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The role of CD30 in the regulation of T cell function Boyle, Julia Katrina 2003

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THE R O L E OF CD30 IN THE R E G U L A T I O N OF T C E L L FUNCTION by JULIA K A T R I N A B O Y L E B . S c , M c G i l l University, 2000  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Microbiology and Immunology) We accept this thesis as cjurJirming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A August 2003 © Julia Katrina Boyle, 2003  U B C Rare Books and Special Collections - Thesis Authorisation Form  Page 1 of 1  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h , Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d without my w r i t t e n p e r m i s s i o n .  Department The U n i v e r s i t y of B r i t i s h Vancouver, Canada  Columbia  http://www.library.ubc.ca/spcoll/thesauth.html  02/09/03  Abstract  CD30 is a member of the TNFR superfamily that was initially identified on Reed-Sternberg cells of Hodgkin's disease and is widely expressed in other lymphomas as well as in a number of autoimmune diseases. On normal cells, CD30 is expressed primarily on activated C D 8 T cells +  and is induced by two distinct pathways, an H-4 dependent pathway and an IL-4-independent pathway via CD28. The precise role of CD30 has been controversial, but it has been implicated in a number of T cell functions, including costimulation, cytokine production, cell survival and cytotoxicity, although much of the published work to date has been carried out in cell lines. In an attempt to elucidate the role of CD30 in normal T cells, the function of primary lymphocytes derived from CD30-deficient mice was studied. In the absence of CD30, proliferation and activation were normal, with CD30 " cells exhibiting levels of proliferation and expression of _/  activation markers comparable to that of wild type cells. As well, among those cytokines examined, production by activated CD30-deficient cells was normal, although production of IL4 was reduced compared to wild type. 2C-transgenic CD30-deficient cells were unable to kill specific target cells to the same extent as wild type effectors, although expression of effector molecules including perforin, granzyme B and FasL was normal, as was killing of targets when the requirement for TCR recognition was bypassed. Finally, although the reduction of effector function suggested that memory development may also be effected, 2C/CD30"'" effectors were able to develop into memory-like cells to the same extent as wild type cells. Although the deletion of CD30 has little effect on a number of T cell functions, particularly activation and proliferation, it appears that CD30 does play a role in the regulation of later events such as cytotoxic effector function and the maintenance of IL-4 production.  11  Table of Contents Abstract  ii  Table of Contents  iii  List of Figures  •'• iv  Acknowledgements  v  1. Introduction 1.1 TNFR Superfamily 1.2 CD30 1.2.1 Identification and Structure 1.2.2 Expression 1.2.3 Signaling 1.2.4 Functional role 1.2.5 CD30L 1.3 Rationale and Experimental Approach  1 1 2 2 3 4 7 11 11  2. Materials and Methods  13  3. Results 3.1 3.2 3.3 3.4 3.5 3.6 3.7  20 20 27 27 31 31 35  CD30 expression Expression of CD30L Effect of CD30 on cell proliferation Expression of activation markers Induction of cytokine production Cytotoxic effector function Expression of cytotoxic effector molecules  38 40  3.9 Memory induction 4. Conclusion 4.1 Summary of results 4.2 Future work  43 43 46  5. References  49  iii  List of Figures 3-1 Expression of CD30 on C D 8 cells  21  3-2 Expression of CD30 on C D 4 cells  22  +  +  3-3 Expression of CD30 on C D 8 Stat6 cells  23  3-4 Expression of CD30 on C D 4 Stat6 cells  24  +  A  +  A  3-5 Expression of CD30 on 2C, 2C/Stat4' and 2C/Stat6- " transgenic cells  26  3-6 Expression of CD30L on C D 4 cells  28  3-7 Expression of CD30L on C D 8 cells  29  3-8 Proliferation of B6 and CD30 - cells  30  A  /  +  +  7  3-9 Expression of activation markers on B6 and CD30" cells  32  3-10 Expression of activation markers on 2C and 2C/CD30" " transgenic cells  33  3-11 Cytokine production by 2C and 2C/CD30 cells  34  3-12 Cytotoxic effector function of 2C and 2C/CD30 " cells  37  A  7  A  7  3-13 Expression of cytotoxic effector molecules by 2C and 2C/CD30 " cells /  39  3-14 Recovery of memory-like cells from T C R a ' " mice injected with activated 2C or 2C/CD30' " /  cells  41  3-15 Expression of memory markers on 2C and 2C/CD30~'~ cells  42  iv  Acknowledgements (  I would like to thank my supervisor, Dr. Hung-Sia Teh for his help and advice throughout my time in graduate studies. This work was supported by a grant from the National Cancer Institute of Canada to Dr. Teh. As well, thank you to Soo Jeet Teh and Becky Dinesin for expert technical assistance and other members of the Teh lab past and present: Oliver Utting, Ed Kim, Darryl Oble, Salim Dhanji, Kasra Mohseni and John Priatel for your friendship, support and assistance. Finally, thank you to my friends and family for your unwavering support.  v  1. Introduction 1.1 T N F R Superfamily The TNFR superfamily is a large and diverse group of receptors that plays a critical role in T cell function. Members of the TNFR superfamily such as TNFR1 (p55), TNFR2 (p75), Fas, CD40, R A N K , OX40, and 4-IBB, have been implicated in the regulation of numerous T cell functions, and are of particular importance in costimulation and regulation of cell survival. The TNFR superfamily can be broken into two main groups: those that contain an intracellular death domain (DD), including TNFR1 and Fas; and those that do not, including TNFR2, CD30, CD40, R A N K , OX40 and 4-IBB. Structurally, all TNFR superfamily proteins are type I transmembrane proteins that associate as trimers. The extracellular domain contains conserved cysteine-rich domains, while the intracellular domain shares little homology apart from the death domain and TRAF binding sites. (Locksley et al., 2001) TNFRs do not possess intrinsic catalytic activity, thus necessitating the use of adaptor proteins for signaling upon ligand engagement. DD-containing members of the TNFR superfamily can associate with adaptors that contain death domains, such as F A D D (Fasassociated D D protein) and T R A D D (TNFR-associated D D protein), leading to the recruitment and activation of caspases and ultimately cell death. Alternatively, all TNFRs, including those that possess death domains, can signal through TRAFs (TNF receptor associated factor). TNFR superfamily members without a death domain interact directly with TRAFs via conserved binding sites, while death domain-containing receptors recruit TRAFs indirectly through TRADD. The six mammalian TRAFs are adaptor proteins that, as their name implies, associate with TNFRs to mediate signaling, leading to activation of transcription factors and generally  1  increasing cell survival, (reviewed in Chung et al., 2002) The two main transcription factors activated by TRAF signaling are N F - K B and AP-1, both of which regulate numerous genes to promote the immune response and cell survival. O f the TRAFs, TRAF2 is the best characterised and the most prevalent, with transcripts detected in almost every tissue. (Rothe et al., 1994) TRAF2 activates both N F - K B and J N K (c-Jun N-terminal kinase), but is only required for I N K activation. (Lee et al., 1997; Yeh et al., 1997; Song et al., 1997) Cell survival and proliferation are both positively regulated by TRAF2, with susceptibility to TNF-induced cell death increased in the absence of TRAF2. As well, TRAF2" " mice are severely runted and lymphopenic, and die /  prematurely, with very few surviving past 3 weeks. (Yeh et al., 1997) 1.2 CD30 1.2.1 Identification and Structure  CD30 was initially identified as a marker for Reed-Sternberg cells in Hodgkin's lymphoma. (Schwab et al., 1982) Subsequently, it has been found to be expressed on normal activated T and B cells, E B V and HTLV-infected T cells, as well as in numerous non-Hodgkin's lymphomas. (Stein et al., 1985) Structurally, CD30 is a type I transmembrane protein that is heavily glycosylated, increasing the putative 64 kDa protein to 120 kDa for human CD30, while murine CD30 cDNA encodes a putative 52 kDa protein that is increased to 110 kDa by glycosylation. The extracellular domain of human CD30 contains six cysteine-rich motifs that are arranged in two cysteine-rich domains. Murine CD30 lacks one of the cysteine-rich motifs and thus only has one complete cysteine-rich domain and one partial cysteine-rich domain. Both murine and human CD30 have long intracellular tails that share little homology with other members