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Characterization of an antigen-specific T helper cell clone and its products Kwong, Pearl Chu 1987

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CHARACTERIZATION OF AN ANTIGEN-SPECIFIC T HELPER CELL CLONE AND ITS PRODUCTS BY Pearl Chu Kwong B.Sc, M.Sc, The University of British Columbia, 1980,1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1987 © Pearl C. Kwong, 1987 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 MICROBIOLOGY The University of British Co lumbia 1956 Main Mal l Vancouver, Canada V6T 1Y3 Date October 8. 1987  DE-6(3/81) ii A B S T R A C T A T helper cell clone, referred to as clone 9, was derived from an allogeneic mixed lymphocyte culture. Clone 9, as well as supernatant factor(s) derived from it, could help the cytotoxic T lymphocyte (CTL) responses of H-2 (D^) responder cells to alloantigens, or they could help the C T L responses of non-D^ responder cells to D* 5 alloantigens. Clone 9 cells or their factor(s) were active only when added during the first 24 hours of a five-day culture period. Clone 9 or its factor(s) could also synergize with interleukin-2 (IL-2)-containing medium in mounting cytotoxic responses to alloantigens. The helper activity in clone 9 supernatant was not due to IL-2 and it was specifically absorbed out by D^-spleen cells. The characterization of the D^-specific helper factor(ASHF) was facilitated by the isolation of a T hybridoma clone (clone 25), obtained from fusion of clone 9 cells with the T cell lymphoma, BW5147, and a B cell hybridoma that produced an I g M monoclonal antibody (clone 30 IgM) which bound A S H F . A n additional monoclonal antibody (F23.1), which recognizes a determinant of the Vg8 family of the T cell receptor, was also particularly useful for the characterization of A S H F . Analysis with these reagents showed that both clone 30 I g M and F23.1 immunoadsorbents could retain A S H F activity. Preabsorption of the A S H F with D^spleen cells prior to affinity purification over a clone 30 I g M column resulted in the absorption of D^-specific helper activity as well as the loss of a 50,000 molecular weight ( M W ) band on S D S - P A G E under reducing conditions. Furthermore, affinity purification of A S H F over the F23.1 immunoadsorbent, but not an irrelevant monoclonal antibody (mAb) column, also yielded a 50,000 M W molecule. Taken together, these findings suggest that the 50,000 M W molecule is a component of the A S H F and it is intimately related to the B chain of the T-cell receptor. The mode of action of clone 9 and its products in the induction b f C T L responses was also investigated. It was found that clone 9 and A S H F could help C T L responses by inducing IL-2 production in B6-stimulated cultures. In addition to A S H F , clone 9 cells also Hi produced an additional factor(s) which participated in the induction of C T L responses. This additional factor(s) was referred to as I L - X . I L - X synergized with excess human recombinant IL-2 in the activation of C T L precursors (CTL-P) in the absence of antigenic stimulation. A model which involves the participation of A S H F , T helper cells, IL-2 and I L - X in the induction of C T L responses is proposed. iv Table of Contents Page Number Abstract i i Table of Contents iv List of Tables v i i List of Figures ix List of Abbreviations x Acknowledgments x i i i Chapter I- Introduction 1 1. Introductory remarks- Immune regulation by T cells 1 2. Methods for isolating antigen-specific T cell clones 2 A . T cell hybridization 3 B . Maintenance of T cells in T cell growth factor (TCGF)-containing media 4 C. Virus-induced transformation 5 3. Antigen-specific T helper (Th) cells 5 A . Role of Th cells in B cell responses 5 i . Cognate interaction 5 i i . Non-cognate interaction 6 i i i . MHC-restricted interaction 6 iv. Idiotype-anti-idiotype interaction 7 B . Role of Th cells in C T L responses 9 C . Activation of Th cells 10 i . Role of accessory cells in Th cell activation ^ , 10 i i . Nature of the T cell antigen receptor 11 Page Number i i i . Role of C D 3 in Th cell activation 13 iv. Role of cell adhesion molecules in T cell activation 16 D . Th cell heterogeneity 16 4. Immune regulation by T-cell derived lymphokines 17 A . Non-antigen-specific lymphokines 18 i . Role of nonspecific lymphokines B cell responses 18 i i . Role of nonspecific lymphokines T cell responses 19 B . Antigen-specific T cell factors 20 i . Antigen-specific helper factors (ASHF) 20 a. Nature of A S H F 20 b. Mode of action of A S H F 22 i i . Antigen-specific T suppressor factors (T $ F) 24 i i i . Relationship between antigen-specific T cell factors and the T cell receptor 25 5. Thesis objectives 26 Chapter II. Characterization of a D^-specific T helper clone 27 1. Introduction 27 2. Materials and methods 27 3. Results 32 4. Discussion 37 5. Summary . 40 6. Appendix: Tables I - X I V 41 v i Page Number Chapter IH. Characterization of a D^-specific helper factor required for the induction of cytotoxic responses to alloantigens with the use of monoclonal antibodies specific for the helper factor or the T cell antigen receptor 55 1. Introduction 55 2. Materials and methods 56 3. Results 60 4. Discusssion 64 5. Summary 68 6. Appendix: Tables X V - X V H and Figures 1 to 5 69 Chapter I V . Mechanism of action of a D^-specific helper clone and factors in the induction of cytotoxic responses to alloantigens 80 1. Introduction 80 2. Materials and methods 81 3. Results 82 4. Discussion 86 5. Summary 90 6. Appendix: Tables X V I I I - X X H and Figures 6-7 91 Chapter V . Conclusions and future directions 99 References ^ 101 List of Tables Page Number Table I. H-2 phenotype of clone 9. 41 Table n. The Thy-1 and Ly t phenotypes of clone 9. 42 Table HI. The helper activity of clone 9 is specific for B 6 responder cells. 43 Table IV . The helper activity of clone 9 is D^-specific. 44 Table V . Activation of antigen-specific C T L by clone 9. 45 Table V I . Requirement of accessory cells for clone 9 function. 46 Table VII . Kinetics of action of clone 9. 47 Table Vin. Helper activity of clone 9 supernatant factor(s) is -specific. 48 Table DC. Activity of clone 9 supernatant in the thymocyte proliferation assay. 49 Table X . Clone 9 supernatant does not support the growth of an IL-2 dependent T cell line. 50 Table X I . Helper activity in clone 9 supernatant is not absorbed by D 2 Con A or L P S blasts. 51 Table X I I . Augmentation of cytotoxic responses by clone 9 factor(s) in the presence of IL-2. 52 Table XIII . Absorption of helper activity in clone 9 supernatant factor(s) by Con A blasts. 53 Table X I V . Enhancement of C T L responses by pulsing Db responder cells with clone 9 supernatant factor(s). ^ 54 Table X V . Neutralization of the helper actionof clone 25 A S H F by a m A b . 75 Table X V I . Clone 30 and ¥23.1 m A b columns can retain clone 25 A S H F . Table X V I I . Biological activity of absorbed A S H F purified over clone 30 m A b column. Table X V I I . Clone 9 and A S H F can help bystander responses. Table X T X . Induction of IL-2 production by clone 9 cells or A S H F Table X X . Supernatants from B6-stimulated clone 9 cultures contain CTL-inducing activity. Table X X I . Supernatants from Con A - stimulated clone 9 cells contain B6-specific helper factors. Table X X I I . Presence of a non-antigen specific helper factor(IL-X) in the supernatants of Con A-stimulated clone 9 cells. IX List of Figures Page Number Figure 1. Helper activity of clone 9 cells in the thymocyte assay. 69-70 Figure 2. Helper activity of clone 25 A S H F in the thymocyte assay. 71-72 Figure 3. Binding of clone 25 A S H F to D b - 7 3 - 7 4 Figure 4. Ge l profile of affinity-purified A S H F that had been pre-absorbed with B 6 or D 2 spleen cells 78 Figure 5. Ge l profile of A S H F purified over different m A b columns 79 Figure 6. Effect of varying the responder cell dose on the helper effect of clone 9 and A S H F 91 -92 Figure 7. A proposed model for the activation of C T L - P by clone 9 cells, A S H F , IL-2 and I L - X 98-99 Chapter I. Introduction 1 1. Introductory remarks- Immune regulation by T cells When the immune system is challenged with antigen, the resulting response involves many complex interactions between different cell populations. Among the cell types involved, T lymphocytes in particular seem to play a central role in most immune responses. Thus, T cells could mediate effector functions such as cytotoxicity and delayed type hypersensitivity (DTH); they are also responsible for regulating both humoral and cell-mediated immune responses (1,2). There are two major types of T cells involved in the regulation of immune responses. A s their names imply, T helper (Th) cells amplify immune responses while T suppressor cells inhibit immune responses. The existence of Th cells was first documented by Claman et al.(3,4), who initially used the term "auxiliary cells" for the thymus-derived cells that synergize with bone marrow-derived cells in the production of antibody responses to sheep red blood cells (SRBC) . Subsequent studies on these "auxiliary cells" (5,6,7) established that thymus-derived cells (or T cells) are necessary to induce antibody responses. The negative counterpart of the T h cell was later discovered by Gershon and Kondo (8). These investigators found that the induction of tolerance in the mouse to a large dose of S R B C was dependent on the presence of T cells, thereby suggesting that T cells are also needed to specifically inhibit the antibody response. This pioneering experiment is the basis for the concept of suppression by T suppressor cells, which was eventually used to explain most of the inhibitory phenomena of the immune response such as the maintenance of self-tolerance (9) and the prevention of allergic (10) and autoimmune diseases (11). Cantor and Boyse (12) were the first to distinguish between Th and T suppressor cells on the basis of their cell surface markers. Using antisera against the polymorphic glycoproteins expressed on the surface of T cells ca l l edLy antigens, Cantor and Boyse showed that T cells expressing the L y 1"23 + phenotype have suppressor activity while T cells expressing the L y l + 2 3 " phenotype exhibit helper activity. Following this initial discovery, T cell subsets were further separated and identified on the basis of other distinct cell surface markers (13-15), drug sensitivities (16-18) and adherence characteristics 2 (19,20). Using cell populations purified by such methods, it was subsequently found that Th cells and T suppressor cells not only regulate antibody responses, but they also regulate cell-mediated responses (1,2,21). Furthermore, these cellular responses appear to be regulated by distinct subpopulations of Th or T suppressor cells (22-24), whose regulatory functions can either be mediated via direct collaboration with other cell types or via antigen-specific or antigen-nonspecific factors (25-28). These studies therefore indicate that T cells are an extremely heterogeneous population with each subpopulation mediating distinct immunological functions; their interactions with each other and with other cell types determine the final outcome of the immune response. 2. Methods for isolating antigen-specific T cell clones The mechanism by which T cells regulate immune responses are complex. If the details of T-cell recognition were to be dissected, it is important that the multiple elements involved in immune responses are isolated and studied independently. In the past, efforts to study immune regulation have been made by enriching for regulatory T cells through positive and negative selection methods (12,29), enrichment of T cells by filtration over a nylon wool column (20), or repeated antigenic stimulation (30). Although these procedures were helpful in providing some insights about T cell function, they are of limited usefulness for the in-depth analysis of the functions of regulatory T cells. Limitations of these procedures include the heterogeneity and the limited lifespan of the isolated T cells. Recent developments in a number of laboratories have provided the means to overcome some of these limitations. These include strategies to immortalize T cells by somatic cell hybridization between normal T lymphocytes and T-cell lymphomas, maintenance of T cells in T cell growth factor (TCGF)- containing media and virus-induced transformation of T cells (31-33). Once the T cells are adapted in tissue culture, these T cells are cloned by limiting dilution or by micromanipulation (31,35). 3 A . T cell hybridization The technique of T-cell hybridization is essentially similar to the somatic cell hybridization pioneered by Kohler and Milstein for the production of B cell hybridomas that secrete monoclonal antibodies of known specificity (36). It involves the fusion of antigen-primed T cells with T cell lymphomas. The resulting hybrids are then selected for their ability to exhibit certain immunological function and for their ability to grow indefinitely in culture (37). The most widely used T cell partner for fusion is BW5147, a Thy 1.1 +, Ly t 1 + 23" lymphoma of A K R (H-2^) origin (37). BW5147 has been fused successfully with either fresh or activated cells, resulting in T cell hybridoma lines which manifest suppressor (38-41), helper (42,43) and cytolytic (44,45) functions. Suppressor T cells are the most common of T cell hybridomas. Except for a few examples (46,47), almost all of the long -term cultures of suppressor T cell clones reported thus far have been immortalized by T cell hybridization (31). B y contrast, the isolation of T h and cytotoxic T lymphocyte (CTL) hybridomas has not been as successful since more tedious selection and enrichment procedures have to be employed before fusion (37,45). T cell hybridoma technology offers a number of distinct advantages (31,35). These hybrids are relatively easy to grow and maintain. Since they can be grown as tumors, they can reach large numbers in a short period of time. Hybrids are also easy to clone by limiting dilution and store well in liquid nitrogen. A common problem associated with T cell hybridomas is their inability to maintain a stable functional phenotype (31,35). Instability seems to be caused by the tendency of T cell hybrids to lose chromosomes which usually leads to the hybrid's loss of immunological function. In order to reduce the risk of losing such valuable hybrids, precautions have been used by freezing down large numbers of the clone early after cloning and frequent recloning to prevent nonfunctional variants from outgrowing the cultures. Other disadvantages associated with this method include the unknown contributions of the fusion partner, the usually low concentration of effector molecules produced by the T hybridomas, their limited usefulness for testing the in vivo function of the T cells in question, and the T cell hybrids may be functionally different from normal T cells. 