8, HSA and Mac-1 recognized by FITC-conjugated anti-rat IgK MAb. The purity of the sorted population was confirmed by FACS analysis of the cells restained with the above MAb. B) Mature SP (TCR ap + CD4+ CD8" or TCR ap + CD4" CD8+) splenic T cells were isolated by passage of nucleated spleen cells through nylon wool columns and subsequent collection of nonadherent cells. Sorted DN spleen cells or nylon wool nonadherent splenic T cells were then stained with either biotinylated anti-TCR ap recognized by SA-PE, anti-TCR Vng (KJ16) recognized by FITC-conjugated anti-rat IgK or FITC-conjugated anti-CD3 MAb. In order to quantify positive staining, gates were set based on the negative control population of sorted cells stained with SA-PE as well as second antibody. DN spleen cells analysed by FACS were 11.0% KJ16+ 23.0% TCR ap + and 57.6% CD3+. Approximately 21% of nylon wool purified mature spleen T cell were KJ16+ whereas 92.4% were TCR ap + and 98.9% were CD3+. 85 Figure 9 Corriirmation that nylon wool purified cells are indeed SP T cells. Nylon wool purified T cells were restained with either Thy-1 or HSA and Mac-1 or CD4 and CD8 MAb recognized by FITC-conjugated anti-rat IgK. In order to quantify positive staining, gates were set based on the negative control population of nylon wool purified spleen cells stained with the FITC conjugated second antibody (superimposed). Approximately 93.7% of nylon wool purified spleen cells were Thy-1+ whereas 39.0% were HSA + and/or Mac-1+ and 94.4 % were CD4+ and/or CD8+. 86 T C R Y 5 « 4 t T C R a(3 32 C D 4 C D 8 H S A M A C - 1 Figure 10 Presence of DNapT cells in the TCR y8" DN spleen cell population. The purification of splenic TCR y8" DN cells involved 2 rounds of panning to deplete those cells stained by rat anti-mouse MAb against CD4, CD8, HSA and Mac-1 on plastic plates precoated with anti-rat IgK MAb. Panned cells were then restained with the above rat anti-mouse MAb recognized by FITC-conjugated anti-rat IgK MAb as well as biotinylated anti-TCR y5 MAb recognized by SA-PE and the resulting negatively stained cells (TCR y8_ CD4" CD8" HSA" Mac-1- or TCR y8" DN) were isolated by cell sorting. Sorted TCR y8" DN cells were then restained with the above antibodies to assess purity as well as with biotinylated anti-TCR ap MAb recognized by SA-PE and analysed by FACS. Approximately 25-33% of TCR y8" DN spleen cells expressed TCR ap based on the results of three experiments. 87 Figure 11 Lymphokine mRNA expression. Total cellular RNA was isolated from 1 X 10^ sorted TCR a p + DN, TCR y8~ DN, CD3" DN and nylon column purified mature spleen T cells following purification (i), or 16 hr incubation in round bottom microtitre wells with immobilized anti-CD3 MAb (ii), IL-7 + IL-1 (iii), media (iv), IL-7 (v) or IL-1 (vi). RNA was converted to cDNA and cytokine specific DNA fragments were amplified by 30 cycles of PCR with 21-mer oligonucleotide primers for IFNy, IL-4, IL-2 and IL-6. PCR amplification of actin specific sequences was used as a positive control. Expected sizes of amplified fragments of actin, IFNy, IL-2, IL-4 and IL-6 were 540, 460, 502, 399 and 638 bp respectively. The amplified DNA was separated by 1% agarose gel electrophoresis and stained with ethidium bromide. 88 b) a) Figure 12 Confirmation of the specificity of the IFNy message by Southern hybridization. Amplified DNA fragments were separated by 1% gel electrophoresis and transferred to a membrane by Southern blotting, followed by hybridization with labeled IFNy cDNA probe and autoradiography. 89 1 CN 00 ro _ J 00 t-n ro +1 + •+ + 1 v© 00 r- CN 00 i 00 ON VO d 00 ro B o ro o ON 00 VO CN © rs +1 + 1 + 1 +1 +1 + 1 + 1 + 1 •oration IL- ro o >/-i ro •<* t~ VO t 1967 00 Incorj idine rS ro 2920 ro •t ~* VO VO VO v> 00 rs O N rS CN m r~ CN ro 1=3 + 1 +1 + 1 + 1 + 1 + 1 + 1 + 1 + 1 + 1 +1 r~ Ov 11550 2952 7583 00 CN ro 2707 2272 6626 2471 12660 00 I 3 3 00 00 00 O N <o ro rs "/-> •o ro >/-> rs <r> rs CN + 1 + 1 + 1 + 1 + 00 •«* O N vo CN m -* o VO ro r» O r~ O N O T) CN VO CN VO CN + 00 - H vo I +1 +1 CN ro + 1 00 CN + 1 — N O ro VO + 1 O + 1 O N + 1 _ ON ® **• •3-o O O O o O O O O © O X X X X X X X X X X X © CN o m V) q rs q q q q ro CN CN ro' ro' ro e "a, E2 < a: 00 8 1 •<* Q <J o •a o "5. * 55 Q o •a C/3 55 Q + ro Q O 2 Q ro Q U Z Q + C O . 8 e 55 Q e 55 Q e 55 Q • C O . (A f 9 H Z Q 90 + 8 • i S 1 I u S I S fn cs I U fN S O O N N O O N O N r-T f W1 fn 00 N O fN +1 + 1 + 1 +1 + v> O N m 00 O N O N O N T f O N fN o JN o t~ r» fn N O 00 m fN + 1 +1 + 1 + 1 + 00 T o 00 00 cn © fn >/"> O N o N O o 00 T f N O 00 cn r f •n cn CN + 1 + 1 + 1 + 1 + 00 N O O N 00 N O fn Tt- IT) m O 00 N O N O cn m •<* 00 •/•> •<* N O 00 O N O N o O N o T f N O o r~ m N O 00 00 +1 + 1 + 1 +1 +1 + 1 + r~- © 00 00 •«* O N cn 00 cn r- CS TT N O 00 r- TJ- r~ N O «/-> N O rs fn cn m cn NO <o NO m + 1 + 1 + 1 + 1 +1 +1 + 1 + 1 +1 + 1 + 1 + 1 o r- <s 00 NO fN fn cn NO ON fN m NO CN fN o © O TJ-o o o o Tt-O o IT) © o o X X X X X X X X X X X X o © o N O © O N © p p fN m CN fN CN C"i cn -* CN e "B. 00 1 _ * .2 5 Z oo Z Q + fn D U Z Q I a u z Q + CO. 8 e DN Z Z DN Q Q i to + to CO. a f-oi u H H H K oo Q • Q + e 00 BC 1 oo Q U • O o CO. a o H J 5 H •z Q + cn. o • cn 91 * * 2 Q ai * 2 Q + CO. a CJ H £ & c o 8 c g 3 CO vo + 1 vo CN •<* vo o 00 co o o co + 1 +1 +1 + 1 +1 + © 00 CO •t- co ON 00 00 «/•> •* 00 CN 00 <o CN VO 00 vo CN 00 VO co CO CO CO ON +1 +1 +1 +| r--ON I C N t ~ CN + 1 ON o + 1 co CO + c Q CO < y ti-C i d -CD i & S anti' ion CN CN +1 CN r-00 VO I 8 o so X X **. °. —< co' 5 Z Q Q 4- • H * * • .2 CJ o £ 8-21 c CO 5 CO ^ "S 'g CN"S » o uv ex 92 3.3 REFERENCES Bruce, J., Symington, F.W., McKearn, T.J. and Sprent, J. (1981) A monoclonal antibody discriminating between subsets of T and B cells. J. Immunol. 127:2496. Ceredig, R., Lynch, F. and Newman, P. (1987) Phenotypic properties, Interleukin-2 production, and developmental origin of a "mature" subpopulation of Lyt-2" L3T4" mouse thymocytes. Immunology 84:8578. Crispe, I.N. and Bevan, M.J. (1987) Expression and functional significance o f the J l Id marker on mouse thymocytes. J. Immunol. 138:2013. Fowlkes, B.J., Kruisbeek, A.M., Ton-That, H., Weston, M.A., Coligan, J.E., Scwartz, R.H. and Pardoll, D.M. (1987) A novel population of T-cell receptor ap bearing thymocytes which predominantly express a single Vp gene family. Nature 329:251. Haskins, K., Hannum, C, White, J., Roehm, N., Kubo, R., Kappler, J. and Marrack, P. (1984) The major histocompatibility complex-restricted antigen receptor on T cells. VI. An antibody to receptor allotype. J. Exp. Med. 160:452. Julius, M.H., Simpson, E. and Herzenberg, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3:645. Papiernik, and Pontoux, (1990) In vivo and in vitro repertoire of CD3 + CD4" CD8" thymocytes. Int. Immunol. 2:407. Suda, T., Murray, R., Guidos, C. and Zlotnik, A. (1990) Growth promoting activity of IL-la, 11-6 and Tumour Necrosis Factora in combination with IL-2, IL-4, or IL-7 on murine thymocytes. J. Immunol. 144:3039. Takahama, Y., Kosugi, A. and Singer, A. (1991) Phenotype, ontogeny, and repertoire of CD4" CD8" T cell receptor cd3+ thymocytes. Variable influence of self-antigens on T cell receptor Vp usage. J. Immunol. 146:1134. Takei, F., Secher, D.S., Milstein, C. and Springer, T. (1981) Use of a monoclonal antibody specifically non-reactive with T cells to delineate lymphocyte subpopulations Immunology 42:371. Vissinga, C.S., Fatur-Saunder, D.J. and Takei, F. (1992) Dual Role of IL-7 in the growth and Differentiation of Immature Thymocytes. Exp. Hematol. 20:998. 93 C H A P T E R IV CONCLUSIONS AND DISCUSSION A unique class of T lymphocytes has been identified in the spleen of normal mice. This novel T cell population, which expresses the a.p TCR/CD3 complex yet lacks the CD4 and CD8 coreceptor molecules as well as HSA and Mac-1, constitutes 0.1-0.3% of total nucleated spleen cells. Of the approximately 0.7-1.1% of nucleated spleen cells which lack CD4 and CD8, 17-22% express the TCR ap. Furthermore, of the 52-60% of DN spleen cell which express the CD3 complex, approximately 30% are of the TCRap + lineage whereas approximately 3 7% express the alternate TCR y8 heterodimers. Phenotypic analysis of splenic DNapT cells demonstrated the expression of Lyt-1, Pgp-1, ICAM-1 and the transferrin receptor whereas minimal to no B220 and YE1/19 expression was observed. Amongst splenic DNapT cells there is a high frequency of Vpg usage (approximately 48%) . This DNapT cell population proliferates in the presence of IL-7 and this response is enhanced by the addition of IL-1. Furthermore, splenic DNapT cells are responsive to MAb which crosslink the TCR associated CD3 complex as well as reagents which bypass the TCR to activate intracellular events. Expression of cytokine mRNA, either constitutively as for IFNy or following TCR activation as for IL-4, suggests an apparent capacity for lymphokine production by this splenic DNapT cell population. Although thymocytes of the DNapT phenotype have been the subject of extensive studies, only recently has attention been turned towards their peripheral counterparts. DNapT cells have been identified in normal human peripheral blood (Shivakumar et al. 1989; Quaratino et al. 1991; Niehues et al. 1994) as well as murine bone marrow (Sykes, 1990; Levitsky et al. 1991), lymph nodes (Guidos et al. 1989; Huang and Crispe, 1992) and liver (Seki et al. 1991; Ohteki et al. 1992). As shown in this thesis, DNapT cells do exist in the spleen of normal mice and constitute 0.1 to 0.3% of total nucleated spleen cells. The existence of splenic DNapT cells has since been confirmed by several other studies. According to Prud'homme et al. (1991), less than 1% of murine spleen cells were of the TCR a p + CD4" CD8" phenotype. Similar to the results shown in Figure 7, this splenic DNapT cell population expressed significant levels of Ly-1 as well as Thy-1 whereas only minimal expression of B220, Mac-1, J l Id and la was observed. In a second study, Levitsky et al. (1991) reported that approximately 0.5 to 2.0% of B cell and macrophage depleted spleen cells isolated from C57BL6 mice were of the TCR a p + CD4" CD8" phenotype. Furthermore, this splenic DNapT cell population was also shown to express a 39 kDa surface molecule referred to as NK1, initially considered to be specific for NK cells (Sentman et al. 1989). Palathumpat et al. (1992) demonstrated that the percentage of DNapT cells increased from 4 to 13 94 % when spleen cells isolated from the C57BL/K9 strain were further purifier! on a Percoll density gradient into fractionated low density cells. Although unsuccessful at the isolation of DNapT cells in the spleens of normal BALB/c and B6 mice, Kubota et al. (1992) demonstrated that IL-3 induced the generation of DNapT cells from the CD4 +/CD8 + T cell-depleted spleen as well as bone marrow of both normal and nude BALB/c mice in primary short term cultures. A significant proportion of these IL-3 induced splenic as well as bone marrow DNapT cells were shown to express the products of Vp 2 genes. Freshly isolated splenic DNapT cells, on the other hand, showed preferential usage of Vpg genes as demonstrated in Figure 8 and since confirmed by Zlotnik etal. (1992). Splenic DNapT cells are considered to be functionally mature as suggested by the absence of expression of the heat stable antigen, a marker characteristically found on immature thymocytes yet absent on mature peripheral T cells (Crispe and Bevan, 1987). In addition, splenic DNapT cells express a functional