of the TNFR superfamily, apart from the sequences for TRAF binding, as will be discussed later. The  2  intracellular domain contains numerous potential N-glycosylation, protein kinase C and casein kinase II phosphorylation sites. Overall, human and murine CD30 are 59% identical and 67% similar. (Bowen et al., 1996; Chiarle et al., 1999a) The extracellular domain of CD30 can be cleaved from the cell surface by TNFctconverting enzyme (TACE). T A C E is a membrane-anchored metalloproteinase-disintegrin that was initially identified as mediating the release of the membrane-bound pro-form of T N F a from the cell surface. Cleavage of CD30 results in the release of the extracellular domain as a 90 kDa soluble form. (Hansen et al., 2000) This soluble form of CD30 is capable of binding to CD30L with high affinity and thus blocking the biological effects of CD30-CD30L interactions, although a 20-fold excess of sCD30 is required to completely block the effects of these interactions. (Hargreaves and Al-Shamkhani, 2002) While the phenomenon of CD30 shedding was identified in cell lines, high levels of sCD30 have been found in the serum of patients with rheumatoid arthritis and other autoimmune disorders, as well as patients infected with HIV. High levels of serum sCD30 tend to be associated with a poor prognosis in patients with HTV. (Pizzolo etal., 1994) 1.2.2 Expression CD30 was initially identified on Reed-Steraberg cells of Hodgkin's lymphoma, as discussed above. It is also expressed on cells in a number of other lymphomas, including anaplastic large cell lymphoma, adult T-cell lymphoma, and some cells in Burkitt's lymphoma, among others. Expression has also been detected in some cases of leukemia caused by HTLV-land in patients infected with a number of viruses, particularly HIV, E B V , HTLV-1, and hepatitis B. Under normal conditions, CD30 is expressed primarily on activated T cells, although some expression  3  can also be detected on B and y5 T cells. In vitro, expression is primarily on C D 8 T cells, with +  only a subset of CD4 cells showing CD30 expression. In both C D 4 and C D 8 cells, CD30 +  +  +  levels peak around 72 hours after stimulation. (Telford et al., 1997) Expression of CD30 is enhanced by IL-4 (Gilfillan et al., 1998) and repressed by JFNy. (Annunziato et al., 1997) As a result, CD30 was thought for a time to be a marker for Th2 cells. (Del Prete et al., 1995a) (Del Prete et al., 1995b) However, CD30 expression is not restricted to cells that are polarized towards a type 2 phenotype. In fact, CD30 expression can be induced in an IL-4-independent manner by means of CD28 crosslinking. (Gilfillan et al., 1998) There have been contradictory reports, using different systems, as to whether CD30 engagement enhances (Rossi et al., 2001) or represses (Bengtsson et al., 2000) the level of expression of CD30. 1.23 Signaling As with other members of the TNFR superfamily, the cytoplasmic domain of CD30 does not possess any intrinsic catalytic activity. Signaling events via CD30 thus require adaptor molecules that can associate with the cytoplasmic tail of the protein. Studies using the yeast two-hybrid system found that CD30 interacts with adaptor proteins from the TRAF family, specifically TRAF1,2, 3, and 5. (Aizawa et al., 1997; Gedrich et al., 1996) CD30 contains two elements at the carboxy-terminal of its cytoplasmic tail that mediate binding to TRAFs. The first, termed D l , has the sequence PEQET and was identified by homology to known TRAF-binding domains of CD40. Deletion or substitution of individual amino acids within the PEQET sequence abolishes binding of TRAF3 but not TRAFI or 2. The second TRAF binding domain, termed D2, was also identified by homology to CD40, and has a sequence of E E E G K E . Deletion of D2 abolishes binding of TRAF2 and 5, while deletion of both sequences completely abolishes all TRAF  4  binding. (Aizawa et al., 1997; Gedrich et al., 1996; Horie et a l , 1998; Lee et al., 1996) Additionally, one report indicates that a 19-amino acid sequence within the cytoplasmic tail of CD30 appears to mediate N F - K B activation independently of TRAF binding, but the effector or effectors involved have not been identified. (Horie et al., 1998) Downstream targets Numerous studies in cell lines have indicated that CD30 signals lead to the activation of N F - K B . A constitutively active construct consisting of a chimera of the extracellular domain of CD8 and the cytoplasmic tail of CD30 was found to lead to a large increase in N F - K B activity over basal levels as measured by both transcription of a luciferase reporter gene construct as well as nuclear translocation of N F - K B proteins. (Lee et al., 1996) Overexpression of CD30 in a cell line was also found to increase N F - K B activity. This response could be inhibited by co-expression of dominant negative forms of TRAF2 and 5. (Aizawa et al., 1997) Until recently, no work had been carried out to elucidate the signaling pathways used by CD30 in normal T cells. However, one report indicated that CD30 crosslinking in normal murine T cells also leads to activation of N F - K B , as measured by nuclear translocation of the protein. Nuclear N F - K B could be detected as early as 30 minutes after stimulation, with levels peaking at 1 hour. Interestingly, co-ligation of TCR and CD30 appeared to delay the kinetics of N F - K B activation. The same report also examined the activation of M A P K s in response to CD30 crosslinking. Both p38 and JNK were phosphorylated following ligation of CD30. This activation occurred independently of TCR co-ligation and could be prevented by the expression of a dominant negative form of TRAF2. (Harlin et al., 2002)  5  Inhibition of signaling Two main mechanisms for downregulating signals from CD30 have been identified. The first, TRIP, was initially isolated as a protein that interacts with TRAF1 and 2. Coprecipitation studies in transfected cell lines showed that TRIP can be found in a complex containing TRAFs and either CD30 or TNFR2. In mice, TRIP is expressed in a number of tissues, including testes, thymus and spleen, and its expression in lymphocytes is downregulated upon activation and entry into cell cycle. Functional assays in transfected cells indicated that TRIP significantly reduced CD30-mediated activation of N F - K B . It has been suggested that the actions of TRIP can explain the divergent effects of CD30, as well as other members of the TNFR superfamily, where the same receptor can lead to either cell survival and proliferation or to cell death, depending on circumstances. (Lee and Choi, 1997) Another mechanism by which CD30 signaling is inhibited is by the depletion of cytoplasmic TRAFs. It has been reported that in cell lines expressing a constitutively active construct of CD30, levels of TRAF 1 and 2 in the cytoplasm are drastically reduced. It was suggested that this reduction was due to the degradation of TRAF proteins, as evidenced by the fact that certain protease inhibitors could block the reduction in TRAF2 levels. Interestingly, while CD30 can interact with TRAF1,2, 3, and 5, only TRAF2 is substantially depleted under the conditions used in the study. (Duckett and Thompson, 1997) However, subsequent studies showed that some of the TRAF proteins were also sequestered in an insoluble fraction that was also found to contain components of the nuclear matrix. (Arch et al., 2000) Cytoplasmic levels of TRAF2 are also reduced in response to antibody crosslinking of endogenously expressed CD30 in certain cell lines, suggesting that TRAF depletion is not simply an artifact of  6  constitutive activation of CD30. (Mir et al., 2000) It is unknown whether this phenomenon also occurs in response to prolonged CD30 signaling in primary lymphocytes. 