4 B . Maintenance of T cells in TCGF-containing media Morgan et al. (48) was the first to demonstrate that T lymphocytes can be cultured ^definitely in the presence of growth factors derived from mitogen-stimulated cultures of normal human lymphocytes. The factor responsible for T cell growth has been termed T C G F or more recently, interleukin 2 (IL-2) (49). IL-2 is a protein with a molecular weight of 15 kilodaltons (kDa) in rats and humans, or 30 kDa in mice, and it is now known to be the major promoter of T cell proliferation (49). The method of growing T cells in IL-2-containing media has been adopted to generate antigen-specific murine T cells with cytotoxic (50-55), helper (54-59), and sometimes suppressor (46) functions. However in some cases, continous growth and maintenance of function of some cells, in particular Th cells, seem to require more than just IL-2. These cells appear to require large doses of antigen and syngeneic irradiated spleen cells (55,59). The use of T C G F and /or repeated antigenic stimulation has been useful for generating T cell lines with cytotoxic and helper activities; isolation of T suppressor cell lines using this method has been more difficult. The in vitro propagation or cloning of T cells in TCGF-supplemented media has several uses (31). It may be of use as an initial step for enriching active cells prior to either fusion or viral transformation. In addition to their usefulness for analysis in vitro , the non-malignant cells obtained through this method have also been used for in vivo reconstitution experiments (60). However, despite their usefulness in many studies, there are certain disadvantages in the use of TCGF-dependent cell lines or clones for study (31). It is difficult to acquire large numbers of cells since culture conditions require many components. The preparation and maintenance of the long-term cell lines are time consuming, tedious and expensive. Special freezing procedures have to be employed to improve viability after thawing. Furthermore, analysis of the T cell clones and their factors are complicated by the presence of a variety of lymphokines in TCGF-amtaining media as well as the presence of irradiated syngeneic feeder spleen cells. C . Virus-induced transformation of T cells 5 Finn et al . (61) developed a method whereby antigen-specific T cells were infected in vitro with radiation leukemia virus (RadLV) and these infected cells were injected intrathymically into syngeneic mice. Within a few months, antigen-specific thymic lymphomas that developed in the majority of the recipients could be cultured and established as permanent cell lines. Using this method, keyhole limpet hemocyanin (KLH)-specific Th lymphomas (61) and hen egg white lysozyme (HEL)-specific T suppressor cell lymphomas (47) were isolated. The applicability of this method, however, has not yet been used to the fullest So far, only very few studies have used this method to isolate T cell clones with specific function(s), presumably because of the low frequency of obtaining transformed cells with the desired function (47,61). On the other hand, the advantages of this method are comparable to those of T-cell hybridization with BW5147 in that the virus-transformed cells also have a capacity to grow indefinitely in culture (31). Furthermore, since only a small viral genome is introduced into the host D N A , the cells obtained have an apparently normal and presumably more stable chromosome karyotype (31,47). 3. Antigen-specific Th cells A . Role of Th cells in B cell responses i . Cognate interaction Following the initial findings that T cell help is required for B cell activation, investigations into the mechanism of Th cell interactions were simultaneously made by Mitchison (62), Rajewsky et al. (63), and Raff et al. (64). B y assessing the anti-hapten secondary antibody responses to carrier-hapten conjugates, these investigators observed that in order to achieve optimal IgG production, T cells need to be primed to the carrier,proteins while B cells need to be primed to the hapten molecules. Furthermore, optimal anti-hapten responses only occurred i f the carrier and hapten were physically linked on the same molecule. Based on these results, it was concluded that T and B cells do not have to recognize the same determinants on an antigenic molecule but that these determinants do have to be physically 6 linked on the same molecular complex for proper Th-B cell collaboration to occur. This type of cellular collaboration has been termed cognate interaction or associative linked recognition since T and B cells are brought into close proximity by an antigenic bridge. The regulatory mechanism by which T cell help is delivered to the B cell is still unclear. It is possible that the T cells may directly interact with the B cells to mediate their helper effects (65). Alternatively, the T cells may produce antigen-specific helper factors which are then focused onto the B cell target by the antigen bridge (66). The requirement for linked or cognate recognition ensures that only the antigen activated cells are helped and prevents the potential danger of triggering other cells recognizing unrelated antigens. This type of interaction is not limited to T helper-B cell collaboration in vivo; it is also found to be essential for the in vitro activation of B cells by antigen (67,68). i i . Non-cognate interaction A second type of Th cell interaction, called non-cognate interaction, does not require an antigen bridge between the Th cells and the B cell target. Instead, T h cells produce nonspecific lymphokines upon presentation of antigen by an accessory cell (25,69). Such nonspecific mediators bind to specific receptors on the surface of antigen-activated B cells, which in turn proliferate and differentiate into mature effector cells. The mode of action of several nonspecific mediators such as IL-2, y-interferon (TPN), interleukin 3 (IL-3) and B-cell stimulating factor-1 (BSF-1) has already been extensively analysed (31,70). The general findings are that their roles in the immune system appear to be widespread; these molecules do not only replace T cells by regulating ongoing immune responses (31,70), but at the same time, they can also recruit more precursor cells from the hematopoietic system (71). This type of indirect interaction has also been observed in other types of immune responses; in particular, it appears to be the major mechanism involved in the helper cell augmentation o f C T L responses (72,73). Thus, while cognate interaction seems to be a major mechanism in the initial step of antigen recognition and precursor cell induction, non-cognate interaction appears to be more crucial in the clonal expansion and differentiation of antigen-specific effector cells. 7 i i i . Major histocompatibility complex (MHC)-restricted interactions Apart from the cognate or non-cognate manner of antigen recognition by Th cells, most T h cells also need to recognize antigens in association with the gene products of the M H C before they can effectively mediate their help. This type of interaction, termed MHC-restricted interaction, was first demonstrated by Kindred and Shreffler (74). These investigators observed that antibody responses in nude mice are reconstituted only i f H-2 compatible, but not H-2 incompatible, donor thymocytes are transferred to the recipient nude mice. Thus, they concluded that effective Th-B cell interaction is only possible i f both T and B cells share M H C determinants. However, this type of interaction does not seem to be an obligatory requirement for all Th-B cell collaboration since another group of workers showed that under certain conditions, histoincompatible Th cells and B cells can effectively cooperate with each other (75). A n explanation for these two conflicting pieces of data was clarified by Katz et al.(75,76), who showed that the mechanism by which histoincompatible Th and B cells collaborate is distinct from the mechanism by which histocompatible Th cells collaborate. In the case of histoincompatible Th cells, cellular collaboration between Th and B cells is possible because the Th cell recognized the allogeneic M H C determinant expressed by the B cells. This mechanism is termed the allogeneic effect and does not require Th cell recognition of any carrier determinants. In the case of histocompatible Th cells, Th cells recognize carrier determinants in the context of syngeneic M H C determinants expressed by the B cells. This latter mechanism is termed physiological cooperaton, presumably because this is how Th -B cell interactions occur normally in vivo. In subsequent experiments, Katz and coworkers performed genetic mapping studies and identified the I -A subregion of the M H C as the gene influencing the restriction element in Th-B cell interactions (77,78). In B cell responses, M H C restricted interactions are not limited to Th -B cell interactions, but they are also observed in Th-accessory cell interactions (69). However, it is still not clear whether MHC^restriction is strictly required for both Th-B cell and Th- accessory cell interactions, or just at the level of Th -accessory cell interactions. Both possibilities may be correct depending on the type of T and B cells participating in the response. It now appears that distinct T and B cell subpopulations may have different genetic requirements in their interactions with other cells (22,79). 8 When the nature of MHC-restricted interactions was investigated with the use of chimeric mice, it was concluded that M H C restriction is not mediated through a like-like interaction between the M H C molecules of the two interacting cells (80), but rather it is mediated through a receptor on the Th cell which recognizes antigen in the context of se l f -MHC molecules (81-83). Furthermore, it was also found that the acquisition of MHC-restricted specificity is influenced in part by the H-2 haplotype expressed in the recipent's thymus (82,84, 87). According to the theory of "Adaptive differentiation of restriction specificity" proposed by Katz (88), the environment in which the precursor stem cells have differentiated determines the physiologic interactive capacity of mature T cells. In other words, the M H C restriction elements that restrict the interaction of T cells with antigen can either contract or expand, depending on the M H C haplotype of the thymic epitheUum in which T cells mature. Presumably such a mechanism of T cell differentiation is important i f self-antigens were to be discriminated from non-self antigens by the organism. A t the present time, there are as yet no convincing data which can explain how thymic education is accomplished. I V . Idiotype-anti-idiotype interactions Another mode of Th cell interaction involves the Th cell recognition of the variable portion of immunoglobulin (Ig) receptors. Several investigators have documented the existence of such interactions (23,89-91). For example, a Th cell distinct from the classical carrier-specific Th cell has been observed to be essential in the induction of anti-phosphoryl choline antibody response which is dominated by the T15 idiotype (23). This T h cell shows specificity for the T15 idiotype as it induces the selective activation of T15-bearing B cells, as well as specificity for a carrier determinant It has been suggested that this T h cell differs from the classical carrier-specific Th cell in that this Th cell recognizes antigens in association with the appropriate idiotype instead of se l f -MHC molecules. Idiotype-anti-idiotype interactions have also been observed in other systems (92,93); in fact, it has been shown to be one of the major pathways used by T suppressor cells in their regulatory process (94,95). The significance of idiotype-anti-idiotype interactions in immune regulation is not yet fully understood but they are presumably involved in the generation of a network of interacting cells (96). 