TCR which can be activated using either anti-TCRaP MAb or the lectin Concanavalin-A (Con-A) (Prud'homme et al. 1991), or by crosslinking of the TCR associated CD3 complex with anti-CD3 MAb (Table II). It is possible that TCR mediated activation of this splenic T cell subset involves signal transduction through the phosphoinositol (PI) pathway as suggested by the responsiveness of splenic DNapT cells to the combination of PMA and ionomycin (Table III). PMA is a phorbol ester which activates PKC whereas ionomycin is a calcium ionophore which induces an increase in [Ca + 2]j , both of which are key events in the PI second messenger pathway. Furthermore splenic DNapT cells are considered to be functionally mature based on their capacity to produce cytokines. As shown in Figure 11, splenic DNapT cells expressed IFNy and IL-4 specific mRNA but neither IL-2 nor IL-6 messages in response to crosslinking of the TCR associated CD3 complex. A recent study has confirmed both IFNy and IL-4 production as well as TNF-a synthesis following anti-CD3 crosslinking by splenic DNapT cells with the use of in vitro bioassays (Zlotnik et al. 1992). Furthermore, IL-2 production was not observed under this condition. Unlike SP T cells which proliferate following IL-2 receptor expression and production of the corresponding cytokine as a consequence of activation of the PI pathway (Weiss et al. 1984; Truneh et al. 1985), the absence of IL-2 production following TCR-CD3 activation of splenic and thymic DNapT cells suggests that the activation of this T cell population may involve an alternate cytokine signalling pathway such as IL-4. To further elucidate the role of IL 'I in TCR signalling of DNapT cells, additional studies are required including the determination of whether MAb directed against IL-4 are capable of blocking the proliferative response of this T cell population to TCR-CD3 activation. Recently, however, Prud'homme et al. (1991) have detected not only IL-4 but also IL-2 production by splenic DNapT cells following activation either by anti-TCR ap MAb or Con A, as determined by the IL-2/IL-4 sensitive cell lines CTLL-2 and CT4S. The absence of 95 CD4 and CD8 expression by DNapT cells further suggests that the TCR signalling pathway employed in this T cell subpopulation may differ from that of classical SP T cells whose CD4 or CD 8 coreceptors have been shown to play a role in T cell activation including signal transduction via an associated cytoplasmic tyrosine kinase p 5 6 ^ (Veillette et al. 1988) as well as augmentation of T cell recognition by binding to monomorphic determinants on class II and class I MHC molecules respectively (Hollander, 1982; Biddison et al 1984). In order to further elucidate the mechanism of TCR signalling in DNapT cells additional studies could therefore include the assessment of p 5 6 ^ expression and protein-tyrosine kinase activity in this T cell population and the possible association of this particular member of the src family with alternative membrane bound molecules. Splenic DNapT cells may represent a population of previously activated T cells. As shown in Figure 7, this splenic subpopulation expresses significant levels of Pgp-1, an adhesion marker previously shown to be upregulated on activated lymphocytes (Budd et al. 1987). In addition, approximately 70% of splenic DNapT cells express the transferrin receptor, recognized by the MAb YE 1/9, which has been previously shown to be expressed on essentially all proliferating cells including activated T lymphocytes (Takei, 1983; Trowbridge and Omary, 1982; Sutherland et al. 1981). Furthermore, approximately 80% of splenic DNapT cells were shown to express ICAM-1, the 86-114 kd glycosylated ligand for LFA-1. Although ICAM-1 is typically expressed at low levels on resting peripheral blood lymphocytes as well as nonhematopoietic ceils, the expression of this adhesion molecule is upregulated following activation in the presence of cytokines including IFNy, IL-1 and TNF (Dustin et al. 1986; Dustin et al. 1988). Phenotypically similar cells isolated from human peripheral blood have also been shown to express markers consistent with previous antigen encounter and activation including CD45RO and HLA-DR respectively (Brooks et al. 1993). Therefore the previous activation of splenic DNapT may provide an explanation as to why this T cell subset constitutively expresses IFNy mRNA (Figure 11). IFNy specific mRNA was detected in freshly isolated DNapT cells and expression of this cytokine mRNA was dependent only on the viability of this splenic subpopulation. Being previously activated may also enable splenic DNapT cells to proliferate in response to IL-7 in the absence of added comitogens (Table I). SP T cells, on the other hand, do not proliferate in response to IL-7 alone (Table I) but require the addition of either PMA or ConA to IL-7 cultures (Morrisey et al. 1989; Chazen et al. 1989). Welch et al. (1989) have previously shown that memory (CD45~) T cells were significantly more responsive to IL-7 than unprimed (CD45+) T cells. The question remains as to the phenotype of the splenic DNapT cells prior to their activation and thus the origin of this unique T cell population. 96 A number of characteristics of splenic DNapT cells suggest that this peripheral T cell population may be related to the subset of DN thymocytes which also express the TCRaP and have been shown to constitute 2-30% of the thymic DN subpopulation depending on the mouse strain studied (Fowlkes et a/. 1987; Howe and MacDonald, 1988; Wilson et al. 1988; Levitsky et al. 1991). Indeed the similarities in surface phenotype extend beyond the expression of the TCR ap and lack of CD4 and CD8 coreceptors. Splenic DNapT cells and their thymic counterparts both lack expression of the HSA, a marker characteristically found on immature thymocytes yet absent on mature peripheral T cells (Crispe and Bevan, 1987). Furthermore, a high percentage (90.9 %) of splenic DNapT cells express Pgp-1 (CD44) (Figure 5), a marker also identified on the majority of TCR a p + DN thymocytes (Suda and Zlotnik, 1993). Both the loss of HSA and subsequent expression of Pgp-1 have been shown to occur following the acquisition of the TCR/CD3 complex at a stage when TN thymocytes branch off from the classical differentiation pathway to become CD3 + DN T cells. Others have shown that splenic DNapT cells and their thymic counterparts also share the expression of NK1.1, a marker originally thought to be expressed exclusively on the natural killer cell lineage (Levisky et al. 1991). Recently it has been shown that the majority of thymic DNapT cells express CD38 (Zlotnik and Bean, 1994). Unlike the majority of murine thymocytes which are CD38", approximately 80% of those thymocytes which express CD38 are of the CD4" CD8" TCR a p + phenotype with the remaining 20% of CD38+ cell being CD8+. It has therefore been suggested that the CD38 molecule may be a unique marker of thymic DNapT cells. Future studies demonstrating CD38 expression on splenic DNapT cells would therefore provide further support for a developmental relationship between this population and the phenotypically similar thymocytes. Vpg overexpression observed in splenic DNapT cells is also a characteristic of thymic TCR a p + DN cells. Approximately 48% of splenic DNapT cells in BALB/c mice expressed either Vpg \ or Vpg 2, whereas the frequency of Vpg usage amongst nylon wool purified SP T cells was only 22% (Figure 8). Similarly, TCR a p + DN thymocytes have also been shown to predominantly express the products of the Vpg genes in at least a twofold higher frequency than in SP T cells (Fowlkes et al. 1987; Ceredig et al. 1987; Papiernik and Pontoux, 1990; Takahama et al. 1991; Zlotnik et al. 1992; Suda and Zlotnik, 1993). This Vpg overexpression was only observed after the first 5 weeks of life and appeared to be superimposed on the developmental appearance of other TCR a P + Vp8~ DN thymocytes. Although the Vpg family consists of three closely related genes, namely Vpg j , Vpg 2 and Vpg 3, the disproportionately high frequency of Vpg + thymocytes has been attributed to the overexpression primarily of Vpg 2- In contrast, approximately 20% of SP thymocytes and peripheral T cells express Vpg. The bias of TCR a p + DN thymocytes and splenic T cells towards Vpg expression suggests a possible role for specific selection processes, 97 whether positive or negative, in the shaping of the TCR repertoire of this population. Unlike HSA" TCR ctp + DN thymocytes, whose Vpg 2 usage was more than three fold compared to SP thymocytes in BALB/c mice, Vpg 2 + thymocytes of the HSA + TCR a.p+ DN phenotype have been shown to be markedly reduced to proportions comparable to that of SP thymocytes (Suda and Zlotnik, 1993). As HSA" TCRa.p+ DN thymocytes are considered to be the progeny of their HSA + precursors, it was therefore suggested that the Vpg 2 overexpression observed for HSA" TCR ot,p+ DN thymocytes was as a result of positive selection with the HSA + TCR ap+ DN subset regarded as a preselection population. A recent study has suggested that the repertoire of TCR ap+ DN thymocytes may be influenced by positive selection events. Bix et al. (1993) observed a more than ten fold reduction in frequency of Vpg 2 + DN thymocytes in class I MHC deficient (P2 microglobulin" or P2m") mice relative to the P2m+ strain. Furthermore, overexpression of Vpg amongst TCR ap+ DN thymocytes was shown to be dependent on class I MHC expression by hematopoietic cells as opposed to thymic epithelial cells as required for selection of SP T cells. Analysis of mice deficient for class II MHC expression due to a disrupted Ap gene revealed only a modest reduction (~ 40 to 50%) of the frequency of TCR a,p+ DN thymocytes, an effect not restricted to Vpg+ cells. Interestingly, the description of a human DNapT cell line specific for the class I-like CD1 molecules prompts the question as to whether this T cell subset undergoes positive selection by nonpolymorphic class I molecules (Porcelli et al. 1989). Therefore, similar to their thymic counterparts, the biased Vpg repertoire expressed by splenic DNapT cells may suggest prior influence by positive selection events. However, employing H-2Q mice, known to selectively increase the expression of Vpi 7 a + CD4 + T cells in the absence of I-E, Huang and Crispe (1992) were unable to demonstrate the influence of positive selection on the usage of the alternate Vp genes in peripheral DNapT cells. Although mature SP T cells expressing either Vpg \ or Vpg are reactive towards Mls a deteirninants and therefore deleted as a consequence of negative selection in M l s a + strains, frequencies of TCR a p + DN thymocytes expressing either Vpg \ or Vpg have not been shown to differ significantly in M l s a + versus Mls a" mice (Egerton and Scollay, 1990; Takahama et al. 1991). Therefore it was suggested that Vpg overexpression was not a consequence of self tolerance. Furthermore, the majority of thymocytes as well as peripheral T cells expressing the transgenic anti-male TCR in male transgenic mice were of the DN phenotype (Kisielow et al. 1988; Teh et al. 1989). This apparent escape from clonal deletion is not thought to be attributed to the absence of CD 8 expression since the majority of thymocytes and peripheral T cells expressing both anti-male TCR and CD8.1 transgenes in double transgenic mice lacked expression of both CD4 and the endogenous CD8.2 and were therefore considered to be DNapT cells (Robey et al. 