1.2.4 Functional role Much of the early work in determining the function of CD30 was carried out in cell lines derived from lymphomas in which CD30 is expressed. This work has yielded many contradictory results. In some cell lines, CD30 engagement results in increased proliferation and cell survival, while in others it leads to cell death. Largely, the results can be broken down as CD30 signals in Hodgkin's disease (HD)-derived cell lines encourage proliferation and increased N F - K B activation. By contrast, CD30 crosslinking in anaplastic large cell lymphoma (ALCL)-derived cell lines tends to lead to reduced proliferation, increased apoptosis and no N F - K B activation. Treatment of HD-derived cell lines with cycloheximide to prevent de novo protein synthesis sensitizes these cell to CD30-induced cell death. (Mir et al., 2000; Gruss et al., 1994; Hsu and Hsu, 2000) The reduction in proliferation in response to CD30 in ALCL-derived cell lines has been shown to be due in part to the upregulation of p 2 1  CIP1/WAF1  , an inhibitor of cell cycle  progression. (Hubinger et al., 2001) In one of the most comprehensive studies on the effects of CD30 on a CD30 tumour cell +  line, antibody crosslinking of CD30 on the cytotoxic large granular lymphoma cell line Y T was shown to lead to the rapid and complete downregulation of surface expression of FasL. Additionally, transcription of granzyme B and perforin mRNA is diminished, but not completely eliminated. As granzyme B and perforin proteins are stored in intracellular granules, this decrease in transcription would not likely have as immediate an effect as the downregulation of FasL. The functional effect of this downregulation of cytotoxic effector molecules is that killing 7  of Fas-expressing target cells is almost completely abolished. In addition, the cells upregulated a number of molecules that would increase susceptibility to apoptosis, including Fas, DR3, and TRAIL, as well as downregulating expression of c-Myc. (Muta et al., 2000) The remainder of this discussion of the function of CD30 will deal primarily with its role in primary T cells. Costimulation While the expression pattern of CD30 precludes it from playing a role in early activation events, evidence suggests that it does play a role in costimulation of T cells. In secondary proliferative responses, in which murine T cells are restimulated after initial activation and a rest period, CD30 crosslinking in conjunction with TCR signaling has a costimulatory effect comparable to that of CD28 activation. This effect is particularly striking under conditions of suboptimal TCR signaling. (Gilfillan et al., 1998) A similar effect can be seen when CD30L-transfected cells are used to stimulate lymphocytes. (Smith et al., 1993) More recently, it has been shown that the majority of dividing human T cells in a mixed-lymphocyte reaction are CD30 , while the non+  dividing population remains CD30". (Chan et al., 2002) However, this study did not distinguish whether CD30 plays a role in promoting increased proliferation, or whether it is simply a marker that is upregulated following cell division. Cytokine production One important function of T cells is the production of cytokines. CD30 has been implicated in regulating the production of a number of cytokines by both C D 4 and y5 T cells. In C D 4 cells, +  +  crosslinking of CD30 in conjunction with TCR engagement has been shown to increase production of IL-4, IL-5, H-10 and IFNy- Furthermore, CD30 signals are capable of leading to 8  increased expression of IL-13 independently of TCR signals. (Harlin et al., 2002) While CD30 engagement on unpolarized cells leads to upregulation of both type 1 and type 2 cytokines, CD30 does not reverse polarization of T cell clones. Namely, CD30 crosslinking upregulates IFNy expression by T h l clones but not Th2 clones. Conversely, production of IL-4 and IL-5 by Th2 clones is upregulated by CD30 signals, while production of these cytokines by T h l clones remains minimal. (Bengtsson et al., 2000) The regulation of cytokine production by CD30 in yb T cells is similar to that seen in C D 4 cells, with co-engagement of T C R and CD30 leading to +  upregulation of LL-4, TPNy and LL-8. (Biswas et al., 2000) CD30 has also been shown to play a role in regulating in vivo cytokine production. In a model of type 1 cytokine-mediated pulmonary inflammation, blockage of CD30-CD30L interactions reduces production of I F N Y  a r ,  d IL-12 by both C D 4 and C D 8 cells, as well as +  +  reducing the size of pulmonary granulomas. (Saraiva et al., 2002) Cell survival Despite lacking a death domain in its cytoplasmic tail, CD30 appears to play a role in increasing the susceptibility of cells to apoptosis. This increase in cell death is not a direct effect of CD30 signals, but is rather due to an increased sensitivity to apoptotic signals via TNFR1. CD30 signals alone have no effect on cell viability, but require the addition of TNFcc to induce cell death. As well, the increase in apoptosis in response to CD30 and T N F a can be negated by the addition of neutralizing antibodies against either T N F a or TNFR1. (Arch et al., 2000; Duckett and Thompson, 1997; Grell et al., 1999) This effect appears to be mediated via the depletion of TRAFs, as cells with low cytoplasmic levels of TRAF2 show higher susceptibility to TNFa-  induced apoptosis. As well, cells transfected with CD30 constructs lacking the TRAF binding sites show much greater resistance to cell death. (Duckett and Thompson, 1997) CD30 also appears to play a role in regulating survival of normal murine T cells in a model of antigen-withdrawal cell death (AWCD). In this system, lymphocytes are cultured with T C R crosslinking and exogenous IL-2 for 2 days, then removed from stimulation and cultured for 4 days more with exogenous IL-2 but no mitogenic stimuli. Blockage of CD30-CD30L interactions drastically reduced the numbers of apoptotic cells in this system. (Telford et a l , 1997) CD30 was thought at one time to play a role in regulating negative selection in the thymus. One paper looked at CD30" " mice on the H - Y transgenic background. H - Y transgenic 7  mice express a TCR specific for a male antigen, thus leading to negative thymic selection in male mice, while female mice undergo normal positive selection. It was reported that H - Y CD30"'" male mice had a defect in negative selection, with a 20-fold increase in double positive thymocytes compared to wild type. (Amakawa et al., 1996) Also, transgenic mice that overexpressed CD30 in the thymus exhibited enhanced negative selection. (Chiarle et al., 1999b) However, it has been shown, both by others and in our lab, that thymic selection in CD30 " mice 7  is normal, with no differences between knockout and wild type mice. (DeYoung et al., 2000) While CD30 mRNA transcripts can be detected in thymocytes, cell surface expression of the protein in the thymus is virtually non-existent. (Bowen et al., 1996; Chiarle et al., 1999b) Given that expression of CD30 is absent on normal thymocytes, it is unlikely to play a role in negative selection, and the enhanced negative selection observed in CD30-transgenic mice is most likely an artifact of the overexpression of the molecule. 10  1.2.5 CD30L CD30L (CD 153) is a member of the TNF superfamily with significant homology to TNFa, TNFp, and CD40L. Structurally, CD30L is a heavily glycosylated type II transmembrane protein with a molecular mass of 40 kDa. CD30L is expressed on a number of cell types, including B cells, neutrophils, eosinophils, and activated T cells, with expression of T cells found primarily on C D 4 cells. (Smith et al., 1993; Shimozato et al., 1999) +  CD30L has a 40 amino acid cytoplasmic tail that appears to be able to transduce signals. Crosslinking of CD30L on neutrophils can induce production of IL-8 as well as a rapid oxidative burst. On T cells, CD30L crosslinking can costimulate proliferation to a certain extent, as well as inducing production of IL-6. (Wiley et al., 1996) Additionally, engagement of CD30L on B cells can inhibit signaling via CD40, downregulate expression of CD40L and inhibit antibody class switching from IgD to IgG. However, in leukemic B cells, CD30L signals increase proliferation and cell survival. (Cerutti et al., 2001) 1.3 Rationale and Experimental Approach Although many studies have already been carried out in an attempt to characterize the function of CD30 in regulating T cell responses, many questions and ambiguities remain. Certain aspects of the functional role played by CD30 have been relatively well defined, in particular the signaling pathways used by CD30, however much of the work has been done in cell lines. While such studies are valuable, it remains to be seen how much of this data is applicable to primary lymphocytes. The published literature to date is also somewhat contradictory at times, and while increasing evidence indicates a stimulatory role for CD30 on normal T cells, as yet no truly coherent model has emerged to describe what role CD30 may play in regulating the immune 11  response. As such, the aim of this research project was to assess the role played by CD30 in a number of aspects of T cell function, in particular activation, proliferation, effector functions and development of T cell memory. Mice deficient in CD30 (CD30 ) were used to study the function of the molecule. 