9 B. Role of Th cells in CTL responses The first evidence to suggest that Th cells can collaborate in the in vitro generation of CTL came from, the studies of Cantor and Boyse, who demonstrated that the addition of Lyt 1 + cells can augment the CTL responses of Lyt 23+ precursor cells to alloantigens (12). In subsequent studies, Pilarski et al. (97,98) demonstrated that antigen-specific, radiation resistant Th cells are required for CTL responses to alloantigens. Moreover, they were able to demonstrate that only Th cells allogeneic with the stimulator cells can provide help in the CTL responses to alloantigens. Pilarski's observations may be similar to the allogeneic effect observed for Th -B cell collaboration, in which non-specific lymphokines may have been produced as a result of the mixed lymphocyte reaction (MLR) between the Th cells and the allogeneic stimulator cells. By contrast, Th cells involved in CTL responses to viral and H-Y antigens (99,100) have been shown to be la-restricted in their interactions. In addition, Keene and Forman (101) presented evidence which shows that CTL responses to Qa-1*5 alloantigen can only be generated in vitro if animals were first primed in vivo with two kinds of antigenic determinants, one determinant recognized by the CTL-P and the other by the Th cell. Successful priming of CTL-P can only be achieved if both antigenic determinants are recognized simultaneously. This type of interaction is reminiscent of the cognate interaction described for B cell responses (62-64). Taken together, these studies suggest that Th cells involved in Th-CTL interactions behave in a manner similar to the Th cells involved in B cell responses. However, it remains to be determined if the Th cell that collaborates with pre-CTL are the same or different from the Th cell that helps B cells. C. Activation of Th cell I. Role of accessory cells in Th cell activation Before Th cells can mediate their effects on other cells, they must first be stimulated by antigen to proliferate and differentiate into an activated state. It is well established that the activation of Th cells requires a direct interaction with accessory cells which presents the 10 antigen on their surfaces (102-103). A s was discussed before, Th-accessory cel l interactions are also MHC-restricted; that is, Th cells must see antigen and self-la determinants simultaneously on the accessory cell in order to be stimulated (104). In general, identity of the M H C , especially of the I -A subregion, between the accessory cell and Th cell is necessary (85,105-108). However, in responses to synthetic polypeptides, where two complementing immune response (Ir) genes are required, identity at both I -A and I-E/C subregions of the M H C is required for maximal stimulation of Th cells (109). Two major functions are performed by accessory cells in the induction of Th cell activity. The first function involves antigen uptake, processing and presentation. Recently, experiments using fixed antigen-presenting cells (110-112) and purified Ia incorporated into planar membranes (113) have provided strong support for the concept that processing of protein antigens by accessory cells is required for T cells to recognize antigen and M H C . In these experiments, these investigators showed that small peptides, derived from fragmentation of soluble protein antigens, can be recognized in the context of Ia by antigen-specific and MHC-restricted T cel l clones whereas the intact proteins are not. Thus, before accessory cells can stimulate T cells in an antigen-specific manner, they have to take up the foreign antigens, proteolytically cleave the antigen into peptide fragments which are then presented on their surface in the context of self-la antigens. There is a growing body of evidence which suggests that a physical complex is formed between Ia and antigen (114-122). Such physical complexes were reported by Erb and Feldmann (114), Puri and Lonai (115) and more recently, Friedman (116) who demonstrated the existence of such complexes in the supernatant fluid of macrophages in the presence of antigen. In addition, more recent investigations were made on the possible interactions between Ia and immunogenic peptides (117-122). B y using methods such as equilibrium dialysis and antigen competition assays, it was shown that the binding of several unrelated peptides to different Ia molecules correlated with their known MHC-restriction patterns. Furthermore, it was found that antigenic competition by different peptides is due to competition for binding to the Ia molecules expressed on accessory cells. The second major function of accessory cells is the production of antigen 11 non-specific regulatory molecules which are required for the maintenance of T cell clonal expansion. One such molecule has been identified as interleukin 1 (IL-1), a 15 k D a protein produced in small amounts by resting macrophages but to a much greater extent when they are stimulated by T h cells (123). IL-1 has been purified to homogeneity and its various activities have been extensively studied (123-129). It has been found that one of the many functions of IL-1 is to induce Ly t l + 2 " 3 " T cells to produce another important mediator, IL-2, which stimulates the proliferation and differentiation of antigen-activated T cells (124-125). This concept was further supported by the observation that some murine cell lines secrete IL-1 in response to IL-1 in the presence of lectins (126-127). In addition, IL-1 has also been found to increase the membrane l ipid viscosity of Lyt 1 + T cell populations, the expression of receptors for processed antigens as well as the expression of EL-1 receptors (128). More recently, a variety of other effects of IL-1 have been described. IL-1 now appears to be involved, directly or indirectly, in every aspect of the immune and inflammatory responses (129). i i . Nature of the T-cell antigen receptor The stimulation of Th cells is initiated in part by the interaction of the T cell receptor with antigen and class II M H C molecules. The structural basis for the MHC-restricted antigen recognition of T cells has long been puzzling immunologists. A t least two types of models have been proposed to explain the dual specificity of T cells. The first model suggests that a single receptor recognizes a complex formed by the antigen and M H C molecule. The second model hypothesizes that the dual specificity of T cells is due to the presence of two receptors; in this case, the antigen and M H C molecules are recognized independently by the two receptors (130-131). Recently, after a long search for the elusive T cell receptor, the structure and the genes encoding the T-cell antigen receptor have been unravelled (132-135). Using mAbs that reacted with antigenic epitopes unique to individual T cell clones, tumors or hybridomas (136-139), several groups have identified the T-cell receptor as an 80-90 kDa heterodimeric transmembrane glycoprotein composed of disulfide-bonded a and B polypeptide chains. The genes corresponding to both a and B have been cloned and sequenced, and the nucleotide sequences indicate that the a and B genes have a high degree of homology with 12 immunoglobulin genes (140-141). They are also composed of distinct segments of variable, diversity, joining and constant regions separated by introns (134,142-146). Furthermore, the genomic organization and mechanism of rearrangement of the a and B genes also show striking similarities with that of the immunoglobulin genes (134,142-148). A third gene, distinct from a and B genes and referred to as the y gene (149), has also been found to have structural similarities with immunoglobulin segments and undergoes gene rearrangement Recently, the product of the y gene has been identified (150) and its function is beginning to be understood (151-154). The isolation of the a and B genes has been crucial in the identification of the ccB heterodimer as the structure which defines the dual specificity of T cells. Yague et al. (155) were able to select for spontaneous variants from a helper T cell hybridoma line which had lost the ability to respond to antigen plus M H C . B y Southern blot analysis, it was found that these variants were lacking in either the a- or the B-chain genes or both. When the a- and B-chain loss mutants were fused, the reactivity of the resulting hybrid to antigen and M H C was restored. These results show that the a- and B-chain genes are necessary for the dual recognition of antigen and M H C . A more direct proof for the role of the aB heterodimer in MHC-restricted antigen recognition was provided by the elegant studies of Dembic et al. (156). These investigators isolated and characterized the a- and B-genes from a donor cytotoxic T cell clone specific for the class I M H C molecule and the hapten fluorescein. The a- and B-genes were then transfected into another cytotoxic T cell clone of different specificity. When the resulting transfectants were tested for their ability to lyse target cells of the donor and recipient T cells, it was found that the transfectants could express the MHC-restricted antigen specificities of both donor and recipient T cells. Thus, these results show thaTme a- and B-chains are sufficient to transfer the MHC-restricted antigen recognition from one cytotoxic T cell to another. A similar conclusion was reached by Saito et al. (157) in their transfection experiments with Th cell clones. In their experiment, functional a- and B-genes from an I-E 13 restricted cytochrome c-specific murine helper T cell clone were transfected into a human Jurkat cell line. Their findings show that the reconstituted a- and B-chains together can transfer specific co-recognition of MHC plus antigen from the murine T cell to the human T cells. These results therefore extend the observations of Dembic et al. (156) in that the a- and B-chains are sufficient to confer co-recognition of antigen and MHC molecules in Th cells. Attempts have also been made to correlate the structure of the a- or B-chain with antigen or MHC recognition by comparing the sequences of a- and B-genes of T cells with different specificities and so far, no simple correlations have been found (158-161). A striking example comes from the observation that cells recognizing class I and class II MHC antigens can utilize the same V or J gene segments (160). iii. The role of the CD3 complex in T cell activation The a- and B-chains of the T-cell receptor are associated with several other membrane proteins, collectively referred to as the CD3 complex. The CD3 complex is composed of three polypeptides which have been termed CD3-y (25 kDa), CD3-8 (20 kDa) and CD3-E (20 kDa) (162-163). Recently, cDNA clones encoding these proteins have been isolated (164-166). The deduced nucleotide sequences of these genes show that CD3 proteins are not homologous to immunoglobulins, indicating that they are not part of the antigen recognition structure of T cells. The CD3 proteins also contain a negatively charged residue in their transmembrane segment It has been speculated that this residue may form a salt bridge with the positvely charged residue in the transmembrane segment of the aB heterodimer (167-168). In addition, the CD3 proteins contain cytoplasmic domains which are much larger when compared to those of the aB heterodimer. It has been suggested that the cytojjlasmic domains may play a role in signal transduction in T cells. Several lines of evidence suggest that the CD3 proteins and the aB heterodimer are intimately associated on the surface of T cells. It has been found that the T-cell receptor comodulated with CD3 antigens on T cell clones (169). Under certain conditions, 14 irnmunoprecipitation of the CD 3 proteins resulted in coprecipitation of the T-cell receptor (170). Treatment of T cells with bifunctional reagents resulted in the cross-linking of CD3 proteins and T-cell receptors (171). T cell mutants independently selected for CD3- or T-cell receptor- loss exhibited a concomitant loss of expression of both CD3 and T-cell receptor, thus indicating that cell surface expression of CD3 requires the co-expression of the cxB heterodimer and vice versa (172). Together, these experiments provide compelling evidence for the existence of the CD3 proteins and the txB heterodimer as a multi-subunit complex on the surface of T cells, with the aB heterodimer recognizing antigen and MHC and the CD3 complex responsible for signal transduction across the membrane. The role of the CD3/antigen receptor complex in T cell activation has been delineated with the use of mAbs to the T-cell antigen receptor or CD3 (173-181). Perturbation of the antigen receptor/CD3 complex with mAbs results in a rapid increase in intracellular calcium concentration ([Ca+^]i) (173,174). Since Ca+^ ionophores can stimulate T cells and lectins can also increase [Ca+2]i, this suggests that changes in [Ca+^]i are physiologically important in T cell activation (173,174). Even when extracellular [Ca+^] was reduced by the addition of a Ca+^ chelator, anti-T-cell receptor antibodies still caused an increase in [Ca+2]i. This change in [Ca+2]i, even in the absence of a Ca+^ gradient across the plasma membrane, must be due to the release of Ca+^ from intracellular stores (175). In the presence of phorbol myristic acetate (PMA), antibodies to either CD3 or the antigen receptor can activate Jurkat cells to produce IL-2. Neither the PMA alone nor the antibodies alone can induce IL-2 production (173,174). This result indicates that the antigen receptor/CD3 complex-mediated increase in [Ca+^]i is not sufficient to induce the production of IL-2 by Jurkat cells. A second signal in the form of PMA, a known activator of protein kinase C, is also required for IL-2 secretion (175-177). Perturbation of the antigen receptor/CD3 complex also generates an increase in inositol triphosphate (TP3), a degradation product of phosphotidyl inositol biphosphate (PIP2) which is a compound normally present in membranes (175). An increase in D^ 's breakdown products, inositol biphosphate (IP2) and inositol phosphate (IP) has also been noted (175). As 15 purified IP3 also releases from intracellular pools of permeabilized Jurkat cells, this therefore suggests that JP3 may act as a Ca + 2 mobilizing signal in intact cells (175). The final effect caused by the perturbation of the antigen receptor/CD3 complex is the increase in IL-2 and y-IFN mRNA transcription which leads to an increase of IL-2 and y-IFN secretion (173,174). However, this increase only occurs in the presence of PMA and free mAbs to the antigen receptor complex or if the mAbs were coupled to sepharose beads. Based on the above findings, a plausible mechanism for signal transduction in T cells has been proposed (180-182). According to this model, there are two essential signals required in the induction of proliferative responses and IL-2 secretion in Th cells. The first signal is provided by the perturbation of the antigen receptor/CD3 complex, which results in the activation of an enzyme called phosphodiesterase. Phosphodiesterase breaks down PIP2 into IP3, and JP3 then acts as a Ca + 2 mobilizing signal, thus causing the release of Ca + 2 from intracellular pools. Alternatively, JP3 can be degraded into IP2, IP and diacylglycerol (DG). If the concentration of DG is high enough, it can activate protein kinase C, an important second messenger in cellular activation mechanisms. The second signal can be provided by PMA or anti-antigen receptor or anti-CD3 coupled to sepharose beads and IL-1. The second signal causes the activation of protein kinase C and leads to an increase in lymphokine mRNA transcription followed by an increase in lymphokine secretion. iv. Role of cell adhesion molecules in T cell activation There are other cell surface molecules on the T cell surface which are apparently involved