1992). It has therefore been suggested that TCR a p + DN cells may be intrinsically indifferent negative selection. However introduction of the bacterial superantigen SEB into the neonatal differentiatiot? environment of both 98 CBA/J and B6 strains, resulted in the markedly reduced frequency of Vpg i , Vpg 2 and Vpg 3 usage amongst DN thymocytes. Furthermore, TCR a p + DN HSA" thymocytes expressing either Vp3' Vpii or Vpi7a, are thought to be influenced by negative selection as suggested by the significant reduction of DN thymocytes bearing either of these Vp TCRs in I-E+ mice relative to I-E" strains (Takahama et al. 1991; Suda and Zlotnik, 1993). Interestingly, the precursors of TCR a p + DN HSA" thymocytes, namely TCR a.p+ DN HSA + thymocytes, cf I-E+ mice have beer, shown to have similar frequencies of Vp3 and Vp \ \ usage as do SP T cells of I-E" strains suggesting that the negative selection of Vp3+ and Vpi \ + DN thymocytes may have occurred during the transition from the HSA + to HSA" phenotype. With respect to TCR Vp expression in peripheral DNapT cells, Prud'homme et al. (1991) have observed low numbers of Vp5+, Vp£+ and Vp 11+ cells of the DN phenotype in the spleens of I-E+ M l s - l a + mice which were comparable to those obtained for untreated splenic T cells. Bone marrow DNapT cells, in contrast resist clonal deletion of their Vp3+, Vp5+, Vpg i + and Vpi i + cells in murine strains where SP T cell expressing any of these Vp genes are deleted (Martinez-A. et al. 1993). Although similar to TCR a p + DN thymocytes in their resistance to the negative selective influences of Mls-l a superantigen on Vpg and Vpg 1 usage, Huang and Crispe (1992) have shown that the percentage of lymph node DNapT cells expressing either Vp5, Vpii or Vpj7a was not reduced in I-E+ strains. Furthermore, Vpi7a+ cell of the DNapT phenotype were actually found to be enriched in the lymph nodes of I-E+ mice. However, significant individual variation in Vp expression amongst lymph node DNapT cells must be taken into consideration before conclusions may be drawn from this study. Analysis of DNapT cells isolated from human peripheral blood has also shown preferential Vp expression, namely Vp2, Vpg, Vpii, Vpi3 or Vpi9, from multiple unrelated donors unlike SP T cells (Porcelli et al. 1993; Brooks et al. 1993; Niehues et al. 1994). Therefore both the lack of CD4 and CD 8 expression as well as the characteristic skewing of the Vp repertoire including potentially autoreactive Vp genes not observed for SP T i/iiiphocytes suggests that cells of the TCR a p + DN phenotype may be governed by selection mechanisms distinct from those influencing the development of conventional T cells. In addition both splenic as well as thymic TCR a p + DN cells are responsive to antigen independent stimulation by IL-7. Although initially identified as a product of stromal cells providing support for the growth of B cells, a role for IL-7 in the development of T cells has since been suggested based on the detection of high levels of IL-7 mRNA in the murine thymus (Henney, 1989) as well as the demonstration of responsiveness to this cytokine by the CD4" CD8" subset of thymocytes (Murray et al. 1989) . It has been subsequently shown that IL-7 plays a dual role in terms of its effect on this DN thymocyte population (Vissinga et al. 1992; Suda and Zlotnik, 1991). More specifically, IL-7 maintains the viability of the CD3* CD4" CD8" subset and therefore allows 99 this thymic population to undergo their precommitted differentiation without the induction of proliferation. In contrast, CD3 + CD4" CD8" thymocytes expressing either TCR ap or y5 respond to IL-7 with the induction of proliferation but not differentiation. Similarly, as shown in Table I, splenic DNapT cells also proliferate in response to IL-7 although to a lesser extent than TCR a p + DN thymocytes. This response, however, appears to be influenced by the presence of monocytes/macrophages as suggested by the decrease in ^H-thymidine incorporation levels observed when DN splenic T cells were further purified by the depletion of Mac-1+ cells. Reconstitution of the proliferative response by the addition of IL-1, a known macrophage product, provides further support for a potential co-stimulatory role of cells of the monocyte/macrophage lineage. Furthermore, monocytes have indeed been reported to produce IL-1 as well as IL-6 and TNFa in response to IL-7 (Alderson et al. 1991). The TCR of both splenic (Table II, Prud'homme et al. 1991) and thymic (MacDonald et al. 1988) TCR a p + DN T cells is thought to be functional based on the ability of MAb which crosslink the TCR associated CD3 complex to activate these two T cells populations. Both splenic (Table III) and thymic (MacDonald et al. 1988) TCR a p + DN T cell subsets can also be activated by a combination of PMA and ionomycin which bypass the TCR by their direct effects on PKC and [Ca+2]j respectively. One final similarity between splenic DNapT cells and their thymic counterparts is the potential for cytokine production. As shown in Figure 11, splenic DNapT cells were capable of both IFNy and IL-4 mRNA expression following the crosslinking of the TCR associated CD3 complex. Under the conditions tested, splenic DNapT cells were not capable of expressing either IL-2 or IL-6 messages. A recent study has demonstrated a similar cytokine pattern for the phenotypically similar subset of thymocytes . More specifically, Zlotnik et al. (1992) observed both IFNy and IL-4 production as well as TNF-a synthesis by HSA + TCR a P + DN thymocytes cells following anti-CD3 crosslinking by the use of in vitro bioassays. Furthermore IL-2 production was not detected under this condition. It has been subsequently shown that IL-4 production by thymic DNapT cells is independent of Vp expression (Suda and Zlotnik, 1993). Whether TCR a p + DN thymocytes constitutively express IFNy specific mRNA, as does their splenic DNapT counterparts, remains to be demonstrated. Therefore, based on similarities with respect to phenotype, Vpg overexpression, responsiveness to IL-7, CD3 crosslinking and a combination of phorbol ester and calcium ionophore as well as potential cytokine production, splenic DNapT cells may be developmentally related to their thymic TCR a P + DN counterparts which have emigrated from the thymus to populate this peripheral 100 organ. Evidence for a thymic dependence of splenic DNapT has been provided by the virtual absence of NK1 + TCRap + T cells, of which a significant proportion are of the DNapT cell phenotype, in the spleen of athymic nude mice and the reconstitution of this splenic subset following fetal thymic engraftment (Levitsky et al. 1991). In addition, although DN T cells expressing a transgenic TCR ap were identified in the murine thymus as well as spleen, bone marrow, and axillary, mesenteric and inguinal lymph nodes, T cells of this phenotype could not be detected in the peripheral tissues of transgenic nude mice (Scott et al. 1989). Furthermore, following intrathymic reconstitution with TN thymocytes precursors, donor derived DNapT cells have been detected in other peripheral organs including lymph nodes and blood suggesting a thymic dependence of this subpopulation (Guidos et al. 1989). In order to provide further support for a developmental relationship between thymic and splenic DNapT cells, additional studies are required including the determination of whether splenic DNapT cells are present following intrathymic reconstitution with either TN or TCR a p + DN thymocytes in either sublethally irradiated or congenic (Thy 1.1 versus Thy 1.2) hosts. The use of retrovirally introduced cytokines such as IL-7 for the expansion of this minor TCRaP + DN thymocyte population would further facilitate the reconstitution studies. How the potential thymic precursors of splenic DNapT cells, namely TCR a p + CD4" CD8" thymocytes, fit into the T cell differentiation pathway has been the subject of numerous studies. TCR a p + DN thymocytes are thought to be composed of late stage differentiated cells based on studies demonstrating the lack of HSA expression, the relatively late appearance during ontogeny and in FTOC (Fowlkes et al. 1987) and the lack of precursor activity following intrathymic transfer (Crispe et al. 1987). Several studies have suggested that TCR a p + CD4" CD8" thymocytes and their peripheral counterparts may be derived from CD4 + CD8 + DP thymocytes which, due to potential self reactivity, have escaped clonal deletion by the downregulation of surface expression of their CD4 and CD8 determinants. In male transgenic mice, for example, the majority of thymocytes and T cells expressing the transgenic anti-male TCR were of the DN phenotype and expressed high levels of the transgenic TCR but were nonetheless tolerant to male self antigen (Teh et al. 1989). Furthermore, the demonstration of demethylation of DNA sequences 5' to the CD 8a gene has implied that TCR a p + DN thymocytes may be derived from a T cell population that had at one time expressed the CD8a gene locus (Wu et al. 1990; Takahama et al. 1991). Cytosine residues of developmentally regulated genes are often demethylated at the time of initial expression and progressive demethylation of several CCGG sites has been observed as T cells develop from immature TN through the DP stage to peripheral mature SP T cells (Carbone et al. 1988). The demethylation pattern observed in TCR a P + DN thymocytes was however distinct from that of either thymic or peripheral CD8 + T cells and therefore provided 101 further evidence that TCR ap+ DN thymocytes may be the progeny of DP thymocytes rather than SP thymocytes or peripheral T cells that have returned to thymus and downregulated CD8 expression. Cyclosporin A treatment of radiation bone marrow chimeras, previously known to arrest development of conventional T cells at the DP thymocyte stage, has also been shown to significantly block the appearance of TCR ap+ DN thymocytes (Takahama et al. 1991). Tiefenthaler et al. (1992) have recently provided direct evidence for the generation of TCR a p + DN thymocytes and their peripheral counterparts from DP thymocytes. Following a 2 day coculture with the stimulatory pair of anti-rat CD2 MAb, OX-54/55, a significant proportion of TCR a p + CD4 + CD8 + thymocytes were shown to have downregulated both CD4 and CD8 while upregulating TCR ap expression. The absence of detectable DNA synthesis ruled out a selective expansion of preexisting CD4l°/" CD^~ cells. Confirmation of this conversion in vivo was provided by the 3 to 4 fold increase in TCR a,p+ DN thymocytes observed in 4 week old Lewis rats following repeated injections with OX-54/55 since birth. Furthermore, while virtually absent in controls, DNapT cells constituted approximately one fourth of all T cells isolated from the lymph nodes of CD2 treated rats. A concomitant depletion of CD4 + T cells was considered to be unrelated to the peripheral accumulation of DNapT cells the proportion of CD4 + T cells, unlike DNapT cells, was also influenced by the nonstimulatory anti-CD2 MAb, OX-34. Recent evidence, however, has suggested that TCR ap+ DN thymocytes may not be the progeny of DP thymocytes but may instead represent a distinct T cell lineage which has branched off from the classical T cell differentiation pathway prior to the DP stage. First of all, in conflict with the suggestion that TCR ap+ DN thymocytes are derived from potentially self reactive DP thymocytes following the downregulation of the CD4 