7  Lymphocytes from these mice were isolated and cultured in vitro for use in functional assays where they could be compared to wild type lymphocytes, with emphasis placed on carrying out the assays under conditions where wild type cells would express CD30. CD30" mice are A  developmentally normal and display no overt phenotype, so peripheral lymphocytes from these mice can be meaningfully compared to wild type cells. For some experiments, the 2C transgenic system was used. The 2C T C R is an M H C class I-restricted TCR that recognises a mitochondrial peptide (p2Ca) presented on L . (Udaka et al., 1993) d  12  2. Materials and Methods 2.1 Mice  Breeders for C57BL/6 (B6, H-2 ) mice were obtained from The Jackson Laboratory (Bar b  Harbor, ME). Breeders for the H-2 2C TCR-transgenic mice were provided by Dr. Denis Loh b  (then at the University of Washington, St. Louis, MO). The H-2 2C TCR-transgenic mice have b  r  been backcrossed for more than ten generations to the C57BL/6 background. BDF1 mice are a first generation cross of B6 with DBA/2 (H-2 ) mice. Breeders for B6-CD30" mice were d  A  provided by Dr. Tak Mak (Ontario Cancer Institute). Breeders for B6-Stat4" " and B6-Stat6~'~ /  mice were provided by Dr. Mike Grusby (Harvard University) and B6-TCRa~~ mice were obtained from the Jackson Laboratory. 2.2 Antibodies and flow cytometry  The following antibodies (Abs) were used: FITC-conjugated mAbs to mouse CD8 (53.67), 2C TCR (1B2), LL-2 (S4B6), and LL-4 (11B11); PE-conjugated mAbs to CD4 (GK1.5), CD8 (53.67) CD30L (RM153), CD25 (PC61), CD44 (Pgp-1), IL-2Rp (TM-|31), CD69 (HL.2F3), CD62L (MEL 14), 1B11, I F N Y (XMG1.2), T N F a and granzyme B (GB12); Tricolor (PE-Cy5)conjugated CD8 (53.67); and biotin-conjugated mAbs to CD30 (mCD30.1), FasL (MFL3), and Fas (Jo2); all antibodies are from from B D PharMingen (San Diego, CA), supplied by Cedarlane Laboratories, Hornby, Canada except for those against CD25, CD69, CD44, and CD62L, supplied by eBioscience, and anti-granzyme B , supplied by Caltag. Cell staining and flow cytometric analysis were performed according to standard procedures. Briefly, cells were incubated with the relevant Abs for at least 15 min at 4°C and subsequently washed twice with FACS medium (PBS and 2% FCS). Cells were then incubated with streptavidin-conjugated  13  Tricolor (PE-Cy5) for at least 15 min. at 4°C in order to detect biotinylated Abs, and also washed twice with FACS medium. Samples were run on a FACScan flow cytometer. The CellQuest software program (BD Biosciences, Mountain View, C A ) was used for data acquisition and analysis. 2.3 Cells and cell culture conditions  Lymph nodes were harvested, and single-cell suspensions were prepared from each of the mouse lines. Single cell suspensions were prepared by passing lymph nodes through a sieve in RPMI 1640 supplemented with with 2% FCS (Life Technologies). For studies of C D 4 C D 8 (CD8) T +  cell subsets, the population was purified from whole lymph node cell suspensions using miniMACS microbeads (Miltenyi Biotec, Auburn, C A ) and mouse biotin-conjugated CD8P (53.58) mAb . The T cell subset was positively selected using a M A C S M S separation column +  and miniMACS magnet according to the manufacturer's protocol (Miltenyi Biotec), achieving >95% purity. For studies involving a mixed population of CD4 CD8" and CD4"CD8 cells, Ig +  +  +  cells were depleted using anti-mouse Ig-coated Dynabeads (Dynal, Oslo, Norway) Cells were cultured at 37°C in 5% C 0 in I M E M (Life Technologies, Burlington, Canada) supplemented 2  with 10% F C S , 5 x 10" u M 2-mercapto-ethanol, and antibiotics (I-medium). 5  Where irradiated splenocytes were used as stimulators, spleens were harvested from BDF1 mice and a single cell suspension generated by passing the spleen through a sieve. Red blood cells were lysed with a solution of Tris-buffered 0.83% NH C1 in PBS on ice for 5 minutes, then 4  2  washed and resuspended in I medium. For irradiation, cells were suspended in cold PBS and placed on ice. Cells were subjected to 2000 Rads in a gamma-cell. To isolate lymphocytes from non-lymphoid organs, mice were sacrificed by C 0 asphyxiation. 2  14  While the heart was still beating but reflexes absent, the sternum was cut and a 26G needle connected to a perfusion apparatus consisting of a 25cc syringe on a stand was inserted into the left ventricle and the right atrium was cut to allow blood to drain out. Perfusion was carried out with 50 U/ml heparin in PBS until organs turned white. Single cell suspension of liver and kidney were prepared by means of chopping them and then passing through a sieve in 20 ml RPMI. The cell suspension was collected in a 50 ml corneal tube and centrifuged at 2000 R P M for 15 minutes. The supernatant was removed by suction and the pellet was resuspended in 15 ml of 35% Percoll (Amersham) in PBS and vortexed to distribute the cell suspension evenly. The cell suspension was then centrifuged at 2000 R P M at room temperature for 20 minutes. At the end of the run, the centrifuge was allowed to stop without using the brakes, and the supernatant was removed by suction, leaving the pellet containing lymphocytes and red blood cells. Red blood cells were lysed and the cell preparation was washed twice in RPMI + 2% FCS before being resuspended in I medium + 10% FCS. 2.4 CD30 expression 2.4.1 Non-transgenic CD4 and CD8 lymphocytes For studies of CD30 and CD30L expression, mixed cultures depleted of Ig cells were stimulated +  with 10 ug/ml plate-bound anti-CD3e (2C11) in a 24-well flat-bottom plate, with an initial cell concentration of l x l O cells/well. Cultures were supplemented with either exogenous LL-2 (20 6  U/ml) or exogenous H-2 (20 U/ml) plus IL-4 (300 U/ml). Soluble anti-CD28 (37.51) mAb was included at a concentration of 10 ug/ml where indicated. Cells were harvested at 24,48,72 and 96 hours of culture and stained with antibodies against CD4, CD8 and either CD30 or CD30L, then analysed by FACS as described in section 2.2. 15  2.4.2 2C transgenic system For studies of CD30 expression in the 2C system, l x l O purified C D 8 cells were stimulated 6  +  with l x l O irradiated BDF1 splenocytes in a 24-well flat-bottom plate for 72 hours. Where 7  indicated, exogenous IL-4 was added to cultures at a concentration of 300 U/ml. Cells were stained with antibodies against 1B2, CD8 and CD30, and expression levels of CD30 were determined by FACS analysis as described in section 2.2. 2.5 Proliferation assays and cell surface marker expression  2.5.1 B6 and CD30~'~ mixed lymphocytes Proliferation assays were performed by incubating 3 x 1 0 - l x l 0 cells with plate-bound anti4  5  CD3s (2C11). Cells were cultured in triplicate in a volume of 0.2 ml in flat-bottom 96-well plates, and 1 uCi [ H]thymidine was added for the last 8 h of a 72-h culture period. In some 3  cultures exogenous fL-2 was added at a concentration of 20 U/ml. To assay cell surface marker expression, 1 x 10 cells were incubated in flat-bottom 24-well plates coated with 10 ug/ml2Cl 1 6  for 72 hours, in the presence of either exogenous IL-2 at a concentration of 20 U/ml or both IL-2 at a concentration of 20 U/ml plus IL-4 at a concentration of 300 U/ml. Cultures were split and fed fresh medium and cytokines as needed. The expression levels of CD25, CD44, and 1B11 were analysed by FACS as described in section 2.2. 2.5.2 2C and 2C/CD3(T - CD8 T cells /  For proliferation assays using 2C cells, l x l O C D 8 cells were cultured with 3 x l 0 mitomycinC4  +  4  inactivated T2-L cells and p2Ca peptide at concentrations ranging from 0-10 uM. Cells were d  cultured in triplicate in a volume of 0.2 ml in round-bottom 96-well plates, and 1 u C i  16  [ H]thymidine was added for the last 8 h of a 72-h culture period. To assay for cell surface 3  marker expression, l x l O 2C C D 8 cells were cultured with l x l 0 irradiated BDF1 splenocytes 6  +  7  in a 24-well flat-bottom plate. Cultures were split and fed with fresh medium as needed. Expression of the cell surface markers CD25, CD44, CD69 and 1B11 was analysed by FACS at 72 hours of culture. 2.6 Intracellular cytokine staining  2C C D 8 cells were cultured as described above. Golgi Stop (BD Pharmingen) was added to the +  cultures for the final 6 hours to prevent secretion of cytokines and enhance staining. For intracellular staining, cells were washed, then fixed and permeablized in 4% paraformaldahyde/0.4% Tween20 in PBS on ice for 30 minutes. The cells were washed, then incubated with the appropriate antibodies in 0.2% Tween20 in PBS on ice for 30 minutes. After washing off excess antibody, the samples were resuspended in FACS medium and analysed on a FACScan flow cytometer using CellQuest software. 2.7 C T L Assay  Cytotoxic effector cells were generated by culturing l x l O 2C or 2C-CD30 ~ lymphocytes with 6  /  l x l O irradiated (2000 Rads) BDF1 spleen cells for 3 days in a 24 well plate (2 ml total volume). 