in T cell activation. By contrast to the T-cell receptor complex, which is important in the antigen-mediated signal transduction, these molecules may be important injnitiating conjugate formation between Th cells and their targets (135,183). Several molecules on the T cell surface have been implicated in conjugate formation and activation of T cells. These include molecules such as LFA-1 (184), CD2 (185), CD4 (murine L3T4) (186) and CD8 (murine Lyt 2) (187). Antibodies to these molecules have been found to either inhibit or induce T cell functions such as proliferation and lymphokine secretion (135,183-187). It has been 16 suggested that such cell adhesion molecules may play a role in increasing the avidity of the ternary complex between antigen receptor, antigen and M H C ; in the case of T h cells, the C D 4 molecules probably bind to the Ia molecules (135). Thus, T h cell activation may occur in the following manner: The first step requires the formation of conjugates between cell surface molecules on T lymphocytes and the target cells. The second step involves the formation of the ternary complex between the T-cell receptor, a class II molecule and antigen. Finally, the third step results in the signal transduction through C D 3 across the plasma membrane and subsequent activation of a second messenger which w i l l mediate the induction of proliferation and lymphokine secretion (135). D . Th cell heterogeneity Several studies indicate that T h cells do not comprise a homogeneous T-cell subset. Th cells are generally subdivided into two categories which are distinguished on the basis of surface phenotype and function (188). One category of Th cells, T h l , is composed of the classical MHC-restricted Lyt 1 + , L 3 T 4 + helper cells which are nylon wool-nonadherent, and lack la-encoded determinants (22,90,189-190). The other Th cell , Th2, reported to be Lyt 1 + , L3T4" (191), are nylon wool adherent (22) and express Ia determinants (22,189,190), but may be MHC-unrestricted in their interaction with B cells (188). The activity of Th2 can often be replaced with lymphokines (189,190) and in at least some systems, Th2 is involved in idiotype-anti-idiotype interactions (89,189-192). T h l and Th2 cells are now available as clones which further confirms the fact that Th cells with distinct functions do exist (193,194). Recently, Mosman et al. (193) studied a panel of T h l and Th2 clones and it was discovered that these Th subsets differ in the lymphokines they produce. T h l do not require supplementation with IL-2 for growth and T h l produces IL-2, y-IFN, colony stimulating factors (CSF) and interleukin 3 (IL-3) in response to antigens such as cMckejured blood cells ( C R B C ) or mouse alloantigens. B y contrast, Th2 is found to be dependent on IL-2 for growth and Th2 produces EL-3 and BSF-1 in response to antigens such as mouse alloantigens, fowl gammaglobulins, and K L H . Clonal populations of Th cells can also exhibit functional heterogeneity. Asano et al. (195) isolated a single Th cell clone which can collaborate via two 17 modes of interactions with B cells depending on the dose of antigen given. A t a low concentration of antigen, the T cell clone cooperates with B cells in an MHC-restricted manner. However, at high concentrations of antigen, this Th cell clone can provide help in a nonspecific way. The interaction of this Th clone with accessory cell was still MHC-restricted but the Th-B cell interaction was not genetically restricted. Thus as a whole, T h cells can operate via different pathways in the activation of precursor B or T cells, and depending on the type and dose of antigen, different Th cells may be activated, or conversely, different modes of help can be mediated by a single Th cell. 3. Immune regulation and T-cell derived factors Th cells can also mediate their action by producing a variety of soluble mediators with distinct biological activities. Some of these mediators can act in an antigen-specific fashion (28,31,37,196-199), and others in a non-antigen-specific manner (70). In the past, the elucidation of the nature and function of these factors has been complicated by the lack of purified sources of factors. But with the advent of T cell c loning, combined with the development of specific assays for lymphokines, it is now possible to purify some of these factors to homogeneity (200-203), clone the genes encoding them (204-207) and to identify the function of lymphokines which are the products of cloned genes (204-207). Consequently, it is now well established that distinct T cell factors can in fact replace regulatory T cells in the activation, proliferation and differentiation of precursor T and B lymphocytes (70,208-211). A . Non-antigen specific lymphokines i . Role of T-cell derived factors in B cell responses Since the discovery of T-cell replacing factors involved in the actuation of B cells by Dutton et al. (25), extensive studies on the regulation of B cell responses have been made and the presence of B cell-specific growth and differentiation factors has been demonstrated (70,208-210,212). Many B S F s have been reported by several investigators in different experimental systems (209), and the exact number of different factors that are involved in the 18 regulation of antibody responses remains to be determined. A t the present time, at least two B S F s have been purified and characterized in detail (202-203,208-210). The first factor is called BSF-1 or interleukin 4. BSF-1 is a T-cell derived lymphokine with a molecular weight of 20 k D a (212). BSF-1 exhibits a variety of functions in the immune response (210). It acts on resting B cells by increasing the levels of expression of MHC-class II molecules on its surface, thereby enhancing the capacity of B cells to act as antigen presenting cells (213). BSF-1 also acts as a co-stimulant with antigens in the proliferative responses of B cells; it stimulates D N A synthesis in cells activated with anti-Ig antibodies (214-215). Furthermore, BSF-1 causes the secretion of IgG j and IgE by B cells stimulated with lipopolysaccharide (LPS) (216-218). Apart from their effects on B cells, BSF-1 also stimulates as well as maintains the growth of normal T cells, some T cell lines, some mast cell lines and several other hematopoietic lineage cells (219-224). The second factor involved in the regulation of B cell responses is BSF-2 (200). B S F - 2 has recently been purified to homogeneity and has been found to have a molecular weight of 21 k D a (203). BSF-2 does not have any growth activity on activated B cells, instead it acts as a B-cel l differentiation factor, which induces activated B cells to become Ig-secreting cells (209). After years of intensive research on the role of T lymphokines in B cell responses, a three-factor requirement scheme has now been proposed for the activation of B cells into antibody-secreting cells (208,209). The first step involves the activation of resting B cells and this is mediated by anti-IgM reagents and B S F - 1 . Following activation, a B - cell derived lymphokine (225) together with BSF-1 cause the clonal expansion of activated B cells. Finally, BSF-2 induces the final maturation of activated B cells into antibody secreting cells. i i . Role of T cell lymphokines in T cell responses __ a s^-- x Similar to B cell responses, the helper T cell requirement in T cell responses can also be replaced by lymphokines (70). One of these lymphokines, referred to as I L - 2 , is the major lymphokine involved in T cell responses (211,226). The primary role of IL-2 is to stimulate T cells to progress from G j to S phase of the cell cyc le , thus ensuring the clonal expansion of 19 antigen reactive T cells (211). Other functions include maintenance of T cell growth in long term cultures (227), induction of C T L responses (228), augmentation of natural killer ( N K ) activity (229), B cell growth and differentiation (230), activation of lymphokine-activated killer cells (231) and the induction of other lymphokines such as y-IFN (232). A s with any polypeptide hormones, IL-2 molecules mediate their effects by binding to specific sites on membrane receptor molecules (233). Although the effects of IL-2 are nonspecific, specificity is nevertheless maintained since only cells specifically activated by antigens can express IL-2 receptors. Therefore in T cell responses, it is the concentration of IL-2 and the number of IL-2 receptors expressed which determines the magnitude and duration of the response to a particular antigen. In addition to IL-2, other regulatory molecules have been implicated as having some influence in T cell responses (232,234-239). The role of y-IFN in the generation of C T L is still not clear but it has been shown to augment C T L responses (234). y-IFN also has an important role in inducing N K activity, macrophage activation and modulates the expression of class II M H C molecules on B cells (235). Colony stimulating factor (CSF), which primarily affects the differentiation of myeloid stem cells (240), has an indirect effect on T cell responses; C S F produced by T cells induces IL-1 production by macrophages which in turn activates Th cells to produce IL-2 (236). More recently, other lymphokines such as receptor inducing factors (RTF) (237) and cytotoxic T cell differentiation factor (CTDF) (238-239) have been found to be involved in the induction of IL-2 receptor expression on antigen (or mitogen)- activated C T L - P and in the differentiation of activated C T L into mature effector cells, respectively. There may be other lymphokines involved in the induction of C T L responses but currently, at least three lymphokines are implicated in cytotoxic responses (237-239,241-242). One mechanism by which C T L s are activated would require the induction of IL-2 receptor expression on C T L - P by antigen. RIF serves to increase IL-2 receptor expression on the antigen-activated C T L - P . IL-2 in turn reacts with the IL-2 receptors and causes clonal proliferation of activated C T L . Finally, C T D F promotes the differentiation of these activated C T L into mature effector cells. B . Antigen-specific factors 20 i . Antigen-specific helper factors (ASHF) a. The nature of A S H F Another effector molecule produced by Th cells upon antigenic stimulation is the A S H F . A S H F s were first discovered in 1972 by Feldmann and Basten who found that culture supernatants from KLH-activated T cells contain an Ig-like molecule which can specifically help dinitrophenyl (DNP)-primed B cells to respond to D N P - K L H in vivo (243). Soon after this, Taussig showed that another A S H F , obtained from T cells "educated" to ( T , G ) - A ~ L , can also exhibit helper activity in vitro (244). B y contrast to Feldmann and Basten's factor, this A S H F does not have an Ig nature (244). Following these initial discoveries, A S H F s have been described in many systems including immune responses to proteins, haptens, synthetic polypeptides, heterologous red blood cells and tumor cells (31,37,196-199). However, the results obtained from different experimental systems are not consistent. For instance, although all of the A S H F were found to have specific antigen binding properties and specific helper activities, controversies still remain with regards to issues such as the antigenic determinants present on the A S H F molecule, the genetic restriction properties of A S H F s as well as the target cells for A S H F s . Some of the problems associated with earlier studies could be caused by the use of heterogeneous T cells as sources of A S H F . Several attempts were made by many investigators to produce monoclonal sources of A S H F s and T cell clones which produced A S H F s specific for ( T , G ) - A - L (42), chicken gamma globulin ( C G G ) (43,115), K L H (245) and streptococcal antigen (246) have been isolated. Eshhar et al. were the first to report the establishment of T cell hybridomas that constitutively produce A S H F (31,37,42). The (T,G)-A~L-specif ic helper factors produced by the hybridoma clone, R9 , are capable of specifically helping the antibody responses of bone marrow cells in vivo and unprimed or hapten-primed B cells in vitro. The (T,G)-A~L-specific helper factor can specifically bind to (T,G)-A—L-sepharose beads and it expresses variable region heavy chain (Vjj) determinants, idiotypic (Id) determinants as well as I -A 2 1 subregion-coded determinants. This A S H F is composed of two non-covalently associated subunits; one bears the Ia determinant and the other expresses the Id determinant. Molecular weight determination by S D S - P A G E indicates that the (T,G)-A~L-specific helper factor is a molecule of 5 0 - 7 0 k D a ( 4 2 ) . Another cloned T cell hybridoma specific for C G G has been characterized by Lonai et al. ( 3 1 , 3 7 , 4 3 , 1 1 5 ) . The CGG-specific helper factor is active in a secondary in vitro antibody response to a hapten-CGG conjugate. B y contrast to the (T,G)-A~L-specific helper factor, the CGG-specific helper factor only binds C G G i f presented by syngeneic macrophages in association with Ia antigens. In addition, IL-1 or IL-1- l ike molecules are also required for specific antigen binding. Apart from binding macrophages, this CGG-specific factor can also be absorbed by B cells provided the B cells share homologous Ia antigens with the factor. Similar to the (T,G)-A~L-specific helper factor, CGG-specific helper factor also expresses Ia determinants. The number of chains of the CGG-specific helper factor was not determined and the molecular weights associated with the CGG-specific helper factor were in the 6 0 - 7 0 kDa and 2 5 - 3 2 k D a range ( 1 1 5 ) . A different kind of A S H F exhibiting antigen specificity for K L H was described by Tada et al. ( 2 4 5 , 2 4 7 , 2 4 8 ) . This helper factor has been named T-cell augmenting factor (TaF) because it can augment the secondary responses of D N P - K L H primed spleen cells by acting on T cells, instead of B cells. TaF cannot replace T helper cell activity and it is thought that the target cell of TaF is a T cell which directly helps B cells to produce antibodies. Although TaF can bind free antigen, its biological activity is I -A restricted. Similar to the aforementioned