and CD8 accessory molecules, was the demonstration that the frequency of TCR ap+ DN thymocytes expressing various anti-sM TCR including either I-E reactive Vpi ^ and Vpi 7 a or Mls-l a reactive Vpg or Vpg \ was not increased in those strains expressing their putative self antigen but was either unaffected or significantly reduced (Egerton and Scollay, 1990; Takahama et al. 1991; Suda and Zlotnik, 1993). In addition, unlike DP thymocytes which are not capable of cytokine production upon stimulation with calcium ionophore and phorbol ester (Fischer et al. 1991), TCR ap+ DN thymocytes have been shown to produce IFNy, IL-4 and TNFa in response to CD3 crosslinking (Zlotnik et al. 1992). Furthermore, although peripheral SP progeny were observed, Guidos et al. (1989b) were unable to obtain detectable levels of CD3 + DN cells following the intrathymic transfer of DP thymocytes. With respect to the association of CD8a demethylation with a potential DP thymic origin for TCR ap+ DN thymocytes, Nikolic-Zugic et al. (1989) have shown that an early thymic subpopulation referred to as TN based on FACS analysis actually express low levels of CD8a. Furthermore, CD8a methylation has indeed been detected in a portion of the TN thymocytes and may correspond 102 to the CD8 l 0 subset (Wu et al. 1990). Although the majority of CD4"^° CD8 1 0 thymocytes, isolated by anti-CD8a MAb coated plates yet identified as CD4" CD8" by FACS, spontaneously differentiated into thymic DP cells following short term culture, a significant proportion of these cells maintained their DN phenotype and acquired CD3 expression (Nikolic-Zugic and Moore, 1989). Therefore the demethylated CD8a genes identified in TCR a.p+ DN thymocytes may actually be reminiscent of a TN phenotype expressed during a previous step in development. Several recent studies have provided support for the TN origin of TCR a p + DN thymocytes (Vissinga et al. 1991; Suda and Zlotnik, 1991; Suda and Zlotnik, 1993). Those TN thymocytes which express CD25 have been shown to consist of precursor cells for all TCR + subsets found within the thymus including CD3 + DN thymocytes as well as DP and SP thymocytes. Following coculture with IL-7, a proportion of the CD25 + TN thymocytes acquired the expression of CD3 associated with either of the ap or y5 TCR. Furthermore the majority of those TCR a p + CD3 + DN thymocytes derived from the CD25 + HSA + CD44" thymocytes were shown to be HSA" CD44+ it was therefore proposed that CD25 + TN thymocytes represent a stage in T cell development when those cells destined to become CD3 + DN branch oft from the classical differentiation pathway to acquire either TCR aP or y5 and differentiate from HSA + CD44" via HSA" CD44" to the HSA" CD44+ phenotype. On the other hand, splenic DNapT cells may represent a heterogeneous population of T lymphocytes. Although the majority of splenic DNapT cells are most likely derived from TCR a.p+ DN thymocyte having emigrated to this peripheral organ, as based on the numerous similarities discussed in this thesis between these two T cell subsets, a small percentage of these splenic T lymphocytes may have alternate origins. It is possible that a minor proportion of splenic DNapT cells may represent a population of spent single positive T cells whose CD4 and CD8 coreceptors have been downregulated. Indeed there are similarities between splenic DNapT cells and mature SP T cells which might support this relationship. In addition to the expression of the TCR a.p, for example, both splenic DNapT and mature SP T cells lack tne heat stable antigen, a marker typically found on immature thymocytes and lost as a consequence of maturation (Crispe and Bevan, 1987). Furthermore, the response of splenic DNapT cells to crosslinking of the TCR associated CD3 complex as assessed by ^ H-thymidine incorporation, is comparable to that of mature SP T cells isolated by the nylon wool column method (Table II). Evidence for the previous activation of splenic DNapT cells including Pgp-1, transferrin receptor and ICAM-1 expression as well as IFNy mRNA expression may provide further support for the possible origin of this splenic subset from spent SP T cells following the down regulation of the CD4 or CD8 coreceptors. Interestingly, Erard et al. (1993) have recently demonstrated the generation of DNapT cells from small CD8 + SP lymph node cells obtained from C57BL/6 mice following 6 day coculture with 103 PMA, ionomycin, IL-2 and IL-4. CD4+ SP T cells under the same culture conditions, however, maintained expression of the CD4 accessory molecule. Following the gradual loss of surface expression of both CD8a and CD8P chains and concomitant decrease of CD8a encoded mRNA but not CD8p message, the CD8" phenotype appeared to be stable as suggested by the lack of CD8 reexpression during an additional 6 days of culture in the absence of IL-4. Unlike their CD8 + SP progenitor cells, this TCRaP + CD4" CD8' T cell population possessed no lytic activity attributed not to the lack of CD8 but to the absence of perforin mRNA induction. Interestingly, this DNapT cell population was capable of a Tj-j2-like pattern of cytokine production mcluding IL-4, IL-5 and IL-10 in addition to a lower level of IFNy secretion and were able to induce the production of immunoglobulin G l (IgGl) from B cells. The CD8 + SP progenitor cells, on the other hand, were only capable of IFNy production and failed to induce detectable antibody production. This switch to non-cytotoxic Tfj2-like DNapT cells has since been extended to viral peptide specific CD8 + SP T cells. Kyburz et al. (1993) demonstrated that in transgenic mice, approximately 30% of CD8 + SP T cells specific for a lymphocytic choriomeningitis viral peptide restricted to class I MHC, switched to the CD8" phenotype upon activation in the presence of IL-4 and were capable of IL-4, IL-5 and IL-10 production following restimulation with the viral peptide. Although splenic DNapT cells are also capable of IL-4 mRNA production in response to activation of the TCR-CD3 complex as shown in this thesis, additional studies including the demonstration of CD8P chain mRNA expression are required before a developmental relationship can be drawn between CD8 + SP T cells and splenic DNapT cells. If splenic DNapT cells are indeed derived from spent SP T cells following the downregulation of the CD4 and CD8 molecules, this T cell subset may be capable of coreceptor reacquisition under the appropriate culture conditions. Prud'homme et al. (1991) demonstrated the acquisition of CD4 by splenic DNapT cells following 4 day coculture with either ConA or anti-TCR ap MAb accompanied by the development of responsiveness to syngeneic and allogeneic MLC. There was a gradual reversion to the DN phenotype in culture following activation. Thymocytes of the TCR a p + CD4" CD8" CD38 + have been shown to transiently express CD8a following activation with solid-phase anti-CD3 MAb for 18 hours (Zlotnik and Bean, 1994). Although it has been suggested that DNapT cells may be derived from SP T cells following the downregulation of the CD4 and CD 8 co-receptors, evidence has been provided to challenge this hypothesis. First of all, the demethylation pattern of the CD8a gene in TCR cu3+ DN thymocytes has been shown to be distinct from that of thymic and peripheral CD8 + SI' T cells. Analysis of P2m+/" chimeras has demonstrated an inverse correlation in the frequency of Vpg + DN thymocytes versus CD8 + SP T cells which further argues against a precursor-product relationship (Bix et al. 1993). Furthermore this thesis provides significant evidence to suggest that splenic 104 DNapT cells represent a unique class of T cells distinct from the what is considered to be the classical type of mature T lymphocyte, namely SP T cells. In contrast to nylon wool nonadherent SP T cells, an unusually high proportion of splenic DNapT cells express a particular member of the Vp family of TCR genes, namely Vpg. Splenic DNapT cells also differ from the nylon wool nonadherent SP T cells with respect to their response to antigen independent activation by IL-7. Whereas splenic DNapT cells proliferate in the presence of IL-7 and this response is enhanced by the addition of IL-1, the proliferation of nylon wool purified SP splenic T cells in the presence of IL-7 is minimal and is not influenced by the addition of IL-1. Furthermore the characteristic pattern of cytokine expression differs between the two types of mature T cells. Whereas both splenic DNapT cells as well as nylon wool purified SP T cells expressed IL-4 mRNA following the crosslinking of TCR associated CD3 complex, IL-2 message was only detected for the latter splenic subset under similar conditions. A more striking difference between splenic DNapT and SP T cells is the pattern of IFNy mRNA expression. Only in response to crosslinking of the TCR associated CD3 complex was significant levels of IFNy message detected for mature SP T cells. With respect to splenic DNapT cells, the expression of IFNy specific message was constitutive and dependent only on the viability of this splenic population. Therefore these results suggest that the majority of splenic DNapT cells are a unique class of mature T cells distinct from the classical type of mature T cell which expresses the SP phenotype. Alternatively, a proportion of splenic DNapT cells may not be of a thymic origin at all but may be the products of an extrathymic differentiation pathway. Interestingly, a significantly higher percentage of bone marrow T cells express the TCR a p + CD4" CD8" phenotype than in either the thymus or spleen suggesting that the bone marrow is a potential site for extrathymic T cell development (Sykes 1990, Palathumpat et al. 1992b; Martinez-A. et al. 1993). Palathumpat et. al. (1992b) provided support for the potential extrathymic origin of bone marrow DNapT cells with the observation of an apparent in vitro conversion of TCR aP" CD4" CD8" bone marrow cells to the TCR a p + DN phenotype. Although TCR rearrangement, as detected by PCR amplification of genomic DNA employing Vp7 and Jp2-Cp2 primers, was not observed in freshly isolated TCR aP' CD4" CD8" marrow cells, P chain rearrangement associated with the differentiation of this marrow subset to the TCR a p + CD4" CD8" phenotype was demonstrated following 48 hr incubation in tissue culture medium suggesting an apparent maturation of bone marrow T cells in the absence of a thymic microenyironment. In a second study, IL-3 v/r.s shown to induce the generation of DNapT cells with predominant Vp2 gene usage from CD4 + /CD8+ depleted bone marrow of normal BALB/c mice (Kubota et al. 1992). Also consistent with the putative lack of dependence of this bone marrow T cell subset on the presence of a mature thymus was the demonstration of the absence of negative selection of normal bone nT~r^ow DNapT cells 105 expressing the self reactive Vp5 receptor in BALB/c mice (Palathumpat et al. 1992b), Vpg \ and Vp3 in DBA/2 and CBA/J mice (Martinez-A. et al. 1993). If the hypothesis suggesting an extrathymic origin for bone marrow D N a p T cells was indeed valid then one would expect that the proportion of D N a p T cells in the bone marrow of athymic nude mice to be similar to that in euthymic strains. Two studies have shown that the proportion of NK1.1+ TCR a p + T cells actually decrease in the bone marrow of nude mice as compared to normal mice (Sykes 1990; Levitsky et al. 1991). The majority of bone marrow NK1.1+ TCR a P + cells, however, have been shown to