7  The cell lines A20, P815 and PJVIA/S were cultured and used as target cells. On day 3, l x l O  6  target cells were labelled with 0.1 uCi Cr. Cytotoxicity was assessed by incubating labelled 51  target cells with effector cells in a 96-well microtiter plate at the indicated effector to target ratios for 4 or 18 hours. Culture supernatants were harvested and chromium release was determined using a gamma counter. Percent specific lysis was calculated by (Sample cpm spontaneous cpm)/(Maximal cpm - spontaneous cpm) x 100%. 17  2.8 Expression of cytotoxic effector molecules  To examine the role of CD30 crosslinking on FasL expression, B6 and CD30 " C D 8 cells were _/  +  activated with plate-bound 2C11 and exogenous cytokines (20 U/ml IL-2 alone or 20 U/ml IL-2 plus 300 U/ml JX-4) for 64 hours. For the final 8 hours of culture, the cells were restimulated with 10 ug/ml plate-bound anti-CD30. Expression of FasL was assessed by FACS as described in section 2.2. 2C cells were activated as described for the C T L assay. Cell surface levels of FasL were assessed by F A C S as described above. Intracellular FACS staining, as described for analysis of cytokine production, was used to look at expression of granzyme B. Cell lysates containing l x l O cells were generated to examine perforin expression. Lysates from unactivated cells were 6  generated for use as a negative control. Lysis buffer: 1% Triton-X-100 lxTNE,pH7.60 10 ug/ml aprotinin 10 ug/ml leupeptin 2 m M PMSF 1 m M sodium orthovanadate 1 m M sodium molybdate Cells were washed with PBS and the supernatant removed. Lysis buffer was added to the cells and the samples were incubated on ice for 10 minutes. The tubes were centrifuged for 10 minutes at 14 000 R P M (10 000 x g) to pellet the cell membranes and nuclei. The supernatants, containing the cell proteins, were transferred to new tubes, SDS sample buffer was added, and the lysates were frozen at -20°C for later use.  18  2.9 Western blotting  Lysates were boiled for 5 minutes, then loaded and run on a 10% SDS-polyacrylamide gel at 110 V until the dye front ran off of the gel. Proteins were transferred to an irnmobilon-polyvinylidene fluoride membrane (Millipore) at 105 V for 1.3 hours. After the transfer was complete, the membrane was blocked with 5% B S A in TBS-T overnight. To look at perforin levels, the membrane was probed with anti-perforin antibody (clone PI-8, Kamiya Biomedical, Seattle, WA) (Kawasaki et al., 1990) The antibody was used at a concentration of 1 jig/ml in TBS-T with 0.5% BSA, and was incubated with the membrane for 3 hours. ProteinG-HRPO was used as a secondary to detect the perforin antibody. 2.10 Memory induction  Unpurified 2C and 2C/CD30"'" lymphocytes were activated with irradiated BDF1 splenocytes for 3 days. At this time, the viable cell population was determined to be over 90% 1B2 CD8 by +  +  FACS. The activated cells were resuspended in PBS, and 5 x l 0 viable 1B2 CD8 cells were 6  +  +  injected into each T C R a mouse. Mice were sacrificed at 2 and 4 weeks post-injection, and /_  lymphocytes were harvested from lymph nodes, spleen, liver and kidneys as described above. To isolate cells from bone marrow, the long bones of the leg (femur and tibia) were flushed with PBS and the wash material collected. The cells were then resuspended in I medium. The percentage of 1B2 CD8 cells recovered, as well as the expression of selected memory markers +  +  (CD44, CD62L, IL-2Rf3) on those cells was assessed by FACS as described in section 2.2.  19  3. Results 3.1 CD30 expression  Due to a large number of contradictory reports in the published literature regarding the expression pattern of CD30, it was necessary to independently determine the conditions necessary for the induction of CD30 expression, as well as the time at which expression levels peak prior to proceeding with any functional assays. When cells were stimulated by TCR crosslinking, CD30 was found to be predominantly expressed on C D 8 T cells, with some +  expression on C D 4 cells. This expression peaks at 72-96 hours of culture, with little detectable +  expression before 48 hours of culture. This is in accordance with previously published work. (Telford et al., 1997) Given the apparent correlation between CD30 expression and H-4 in the published literature, I then went on to investigate the cytokine requirements for induction of CD30. In mixed cultures (containing both C D 4 and C D 8 cells) that were stimulated with anti+  +  CD3 mAb (2C11), IL-2 alone was sufficient to induce CD30 expression at a high level on C D 8  +  cells (Fig. 3-1), while inducing lower levels of CD30 expression on C D 4 cells. The presence of +  both exogenous IL-2 and IL-4 enhanced CD30 expression levels on both C D 4 and C D 8 cells. +  +  However, in cultures containing only purified C D 8 cells, the addition of both IL-2 and IL-4 is +  required for CD30 expression. The reasons behind this difference remain unclear, but it could be as simple as production of IL-4 by C D 4 cells in a mixed culture. Another possibility is related +  to the preferential expression of CD30 ligand on C D 4 cells, as discussed below. It has been +  demonstrated that cells expressing low levels of CD30 can be induced to upregulate expression upon co-culture with CD30L-expressing cells. (Rossi et al., 2001) Costimulation via the addition  20  Figure 3-1 Expression of CD30 on C D 8 B6 cells. B6 and C D 3 0 c e l l s were stimulated with 2C11 and exogenous IL-2 ± IL-4 and stained for CD30 expression at 72 and 96 hours of culture. Soluble anti-CD28 was also added to some cultures. Results shown are gated on CD4"CD8 cells. Filled purple histogram: No anti-CD28. Bold green histogram: + anti-CD28. Dashed pink histogram: CD30"" cells. +  +  21  Figure 3-2 Expression of CD30 on C D 4 B6 cells. B6 and C D 3 0 c e l l s were stimulated with 2C11 and exogenous LL-2 ± IL-4 and stained for CD30 expression at 72 and 96 hours of culture. Soluble anti-CD28 was also added to some cultures. Results shown are gated on CD4 CD8" cells. Filled purple histogram: No anti-CD28. Bold green histogram: + anti-CD28. Dashed pink histogram: CD30"" cells. +  +  22  72 hours  96  tours  Figure 3-3 Expression of CD30 on C D 8 Stat6' cells. CD30 and Stat6 cells were stimulated with 2C11 and exogenous IL-2 ± IL-4 and stained for CD30 expression at 72 and 96 hours of culture. Soluble anti-CD28 was also added to some cultures. Results shown are gated on CD4" C D 8 cells. Filled purple histogram: No anti-CD28. Bold green histogram: + anti-CD28. Dashed pink histogram: CD30"" cells. +  7  +  23  72 hours  96 hours  Figure 3-4 Expression of CD30 on C D 4 Stat6 cells. CD30 and Stat6 cells were stimulated with 2C11 and exogenous IL-2 ± IL-4 and stained for CD30 expression at 72 and 96 hours of culture. Soluble anti-CD28 was also added to some cultures. Results shown are gated on CD4CD8" cells. Filled purple histogram: No anti-CD28. Bold green histogram: + anti-CD28. Dashed pink histogram: CD30" ~ cells. +  A  24  of soluble anti-CD28 to cultures did not increase CD30 expression levels on C D 8 T cells, +  indicating that perhaps the level of expression obtained by TCR crosslinking and the addition of cytokines is the maximal level of expression, but did sustain CD30 expression for a longer period in the absence of exogenous IL-4. (Fig. 3-1, upper panel) On C D 4 cells, soluble anti+  CD28 in conjunction with TCR crosslinking had the effect of increasing CD30 expression levels in the presence of either IL-2 alone or LL-2 and IL-4, and also appeared to sustain higher levels of CD30 at 96 hours of culture. (Fig. 3-2) To evaluate the role of IL-4 in the induction of CD30 expression, Stat6 " lymphocytes v  were used. Stat6 is a signal transduction molecule involved in signaling from the IL-4 receptor; thus, in its absence, cells cannot respond to EL-4. (Kaplan et al., 1996) CD30 expression could not be induced on Stat6''' cells stimulated by means of T C R crosslinking alone, with the addition of exogenous cytokines to the culture (LL-2 and IL-4) having no effect. However, the addition of soluble anti-CD28 to cultures was able to induce expression of CD30 on both C D 8 (Fig. 3-3) +  and, at lower levels, on CD4 cells. (Fig. 3-4) This indicates that two pathways exist for the +  induction of CD30 expression, one LL-4 dependent and one IL-4 independent but dependent on CD28 stimulation. The expression of CD30 on 2C transgenic cells was also examined. In this system, cells are activated using APCs expressing the specific peptide recognized by the 2C TCR, thus providing a more physiological model of activation compared to cells that are activated with plate-bound anti-TCR antibody. Expression levels of CD30 were evaluated on wild type 2C, 2C/Stat4 " and 2C/Stat6~~ cells. Stat4 is involved in signaling via the IL-12 receptor (Cho et al., /  /  1996) and the 2C/Stat4" " was used as a control for cells that could respond to LL-4 but lacked a /  25  Stat molecule. In the absence of exogenous IL-4 2C, 2C/Stat4"" and 2C/Stat6"" all expressed equivalent levels of CD30 after 72 hours of culture with antigen-expressing APCs. (Fig 3-5 top row) As expected, the addition of IL-4 increased CD30 expression on 2C and 2C/Stat4"~ but not 2C/Stat6" cells. (Fig. 3-5 bottom row) This suggests that the two pathways of CD30 induction can act in a synergistic manner.  