A S H F s , TaF also expresses M H C determinants and V J J determinants. In addition, TaF also expresses a determinant that is detected by a m A b which is supposedly reactive to the constant region of the T-cell receptor (Tind). Furthermore, TaF is a two-chain molecule with a molecular weight of 7 0 kDa; one chain contains the V J J and Tind determinants'and the other contains the l a determinant More recently, a streptococcal antigen-specific helper factor from a monkey and mouse cell line has been characterized ( 2 4 6 ) . This helper factor contains determinants which 22 are homologous to 62 microglobulin. In addition, this helper factor can also bind to a rabbit anti-mouse helper factor antibody column and can react to antisera directed against I -A or I-E. The molecular weight has been found to be 70 kDa but the subunit structure of this factor was not determined. In light of the information gathered from A S H F studies, some generalizations and speculations on the structure have been made (31,37,196-199). A S H F s bind antigen either in a free form or in an MHC-restricted manner. Except for TaF, all of the A S H F replace T cells in helping antibody responses. The molecular weight of A S H F seems to be in the range of 50-70 kDa. A S H F s contain M H C determinants which can either be encoded by I-E and/or I-A. The A S H F is composed of two chains; one chain containing M H C determinants and the other containing V J J determinants. Since all of the factors tested reacted with antisera specific for V J J or Id determinants and it is now established that T cells do not use V J J genes for their antigen recognition structures (132-133), it can therefore be assumed that A S H F s must share common ancestral origins with immunoglobulins. b. Mode of action of A S H F Although some efforts have been attempted to study the mode of action of A S H F , this issue is still unresolved. Based on the limited data available on the mode of action of A S H F , different models have been offered to explain several ways by which A S H F s help in immune responses. One model, proposed by Feldmann (66), suggests that A S H F s which are cytophilic to macrophages can act by "focusing" the antigens onto B cells. In this case, the role of the Ia determinants is speculated to be important as an interaction molecule with the macrophage. Therefore it is suggested that macrophages, but not B cells, have to share M H C identity with the A S H F (66). Another model implies that there is an acceptorjsite for A S H F on B cells and that the A S H F and B cells interact v ia the antigen bridge, the result of which leads to B cell activation (249). The role of antigen in such a scheme is suggested to be involved in concentrating the factors and focusing the factors onto the receptors, thereby facilitating the interaction of the A S H F s with their acceptor site on the B cells (250). A totally different 23 scheme was suggested by Tokuhisa et al. (247) to explain the mode of action of their TaF. In their model, there are at least two types of helper factors and cell types involved. The first Th cell, a Lyt-1+ T cell, produces TaF upon antigen stimulation. TaF then acts on the second Th cell in an la-restricted manner. The second Th cell is triggered by both antigen and TaF to release the classical ASHF. This ASHF in turn acts on a macrophage or B cell to mediate its classical helper effect This model is similar to that elucidated for T suppressor cells and their factors (TsF) where there are at least three types of TsF with distinct structures and functions involved in the suppression of an immune response (251). However, before it can be judged that a parallel mechanism is at work in the helper regulatory pathway, more work is required in the characterization of ASHFs and their function in immunoregulation. ii. Antigen-specific suppressor factors (TsF) To date, TsFs have been characterized in much greater detail than ASHFs (31,37,199). Because of the technology of T cell immortalization, progress in this field has been so fast that TsFs have been isolated in virtually every aspect of the immune response and for almost all classes of antigens (31,37,196-199). Among the TsFs that have been isolated, the TsF for GAT (252-254), SRBC (255-256), KLH (257-259) and the hapten NP (260-262) have been studied most extensively. In general, TsF mediating suppression of IgG responses are Igh+or Id+, IgC" and 1-1+ (252-259). Except for the KLH-specific TsF (257-259), all of these TsFs do not exhibit MHC restriction (252-256). By contrast, TsFs mediating DTH suppression (260-262) are composed of TsFs with distinct structures and functions. TsFl is Id+, I-J+ and is Igh-restricted. TsF2 is I-J+ and exhibits both Igh and I-J restriction. TsF3 is similar to TsFl, except that it exhibits both Igh and I-J restriction. Apart from expressing different determinants, different TsF species have been found to have variable molecular weights and contain different numbers of subunits. The GAT-specific TsF appears to be a 29 kDa molecule consisting of only one chain (252-254). The SRBC-specific TsF appears to be comprised of two covalently linked subunits; one (45 kDa) suppresses antibody responses nonspecifically and does not bind antigen, and the other (24 kDa) binds antigen but lacks biological activity (255-256). The KLH-specific TsF (257-259), on the other hand, has two 24 non-covalently linked chains with one chain binding antigen and the other expressing I-J determinants. The apparent molecular weight of this TsF is between 42-68 kDa. A plausible reason for the many seemingly contradictory characteristics of TsF is that there may be distinct types of TsF at play in the various experimental systems being studied (263). Evidence showing that such may be the case has been demonstrated in some systems. In the G A T system (252-254), it was shown that the single chain, non-MHC restricted TsF can actually induce the production of a two-chain, MHC-restricted TsF. Similarly, in the NP system (260-262), an antigen-specific Id + TsF called inducer TsFl was found to induce the production of an anti-Id TsF2, which in turn induces an antigen-specific TsF3, the factor responsible for directly suppressing the Th or the B cell. Thus, in light of these findings, it has been concluded that a suppressor cell cascade is present among distinct T suppressor cells and their respective TsFs, and the multiple interactions between these elements are formed to provide a sophisticated means of regulating the immune system. iii.Antigen-specific T cell factors and T-cell antigen receptors T cell factors were originally assumed to be the secreted form of T cell receptors because they offer many characteristics which make them suitable candidates for the T-cell receptor (27,28,196-199). For example, both T cells and their factors recognize antigen, either in free form or associated with M H C antigens (27,28,196-199). Both may bear idiotypes or Vpj determinants, suggesting that T cells may use V J J genes to encode for their antigen recognition structures (27,28,196-199). Both T cells and their factors were found to bear Ia determinants (22,27,28,196-199). Anti-la antibodies have been shown to block antigen binding to la-positive T cells (43,188). Thus, it was tentatively assumed that la-bearing molecules may be related to the T-cell receptor. Unfortunately, despite all these similarities, efforts to study the nature of the T-cell receptors and their genes through thejuseof antigen-specific T cell factors have only met with failure. Recently, the nature of the T-cell receptor and its genes have been characterized (132-135). It is now clear that T-cell receptors contain variable portions which are homologous to immunoglobulin Vpj determinants. T-cell receptors do not bear Ia determinants. These 25 molecules have a molecular weight of about 90 kDa and contain two glycosylated polypeptide chains a and B, both of which contain a variable region. Furthermore, unlike Ig genes which encode for constant region isotypes that have different effector functions, T-cell receptor genes do not appear to transcribe mRNAs with constant region isotypes (264). In contrast, antigen-specific T cell factors contain Ia determinants, have molecular weights of 50-70 kDa and possess two chains, one bearing the V J J determinant and the other the Ia determinant (199). Therefore, in light of these differences and the fact that all the evidence pointing to the possibe relationship between the T-cell receptor and T cell factors were mostly based on serological data, further data are required before we can determine the relationship between antigen-specific T cell factors and T-cell receptors. 26 4. Thesis objectives From the above account, it is clear that ASHF-producing T cell clones are still relatively rare. At the time that this project was initiated, practically all of the monoclonal ASHFs under investigation have been involved in enhancing B cell responses. Moreover, the mechanism whereby these ASHFs deliver their help is still speculative. Therefore for the purpose of this Ph.D. thesis, I have taken upon the task of isolating ASHF-producing clones from a mixed lymphocyte reaction (MLR) (Chapter 2). One of these clones, with specificity for D*\ was characterized and later fused with a T lymphoma line to generate ASHF-producing T cell hybridomas (Chapter 3). The ASHF produced by a cloned T cell hybridoma was characterized with the use of mAbs raised against the putative ASHF and the Vg g family of the T-cell receptor (Chapter 3). Finally, the role of the Db-specific Th cell clone and its soluble products, which include ASHF and a non-antigen-specific helper factor distinct from IL-2, in the induction of C T L responses was investigated (Chapter 4). 27 Chapter 2. Characterization of a D^ specific T helper clone 1. Introduction One common method of propagating Th cell clones in vitro is to expand the antigen -specific Th cells in medium supplemented with TCGF and antigen (53-57), and to subsequently clone the Th cells by limiting dilution in the presence of both TCGF and antigen (53,55). By using this method, Apte et al (57) have obtained a Th cell line that produces ASHFs bearing H-2 and Vpj determinants. Helper factors produced by this line enhance antibody responses of antigen-primed B cells. Others (53-55,57), using a similar method, have reported the isolation of another type of T helper clone that responds to stimulation with antigen by producing antigen non-specific growth promoting factors. These clones or their factors help antibody and CTL responses by promoting the proliferation of activated B and T cells. In this chapter, the characterization of a Th cell clone derived from a mixed lymphocyte culture is described. By contrast to other Th cells which function by initially recognizing the stimulating antigen, this novel IL-2 dependent H-2^  T cell clone augmented CTL responses via the recognition of antigens on the responder cells. 2. Materials and methods a. Animals. C57BL/6 (B6), DBA/2 (D2), AKR and CBA/J (CBA) mice were purchased from the Jackson Laboratory, Bar Harbor, Maine. C57BL/10 (BIO), BIO.A, BIO.A (2R) and B10. A (3R) were bred in the animal facility in this department. Wistar rats were obtained from the Animal Care Center at the University of British Columbia. b. Culture Medium.The culture medium used was RPMI1640 (GD3CO) supplemented with 28 25mM NaHCO^ 10% fetal calf serum (Animal Health Laboratories, Toronto), 10 mM HEPES buffer, pH 7.2,5 x 10"^  M 2-mercaptoethanol, 50 units/ml and 50 |ig/ml of penicillin-streptomycin (GIBCO) and 30 Ug/ml glutamine. Al l cultures were incubated at 37° in humid 5% CO2 in air. c. Sources of IL-2. Rat T C G F was prepared according to the method of Gillis et al. (227). Briefly, NH4CI treated spleen cells from 2-3 months old Wistar rats were cultured at lofyml with 5 ug/ml Con A for 48 hours in culture medium. The culture supernatants (Con A SN) were harvested, filter sterilized and stored at -20°C until use. BL-2-containing supernatants were also prepared from a variant of the murine T lymphoma line, EL4-IL-2, as described (265). The EL4-IL-2 cells were grown in complete medium to a final concentration of about 1 x 10^ cells/ml. Large numbers of this variant were obtained by growing the tumor cells in the ascitic form in B6 mice. For factor production, the EL4-IL-2 cells were cultured at 10 6 cells/ml in medium supplemented with 5% horse serum (GIBCO) and 10 ng/ml PMA (Sigma Chemical Co., St. Louis, MO.) for 24 hours at 37°C. The supernatant (EL4.PMA) was filter-sterilized and stored at -20°C. The activities of the IL-2 preparations were calibrated by testing for their ability to promote the proliferation of an IL-2-dependent murine T cell Une, M T L 2.8.1 (266). An IL-2 unit of activity was defined as the amount of IL-2 that was required to give 50% of the maximal proliferative response of M T L 2.8.1 cultured at 2.5 x 10* cells/ml for 2 days; 1 [id of [ 3 H ] Thymidine (TdR) was added for each 0.20 ml of culture during the last 16 hr of the culture period. Typically, crude EL4.PMA and Con A SN had EL-2 activities of about 500 to 1000 U/ml and 30-60 U/ml, respectively. The EL4-IL-2 variant and M T L 2.8.1 cell lines were obtained from Dr. Vern Paetkau of the Department of Biochemistry, University of Alberta, Edmonton, Canada. d. Generation of long term cultures and maintenance of clones. A primary M L R was performed by culturing 5 x B6 responding spleen cells with the same number of 2000 rads irradiated (60 Co source Gamma Cell 220, Atomic Energy of Canada, Ltd.) D2 spleen cells in a total 29 volume of 50 m l in Falcon 3024 flasks. Four days later, the cells were harvested and 5 x 1 0 ^ primed responder cells were restimulated with the same number of irradiated D 2 spleen cells. After three days of incubation, 10^ M L R blasts were cultured in Falcon tubes (no. 2057) containing 1 m l of C M (25% Con A S N , 50 m M alpha methyl mannoside in complete medium). Cultures were fed with C M every 3-4 days and were restimulated with 10^ irradiated D 2 spleen cells weekly. Cultures were reseeded when the cell density approached 10*\ One month after the initiation of the cultures the density of D2 stimulator cells added weekly was increased to 2.5 x 1 0 6 cells per tube and 10^ irradiated (2000 rads) B 6 spleen cells were also added weekly to serve as accessory cells. After two more months in culture the long term cells were cloned by limiting dilution in 96 well Costar flat bottom plates. The cells to be cloned were diluted to an average of 0.3 cells per well . 