express CD4 or CD 8 and therefore the reduction of this population in nude mice can not necessarily be extrapolated to the DN subset. Subsequent studies, however, have demonstrated that unlike the percentage of typical TCR a p + CD4 + and CD8 + SP T cells which was markedly reduced in the spleens of nude mice, the proportion of DNapT cells in the bone marrow of this athymic murine strain was indeed similar to that of normal euthymic mice (Palathumpat et al. 1992b; Kubota et al. 1992). It is possible that a proportion of splenic D N a p T cells may be derived from the phenotypically similar cells identified in murine bone marrow. Similar to their splenic counterparts, bone marrow D N a p T cells are Ly-1 + Thy-1+ and lack HSA and B220 (Figure 6; Martinez-A. et al. 1993). Kubota et al. (1992) have also demonstrated the IL-3 induced generation of D N a p T cells from both CD4+/CD8+ depleted bone marrow as well as spleen of normal and nude mice. Furthermore, Palathumpat et al. (1992b) demonstrated that the percentage of D N a p T cells in both the bone marrow and spleen of nude mice were comparable to that of normal mice. In addition, the neonatal spleen contained a similar percentage of DNapT cells but a significantly reduced number of typical T cells when compared to adult mice suggesting the presence of D N a p T cells in the murine spleen prior to the emigration of SP T cells from the thymus. However, unlike splenic DNapT cells where almost one half of the population expresses Vpg genes, the frequency of Vpg usage for bone marrow cells of this phenotype has been shown to be only 14-35% (Martinez-A. et al. 1993). In addition, whereas splenic D N a p T cells expressing either Vp5, Vpg or V p i j are reduced in I-E+ M l s - l a + strains (Prud'homme et al. 1991), phenotypically similar bone marrow cells expressing these Vp genes appear to be resistant to clonal deletion (Palathumpat et al. 1992b; Martinez-A. et al. 1993). Although both are responsive to stimulation with anti-CD3 MAb (Figure 11; Marinez et al. 1993) suggesting functional TCR, the patterns of cytokine production are somewhat different. Whereas bone marrow D N a p T cells secrete IL-2, IL-3, IL-4 and IL-5 in response to anti-Vp5 and anti-Vpii, (Martinez-A. et. al. 1993) the corresponding splenic subset produce IFNy, IL-4 and T N F a following activation of the TCR associated CD3 complex (Figure 11; Zlotnik et al. 1992). Additional studies to further elucidate the developmental relationship between bone marrow and splenic DNapT cell could include the determination of the presence DNapT spleen cells following reconstitution of sublethally irradiated or congenic hosts with bone marrow T C R a P + DN T cells. Furthermore it 106 would be of interest to determine whether DNapT cells exist in the bone marrow and spleen of mice deficient in IL-3 production such as an IL-3 gene knockout murine strain. The liver has been proposed as an alternate site for development of DNapT cells. Seki et al. (1991) have shown that while a single peak pattern of bright TCR ap and V p expression was observed in intestinal epithelia as well as mononuclear cells of lymph nodes, spleen and blood of normal mice, a two peak pattern, specifically intermediate and high expression of both TCR ap V p was detected in the liver. Further characterization of such intermediate hepatic cells revealed that although a large proportion of V p + T cells with intermediate TCR also expressed either CD4 or CD8, a significant percentage of TCR a p m t e r m e d i a t e (TCR <x$mt) cells were of the DN phenotype. Although TCR a p m l cells constitute a small proportion of cells in the liver of young mice (6-8 weeks of age), Ohteki et al. (1992) demonstrated a significant expansion of this population as a consequence of aging with DNapT cells assuming the predominant phenotype in the older mice (60 weeks of age). At older ages when the thymus is characteristically involuted, this extrathymic pathway was in its most activated state and the apparent TCR a p m t progeny of this pathway began to appear in the periphery, first in the peripheral blood and then eventually in the spleen and lymph nodes. Confirmation that the liver is a site of extrathymic differentiation was provided by the observation of only TCR a p m t cells including DNapT cells in the liver of congenitally athymic nude mice. Furthermore, an age dependent increase in the proportion of this hepatic population was also observed in the athymic mice. The expansion of DNapT cells, including those bearing self reactive TCRs, has also been demonstrated in the liver of normal mice following bacterial stimulation (Abo et al. 1991). This proposed extrathymic pathway has also been shown to be activated following estrogen administration at the expense of intrathymic T cell development (Okuyama et al. 1992). Subcutaneous injection of 1 mg of estrogen resulted in the increase of hepatic mononuclear cells enriched for TCR a p m l cells including V p + cells and forbidden clones while the thymus underwent estrogen induces atrophy with depletion of TCR apdull CD4+ CD8 + thymocytes. Interestingly, the liver has been shown to be a major site for the proliferation of DNapT cells in numerous autoimmune mice including MRL-lpr/lpr, C3H/HeJ-gld/gld, BXSB, NOD and NBZBAV Fi strains (Ohteki et al. 1990, Masuda et al. 1991). Following the onset of the autoimmune disease, DNapT cells expressing both Pgp-1 and B220 have been shown to accumulate in the liver of Ipr mice where they reside in hepatic sinusoids in close association with Kupffer and endothelial cells. While DNapT cells accounted for greater than 50% of total hepatic TCR a p + cells, the vast majority of TCR ap expressing T cells in the lymph nodes and spleen of Ipr mice were of the DNapT phenotype. Assessment of CD3 depleted hepatic MNC cultured for three days revealed a prominent increase in expression of TCR ap and CD3 but not CD4 or CD8 and suggested the possible extrathymic development of DNapT cells 107 null cell precursors. Furthermore, in vivo intravenous injection of 3H-mymidine demonstrated peak ^ H-TdR incorporation by hepatic MNC on day 1 at levels almost comparable to that of bone marrow. A peak of radioactivity on day 5 in lymph node MNC provided support for the possible migration of hepatic derived DNapT cells to peripheral tissues and the resultant lymphadenopathy in MRL-lpr/lpr mice. Although these studies have implicated the hyperactivity of this extrathymic T cell development pathway in the pathogenesis of the marked T lymphocyte accumulation observed in Ipr mice, a recent study has proposed an alternate role for the liver in this autoimmune disorder. Employing a modified separation procedure which combined mild enzymatic digestion, FCS gradient centrifugatidn and panning, Huang et al. (1994) were able to increase the liver T cell yield over Ficoll density gradient centrifugation and were therefore able lo demonstrate that the predominant intrahepatic lymphocyte in the normal adult mouse were of the TCR ap m t CD4" CD8" B220+ HSA+ phenotype. Furthermore, this hepatic TCRapm t DN T cell subset, whose phenotype is similar to those T cells massively expanded in Ipr mice, were shown to be actively proliferating and undergoing apoptosis. Together with the absence of significant RAG-1 and RAG-2 expression required for TCR a and P chain rearrangement (Crispe and Huang, 1994), it was therefore suggested that the liver is a site for T cell destruction as opposed to T cell development and that failure of T cells to undergo apoptosis possibly due to the insertional mutation in the Fas gene (Watanabe-Fukunaga et al. 1992) may result in the accumulation of TCR a P m t CD4* CD8" B220 + T cells in the liver and subsequent spill over into the periphery of Ipr mice. Therefore based on the demonstration of a B220 + HSA + phenotype as well as significant apoptotic activity, it is unlikely that hepatic DNctPT cells represent precursors of splenic DNapT cells which are HSA" B220" (Figure 7; Prud'homme et al. 1991). In order to confirm that hepatic and splenic DNapT cells are indeed developmentally unrelated, sublethally irradiated or congenic hosts could be assessed for the absence of splenic DNapT cells following reconstitution by hepatic TCRaP + DN T cells. Furthermore the failure of bacterial stimulation or subcutaneous estrogen injection to induce the expansion of splenic DNapT cells would provide further evidence for a developmental pathway distinct from that of phenotypically similar hepatic cells. Although both splenic DNapT cells and TCR y5 + DN T cells share tne HSA" CD3+ DN phenotype as well as the ability to respond to antigen independent activation of IL-7 with or without the addition of IL-1, one significant difference has been observed between these two T lymphocyte subsets. DNapT cells proliferated vigorously in the presence of immobilized anti-CD3 MAb, a response comparable to that of nylon wool purified SP T cells. TCR y8 + DN spleen cells, on the other hand, were nonresponsive to anti-CD3 crosslinking despite the expression of the TCR/CD3 complex. It has recently been shown that TCR/CD3 signaling by crosslinking with immobilized MAb specific for either TCR y5 or CD3 complex in the presence of IL-2 induces 108 death by apoptosis in nontransformed IL-2-dependent TCR y 5 + DN T cells isolated from normal human peripheral blood (Janssen et al. 1991). It would therefore be interesting to determine whether TCR y 8 + DN spleen cells, which are nonresponsive when cultured with immobilized anti-CD3, actually undergo apoptosis under these conditions. Interestingly, the addition of IL-1 to TCR y 8 + DN cells cultured with immobilized anti-CD3 appears to restore the responsiveness of this splenic subset towards the activation of the TCR/CD3 complex. Therefore whereas anti-CD3 crosslinking is sufficient to stimulate splenic DNapT cells to proliferate, the activation of TCR yS + DN spleen cells appears to require a comitogenic stimulus. Splenic DNapT cells are considered to be functionally mature as suggested by the absence of expression of the heat stable antigen, a responsiveness to crosslinking of the TCR associated CD3 complex which is comparable to that of nylon wool nonadherent mature SP T cells and an ability to express cytokine specific messages. Although the functional significance of this mature T cell population in the immune response remains to be established, several possible functions have been proposed. DNapT cells have been suggested to play a key role in the suppression of immune responses. Cloned cell lines of the DNapT phenotype derived from the spleens of neonatal or adult mice recovering from either irradiation or bone marrow transplantation have been shown to inhibit both GVHD and MLR (Hertel-Wulff et al. 1984, Schwadron et al. 1985, Strober et al. 1987, Sykes et al. 1990). Lethal GVHD suppression was also performed by DNapT cells obtained from the spleens of Mls-1^ mice following immunization with Mls-l a cells (Bruley-Rosset et al. 1990). Furthermore, suppression of acute GVHD has been detected in the low density fraction of Percoll density gradient fractionated spleen cells from normal mice and was correlated with the depletion of typical SP T cells and the enrichment of DNapT cells (Palathumpat et al. 1992). Similarly, fractionation of normal bone marrow cells yielded low density fractions enriched for DNapT cells with natural suppressor activity and capable of inhibiting both MLR and GVHD (Benveniste et al. 