CD30  Figure 3-5 Expression of CD30 on 2C, 2C/Stat4 and 2C/Stat6' transgenic cells. 2C, 2C/Stat4- " and 2C/Stat6"" CD8+ cells were stimulated with irradiated BDF1 splenocytes ± exogenous IL-4 for 72 hours, at which time CD30 expression levels were assessed by FACS Filled purple histogram: Cells stained for CD30. Bold green histogram: Unstained control. y  /  26  3.2 Expression of CD30L  Expression of CD30L was examined, and found to be present predominantly on C D 4 cells (Fig. +  3-6), although expression can also be detected on C D 8 cells. (Fig. 3-7) CD30L is detectable +  starting at around 24 hours of culture, with levels peaking around 48 hours. As with CD30, expression of CD30L is absent on resting wild type cells, although low levels can be detected on immediately ex vivo CD30" cells. While CD30L expression levels on activated wild type and A  knockout lymphocytes is comparable at early time points, CD30L remains elevated on CD30"'" cells past the time at which expression on wild type cells is absent. This, together with the coincident downregulation of CD30L and upregulation of CD30, suggests that CD30-CD30L interactions may play a role in regulating the expression of CD30L. 3.3 Effect of CD30 on cell proliferation  While the expression profile of CD30 indicates that it is unlikely to play a role in regulating the initial activation or proliferation of T cells, published reports have suggested a costimulatory role for CD30. As such, we wanted to confirm whether the CD30" lymphocytes showed any A  difference in their proliferative potential as compared to wild type. In a standard proliferation assay, neither B6-CD30"'" cells activated by TCR crosslinking nor 2C/CD30~'~ lymphocytes activated by specific peptide showed any proliferative defect over 72 hours, as measured by incorporation of H-labelled thymidine. (Fig. 3-8) 3  27  Figure 3-6 Expression of CD30L on C D 4 cells B6 and CD30"'" cells were grown in mixed culture and stimulated with 2C11 and supplemented with IL-2 alone or JL-2 and IL-4, as indicated. Staining for CD30L was carried out immediately ex vivo and at 24,48, and 72 hours of culture. Results are gated on CD4CD8" cells. Filled purple histogram: B6. Bold green histogram: CD30"". Dashed pink histogram: unstained control. +  28  Ex vivo  v>  IL  IL-2 + IL-4  2 a  '  24 tours  -ii imnlitfiij Vf  ID  «0*  10''  aiwiri  ' " ^ ' V i i i i ; ..I IU  10  1U  Id  IU  1W  -1 48 hours  t *-*"w' -v . v  #l  _  72 hours  Figure 3-7 Expression of CD30L on C D 8 cells B6 and CD30"" cells were grown in mixed culture and stimulated with 2C11 and supplemented with IL-2 alone or IL-2 and IL-4, as indicated. Staining for CD30L was carried out immediately ex vivo and at 24,48, and 72 hours of culture. Results are gated on CD4"CD8 cells. Filled purple histogram: B6. Bold green histogram: C D 3 0 ' . Dashed pink histogram: unstained control. +  +  29  50000 00 -r  A)  45000 00 -  -thymidine uptake (CPU  40000 00 -  X  35000 00 30000 00 25000 00 -  oo -  20000  15000 00 10000  oo -  5000 00 0 00 3x10 3 A  1x10 5 A  3x10 4  A  A  N u m b e r of c e l l s / w e l l  120000.00  B)  1x10 4  Q. 100000.00  o c I o  80000.00  a. i_  o u c  60000.00  <D  c 5  40000.00  Z  20000.00  1 >»  0.00 1.0  0.1  0.01  0.001  0  Amount of p2Ca peptide ( M) U  Figure 3-8 Proliferation of B6 and CD30' cells A) Proliferation of B6 and CD30 lymphocytes. B6 and CD30" " cells were cultured with 10 ug/ml plate-bound anti-CD3 antibody (2C11) in a 96 well flat bottom plate at the indicated cell concentrations. Exogenous IL-2 (20 U/ml) was added to cultures where indicated. 1 u.Ci [ H] thymidine was added to each well for the final 8 hours of a 72-hour culture period. Results are representative of three separate experiments. B) Proliferation of 2C and 2C/CD30"'" lymphocytes. 2C and 2C/CD30-/- cells were cultured with mitomycin C-inactivated T2-L cells plus the indicated concentration of p2Ca peptide. 1 uCi [ H] thymidine was added to each well for the final 8 hours of a 72-hour culture period. Results are representative of three separate experiments. 7  7  3  d  3  30  3.4 Expression of activation markers  The upregulation of certain cell surface markers is considered to be a hallmark of T cell activation. These include the alpha chain of the LL-2 receptor (CD25); CD44, an adhesion molecule involved in leukocyte migration; and IB 11, an adhesion molecule whose expression appears to be correlated with cytotoxicity. Activated B6 and CD30" "cells stimulated with plate /  bound 2C11 were assessed for their expression of these markers at 72 hours of stimulation in order to determine whether the lack of CD30 had any impact on cell activation. In this system, the expression of CD25 (Fig. 3-9A, B), CD44 (Fig. 3-9C, D) and 1B11 (Fig. 3-9E, F) was unaffected by the lack of CD30, whether in the presence or absence of exogenous LL-4. Given that proliferation of CD30" cells is normal, this result was not unexpected. A  Expression of activation markers on 2C and 2C/CD30"'" cells stimulated with antigenexpressing APCs was also evaluated. (Fig. 3-10) In contrast to B6 cells, expression of CD25 and CD69 was slightly increased on antigen-activated CD30-deficient cells. There were also more antigen-activated CD30" " cells that expressed high levels of CD44 and 1B11 relative to similarly /  activated C D 3 0  +/+  cells. This was somewhat surprising, as 2C/CD30" " proliferate to the same 7  extent as wild type, as discussed above. 3.5 Induction of cytokine production  Activated T cells produce large quantities of numerous cytokines, and published reports have indicated that CD30 crosslinking influences cytokine production by C D 4 (Bengtsson et al., +  2000) and yt> T cells. (Biswas et al., 2000) Therefore, we wanted to determine whether CD30 plays a similar role in C D 8 cells. Activated 2C and 2C/CD30"'" C D 8 lymphocytes were stained +  +  31  Figure 3-9 Expression of activation markers on B6 and CD30"' cells. B6 and CD30 CD8 cells were activated with 2C11 plus exogenous IL-2 ± IL-4 for 72 hours, at which time expression of CD25 (A-B), CD44 (C-D) and IB 11 (E-F) was assessed by FACS. A , C , and E: Cells cultured with IL-2; B, D, and F: Cells cultured with LL-2 + IL-4. Shaded purple histogram: B6. Bold green histogram: C D 3 0 ' . Dashed pink histogram: unstained control.  32  Figure 3-10 Expression of activation markers on 2C and 2 C / C D 3 0 ' transgenic cells. Cells were activated with irradiated BDF1 splenocytes for 72 hours and stained for A) CD25, B) CD44, C) CD69, and D) 1B11. Filled purple histogram: 2C. Bold green histogram: 2C/CD30"". Dashed pink histogram: Unstained control.  33  Figure 3-11 Cytokine production by 2C and 2C/CD30' cells. Intracellular expression of the cytokines IL-2, IL-4, IFNy and T N F a by 2C and 2C/CD30" cells activated with irradiated BDF1 splenocytes was assessed at 24 (A-D) and 72 (E-H) hours of culture. Cells were treated with GolgiStop for 5 hours prior to staining for IL-2 (A and E), IL-4 (B and F), IFNy (C and G) and T N F a (D and H). Filled purple histogram: 2C. Bold green histogram: 2C/CD30"'". Dashed pink histogram: Unstained control.  34  for LL-2, LL-4, IFNy, and T N F a expression at 24 and 72 hours of culture. (Fig. 3-11) No differences in the production of LFNy or T N F a could be detected at any time point examined. Production of EL-2 and LL-4 was the same between wild type and knockout at 24 hours, while at 72 hours CD30" cells exhibited a reduction in expression of LL-4. (Fig. 3-1 IF) This suggests that A  CD30 may play a role in sustaining the production of LL-4 as well as potentially playing a role in the polarization of activated cells towards a type 2 phenotype. 