1 x 10^ irradiated D 2 spleen cells were added to each well . The wells were fed on days 5 and 10 with C M . On day 14, wells with growth were identified by observation under an inverted microscope. Cytolytic clones were screened by using the ^Cr release assay (267). Non-cytolytic clones were tested for their abilities to help a primary B 6 anti-D2 cytotoxic response. Clones with detectable functions were maintained in flasks (Falcon 3012) and reseeded weekly at 1 0 5 cells per flask in 10 m l medium containing C M , 2.5 x 10^ D2 irradiated spleen cells and 10^ B 6 irradiated spleen cells. Using this method, four H-2^-specific cytolytic clones and three clones with helper activity for the B6 anti-D2 C T L response were isolated; only the helper clones were further characterized. O f the three helper clones isolated, clone 9 was the most stable. Clone 9 had been periodically subcloned at 0.3 cells/well. Both clone 9 as well as its subclones were used for this study. Since results obtained with both clone 9 and its subclones were similar, both clone 9 as well as its subclones have been referred to as clone 9. Clone 9 had a cell doubling time of approximately 36 hours. It required Con A S N , D 2 and B 6 irradiated spleen cells for its long term growth. Furthermore, helper activity could be recovered from cells that had been stored in liquid nitrogen. For freezing of cells, the method of Dr . H . Mostowski of the National Institute of Health, Bethesda, Maryland was used. Cells were washed twice in complete medium and at least 3 x 10^ cells were resuspended in 0.5 m l of 10% Dextran (Sigma)-90% fetal calf serum solution. The cells were incubated for 30 minutes to 1 hour at room temperature. Then 0.5 ml 30 of an ice cold mixture of 10% dextran, 15% DMSO and 75% FCS were added to the cells dropwise while vortexing at a low speed. The mixture was transferred to a freezing vial. The vial was stored in a styrofoam box at -70°C for 24 hours and then transferred to liquid nitrogen. To thaw, vials were warmed rapidly in a 3 7 ° C water bath. Cells were washed twice with complete medium and resuspended in 2 ml of medium consisting of C M , 10^ B6 and 2.5 x 10^ D2 irradiated spleen cells. The cells were fed with C M after 2-3 days. When sufficient growth was detected, the cells were transferred to flasks. To prevent loss of activity by overgrowth of inactive cells or by microbial infection, clone 9 cell cultures were periodically recloned and tested for mycoplasma infection with the Mycotect kit (BRL). * e. Cell surface marker analvsis.The H-2, CD4, Lyt-1, Lyt-2 and Thy-1 markers of clone 9 were analysed by antibody-dependent complement-mediated cytotoxicity assay. Anti-D d ((BIO.AKM x A.SW)F 1 anti-ATH), anti-Kd ((BIO x A)F1 anti-B10.D2), anti-Db ((B10.A (5R) x A)Fj anti-BlO.A (2R), anti-Kb ((A x B10.D2)F1 anti-B10.A(5R)), anti-Lyt-1.1 ((Balb/C By xB10)F 1 anti-B6-Lyt-l.l), and anti-Lyt-2.1 (B10.K anti-B6.H-2k.CE) sera were obtained from the National Institute of Allergy and Infectious Diseases, Bethesda, M D . The monoclonal anti-Thy-1.2 antibody was a rat anti-mouse Thy-1.2 mAb prepared as described (268). The rat monoclonal IgC^b antibody, G K 1.5, developed by Dialynas et al. (186) was obtained from the American Type Culture Collection. This mAb is specific for the CD4 cell surface molecule, a marker for helper T cells (186). For treatment of cells with these antibodies, 5 x 10 5 cells were incubated with 0.1 ml of the appropriate antibody for 30 min at room temperature. The cells were then washed twice and incubated for 40 min at 3 7 ° C with 0.1 ml "low tox" rabbit complement (Cedarlane Laboratories, Ontario) diluted eight times with RPMI1640 plus 10 mM HEPES. The cells were washed once and the numbers of live and dead cells were scored by dye exclusion; an average of 200 cells was counted. f. Assay for helper activity. Clones were washed thoroughly with prewarmed medium and 104 31 irradiated (2000R) cells were mixed with 3-5 x IO 3 B6 responder spleen or lymph node (LN) cells and 10^ irradiated D2 stimulator spleen cells. These cells were cultured in 96-well V-bottom microtiter trays (Flow Laboratories, Cat. No. 76-023-05) in a volume of 0.2 ml. After 5 days the cultures were assayed for C T L activity using 2 x 10 3 -^Cr-labelled target cells as previously described (267). Target cells used included P815 mastocytoma cells (H-2d) or RDM-4 (H-2^) cells, maintained weekly by intraperitoneal passage through D2 mice or A K R mice, respectively, and LPS blasts, which were prepared by incubating 107 spleen cells with 20 ug/ml LPS (Sigma) in 10 ml complete medium for 48 hours. After 3-4 hours of incubation with the target cells, the cultures were centrifuged, and 0.1 ml was removed and counted in a gamma counter. Ninety percent of the total release count was taken as the value for maximal release. Data are presented as % specific -^Cr release (experimental count-spontaneous release/maximal release count -spontaneous count)x 100. Spontaneous release was determined in supernatants of wells containing stimulator cells only. In all experiments the spontaneous release of the various targets ranged from 10-25% of maximum releasable counts in a 3-4 hour ^ C r release assay. Data are means of six to eight cultures and experiments were always repeated at least twice for reproducibility. A more sensitive assay for the detection of clone 9 helper function was subsequently developed. This involved mixing 5 x 103 responder B6 cells, 10* irradiated stimulator cells and 10* helper cells in V-bottom wells in a volume of 0.2 ml. After 1 day, a final concentration of 10-20% Con A SN and 50 mM alpha methyl mannoside were added to the cultures. On day 5, cultures were assayed for C T L activity using a -^Cr release assay. g. Clone 9 supernatant factors. 7 day cultures of clone 9 were seeded at 2 x 10^ cells in 2 ml complete medium for 48 hours. The supernatant was harvested and concentrated 10-20 times by UM10 Amicon ultrafiltration before use. h. Detection of IL-2 activity in clone 9 supernatant. Two assays were used: a) 5 x 10 3 M T L 2.8.1 cells were incubated with Con A SN or clone 9 supernatant After 24-48 hours the cultures were pulsed with 1 jtCi 3 H-TdR for 6 hours, harvested onto glass fiber filters and 32 counted in a scintillation counter, b) 10 5 thymocytes and 2 | i g / m l Con A were incubated with Con A SN or clone 9 supernatant in round bottom microtiter wells. On day 3, cultures were pulsed with ^H-TdR for 6 hours; then harvested and counted (269). i. Absorption of helper activity with Con A blasts. Con A blasts were prepared by incubating IO** spleen cells per ml with 2 n g / m l Con A for 48 hours. 0.5 ml of clone 9 supernatant was absorbed twice with 5 x 10^ Con A blasts at 3 7 ° C for 1 hour. j . Pulsing of responder cells with clone 9 supernatant. 10^ responder cells were incubated with 1 ml of supernatant factors for 1 hour at room temperature. The cells were then washed and used as responders in the helper assay. 3. Results a. Generation of Clone 9 cells. A primary mixed lymphocyte reaction was performed by culturing B6 responder spleen cells with irradiated D2 spleen cells. Antigen-specific cells were enriched by restimulating cells from the primary culture with irradiated D2 and B6 spleen cells in IL-2-containing medium. After three months in culture, these antigen-primed cells were cloned by limiting dilution. The clones were expanded and screened for cytolytic activity or for their helper activity in a primary B6 anti-D2 cytotoxic response. Using this method, four H-2d-specific cytolytic clones and three clones with helper activity for the B6 anti-D2 response were isolated. Of the three helper clones isolated, clone 9 was found to have a cell doubling time of approximately 36 hours and it required Con A SN, D2 and B6 irradiated spleen cells for optimal long term growth. b. Cell surface markers on clone 9. The H-2, Thy-1, Lyt-1, Lyt-2 and CD4 phenotypes of clone 9 were determined by antibody-dependent complement-mediated cytolysis. The results in Table I (p.41) indicate that clone 9 is of H-2^ origin. Thus, clone 9 was killed by K d - a n d 33 Dd-specific antisera but not by K b - and Db-specific antisera. The EL4 (H-2b) and P815 (H-2d) tumor cells were included as positive and negative controls for the two antisera. The fact that clone 9 was found to be of the H-2 d phenotype was unexpected inasmuch as the mixed lymphocyte culture from which clone 9 was derived was supposedly of a B6 (H-2b) anti-D2 (H-2d) origin. This suggests that during the establishment of the clones some D2 stimulator cells must have survived the irradiation and became propagated as D2 T cells specific for B6, which were added as a source of accessory cells. The data in Table II (p.42) show that clone 9 is Thy-1+, Lyt-1" and Lyt-2". When clone 9 cells were tested for the T helper cell-associated marker, CD4, clone 9 cells were found to be 95% positive for the marker. Thus, clone 9 is an H-2d, Thy 1+, Lyt V2; CD4+ helper cell clone. c. Clone 9 is specific for the D - alloantigen. The ability of clone 9 to augment the CTL responses of B6 (H-2b) and AKR (H-2 )^ spleen cells was determined. Table HI (p.43) shows that clone 9 helped the response of B6 but not AKR LN cells to H-2 d alloantigens. However, clone 9 also helped B6 LN cells to mount a cytotoxic response to AKR (H-2 )^. This suggests that clone 9 may interact directly with the responder, but not the stimulator, cells. The fine antigenic specificity of clone 9 was mapped by detennining its helper effect on responder cells derived from different BIO recombinant mice. Table IV (p.44) shows that clone 9 only helped the responses of BIO, BIO.A (2R) and B6 but not BIO.A or BIO.A (3R) cells. Since B6, BIO and BIO.A (2R) cells express the D b alloantigens and BIO.A and BIO.A (2R) are D d and D b , respectively but identical otherwise, this suggests that clone 9 was specific for D b . When BIO.A or BIO.A (3R) responder cells were used, CTL responses could be detected when the cultures were supplemented with Con A SN, suggesting that the lack of help by clone 9 cells was not due to the lack of relevant CTL-P in these cultures. An alternative explanation for these data is that under this set of experimental conditions, i.e. low stimulator and responder cell doses, BIO.A and BIO.A (3R) are non-responders in CTL responses against K d and hence cannot be helped by clone 9 cells in mounting CTL responses to K d . However, this is unlikely inasmuch as it was shown in later experiments that helper factors derived from clone 9 cells were also incapable of helping CTL responses of B10. A responders to other than H-2 d 34 alloantigens (see Table XTV,p.54). d. Antigen specificity of C T L generated in clone 9-supplemented culmres. It has been reported recently that the inclusion of IL-2-containing supernatants in mixed lymphocyte cultures led to the induction of nonspecific C T L (268). These nonspecific C T L could kill a broad range of target cells, including syngeneic Con A- or LPS- induced splenic blasts. In view of this finding it was important to determine the specificity of C T L produced as a result of the helper action of clone 9. This was done by splitting each culture into 3 equal portions at the end of the culture period and assaying each portion against the specific target (D2 LPS blasts), the self target (B6 LPS blasts) and a third party target (CBA LPS blasts). As shown in Table V (p.45), C T L derived from cultures supplemented with clone 9 were specific for the stimulating antigen. By contrast, cultures supplemented with EL4.PMA gave high lysis values for all three targets; however, preference for the stimulator target was still observed. This experiment therefore illustrates that the C T L produced with clone 9 help were more specific for the stimulating alloantigen than C T L generated in the presence of EL4.PMA. Furthermore, this experiment shows that clone 9 is not a polyclonal activator of C T L . e. Requirements for clone 9 function. When the dose of D2 stimulator cells in the helper assay was reduced from 10^ to 10^ and the other conditions were kept the same, there was no C T L response generated (Table VI, p.46, compare line 3 with 10). This suggested that clone 9 might require accessory cells from the stimulator population for its function. As shown in Table VI (p.46), the addition of either irradiated B6 or A K R splenocytes enhanced the activity of clone 9. These results are consistent with the need for a genetically non-restricted accessory cell population. A further requirement for clone 9 function was that the helper cells must be added during the initiation period of the culture. Clone 9 only helped the C T L responses if it was added on day 0 but not on days 1 to 4 during a 5-day culture period (Table V H , p.47). f. Clone 9 supernatant contains helper activity. Supernatant factor(s) from clone 9 was obtained from 48 hr cultures of clone 9 seeded at 1-2 x lofyml in complete medium. This supernatant 35 was concentrated at least 10-fold before being tested for helper activity. The data in Table VLTI (p.48) demonstrate the presence of helper activity in clone 9 supernatant and map its specificity to D b . Thus only BIO.A (2R) was helped by clone 9 supernatant but not BIO.A or BIO.A (3R). The lack of C T L responses in the B10.A and B10.A (3R) groups could not be explained on the basis that relevant CTL-P were lacking in these cultures since C T L responses were detected when the cultures were supplemented with ConA SN. As was the case with clone 9 cells, the supernatant also had to be added on day 0 of a 5-day culture period in order to help the C T L response (data not shown). g. Clone 9 supernatant does not contain IL-2. The possibility that the active moiety in clone 9 supernatant was IL-2 was eliminated because of the following observations: a) It did not exhibit any potentiating effect in the thymocyte