1990, Palathumpat et al. 1992). In further support of a suppressor like function for DNapT cells, consistent increases in splenic CD3 + CD4" CD8" cells have been demonstrated in animals receiving GVH-inducing inocula (Abraham et al. 1992). Further enhancement of the percentage of this splenic subpopulation occurred when recipients of allogeneic inocula were treated with IL-2. Early administration of IL-2 following bone marrow transplantation has indeed been demonstrated to provide potent prophylaxis against GVHD mortality without preventing alloengraftment or reducing graft versus leukemia effects (Sykes et al. 1990b,c). A recent study has demonstrated the switch of CD8 + SP T cells, following activation in the presence of IL-4, to noncytolytic DNapT cells capable of T]-[2 cytokine production, including IL-4, IL-5 and IL-10, and of induction of 109 immunoglobulin production by B cells (Erard et al. 1993). It has been suggested that this IL-4 induced diversion of resting CD8 + SP T cells away from the IFNy producing cytotoxic pathway may represent one mechanism for suppression of CD8 cytotoxic responses. Furthermore, the Tf^2 cytokine IL-4 and IL-10, as produced by these noncytolytic DNapT cells, are considered to be suppressor factors for Tj-jl type responses (Modlin and Nutman, 1993). Interestingly, following HTV-1 infection, progression from the protective phase of the disease, chuiccterized by a high frequency of HTV-1 specific CD8 + SP T cells, to AIDS is correlated with a significant reduction of these HTV-1 specific cytotoxic T cells and a concomitant increase in Tj-j2 cytokine activity (Riddell etal. 1993, Carmichael et al. 1993, Clerici and Shearer 1993). Furthermore, a significant increase in the absolute number of CD3 + CD4" CD8" T cells in the peripheral blood of HTV-1 infected individuals has been shown to occur in the late stages of AIDS (Margolick et al. 1989). Whether the redirection of CD8 + SP T cells down a noncytolytic Tj-j2-like DNapT cell pathway plays a role in the progression from HTV-1 infection to AIDS remains to be clarified. Conversely, a role for DNapT cells in cytotoxic responses and allogeneic tumour rejection has also been suggested. CTL with the TCRaP + CD4" CD8" phenotype have been generated from MLTC spleen cells of anti-CD8 MAb treated B6 mice (Mieno et al. 1991). Similarly, rejection of an LV variant line of FBL-3 leukemia in CD8 depleted syngeneic B6 mice was also shown to be mediated by DNapT cells as well as CD4 + CTL generated by the activation of CD4 + helper T cells (Yoshimura et al. 1993). Furthermore, IL-2 dependent T cell lines of DNapT phenotype with non-MHC-restricted cytolytic activity against tumour targets, syngeneic and allogeneic fibroblasts and PHA blasts from allogeneic donours have been isolated from the peripheral blood of a patient with combined immunodeficiency and tissue injury resembling GVH who had a massive expansion of this cell type (Brooks et al. 1990). Following activation by mitogens or PMA and IL-1, these cell lines were capable of IFNy as well as TNFa and TNFp production. Additional studies are required in order to determine whether freshly purified splenic DNapT cells are capable of cytotoxic function against, for example, Yac-1 target cells. Alternatively, splenic DNapT cells may play a potentially important role in the development of Tfj2 immune responses. Although it has been previously shown that IL-4 plays a pivotal role in the differentiation of Tj-jO cells to the Tjj2 phenotype (Swain et al. 1990), the source of this cytokine is unknown. Based on the demonstration of IL-4 production by splenic DNapT cells as shown in Figure 11 and by Prud'homme et al. (1991) and Zlotnik et al. (1992), it is possible that this splenic T cell subset may be the source of IL-4 during the initial stage of development of the Tj-r2 response in vivo. 110 Several studies have suggested that DNapT cells are autoreactive and have implicated this phenotypically unique T cell population in the development of autoimmune diseases. Following activation with either anti-TCR aJ3 MAb or Con A, splenic DNapT cells acquired high reactivity in both syngeneic and allogeneic MLC (Prud'homme et al. 1991). Interestingly, this autoreactivity corresponded to the acquisition of CD4 expression with gradual reversion to the DN phenotype following the arrest of activation. A marked expansion of DNapT cells has been observed in the peripheral blood of patients with autommune disorders including systemic sclerosis (Sakamoto et al. 1992) and systemic lupus erythematosus (SLE) (Shivakumar et al. 1989). Along with CD4 + SP T cells, these TCR expressing double negative T cells were responsible for the induction of oligoclonal production of highly cationic anti-DNA autoantibodies. Studies of the murine model for SLE have also implicated peripheral DNapT cells in the development of autoimmune disorders. DNapT cells have been shown to accumulate in the massively enlarged lymph nodes and spleen of the autoimmune lymphoproliferative murine strain MRL-lpr/lpr (Wolfsy et al. 1984; Nemanzee et al. 1985). Strains which carry the mutant autosomal gene, Ipr, spontaneously develop a severe and rapidly progressive autoimmune disease characterized by the production of a variety of autoantibodies, immune complex-mediated glomerulonephritis and vasculitis in addition to the massive lymphadenopathy (Andrews et al. 1978; Theofilopoulos and Dixon, 1981). It has been suggested that the massive accumulation of abnormal Ipr T cells may be due to a failure to undergo apoptosis secondary to an insertional mutation in the Fas gene (Watanabe-Fukunaga et al. 1992). Both the lymphoproliferation and autoimmunity observed in Ipr mice are thought to be thymic dependent based on the abrogation of these abnormalities in neonatally thymectomized mice (Hang et al. 1984). In addition, cells of the atypical B220 + Pgp-1+ phenotype have also been identified within both of the DN and DP thymocyte subpopulations of this autoimmune murine strain (Tutt Landolfi et al. 1993). Furthermore, Ipr DNapT cells have demethylated CD8a sequences and are thought to be influenced by thymic negative selection as suggested by the absence of Vpg \ + and Vpg"1" cells amongst DNapT lymph node cells of M l s - l a + AKR Ipr mice (Singer et al. 1989). A number of similarities exist between the DNapT cells which reside in the spleen of normal mice and the abnormally expanded T cell population in Ipr mice. Similar to DNapT cells in the spleens of normal mice, the abnormally expanded cells of Ipr mice are also of the TCR a p + DN phenotype and express Lyt-1 and Pgp-1 but not HSA (Nemazee et al. 1985; Davidson et al. 1986). Furthermore, TCR gene expression in DNapT cells of both normal and Ipr mice is skewed in favour of the Vpg family (Singer et al. 1986). This preferential usage of Vpg genes is also characteristic of other murine models of autoimmune disorders including collagen-induced arthritis (Osman et al. 1993) and diabetes (Fukuda et al. 1989). However, there are differences between splenic DNapT cells of normal mice and the abnormal Ipr T cells, particularly with respect to surface phenotype. Unlike splenic DNapT cells of normal mice, the Ill majority of abnormal subset of T cells in Ipr mice express B220, an isoform of the common leukocyte antigen Ly-5 whose expression is normally restricted to cells of the B lineage (Coffman and Weissman, 1981). Furthermore abnormal Ipr T cells and normal splenic DNapT cells also show a reciprocal expression pattern for the YE1/19.1 and YE1/9.9 (Takei, 1984; Davidson et al. 1986) . Whereas abnormal Ipr T cells express YE1/19.1, an antigen similar to the plasma cell antigen PC-1 (Takahashi et al. 1970), splenic DNapT cells of normal mice are YE1/19". In contrast, abnormal Ipr T cells lack the expression of YE 1/9.9 whereas normal splenic DNapT cells are YEl/9 +. The MAb YE 1/9.9 is specific for the transferrin receptor which is expressed on essentially all proliferating cells including activated T lymphocytes (Takei 1983; Trowbridge and Omary, 1982; Sutherland et al. 1981). DNapT cells of normal and Ipr mice also differ in their response to TCR-CD3 engagement. Although abnormal Ipr cells undergo a rapid increase in intracellular calcium with the generation of inositol phosphates and expression of interleukin-2 receptors, unlike normal splenic DNapT cells the atypical Ipr cells do not proliferate in response to TCR-CD3 activation due to the absence of IL-2 production (Davignon et al. 1985). Although the role of DNapT cells in the development of autoimmune disorders has yet to be established, the constitutive expression of IFNy message by this phenotypically unique T cell population provides support for an association with autoimmunity. IFNy has been shown to promote B cell growth (Morikawa et al. 1987) and induce IgG2a production (Snapper and Paul, 1987) as well as class II MHC expression on the surface of macrophages and B cells (Goedegebuure et al. 1989). Increased levels of class II MHC expression as well as increased amounts of Ig including the IgG2a isotype have observed in Ipr mice. ConA-stimulated Lyt-l +2" T cells isolated from the spleens of Ipr mice are capable of significant IFNy production despite hypoproduction of IL-2 (Santoro et al. 1983). In addition, RNA protection assays have demonstrated that abnormal Ipr T cells isolated either as CD4" CD8" and B220+/Thy-1+ subsets spontaneously transcribe IFNy as well as TNF-a genes in vivo (Murray and Martens, 1989). Furthermore PCR analysis has confirmed the expression of genes encoding IFNy as well as TNF-a, TNF-P and IL-6 by the CD4" CD8" Thy-1+ B220+ subset of T cells in Ipr mice (Murray et al. 1990). IFNy has also been shown to accelerate the progression of autoimmune disease in New Zealand Black mice (Heremans et al. 1978) and can induce immune complex glomerulonephritis in newborn mice (Gresser et al. 1976). Furthermore, disturbances in IFNy activity have been observed in patients with autoimmune disorders including SLE, rheumatoid arthritis, scleroderma and Sjogren's syndrome (Hooks et al. 1979). Since the splenic DNapT cell population, which constitutively expresses IFNy mRNA, is expanded by IL-7 and IL-1, this stimulation pathway may lead to antigen-independent production of IFNy. On the other hand, antigen-dependent activation of splenic DNapT cells may lead to the production of IL-4. In addition to the proliferative effects 112 on activated B cells (Kishimoto, 1985), IL-4 has also been shown to induce the expression of class II MHC genes on the surface of resting human and murine B cells (Smith and Rennick, 1986; Rousset et al. 1988) as well as enhance the production of Ig including IgGi by activated B cells (Coffman and Carty, 1986). IL-4 as well as IFNy may therefore be potential sources of the B cell differentiation factor (BCDF) activity that is spontaneously produced in the autoimmune Ipr mice (Prud'homme et al. 1983) and leads to polyclonal B cell activation and ultimately production of autoantibodies. Uncontrolled production of IL-4 as well as IFNy by splenic DNapT cells may also contribute to the increased expression of class II MHC which could induce the proliferation of CD4 + autoreactive T cells. Additional studies are required in order to determine whether splenic DNapT cells are in fact involved in the development of autoimmune diseases. It would be of interest to determine whether mice in which the DNapT cell population has been expanded by, for example, infection by an IL-7 retrovirus develop autoimmune characteristics. Based on the results of a previous study which has demonstrated the ability of IL-3 to induce the generation of DNapT from CD4 +/8 + depleted bone marrow and spleen cells (Kubota et al. 1992), 11-3 overexpressing mice could be assessed for the possible expansion of bone marrow and splenic DNapT cells and concurrent development of autoimmune characteristics. Futhermore Ipr mice could be assessed for possible IL-3 overexpression as a source for the abnormally expanded TCR a p + DN T cell population. A subsequent study could include the assessment of potential suppression of autoimmune manifestations in Ipr mice following IL-3 gene knockout. The role of DNapT cells, whether it be in the development of autoimmune disorders, cytotoxic responses and tumour rejection or the suppression of immune responses, will no doubt be the subject of future studies. 