3.6 Cytotoxic effector function  The expression pattern of CD30, with expression peaking at 72 hours following initial stimulation, indicated that it was likely to play a role in regulating T cell function around this time. As well, one published report suggested a role for CD30 in downregulating effector function in a cytotoxic cell line. (Muta et al., 2000) As such, the ability of 2C/CD30" C D 8 cells A  +  to kill specific targets was tested and compared to that of wild type. As targets, two tumour cell lines were used: A20, a Fas B cell lymphoma cell line of Balb/c origin, and P815, a Fas" +  mastocytoma line derived from a DBA/2 mouse. Both Balb/c and DBA/2 mice express the M H C class I molecule L , and so are recognized by the 2C TCR. In both a 4-hour C T L assay (P815 d  target) and an 18-hour C T L assay (A20 target) 2C/CD30~ " effectors exhibited an approximate /  10-fold reduction in killing as compared to 2C despite the somewhat higher level of activation of the 2C/CD30 " cells. (Fig. 3-12A, B) However, when PHA was used to bypass the requirement 7  for TCR recognition, no difference in killing between wild type and knockout could be detected. (Fig. 3-12C) These results suggest that CD30 may play a role in either enhancing TCR signals or in stabilizing interactions between the effector and the target cell and that this role is bypassed when P H A is added. The P H A result also suggests that 2C/CD30" " killers possess the same 7  35  potential to kill target cells as antigen-activated wild type 2C cells.  A)  80.00 -• 70.00 -  0.00 H  "  1  30:1  '  10:1  B)  3:1  1:1  Effector:Target ratio  0.00 4-  1  10:1  1  3:1  1  1:1  Effector: Target ratio  0.3:1  45.00  C)  -2C + PHA 40.00  -2C/CD30/- + PHA -2C  35.00 30.00 u  -2C/CD30-  25.00  o 20.00 i Q.  *  15.00 10.00 5.00 0.00  10:1  30:1  -5.00  1:1  3:1  Effector: Target ratio  Figure 3-12 Cytotoxic effector function of 2C and 2C/CD30" " cells. Effector cells were 7  generated by culturing 2C or 2C-CD30" " cells with irradiated BDF1 spleen cells for 3 days. A) Killing of the Fas-negative P815 cell line. B) Killing of the Fas-positive A20 cell line. C) Killing of antigen-negative RMA/S cells with and without the addition of PHA. Target cells were labeled with Cr, then incubated with effector cells in a 96 well round bottom plate at the indicated effector to target ratios. Cell lysis was determined by measuring ' C r release into culture supernatants. Results are representative of at least two separate experiments. 7  51  5  37  3.7 Expression of cytotoxic effector molecules  FasL is a member of the TNF superfamily expressed by C D 8 T cells that is involved in +  induction of apoptosis in target cells by means of interactions with its receptor, Fas, a death domain containing member of the TNF receptor superfamily. One previously published report indicated that CD30 crosslinking in a tumour cell line resulted in downregulation of FasL expression, contributing to a decrease in cytotoxic effector activity. (Muta et al., 2000) Therefore, we investigated whether this result would hold true in primary lymphocytes. Surprisingly, CD30 crosslinking on C D 8 cells activated by TCR crosslinking in the presence of +  LL-2 and LL-4 appears to lead to an upregulation of FasL expression. (Fig. 3-13 A ) As expected, CD30 crosslinking had no effect on CD30" cells. In the 2C transgenic system, however, both A  wild type and knockout cells express equally high levels of FasL upon activation with specific antigen, even in the absence of CD30 crosslinking. (Fig.3-13B) Perforin and granzyme B are key components of the cytotoxic granules that are involved in the killing of target cells. As 2C/CD30" " effectors exhibited a comparable decrease in killing 7  of both Fas and Fas" targets, it seemed reasonable that any defect in these cells would lie in the +  granule-dependent cytotoxic pathway. Expression of perforin was assessed by means of Western blot. No difference in perforin levels could be detected between day 3 activated 2C and 2C/CD30''" cells. (Fig. 3-13C) As well, granzyme B levels were comparable between wild type and knockout when assessed by means of intracellular staining. (Fig. 3-13D) Together, these results indicate that the reduction in killing by 2C/CD30 " CD8 T cells is not due to a defect in 7  activation or effector differentiation despite the observed role for CD30 in the upregulation of FasL expression on CD8 cells activated by TCR crosslinking. 38  £ y vivo  C) HiC  Day 3  CD30-'- I 2C •mm  CQ30-/m  ••  I Perforin  Figure 3-13 Expression of cytotoxic effector molecules by 2C and 2C/CD30"' cells. A ) Effect of CD30 crosslinking on FasL expression. B6 CD8 lymphocytes were activated with platebound 2C11 and exogenous IL-2 + LL-4 for 72 hours, then restimulated with plate-bound antiCD30 for 8 hours. FasL expression levels were determined by FACS analysis. Filled histogram: No anti-CD30. Bold histogram: + anti-CD30. Dashed histogram: Unstained control. B)Expression of FasL on 2C cells. Activated 2C and 2C/CD30"'" cells were stained for FasL expression. Filled histogram: 2C Bold histogram: 2C/CD30 ~" Dashed histogram: Unstained control C) Western blot for perforin expression. Whole cell lysates from day 3 activated 2C and 2C/CD30"" cells were resolved by SDS-PAGE and transferred to a membrane. The membrane was probed with rat anti-mouse perforin, with proteinG-HRPO used as a secondary. Lysates from unstimulated (ex vivo) cells were used as a negative control. The membrane was then stripped and reprobed for Erkl to ensure equal loading of samples. D) Granzyme B expression. Expression of granzyme B was assessed by intracellular FACS of activated 2C and 2C/CD30"'" cells. Filled histogram: 2C Bold histogram: 2C/CD30Dashed histogram: Unstained control. +  39  3.9 Memory induction Decreased effector function by antigen-activated CD30 " CD8 T cells suggested that later V  functions may also be affected. Therefore it is of interest to determine whether CD30 could also affect the production of memory CD8 T cells. For these studies, 2C and 2C7CD30 " cells were 7  activated in vitro with antigen for three days. 5 x l 0 antigen-activated 1B2 CD8 cells were then 6  +  +  injected into TCRce"" mice. The use of a lymphopenic recipient allowed for greater recovery of the injected cells as there were no endogenous T cells to compete for niches within the tissues, although the absence of C D 4 cells in the recipient could also have a negative effect on the +  development of memory cells. After four weeks, the recipients were sacrificed and the number of 1B2 CD8 cells persisting in lymphoid (peripheral lymph nodes, mesenteric lymph nodes, +  +  spleen and bone marrow) and non-lymphoid (liver and kidney) organs was evaluated. Recovery of transgenic cells was comparable between recipients of wild type versus knockout cells. (Fig. 3-14) Expression of the memory markers CD44, LL-2RP and CD62L was also comparable between wild type and knockout cells. (Fig. 3-15)  40  1.00E+07 a i_>  a > > o  g1.00E+06  I •  »  "35 o  • 2C • 2C/CD30-/-  +  Q1.00E+05  o OQ  1.00E+04  I  •  •  • •  PLN  MLN  na>  E 3  z  1.00E+03 Spleen  Bone Marrow  Liver  Kidney  Figure 3-14 Recovery of memory-like cells from TCRa" " mice injected with activated 2C or 2C/CD30 cells. Activated 2C or 2C/CD30"'" cells were injected into T C R a mice. After 4 weeks, lymphocytes were harvested from the indicated tissues and stained for expression of the 2C TCR and CD8 to determine the number of transgenic cells persisting at this time. Results are pooled from two separate experiments. 7  7  A  41  o  Lymph  node 1  A)'1  ° -a  ~  Spleen  j  — • ~i ~  Liver  "\  Figure 3-15 Expression of memory markers on 2C and 2C/CD30' cells. Memory-like cells recovered from TCRa" mice after 4 weeks were stained for expression of A) CD44; B)CD62L; and C) IL-2Rp. Results shown are representative of two experiments and are gated on 1B2 CD8 cells. Filled histogram: 2C; bold histogram: 2C/CD30"'". A  +  42  4. Conclusion 4.1 Summary of results The expression pattern of CD30, with induction via an LL-4-dependent or an IL-4-independent pathway, has been clearly defined and is consistent with previously published work. (Gilfillan et al., 1998) Expression of CD30 on non-transgenic cells activated by plate-bound anti-TCR antibody is predominantly on CD8 cells with lower levels on CD4 cells. Induction of CD30 expression in this system requires the presence of LL-2 and either EL-4 or CD28 crosslinking, with CD30 expression peaking at around 72 hours. Stat6-deficient cells are unable to respond to LL-4 and express CD30 in response to CD28 crosslinking suggesting an LL-4-independent mechanism for the expression of CD30 and that CD28 signals do not merely increase cytokine production by the cells, bypassing the requirement for exogenous LL-4 in the culture medium. In the 2C transgenic system, antigen-specific stimulation with APCs is sufficient to induce CD30 expression on 2C, 2C/Stat4 - and 2C/Stat6 CD8 T cells, while CD30 levels on2C and2C/Stat4" /  A  '' cells can be enhanced by the addition of exogenous LL-4. As with non-transgenic cells, CD30 levels in the 2C system peak at 72 hours and start to fall off between 96 and 120 hours. By contrast, CD30L is expressed primarily on C D 4 