proliferation assay (Table LX, p.49). In previous studies (269), EL-2 was found to augment proliferative responses of Con A-stimulated thymocytes cultured at less than 10^ cells per ml. b) It did not support the proliferation of an IL-2-dependent T cell clone (Table X , p.50). In this study only Con A SN or EL4.PMA, but not clone 9 supernatant, was able to support the growth of M T L 2.8.1 cells, c) Its activity was not absorbed out by activated T cells (Table XI, p.51). Thus, the helper activity of Con A SN, but not clone 9 supernatant, was absorbed out by D2 Con A blasts. As expected absorption with D2 LPS blasts did not remove the helper activity in either Con A SN or clone 9 supernatant. h. Augmentation of cytotoxic responses by clone 9 supernatant in the presence of IL-2. The results above suggested that clone 9 required accessory cells for its function and showed that it did not mediate its helper function via IL-2. Since accessory cells are required for IL-2 production (270) it was possible that the accessory cell requirement for clone 9 might be replaced by IL-2. If this were the case, one would expect to observe a synergistic effect between clone 9 supernatant and Con A SN. This was tested and the results are shown in Table XII (p.52). One sees that when the stimulator dose was only 10* no response was observed in cultures supplemented only with clone 9 supernatant (Une 4). When Con A SN alone was 36 added a slight response (7.0%) was observed (line 7). However, when clone 9 supernatant was added together with Con A SN, a synergistic response (59.6%) was observed (line 8). It should be noted that augmentation by clone 9 supernatant was seen only in antigen-stimulated cultures (compare lines 6 and 8) and in cases where the responder cells were of B6 origin (Experiment 2). In this experiment synergy between Con A SN and clone 9 supernatant was observed in the B6 anti-CBA but not in the D2 anti-CBA C T L response (compare lines 10-12 and 14-16). i. The helper activity of clone 9 supernatant is specifically absorbed out by D^-bearing cells. When clone 9 supernatant was absorbed with BIO.A or BIO.A (2R) Con A blasts and then tested for its ability to augment C T L responses in the presence of Con A SN, it was found in two out of two experiments that the helper activity was completely (ExpL 1) or partially (ExpL 2) absorbed out by BIO.A (2R) blasts but not by B10.A blasts (Table XUI, p.53). This result indicates that the helper activity is capable of specific binding to D b alloantigens. In a third experiment the helper activity in clone 9 supernatant was absorbed by B6 but not D2 Con A blasts (Table XIII, p.53), a finding which is consistent with the interpretation that the helper activity is H-2^-specific. Another experiment that shows the same conclusion is illustrated in Table XTV (p.54). In this case, BIO.A or B10.A(2R) L N cells were incubated with clone 9 supernatant for 1 hour at room temperature and the cells were washed before being used as responder cells. The results show that the C T L responses of B10.A(2R), but not BIO.A, were augmented by pulsing with clone 9 supernatant. This again suggests that the helper activity in clone 9 supernatant binds to D b . It should be noted that both BIO.A ( K k D d ) and B10.A(2R) (K^D^) differs from CBA (K^D^) at the D-end of the MHC. Thus, in order to observe synergy between clone 9 supernatant and Con A SN, it is not essential for the responder and stimulator cells to differ at both the K and the D region of the MHC. Consequently, the lack of synergy between Con A SN and clone 9 supernatant when BIO.A (K^D d) responder cells and DBA/2 ( K d D d ) stimulator cells were used (see Table VIII, p.48) cannot be explained by the fact that the responder and stimulator cells are not entirely MHC-different Rather, this study further illustrates that clone 9 and its supernatant factor specifically helps D D responder cells. 37 3. Discussion In the present study, the characterization of a T cell clone which could participate in the activation of D b CTL-P was described. Clone 9 was derived from a mixed lymphocyte culture five years ago and had since been maintained in the presence of Con A SN, irradiated stimulator and syngeneic spleen cells. Although the original intention was to isolate B6 helper clones from a B6 anti-D2 MLR, clone 9 turned out to be of D2 origin. The most likely explanation for this is that clone 9 cells must have been derived from a D2 spleen cell which had survived the 2000 rads radiation dose. Clone 9 is therefore an allo-class -I-specific helper clone because it is a helper clone of H-2 d phenotype with a specificity against H-2 D b . Thus, in situations where irradiated stimulator cells are required for the propagation of the T cell clones, it is important to check the H-2 phenotype of the clones before proceeding with further characterization. To prevent the overgrowth of clone 9 by the irradiated stimulator and syngeneic spleen cells, precautions were made by periodically re-cloning clone 9 cultures and characterizing the subclones for antigen-specific helper activity in the induction of C T L responses. Furthermore, many batches of active clone 9 cells were kept in frozen storage until use. In recent years, several cloned lines of T helper cells have been isolated and their characteristics seem to differ from those of clone 9. Generally these helpers bear Lyt-1 antigens (271-274). They proliferate in the presence of antigen (55,59,275,276) and they usually recognize the antigen in the context of self Ia antigens (55,56,58,275-277). Upon antigenic stimulation, some helpers produce non-specific factors (54,55,59,272,275,276,279) which promote the amplification of B (275-279) and T cell (54,55,59,272,276) responses. Al l other T helper clones reported so far appear to help C T L responses by producing IL-2 (59,272). By contrast, clone 9 helped C T L responses by specifically recognizing D b responder cells. Plate et al. (280) had reported that allo-class I-specific helper molecules can also be produced by the draining lymph node cells a few hours following the implantation of a skin allograft. It is not 38 clear whether these two class I-specific helper factors) are related or different from each other. Recent studies suggest that CTL-P, upon interaction with antigen (or mitogen) and an early acting factor (referred to as RIF or T cell cytotoxicity inducing factor 1 (TCF1)), express receptors for IL-2 (73,241,242,281-285). IL-2 and a late acting factor (referred to as CTDF) are required for the proliferation and differentiation of IL-2R4" CTL-P into effector CTL, respectively (211,239,242,281-285). From the studies made so far with clone 9, it is still not clear as to how the helper action of clone 9 fits into the above scheme of T cell activation. However, the following points are established: 1) clone 9 was required only at the initiation of the culture, 2) the supernatant from clone 9 did not exhibit any properties similar to IL-2, 3) the target cell for clone 9 resides in the responder population, 4) the clone and its helper factor(s) both recognize D b and 5) the clone and its factor can synergize with IL-2-containing medium in cytotoxic responses to alloantigens. This mode of action of clone 9 is similar to the B cell helper clones described by Julius et al. (287) and Zubler et al. (277). In their studies with B cells, they found that in addition to the antigen signal, a specific Th signal, which can be provided by la-restricted Th cells specific for cell surface antigens on the B cells, is also required to activate resting B cells. Similarly, CTL-P may also require a specific helper signal for their activation. Thus, clone 9 or its factor(s) may act in concert with specific alloantigens in the activation of CTL-P. One could speculate that antigen-activated CTL-P might express more IL-2 receptors in the presence of clone 9 cells or its factor(s). This could account for the synergy observed between clone 9 or clone 9 factor(s) with IL-2-containing medium. Keene and Forman (101) reported that there is an obligatory requirement for two types of T helper cells in the sensitization of CTL-P in vivo: One helper population appears to activate resting CTL-P and another helper population appears to activate antigen-primed CTL-P. Clone 9 may represent one of these cell types. Alternatively, since it was not formally demonstrated that clone 9 directly interacted with CTL-P, one could not rule out the possibiUty that clone 9 could help the activation of CTL-P indirectly by interacting with other lymphocytes. The existence of helper clones specific for allogeneic class I molecules is an intriguing observation. Most Th cells appear to be restricted to self-class II molecules, i.e., Th 39 cells recognize nominal antigen in the context of self-class II molecules. However, it is conceivable that helper clones such as clone 9 are in fact restricted to a self-class II molecule and the interaction with D b represents a fortuitous cross-reaction between a M H C class II molecule plus antigen with D b . Alternatively, it is possible that clone 9 is a truly allo-class I-specific helper clone and is different from the self-MHC-restricted helper cells. Another characteristic of clone 9 and its supernatant factor is that their enhancing effect on C T L responses of D b responder cells to alloantigens, although reproducible, was rather small (see Table VHI, p.48, for example). This could be due to suboptimal culture conditions or that clone 9 and its factor could only act on a small subpopulation of D b CTL-P specific for alloantigens. Efforts to improve the culture conditions for the helper assay will be described in the following chapter. In addition, T cell hybridomas were generated by fusing clone 9 with the A K R lymphoma, BW5147. Supernatant factors from cloned lines of the hybridoma cells also specifically enhanced the C T L responses of D b responder cells to alloantigens (see Chapter 3). Further characterization of the helper factor from cloned lines will also be described in the next chapter. 40 5. Summary A T cell helper clone was derived five years ago from a mixed lymphocyte culture. This clone, referred to as clone 9, was propagated in IL-2-containing medium in the presence of irradiated stimulator and irradiated syngeneic spleen cells. Clone 9 was of H-2 d origin and was found to be Thy-1 + and Lyt-1"2". Clone 9, as well as supernatant factor(s) derived from it, were able to enhance primary cytotoxic responses of D b responder cells to alloantigens. Furthermore, clone 9 cells or its factor(s) were only active when added during the first 24 hours of a 5-day culture period. When a low stimulator cell dose (10^ cells per 0.2 ml culture) was used, it was possible to demonstrate that clone 9 also required a source of irradiated allogeneic splenic accessory cells to exert its helper action. Under these conditions clone 9 or its factor(s) could also synergize with IL-2-containing medium in mounting cytotoxic responses to alloantigens. Synergy between IL-2-containing medium and clone 9 or its factor(s) was observed only when D b responder cells were used. The helper activity in clone 9 supernatant was also specifically absorbed out by Con A-stimulated D b spleen cell blasts. Preincubation with clone 9 supernatant for 1 hr at room temperature also led to enhanced cytotoxic responses of D b responder cells to alloantigens. Clone 9 supernatant was also found to be devoid of detectable IL-2 activity. Thus, clone 9 or its helper factor(s) appear to exert its helper activity by an early interaction with D b responder cells. 41 Table I. H-2 phenotype of clone 9 CELL ANTISERA** CYTOTOXIC INDEXC KLA (H-2b) BL4 anti-D d 0.34 BL4 antl-K d 0.19 EL4 antl-K b 0.75 EL4 anti-D b 0.84 P815 (H-2d> • _ P815 antl-D d 0.84 P815 antl-K d 0.62 P815 antl-K b 0.01 P815 antl-D b -0.01 Clone 9 Clone 9 antl-D d 1.04 Clone 9 anti-K d 0.66 Clone 9 anti-K b 0.02 Clone 9 antl-D b -0.03 a. 5xl(P cells were incubated with 0.1 ml of the appropriate antisera at a final concentration of 1 in 50 for 30 minutes at room temperature. The cells were washed once and incubated with 0.1 ml or a 1/8 diluted low tox rabbit complement for 40 minutes at 37°C. b. Refer to Materials and Methods for details (p.30). c. Cytotoxic Index= fraction killed in experimental group - fraction killed in complement group / 1 - fraction killed in complement group. Table II. The Thy-1 and Lyt phenotypes of clone 9* CELL ANTISERA CYTOTOXIC INDEX B6 LN (Lyt-1.2) anti-Lyt-1.1 0.02 D2 LN (Lyt-1.1) M 0.45 Clone 9 M -0.03 B6 LN (Lyt-2.2) anti-Lyt-2.1 0.02 D2 LN (Lyt-2.1) " 0.64 Clone 9 M 0.02 EL4 (Thy-1.2+) anti-Thy-1.2 0.92 P815 (Thy-1.2~) M 0.02 Clone 9 " 0.90 a. The procedure used was similar to that described in Table I (p.41). b. Refer to Materials and Methods for details (p.30). Table IU. The helper activity of clone 9 is specific for B6 responder cellsa. 43 3 x 10 3 1 x 105 C L O N E 9 % SPECIFIC RESPONDER STIMULATOR (2000R) LYSIS L N CELLS SPLEEN CELLS (2000R) B6(H-2 b) - - 0.0 ± 0 . 2 B6 D2(H-2 d) - 2.3 ± 0 . 8 B6 D2 10 4 36.4 ± 6 . 9 AKR(H-2 k ) - - -0- 4 ± 0-5 A K R D2 - -1.5 ± 0 . 4 A K R D2 1 Q 4 -1.4 ± 0 . 4 B6 - - 0.0 ± 1 . 9 B6 A K R - -2.3 ± 2 . 1 B6 A K R 10 4 30.0 ± 5 . 1 a. Cultures were set up in conical bottom microtiter trays in a volume of 0.20 ml. After 5 days the cultures were assayed for C T L activity using 2 x 103 ^ C r - labelled P815 (H-2d) (lines 1-6) or RDM-4 (H-2^-) cells (lines 7-9). After 3-4 hours of incubation with the target cells, the cultures were centrifuged, and 0.1 ml of supernatant was removed and counted in a gamma counter. Ninety percent of the total release count was taken as the value for maximal release. Data are presented as % specific lysis =(Experimental count - spontaneous release count / maximal release count - spontaneous release count) x 100. Spontaneous release was determined in supernatants of wells containing stimulator cells only. The spontaneous release of the various targets ranged from 10-25 % of the maximum releasable counts. 