113 References Abraham, V.S., Sachs, D.H. and Sykes, M. (1992) Mechanism of protection from graft-versus host disease mortality by IL-2. Ill Early reductions in donor T cell subsets and expansion of a CD3+ CD4" CD8- cell population. J. Immunol. 148:3746. Abo, T., Ohteki, T., Seki, S., Koyamada, N., Yoshikai, Y., Masuda, T., Rikiishi, H. and Kumagai, K. (1991) The appearance of T cells bearing self reactive T cell receptor in the livers of mice infected with bacteria. J. Exp. Med. 174:417. Alderson, M.R., Tough, T.W., Ziegler, S.F. and Grabstein, K.H. (1991) Interleukin 7 induces cytokine secretion and tumoricidal activity by human peripheral blood monocytes. J. Exp. Med. 173:923. Andrews, B.S., Eisenberg, R.A., Theofilopoulos, A.N., Izui, S. Wilson, C.B., McConahey, P.J., Murphy, E.D., Roths, J.B. and Dixon, F.J. (1981) Etiopathogenesis of murine SLE. Immunol. Rev. 55:179. Benveniste, P., Chadwick, B.S., Miller, R.G. and Reimann, J. (1990) Characterization of cells with T cell markers in athymic nude mice bone marrow and of their in v/Yra-derived clonal progeny. Comparison with euthymic bone marrow. J. Immunol. 144:411. Biddison, W.E., Rao, P.E., Talle M.A., Goldstein, G. and Shaw, S. (1984) Possible involvement of the T4 molecule in T cell recognition of class II HLA antigens. Evidence from studies of CTL: target binding. J. Exp. Med. 159:783. Bix, M., Coles, M. and Raulet, D. (1993) Positive selection of Vng + CD4" CD8" thymocytes by class I molecules expressed by hematopoietic cells. J. Exp. Med. 178:901. Brooks, E.G., Wirt, D.P., Goldblum, R.M., Vaidya, S., Asuncion, M.T., Patterson, J.C., Ware, C.F. and Klimpel, G.R. (1990) Double negative (CD4" CD8") T cells with an a/p T cell receptor. Non-MHC-restricted cytolytic activity and lymphokine production. J. Immunol. 144:4507. Brooks, E.G., Balk, S.P., Aupeix, K., Colonna, M., Strominger, J.L. and Groh-Spies, V. (1993) Human T-cell receptor (TCR) a/p+ CD4" CD8" T cells express oligoclonal TCRs, share junctional motifs across TCR Vp-gene families, and phenotypically resemble memory T cells. Proc. Natl. Acad. Sci. 90:11787. Bruley-Rosset, M., Miconnet, I., Canon, C. and Halle-pannenko, O. (1990) Mls a generated suppressor . 1. Suppression is mediated by double negative (CD3 + CD5 + CD4" CD8") a/p T cell receptor-bearing cells. J. Immunol. 145:4046. Budd, C.R., Cerottini, J.C., Horvath, C, Bron, C, Pedraini, T., Howe, R.C. and MacDonald, H.R. (1987) Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation. J. Immunol. 138:3120. Carbone, A.M., Marrack, P. and Kappler, J.W. (1988) Demethylated CD8 gene in CD4+ T cells suggests that CD4 + cells develop from CD8 + precursors. Science 242:1174. 114 Carmichael, A., Jin, X., Sissons, P. and Borysiewicz L. (1993) Quantitative analysis of the human immunodeficiency virus type 1 (HTV-1 )-specific cytotoxic T lymphocyte (CTL) response at different stages of HTV-1 infection: Differential CTL responses to HTV-1 and Epstein-Barr Virus in late disease. J. Exp. Med. 177:249. Ceredig, R., Lynch, F. and Newman, P. (1987) Phenotypic properties, interleukin-2 production, and developmental origin of a "mature" subpopulation of Lyt-27L3T4" mouse thymocytes. Proc. Natl. Acad Sci. USA. 84:8578. Chazen, G.D., Pereira, G.M.B., LeGros, G., Gillis, S. and Shevach, E.M.(1989) Interleukin 7 is a T-cell growth factor. Proc. Nat. Acad. Sci. USA 86:5923. Clerici, M. and Shearer, G.M. (1993) A Tj j l to TJJ2 switch is a critical step in the etiology of HTV infection. Immunol. Today 14:107. Coffman, R.L. and Carty, J. (1986) T cell activity that enhances polyclonal IgE production and its inhibition by interferon-y. J. Immunol. 136:949. Coffman, R.L. and Weissman, I.L. (1981) B220: a B cell specific member of the T200 glycoprotein family. Nature 289:681. Crispe, I.N. and Bevan, M.J. (1987) Expression and functional significance of the J l Id marker on mouse thymocytes. J. Immunol. 138:2013. Crispe, I.N., Moore, M.W., Husmann, L.A., Smith, L., Bevan, M.J. and Shimonkevit, R.P. (1987) Differentiation potential of subsets of CD4" CD8" thymocytes. Nature 329:336. Crispe, I.N. and Huang, L. (1994) Neonatal, moribund and undead T cells: the role of the liver in T cell development. Semin. Immunol. 6:39. Davidson, W.F., Dumont, F.J., Bedigian, H.G., Fowlkes, B.J., and Morse, H.C. (1986) Phenotypic, functional, and molecular genetic comparisons of the abnormal lymphoid cells of C3H-lpr/lpr and CeH-gld/gld mice. J. Immunol. 136:11. Davignon, J-L., Budd, R.C., Cereig, R., Piguet, P.F., MacDonald, H.R., Cerottini, J-C, Vassalli, P. and Izui, S. (1985) Functional analysis of T cells bearing the Ipr gene. J. Immunol. 135:2423 Egerton, M. and Scollay, R. (1990) Intrathymic selection of murine TCR a p + CD4" CD8~ thymocytes. Int. Immunol. 2:157. Erard, F., Wild, M.-T., Garcia-Sanz, J.A. and Le Gros, G. (1993) Switch of CD8 T cells to noncytolytic CD8" CD4" cells that make Tj-[2 cytokines and help B cells. Science 260:1802. Fischer, M., MacNeil, I., Suda, T., Cupp, J.E., Shortman, K. and Zlotnik, A. (1991) Cytokine production by mature and immature thymocytes. J. Immunol. 146:3452. 115 Fowlkes, B.J., Kruisbeek, A.M., Ton-That, H., Weston, M.A., Coligan, J.E., Schwartz, R.H. and Pardoll, D.M. (1987) A novel population of T-cell receptor a.p bearing thymocytes which predominantly express a single Vp gene family. Nature 329:251. Fukuda, M., Horio, F., Kubo, R. and Hattori, M. (1989) Monoclonal antibody F23.1 against T cells bearing Vpg TCR elements can prevent the development of insulitis in NOD mice. Diabetes 38:12A. Goedegebuure, P.S., Segal, D.M., Braakman, E., Vreugdenhil, R.J., Van Krimpen, B.A., Van de Griend, R.J. and Bolhuis, R.L.H. (1989) Induction of lysis by T cell receptor y5 +/CD3 + T lymphocytes via CD2 requires triggering via the T l 1.1 epitope only. J. Immunol. 142:1797. Gresser, I., Morel-Maroger, L., Maury, C, Tovey, M.G. and Pontillon, F. (1976) Progressive glomerulonephritis in mice treated with interferon preparations at birth. Nature 263:420. Guidos, C.J., Weissman, LL. and Adkins, B. (1989a) Developmental potential of CD4"8" thymocytes: Peripheral progeny include mature CD4"8" T cells bearing ap T cell receptor. J. Immunol. 142:3773. Guidos, C.J., Weissman, I.L. and Adkins, B. (1989b) Intrathymic maturation of murine T lymphocytes from CD8 + precursors. Proc. Natl. Acad. Sci. USA 86:7542. Hang, L., Theofilopoulos, A.N., Balderas, R.S., Francis, S.J. and Dixon, F.J. (1984) The effect of thymectomy on lupus-prone mice. J. Immunol. 132:1809. Henney, C.S. (1989) Interleukin-7: effects on early events in lymphopoiesis. Immunol. Today 10:170. Heremans, H., Billiau, A., Colombatti, A., Hilgers, J. and Desomer, P. (1978) Interferon treatment of NZB mice: accelerated progression of autoimmune disease. Infect. Immunol. 21:925. Hertel-Wulff, B., Okada, S., Oseroff, A. and Strober, S. (1984) In vitro propagation and cloning of murine natural suppressor (NS) cells. J. Immunol. 133:2791. Hollander, N. (1982) Effects of anti-Lyt2 antibodies on T cell function. Immunol. Rev. 68:43. Hooks, J.J., Moutsopoulos, H.M., Geis, S.A., Stahl, N.I., Decker, J.L. and Notkins, A.L. (1979) Immune interferon in the circulation of patients with autoimmune disease. N. Engl. J.Med. 301:5. Howe , R.C. and MacDonald, H.R. (1988) Heterogeniety of immature (Lyt-27L3T4_) thymocytes: Identification of four major phenotypically distinct subsets differing in cell cycle status and in vitro activation requirements. J. Immunol. 140:1047. Huang, L. and Crispe, LN. (1992) Distinctive selection mechanisms govern the T cell receptor repertoire of peripheral CD4" CD8" ap T cells. J. Exp. Med. 176:699. 116 Huang, L., Sye, K. and Crispe, I.N. (1994) Proliferation and apoptosis of B220+ CD4" CD8" TCR apmt T cells in the liver of normal adult mice: implication for Ipr pathogenesis. Int. Immunol. 6:533. Janssen, 0., Wesselborg, S., Heckl-Ostreicher, B., Pechhold, K., Bender, A., Schondelmaier, S., Moldenhauer, G. and Kabelitz, D. (1991) T cell receptor/CD3 signaling induces death by apoptosis in human T cell receptor y8 + T cells. J. Immunol. 146:35. Kishimoto, T. (1985) Factors affecting B-cell growth and differentiation. Ann. Rev. Immunol. 3:133. Kisielow, P., Bluthmann, H, Staerz, U.D., Steinmetz, M. and von Boehmer, H. (1988) Tolerance in T cell receptor transgenic mice involves deletion of nonmature CD4 +8 + thymocytes. Nature 333:742. Kubota, H, Okazaki, H, Onuma, M., Kano, S., Hattori, M. and Minato, N. (1992) CD3+4'8- ap T cell population with biased T cell receptor V gene usage: Presence in bone marrow and possible involvement of IL-3 for their extrathymic development. J. Immunol. 149:1143. Kyburz, D., Aichel, P., Spenser, D.E., Hengartner, H., Zinkernagel, R.M. and Pircher, H. (1993) T cell immunity after a viral infection versus T cell tolerance induced by soluble viral peptides. Eur. J. Immunol. 23:1956. Levitsky, H. Y., Golumbek, P.T. and Pardoll, D. M. (1991) The fate of CD4"8- T cell receptor-ap+thymocytes. J. Immunol. 146:1113. MacDonald, H.R., Howe, R.C, Pedrazzini, T., Lees, R.K., Budd, R.C, Sctwider, R., Liao, N.S., Zinkernagel, R.M., Louis, J.A., Raulet, D.H., Hengartner, H. and Miescher, G. (1988) T-cell lineage repertoire selection and tolerance induction. Immunol. Rev. 104:157. Margolick, J.B., Carey, V., Munoz, A., Polk, B.F., Giogi, J.V., Baurer, K.D., Kaslow, R. and Rinaldo, C. (1989) Development of antibodies to HIV-1 is associated with an increase in circulating CD3 + CD4" CD8" lymphocytes. Clinical Immunol. Immunopathol. 51:348. Martinez-A.,C, Marcos, M.A.R., de Alboran, I.M., Alonso, J.M., de Cid, R., Kroemer, G. and Coutinho, A. (1993) Functional double-negative T cells in the periphery express T cell receptor Vp gene products that cause deletion of single-positive T cells. Eur. J. Immunol. 23:250. Masuda, T., Ohteki, T., Abo, T., Seki, S., Nose, M., Nagura, H. and Kumagai, K. (1991) Expansion of the population of double negative CD4" 8" Tap-cells in the liver is a common feature of autoimmune mice. J. Immunol. 147:2907. Mieno, M., Suto, R., Obata, Y., Udono, H, Takahashi, T., Shiku, H. and Nakayama, E. (1991) CD4" CD8" T cell receptor ap T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumour rejection. J. Exp. 174:193. Modlin, R.L. and Nutman, T.B. (1993) Cytokines and negative immune ;egulation in human infections. Curr. Opin. Immunol. 5:511. 117 Morikawa, K., Kubagawa, H., Suzuki, T. and Cooper, M.D. (1987) Recombinant interferon enhances the proliferative response of human B cells. J. Immunol. 