cells. Expression levels peak at 48 +  hours and then fall off sharply to undetectable levels by 72 hours when cells are activated with plate-bound anti-TCR antibody and supplemented with either exogenous LL-2 alone or both LL-2 and LL-4. The expression patterns of CD30 and CD30L suggest a feedback pattern between the two molecules, with expression of CD30L disappearing at around the same time as CD30 appears. As well, levels of CD30L remain elevated on CD30" " cells where CD30-CD30L 7  interactions are absent. It is likely that interaction between CD30 and CD30L leads to 43  internalization of CD30L. A less likely possibility is that release of CD30 in a soluble form (Hansen et al., 2000) leads to the binding of sCD30 to CD30L and thus blocks the binding of antibody to CD30L, preventing its detection. The relatively late expression of CD30 suggests that it is unlikely to play a role in early events of activation and proliferation. Over 72 hours, proliferation in response to either TCR crosslinking or specific antigen on APCs is unaffected in CD30-deficient CD8 T cells. While published reports have implicated CD30 in costimulation, the normal proliferation of CD30"  /_  cells, whether induced by antibody crosslinking of the TCR or by encounter of specific antigen on APCs, does not contradict this. Rather, it suggests that whatever role CD30 plays in costimulation is not essential, and that other molecules can compensate for its absence. As well, the expression pattern of CD30 suggests that it is more likely to play a role in maintaining sustained activation than in the initiation of an immune response. Expression of the activation markers CD25, CD44 and IB 11 on cells stimulated by TCR crosslinking is the same on both wild type and CD30' " cells at 72 hours of culture. However, 7  when 2C cells are activated in an antigen-dependent manner, expression of CD69 and CD25 is higher on 2C/CD30" " cells than on wild type 2C cells, while CD44 and 1B11 expression levels /  are more comparable between the two cell types. Production of cytokines by 2C/CD30 " cells activated by specific antigen was not greatly /  affected, with the only real observed reduction being in the production of IL-4 at 72 hours. Given the association between IL-4 and CD30 expression, this finding was not surprising, but does indicate that CD30 may be playing a role in polarizing cells towards a type 2 phenotype. When cytokine production is examined at an early time point, before CD30 expression, levels of EL-4  are equal between wild type and knockout. However, by 72 hours, when CD30 levels are at their peak, levels of LL-4 are substantially lower in knockout cells than in wild type. This suggests that CD30 may play a role in sustaining production of this cytokine. This result is somewhat puzzling in conjunction with the finding that 2C7CD30 " exhibit reduced killing however, as LL-4 has been 7  shown to reduce both cytotoxic activity and expression of perforin and granzyme. (Aung and Graham, 2000; Kienzle et al., 2002) One intriguing finding was the decreased ability of CD30 " effectors to kill specific 7  targets despite having divided to the same extent as wild type cells, as proliferation and acquisition of effector functions have generally been considered to be linked. Interestingly, it has recently been shown that deletion of the TNFR superfamily member 4-IBB has a similar effect, where proliferation of 4-IBB" " cells is even somewhat enhanced but the ability to kill is reduced. 7  (Kwon et al., 2002) However, the fact that activated 2C/CD30"'" cells express the effector molecules FasL, perforin and granzyme B to the same extent as wild type cells indicates that the observed reduction in killing is not due to a defect in effector differentiation. As well, the defect in killing can be overcome when the requirement for TCR engagement is bypassed by using P H A to bring the cells together. This suggests that release of cytotoxic granules is normal in CD30-deficient cells. It is thus likely that CD30 plays a role in cytotoxicity either by signaling in a manner that enhances TCR-mediated signals upon target recognition or by stabilizing interactions between the effector and the target cell, which was overcome by the addition of PHA. Given that CD30 crosslinking on non-transgenic cells leads to the upregulation of FasL expression, it is possible that CD30 does play some role in the development of cytotoxic effector function depending on circumstances, but that this requirement is bypassed by the high affinity 45  interaction of the 2C TCR with its Iigand. Finally, the absence of CD30 had no effect on the development of memory-like cells generated by the adoptive transfer of activated C D 8 lymphocytes into TCRct"'" recipients, both +  in terms of persistence of the cells and in the expression of memory markers. Given that CD30"'" appear to differentiate into effectors efficiently, despite their reduced killing ability, this result was not entirely unexpected, despite the published association of CD30 expression with memory phenotype in human peripheral blood cells. (Ellis et al., 1993) It is possible that development of memory was affected by the absence of CD4 T cell help in either the initial activation of the injected cells in vitro or in the TCRa"" recipient, which has recently been shown to be essential for the development of normal CD8 T cell memory. (Janssen et al., 2003) 4.2 Future work While CD30 lymphocytes are reasonably well characterized, some work remains to be done. /_  Despite the lack of any effect on the initial activation and proliferation of C D 8 T cells, it is +  possible that CD30 may play a role in either the maintenance of long-term activation or in regulating cell survival. As CD30 has been shown to play both a costimulatory role in secondary proliferation (Gilfillan et al., 1998) and in encouraging apoptosis (Telford et al., 1997) in primary lymphocytes, it is uncertain whether CD30" " would exhibit reduced proliferation or 7  increased cell survival upon restimulation. In addition, the precise nature of the observed defect in killing by 2C/CD30" ~ /  lymphocytes has yet to be determined. Based on expression of activation markers, 2C/CD30"'" CD8 T cells are activated to an equal or greater extent than are 2C CD8 T cells and express equivalent levels of effector molecules. However, these cells are unable to kill specific targets as  46  efficiently as wild type effectors for reasons that remain unclear. Recognition and killing of a target cell by cytotoxic effectors involves several steps, and it will be necessary to determine at which step the defect lies in order to discover the cause of the reduced cytotoxic effector function by 2C/CD30" cells. The initial contact between the effector and the target cell is A  mediated by non-specific adhesion molecules such as LFA-1 and I C A M . After this initial interaction, the cells are bound more tightly together by the binding of the TCR to the M H C peptide complex on the surface of the target cell and the recruitment of various accessory molecules that are localized to lipid rafts to form the immunological synapse. These accessory molecules serve to both stabilize the interaction between the two cells and as costimulatory receptors. Signals transmitted by the TCR and other receptors in the synapse lead to the reorganization of cytoskeleton of the effector cell in order to orient the cytotoxic granules towards the target cell, and ultimately to the release of the contents of the cytotoxic granules, thus killing the target, (reviewed in Russell and Ley, 2002) It is likely that the role played by CD30 in cytotoxicity is in either the stabilization of the contact between the effector and the target cell, or in transmitting costimulatory signals. While it is presently unknown whether CD30 can localize to lipid rafts, other members of the TNFR superfamily, including TNFR1 (Cottin et al., 2002) and CD40 (Arron et al., 2002) have been shown to do so, indicating that CD30 is likely to do the same and could thus be expected to be found in the immunological synapse. Finally, while there was no difference in memory induction in the model used here, it is possible that CD30 may play a role in development of memory under more physiological conditions. As CD30L is expressed primarily on C D 4 cells, which are absent in T C R a " mice, it +  7  is likely that the injected cells did not encounter any CD30L in the host animal, which may or  47  may not have an effect on the development of memory cells.  5. References Aizawa, S., Nakano, H., Ishida, T., Horie, R., Nagai, M . , Ito, K., Yagita, H., Okumura, K., Inoue, J., and Watanabe, T. (1997). 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