139:761. Morrissey, P.J., Goodwin, R.G., Nordan, R.P., Anderson, D., Grabstein, K.H., Cosman, D., Sims, J., Lupton, S., Acres, B., Reed, S.G., Mochizuki, D., Eisenman, J., Conlon, P.J. and Namen, A.E (1989) Recombinant interleukin 7, pre-B cell growth factor, has costimulatory activity on purified mature T cells. J. Exp. Med. 169:707. Murray, L. and Martins, C. (1989) The abnormal T lymphocytes in Ipr mice transcribe interferon-y and tumor necrosis factor-a genes spontaneously in vivo. Eur. J. Immunol. 19:563. Murray, L., Lee, R. and Martens, C. (1990) In vivo cytokine gene expression in T cell subsets of the autoimmune MRL/Mp-lpr/lpr mouse. Eur. J. Immunol. 20:163. Murray, , R., Wrighton, N., Lee, F. and Zlotnik, A. (1989) Interleukin-7 is a thymocyte growth and maintenance factor for mature and immature thymocytes. Int. Immunol. 1:526. Nemanzee, D.A., Studer, S., Steinmetz, M., Dembie, Z. and Kiefer, M. (1985) The lymphoproliferating cells of MRL-lpr/lpr mice are a polyclonal population that bear the T lymphocyte receptor for antigen. Eur. J. Immunol. 15:760. Niehues, T., Gulwani-Akolkar, B., Akolkar, P.N., Tax, W.and Silver, J. (1994) Unique phenotype and distinct TCR Vp repertoire in human peripheral blood a.pTCR+, CD4" and CD 8" double negative T cells. J. Immunol. 173:1072. Nikolic-Zugic, J. and Moore, M.W. (1989) T cell receptor expression on immature thymocytes with in vivo and in vitro precursor potential. Eur. J. Immunol. 19:1957. Nikolic-Zugic, J., Moore, M.W. and Bevan, M.J. (1989) Characterization of the subset of immature thymocytes which undergo rapid in vitro differentiation. Eur. J. Immunol. 19:649. Ohteki. T., Seki, S., Abo, T. and Kumagai, K. (1990) Liver is a possible site for the proliferation of abnormal CD3+4"8" double negative lymphocytes in autoimmune MRL-lpr/lpr mice. J. Exp. Med. 172:7. Ohteki, T., Okuyama, R., Seki, S., Abo, T., Sugiure, K., Kusumi, A., Ohmori, T., Watanabe, H. and Kumagai, K. (1992) Age-dependent increase of extrathymic T cells in the liver and their appearance in the periphery of older mice. J. Immunol. 149:1562. Okuyama, R., Abo, T., Seki, S., Ohteki, T., Sugiura, K., Kusumi, A. and Kumagai, K. (1992) Estrogen administration activates extrathymic T cell differentiation in the liver. J. Exp. Med. 175:661. Osman, G.E., Toda, M., Kanagawa, O. and Hood, L.E. (1993) Characterization of the T cell receptor repertoire causing collagen arthritis in mice. J. Exp. Med. 177:387. 118 Palathumpat, V., Dejbakhsh-Jones, S., Holm, B. and Strober, S. (1992a) Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease. J. Immunol. 149:808. Palathumpat, V., Dejbakhsh-Jones, S., Holm, B., Wang, H, Liang, 0. and Strober, S. (1992b) Studies of CD4" CD8" aP bone marrow T cells with suppressor activity. J. Immunol. 148:373. Papiernik, M. and Pontoux, (1990) In vivo and in vitro repertoire of CD3 + CD4" CD8" thymocytes. Intl. Immunol. 2:407. Porcelli, S., Brenner, M., Greenstein, J., Balk, S., Terhorst, C. and Bleicher, P.A. (1989) Recognition of cluster of differentiation antigens by human CD4" CD8" cytolytic lymphocytes. Nature 341:447. Porcelli, S., Yockey, C.E., Brenner, M. and Balk, S. (1993) Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4"8" a/p T cells demonstrates preferential use of several Vp genes and an invariant TCR a chain. J. Exp. Med. 178:1. Prud'homme, G.J., Park, C.L., Fieser, T.M., Kofler, R., Dixon, F.J. and Theofilopoulos, A.N. (1983) Identification of a B cell differentiation factor(s) spontaneously produced by proliferating T cells in murine lupus strains of the Ipr/lpr genotype. J. Exp. Med. 157:730. Prud'homme, G.J., Bocarro, D.C. and Luke, E.C.H. (1991) Clonal deletion and autoreactivity in extrathymic CD4" CD8" (Double Negative) T cell receptor-ap T cells. J. Immunol. 147:3314. Quaratino, S., Murison, G., Knyba, R.E., Verhoff, A. and Londei, M. (1991) Human CD4" CD8" a p + T cells express a functional T cell receptor and can be activated by superantigens. J. Immunol. 147:3319. Riddell, S.R., Gilbert, M.J. and Grennberg, P.D. (1993) Cytotoxic T cell therapy of cytomegalovirus and HTV infection. Curr. Opin. Immunol. 5:484. • Robey, E.A., Ramsdell, F., Gordon, J.W., Mamalaki, C, Kioussis, D., Youn, H.J., Gottlieb, P.D., Axel, R. and Fowlkes, B.J. (1992) A self reactive T cell population that is not subject to negative selection. Int. Immunol. 4.969. Rousset, F., Malefijt, F.W., Slierendregt, B., Aubry, J-P.,Bonnefoy, J-Y., Defiance, T., Banchereau, J. and deVries, J. (1988) Regulation of Fc receptor for IgE (CD23) and class II MHC antigen expression on Burkitt's lymphoma cell lines by human IL-4 and IFN-y. J. Immunol. 140:2625. Sakamot, A., Sumida, T., Maeda, T., Itoh, M., Asai, T., Takahashi, H, Yoshida, S., Koike, T., Tomioka, Hand Yoshida, S. (1992) T cell receptor Vp repertoire of double negative a/p T cells in patients with systemic sclerosis. Arthrit. Rheumat. 35:944. Santoro, T.J., Benjamin, W.R., Oppenheim, J.J. and Steinberg, A.D. (1983) The cellular basis for immune interferon production in autoimmune MRL-lpr/lpr mice. J. Immunol. 131:265. 119 Schwadron, R.B., Gandour, D.M. and Strober, S. (1985) Cloned natural suppressor cell lines derived from the spleen of neonatal mice. J. Exp. Med. 162:297. Scott, B., Bluthmann, H., Teh, H.S. (1989) Nature 338:591. Seki, S., Abo, T., Ohteki, T., Sugiura, K. and Kumagai, K. (1991) Unusual ap-T cells expanded in autoimmune Ipr mice are probably a counterpart of normal T cells ii . the liver. J. Immunol. 147:1214. Sentman, C.L., Kumar, V., Koo, G. and Bennett, M. (1989) Pan natural killer cell monoclonal antibodies and their relationship to the NK1.1 antigen. Hybridoma 8:605. Shivakumar, S., Tsokos, G. and Datta, S.K., (1989) T cell receptor a/p expressing double-negative (CD47CD8") and CD4 + T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis. J. Immunol. 143:103. Singer, P.A., Balderas, R.S., McEvilly, R.J., Bobart, M. and Theofilopoulos, A.N. (1989) Tolerance-related Vp clonal deletions in normal CD4"8", TCR a p + and abnormal Ipr and gld cell populations. J. Exp. Med. 170:1869. Smith, CA. and Rennick, D.M. (1986) Characterization of a murine lymphokine distinct from interleukin 2 and interleukin 3 (IL-3) possessing a T-cell growth activity and a mast cell growth factor activity synergizes with IL-3. Proc. Natl. Acad. Sci. USA 83:1857. Snapper, CM. and Paul, W.E. (1987) Interferon-y and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944. Strober, S., Palathumpat, V., Schwadron, R. and Hertel-Wulff (1987) Cloned natural suppressor cells prevent lethal graft-versus-host disease. J. Immunol. 138:699. Suda, T. and Zlotnik, A. (1991) IL-7 maintains the T cell precursor potential of CD3" CD4" CD8" thymocytes. J. Immunol. 146:3068. Suda, T. and Zlotnik, A. (1993) Origin, differentiation, and repertoire selection of CD3 + CD4" CD8" thymocytes bearing either ap or y5 T cell receptors. J. Immunol. 150:447. Sutherland, R., Delia, D., Schneider, C, Newman, R., Kemshead, J. and Greaves, M. (1981) Ubiquitous cell-surface glycoprotein on tumour cells is proliferation-associated receptor for transferrin. Proc. Natl. Acad. Sci. USA 78:4515. Swain, S.L., Weinberg, A.D., English, M. and Huston, G. (1990) IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145:3706. Sykes, M. (1990) Unusual T cell populations in adult murine bone marrow: prevelance of CD3 + CD4" CD8" and apTCR cells. J. Immunol. 145:3209. Sykes, M., Hoyles, KA., Romick, M.L. and Sachs, D.H. (1990a) In vitro and in vivo analysis of bone marrow derived CD3+ CD4" CD8" NK1.1+ cell lines. Cell Immunol. 129:478. 120 Sykes, M., Romick, M.L., Hoyles, K.A. and Sachs, D.H. (1990b) In vivo ao!ministration of interleukin-2 plus T cell-depleted syngeneic marrow prevents graft-versus-host mortality and permits alloengraftment. J. Exp. Med. 171:645. Sykes, M., Romick, M. and Sachs, D.H. (1990c) Interleukin-2 prevents graft-versus-host disease without diminishing the graft-versus-leukemia effect of allogeneic lymphocytes. Proc. Natl. Acad. Sci. USA 87:5633. Takahama, Y., Kosugi, A. and Singer, A. (1991) Phenotype, ontogeny, and repertoire of CD4" CD8" T cell receptor a p + thymocytes. Variable influence of self-antigen? on T cell receptor Vp usage. J. Immunol. 146:1134. Takahashi, T., Old, L.J. and Boyse, E.A. (1970) Surface alloantigens of plasma cells. J. Exp. Med. 131:1325. Takei, F. (1983) Two surface antigens expressed on proliferating mouse T lymphocytes defined by rat monoclonal antibodies. J. Immunol. 130:2794. Takei, F. (1984) Unique surface phenotype of T cells in lymphoproliferative autoimmune MKLMP-lpr/lpr mice. J. Immunol. 133:1951. Teh, H.S., Kishi, H, Scott, B. and von Boehemer, H. (1989) Detection of autospecific T cells in T cell receptor (TCR) transgenic mice spares cells with normal TCR levels and low levels of CD8 molecules. J. Exp. Med. 169:795. Theofilopoulos, A.N. and Dixon, F.J. (1981) Etiopathogenesis of murine SLE. Immunol. Rev. 55:179. Tiefenthaler, G., Hanke, T. and Hunig, T. (1992) Maturation of CD4"8" a/p TCR+ T cells induced by CD2-stimulation in vivo and in vitro. Int. Immunol. 4:825. Trowbridge, I.S. and Omary, M.B. (1981) Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin. Proc. Natl. Acad. Sci. USA 78:3039. Truneh, A., Albert, F., Golstein, P. and Schmitt-Verhulst, A.M. (1985) Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester. Nature 313:318. Tutt Landolfi, M., Van Houten, N., Russel, J.Q., Scollay, R., Parnes, J.R. and Budd, R.C. (1993) CD2" CD4" CD8" lymph node T lymphocytes in MRL Ipr/lpr mice are derived from a CD2 + CD4 + CD8 + thymic precursor. J. Immunol. 151:1086. Veillette, A., Bookman, M.A., Horak, E.M. and Bolen, J.B. (1988) The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56/c^. Cell 55:301. Vissinga, C.S. Fatur-Saunders, D.J. and Takei, F. (1992) Dual role of IL-7 in the growth and differentiation of immature thymocytes. Exp. Hematol. 20:998. 121 Watanabe-Fukunaga, R., Brannan, C.I., Copeland, N.G., Jenkins, N.A. and Nagata, S. (1992) Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314. Welch, P.A., Namen, A.E., Goodwin, R.G., Armitage, R. and Cooper, M.D. (1989) Human IL-7: a novel T cell growth factor. J. Immunol. 143:3562. Weiss, A., Imboden, J., Schoback, D. and Stobo, J. (1984) Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium. Proc. Natl. Acad. Sci. USA 81:4169. Wilson, A., Ewing, T., Owens, T., Scollay, R. and Shortman, K. (1988) T cell antigen receptor expression by subsets of Ly-2" L3T4" (CD4" CD8") thymocytes. J. Immunol. 140:1470. Wolfsy, D., Hardy, R.R. and Seaman, W.E. (1984) The proliferating cells in autoimmune MRL/lpr mice lack L3T4, an antigen on "helper" T cells that is involved in the response to class II major histocompatibility antigens. J. Immunol. 132:2686. Wu, L., Pearse, M., Egerton, M., Petrie, H. and Scollay, R. (1990) CD4" CD8" thymocytes that express the T cell receptor may have previously expressed CD8. Int. Immuno' 2:51. Yoshimura, A., Shiku, H. and Nakayama, E. (1993) Rejection of an I A + variant line of FBL-3 leukemia by cytotoxic T lymphocytes with CD4 + and CD4" CD8' T cell receptor-ap phenotypes generated in CD8-depleted C57BL/6 mice. J. Immunol. 150:4900. Zlotnik, A. and Bean, A.G.D. (1994) Production of IL-4 by non-Th2 T-cell subsets: possible role of CD4" CD8" a.pTCR+ and CD4 + subset T cells in T helper subset regulation. 53rd Forum in Immunology 606. Zlotnik, A., Godfrey, D.L, Fischer, M. and Suda, T. (1992) Cytokine production by mature and immature CD4" CD8" T cells: ap-T cell receptor+ CD4" CD8" T cells produce IL-4. J. Immunol. 149:1211. 

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