44 Table IV. The helper activity of clone 9 is Db-specifica 3 x 10 3 H-•2 ALLELES 10 5 D2 io4 • ConA SN b Z SPECIFIC LYSIS RESPONDER STIMULATOR CLONE 9 (10Z) AGAINST P815 LN CELLS K IA IE D SPLEEN CELLS (2000 R) EXPT 1 EXPT 2 B6 b b b b _ 3.6 ± 2.3 0.4 ± 1.1 B6 b b b b + - - 5.5 ± 1.8 2.6 ± 1.0 B6 b b b b + + - 56.4 ± 6.5 28.7 ± 5.4 B6 b b b b + • n.d.' c 21.0 t 3.0 BIO b b b b _ 0.0 t 1.1 n.d. BIO b b b b + - - 6.2 ± 2.6 n.d. BIO b b b b • + 39.3 ± 5.6 n.d. BIO.A k k k d _ -0.1 t 0.2 1.9 ± 1.2 BIO.A k k k d + - - -0.5 ± 0.6 2.2 ± 0.8 BIO.A k k k d + - 0.5 t 1.0 6.0 ± 1.0 BIO.A k k k d + — •f n.d. 10.8 ± 0.9 B10.A(2R) k k k b _ -1.3 ± 0.4 0.2 ± 0.8 B10.A(2R) k k k b + - - 0.7 ± 1.2 1.6 ± 0.7 B10.A(2R) k k k b • - 23.4 ± 4.3 34.1 ± 4.8 B10.A(2R) k k k b + • n.d. 12.2 ± 5.1 B10.A(3R) b b k d -1.6 ± 0.7 0.7 ± 1.2 B10.A(3R) b b k d + - - -0.8 ± 0.5 0.5 ± 3.0 B10.A(3R) b b k d + + - 6.0 t 2.9 0.9 ± 2.3 B10.A(3R) b b k d + - • n.d. 15.6 ± 3.0 a. Culture conditions were similar to those in Table m (p.43) b. On day 1, the cultures were supplemented with 10% (v/v) Con A SN and 50 mM alpha methyl mannoside. c. n.d. = not determined. Table V. Activation of antigen specific C T L by clone 9 a 45 B6 RESPONDER D2 STIMULATOR HELP % SPECIFIC LYSIS AGAINST LN CELLS SPLEEN CELLS B6(H-2b) CBA(H-2k> D2(H-2d) • (2000 R) 5 x IO 3 5 x IO 3 5 x 10 5 x 10 3 5 x 10! 5 x 10 3 5 x 10: 0.0 ± 1.0 -5.7 ± 1.6 10 A clone 9 b -0.3 ± 1.7 5% BLA 19.A ± 3.8 fa c t o r 0 0.0 ± 2.1 0.0 ± 1.0 3.5 ± 2.1 3.9 ± 3.5 10.8 t 3.5 A7.0 ± A.8 A3.3 ± A.2 77.5 ± 6.6 a. Cultures were set up in conical bottom wells in a volume of 0.2 ml. On day 5, cultures were split and tested against 2 x 10 3 5 1Cr-labelled B6, C B A and D2 LPS blasts. b. Clone 9 cells were irradiated with 2000 rads. c. The EL4 factor was obtained by stimulating an EL4 variant with phorbol myristic acetate for 24 hours at 37°C. The supernatant was filter-sterilized and the IL-2 activity of the supernatant was calibrated by testing for its ability to promote the proliferation of an IL-2 dependent murine T cell line. The IL-2 titre of this supernant contained 600 U/ml. Refer to materials and methods for more details (p.28). 46 Table VI. Requirement of accessory cells for clone 9 function3 B6 RESPONDER D2 STIMULATOR SPLENOCYTES CLONE 9 PERCENT SPECIFIC LN CELLS SPLEEN CELLS <2000 R) (2000 R) LYSIS AGAINST (2000 R) 3 X i o 3 i o 5 2.2 ± 0.8 3 X IO 3 i o 5 - • i o 4 11.8 ± 1.9 3 X i o 3 i o 5 5 X i o 4 B6 - 6.5 ± 1.8 3 X i o 3 i o 5 5 x i o 4 B6 i o 4 32.6 ± 6.2 3 X i o 3 i o 5 5 x i o 4 AKR - 8.5 ± 3.7 3 X i o 3 i o 5 5 X i o 4 AKR i o 4 24.2 ± 5.3 3 X i o 3 i o 4 — - 0.0 ± 0.2 3 X i o 3 i o 4 - i o 4 -1.0 t 0.2 3 X i o 3 i o 4 5 X i o 4 B6 - 0.7 ± 0.4 3 X i o 3 i o 4 5 X i o 4 B6 i o 4 16.0 ± 4.1 a. Culture conditions were the same as in Table IU (p.43). 47 Table VTI. Kinetics of action of clone 9 B6 RESPONDER D2 STIMULATOR CLONE 9 DAY OF CLONE 9 PERCENT SPECIFIC SPLEEN CELLS SPLEEN CELLS (2000 R) (2000 R) ADDITION LYSIS AGAINST P815 3 x i o 3 0.0 ± 0.4 3 x i o 3 i o 5 - -1.0 ± 0.2 3 x i o 3 i o 5 i o 4 day 0 62.4 ± 6.2 3 x i o 3 10 5 i o 4 day 1 2.2 ± 3.6 3 x i o 3 i o 5 i o 4 day 2 1.8 ± 1.2 3 x i o 3 i o 5 i o A day 3 8.9 ± 5.1 3 x i o 3 i o 5 • i o 4 day 4 1.7 t 0.8 a. Culture conditions were similar to those in Table IE (p.43), except that clone 9 cells were added on different days of a 5-day response. Table VOL Helper activity of clone 9 supernatant factor(s) is DD-specific 48 10 5 D2 ConA SNb Z SPECIFIC LYSIS RESPONDER STIMULATOR CLONE 9 AGAINST P815 LN CELLS SPLEEN CELLS SUPERNATANT" (10Z) 5 x 103 (2000 R) (1/5) EXPT 1 EXPT 2 .. EXPT 3 B6 - 3.6 ± 2.3 1.1 ± 0.6 1.0 ± 2.4 B6 • - - 5.5 ± 1.8 0.6 t 0.9 2.7 ± 0.6 B6 + - 24.3 ± 4.6 10.5 ± 2.2 12.1 t 1.4 B6 • n.d. n.d 53.2 ± 3.7 BIO.A - -0.1 ± 0.2 3.2 ± 0.8 . 1.6 t 0.2 BIO.A + - - -0.5 ± 0.6 1.4 ± 0.5 0.5 t 0.4 BIO.A + 1.2 ± 1.1 -0.6 ± 0.5 2.7 ± 1.0 BIO.A • + n.d. n.d. 16.2 ± 1.7 B10.A(2R) _ -1.3 t 1.4 3.8 ± 0.9 0.4 t 0.5 B10.A(2R) + - - 0.7 t 1.2 -0.2 ± 0.4 0.4 ± 0.4 B10.A(2R) • •f - 8.0 ± 3.0 12.1 ± 2 .0 13.7 t 1.1 B10.A(2R) — n.d. n.d. 45.9 ± 3.5 B10.AOR) — -1.6. ± 0.7 0.7 ± 0.6 2.1 ± 0.6 B10.A(3R) - - -0.8 ± 0.5 -0.8 ± 0.7 2.7 ± 0.7 B10.A(3R) + • - 2.6 ± 3.6 -1.2 ± 0.7 5.4 ± 2.0 B10.A(3R) + n.d. n.d. 31.2 * 5.7 a. Clone 9 supernatant was obtained by culturing 2 x 10° clone 9 cells in 2 ml of complete medium for 48 hours. All of the supernatants used in this experiment were concentrated 10 times by ultrafiltration. b. On day 1, the cultures were supplemented with 10% (v/v) Con A SN and 50 mM alpha methyl mannoside. 49 Table IX. Activity of clone 9 supernatant factors) in the thymocyte proliferation assay a B6 THYMOCYTES 2 yg Con A/ml FACTOR COUNTS PER MINUTE (M ± S.E.M.) i o 5 389 ± 81 io 5 + - 978 ± 24 io 5 + 1/4 ConA SN 22594 ± 273 io 5 + 1/8 ConA SN 14633 ± 1078 io 5 + 1/16 ConA SN 7334 ± 178 io 5 + 1/4 clone 9 supernatant** 856 ± 134 io 5 + 1/8 clone 9 supernatant 924 ± 104 io 5 + 1/16 clone 9 supernatant 758 ± 48 0 a. 10^ thymocytes and 2 jig/ml Con A were incubated with Con A SN or clone 9 supernatant in round bottom microliter wells. On day 3, cultures were pulsed with 3 H-TdR for 6 hours; then harvested and counted For further details, see materials and methods section on page 31. b. Clone 9 supernatant was prepared as described in Table VIQ (p.48). 50 Table X. Clone 9 supernatant does not support the growth of an IL-2 dependent T cell linea MTL 2.8.1* FACTOR CPM (M ± S.E.M.) 5 X 10 3 - 2781 ± 314 5 x IO 3 1/4 ConA SN 57237 ± 1040 5 x 10 3 1/8 ConA SN" 52092 ± 956 5 x 10 3 1/16 ConA SN 39938 ± 1115 5 x IO 3 1/32 ConA SN 22077 ± 452 5 x 10 3 b 1/4 clone 9 SN 4944 ± 231 5 x IO 3 1/8 clone 9 SN 3818 ± 357 5 x 10 3 1/16 clone 9 SN 4580 ± 792 5 x 10 3 1/100 BL4 SN 93096 ± 2307 5 x 10 3 1/300 EL4 SN 79516 ± 2704 5 x IO 3 1/1000 EL4 SN 45337 ± 1306 5 x IO 3 1/3000 EL4 SN 17912 ± 769 a. Triplicate cultures were set up in round bottom microtiter plates in a volume of 0.20 ml. The cultures were harvested after 48 hr, 1 nCi 3 H-TdR was added during the last 16 hr. b. Clone 9 supernatant was prepared as described in Table VITJ(p.48). 51 Table XI. Helper activity in clone 9 supernatant is not absorbed by D2 Con A or LPS blastsa B6 RESPONDER D2 STIMULATOR FACTOR ABSORPTION PERCENT LN CELLS SPLEEN CELLS WITH: SPECIFIC (2000R) LYSIS 3 X i o 3 * 0.0 ± 0.2 3 X IO 3 i o 5 - - -0.8 ± 0.02 3 X IO 3 i o 5 ConA SNb - 13.7 ± 0.8 3 X IO 3 i o 5 ConA SNb Con A blasts 0.5 t 0.2 3 X IO 3 i o 5 ConA SNb LPS . d blasts 16.0 ± 1.0 3 X i o 3 i o 5 clone 9 supernatant 0 - 18.0 ± 1.6 3 X i o 3 i o 5 Con A blasts 15.9 ± 1.7 3 X i o 3 i o 5 LPS blasts 15.8 ± 1.7 a. 0.3 ml of Con A SN or 10X concentrated clone 9 supernatant were absorbed with 2 x 10? Con A or LPS blasts on ice for 6 hours. b. Con A SN was used at a final concentration of 5% (v/v). c. Clone 9 supernatant was used at a final concentration of 10% (v/v). d. Con A blasts were prepared by culturing 10^ D2 spleen cells/ml with 2 ng/ml Con A for 48 hrs. LPS blasts were prepared by culturing 10^ D2 spleen cells with 20 |ig/ml LPS for 48 hrs. 52 Table XII. Augmentation of cytotoxic response by clone 9 factor(s) in the presence of IL-2 a 5 x 10 3 10A STIMULATOR CLONE 9 ConA SN PERCENT SPECIFIC LYSIS RESPONDER SPLEEN CELLS SUPERNATANT (20Z) AGAINST LN CELLS (2000R) D2 B6 CBA EXPT 1 B6 - - - 0.0 ± 1.7 Not done Not done B6 - 5Z - -4.8 t 0.7 tt it B6 D2 - - -4.0 t 1.2 tt it B6 D2 5Z - -2.2 ± 0.6 tt it B6 - _ + -0.8 t 1.1 Not done Not done B6 - 5Z • -6.3 ± 1.0 u I I B6 D2 - • 7.0 ± 4.0 ti •i B6 D2 5Z 59.6 t 9.4 it ii EXPT 2 B6 CBA - - 0.0 t 0.6 5.5 ± 2.2 0.0 ± 0.6 B6 CBA 10Z - -3.2 t 0.6 0.8 * 0.2 -0.8 ± 0.5 B6 CBA - • 0.0 t 0.8 0.1 t 2.3 16.5 ± 2.8 B6 CBA 10Z • 4.4 ± 1.0 -1.6 t 0.6 27.0 ± 4.2 D2 CBA — - 3.8 t 0.5 -2.1 ± 0.1 1.2 ± 0.8 D2 CBA 10Z - 2.6 t 0.6 -2.0 * 0.6 1.2 ± 1.1 D2 CBA - • 2.0 t 0.9 4.1 t 3.3 10.0 ± 1.0 D2 CBA 10Z 2.9 t 1.4 4.9 ± 1.5 13.7 ± 2.4 a. Cultures were set up in V bottom wells in a volume of 0.2 ml. On day 1, a final concentration of 20% Con A SN and 50 mM alpha methyl mannoside were added to the cultures. On day 5, cultures were assayed for cytolytic activity against 2 x l 0 3 5 1 C r -labelled D2 (H-2d), B6 (H-2b) or C B A (H-2k) LPS blasts. 53 Table XIU. Absorption of helper activity in clone 9 supernatant factor(s) by D" Con A blastsa RESPONDER B6 LN CKLLS D2 STIMULATOR SPLEEN CELLS (2000 R> TRBATMENT OF CLONK 9 SUPERNATANT^ ' ConA SN PERCENT SPECIFIC LYSIS AGAINST D2 EXPT. 1 EXPT. 2 5 x 10 5 x 10 3 5 x 10 3 5 x 10 3 5 x IO 3 10 io 4 i o 4 i o 4 !0 4 • unabsorbed + absorbed with BIO.A • absorbed with B10.AX2R) • 0.0 t 0.3 21.2 t 1.8 39.8 ± 5.4 32.9 t 5.3 23.1 t 2.3 0.0 t 0.2 12.7 t 1.8 41.5 ± 2.3 40.5 t 3.0 28.9 ± 3.1 5 x 10 5 x IO3 5 x IO 3 5 x 10 3 5 x 10 3 10 IO4 io 4 io 4 i o 4 unabsorbed absorbed with D2 absorbed with B6 •f •f • • 0.0 t 1.3 22.0 ± 3.7 32.1 i 4.2 40.0 t 3.8 21.5 ± 4.8 a. Culture conditions were similar to those described in Table XLT (p. 52). b. 0.5 ml supernatant was absorbed twice with 5 x 10 7 Con A blasts at 3 7 ° C for 1 hour. The supernatants were tested at a final concentration of 1/10 in the helper assay. c. A final concentration of 20% Con A SN and 50 mM alpha methyl mannoside was added on day 1. 54 Table XIV. Enhancement of C T L response by pulsing D b responder cells with clone 9 supernatant factor(s) a 5 X 10 RESPONDER LN CELLS TREATMENT OF RESPONDERSb 10 STIMULATOR SPLEEN CELLS (2000 R) % SPECIFIC LYSIS AGAINST CBA -ConA SN +ConA SN BIO.A BIO.A BIO.A 10 LN + 1/1 supernatant 6 10 LN + 1/2 supernatant CBA CBA CBA 1.0 ± 0.8 2.6 ± 0.4 2.6 ± 0.4 6.2 ± 1.1 6.0 ± 2.1 6.7 t 2.5 BIO.A (2R) - CBA 6 BIO.A (2R) 10 LN + 1/1 supernatant CBA BIO.A (2R) 106LN + 1/2 supernatant CBA 0.3 ± 0.3 -0.2 ± 0.3 -1.0 ± 0.7 13.8 ± 2.4 34.9 ± 4.8 20.8 ± 5.4 a. Culture conditions were similar to those described in Table X U (p. 52). b. 10 6 responder L N cells were pulsed with 1 ml of supernatant factors for 1 hour at room temperature. The cells were washed once and 5 x 103 responder cells were cultured with 10^ irradiated CBA spleen cells. c. A final concentration of 20% Con A SN and 50 nM alpha methyl mannoside was added on day 1. 55 Chapter 3. Characterization of a pb-specific helper factor required for the induction of  cytotoxic responses to alloantigens with the use of monoclonal antibodies specific for the helper factor or the T-cell antigen receptor 1. Introduction Th cells and their soluble products play an essential role in the induction of C T L responses to alloantigens (49,97,101). Previous studies suggest that the activation of C T L requires at least three signals. The first signal is provided by antigen (281,283,288). Within 5-12 hr of interaction with antigen, CTL-Ps express receptors for IL-2, the second signal (233,289). Upon binding of IL-2 to JJL-2 receptors, 'poised'T cells are stimulated into proliferation (282,289). The third signal is provided by maturation factors that are required for inducing the differentiation of C T L into mature effector cells (238,239). Subsequent studies suggest that, in addition to IL-2, an early acting factor, referred to as TCF1 (284-286), or RTF (237), may also be required for the induction of cytotoxic responses; TCF1 or RTF is postulated to be required for the induction of IL-2 receptors on antigen-activated CTL-P (237,286). However, more recent studies have indicated that highly purified Lyt-2 + cells only require leucoagglutinin (241) or antigen (242) and recombinant IL-2 to mount a cytotoxic response. Despite this controversy with regard to the role and number of non-antigen -specific factors that are required for the growth and differentiation of antigen-activated CTL-P, it is nevertheless clear that non-antigen-specific factors, and in particular JJL-2, play an essential role in cytotoxic responses. By contrast, little is known about the role of antigen-specific helper factors (ASHF) in the induction of cytotoxic responses. Several studies have suggested that ASHF may also be involved in the induction of cytotoxic responses (280,290,291). In the previous chapter, the isolation of a Th clone, referred to as clone 9, from an H-2 d anti-H-2b mixed lymphocyte culture was described (291). Clone 9 produced an ASHF that could synergize with IL-2-containing medium in augmenting C T L responses to alloantigens; this ASHF was specifically absorbed out by BIO.A (B10.A(2R)), but not by B10.A, spleen cells. 56 In an attempt to characterize this ASHF further, clone 9 cells were fused with the A K R thymoma, BW5147, and a T cell hybridoma line that secreted the DD-specific helper factor was isolated. In addition, a B cell hybridoma line that produced an IgM mAb to this factor was constructed. Using a helper-deficient thymocyte culture system (97), it was possible to demonstrate that this ASHF is required for the induction of cytotoxic responses. Studies with the F23.1 mAb (292), which is specific for the B chain (293) of the T-cell receptor, indicate that this ASHF bears an antigenic determinant that is recognized by this mAb. The bioassay and the antigen-binding properties of this ASHF are also described in this chapter. 2. Materials and methods a. Mice. CBA, B6, D2, B10.A (2R), B10.A and (AKR x DBA)Fj mice were purchased from the Jackson Laboratory, Bar Harbor, Maine. b. Culture medium. The culture medium used was Iscove's Modified Dulbecco's medium (Gibco, Grand Island, New York), supplemented with 10% fetal calf serum (Bockneck, Rexdale, Ontario), 5 x 10"^  M 2-mercaptoethanol, 50 U/ml of penicillin and 50 |ig/ml streptomycin (Gibco). All cultures were incubated at 37°C in 5% C O 2 in humid air. c. Construction of clone 9 hybrids. Clone 9 cells (H-2d) were fused with BW5147 cells (H-2k) at a ratio of 1:1 using polyethylene glycol as previously described (294). Hybridoma cultures were assayed for their ability to enhance the C T L responses of B6 spleen cells to alloantigens (291). Selected wells were grown and cloned by limiting dilution at 0.3 cells/well. The hybridoma clones were subcloned at least once at 0.3 cells/well. One of the hybridoma clones, designated as clone 25, was used as the source of ASHF in all the experiments reported here. 57 d. ASHF production. (AKR x DB A/2)Fi mice were injected intraperitoneally with 1 ml of 0.5% trypan blue. A day later, these mice were injected intraperitoneally with 5 x 10 6 exponentially growing clone 25 cells. Two weeks later, ascites were collected from the peritoneal cavity of these mice. The ascitic fluid was cleared by centrifugation and treated with leupeptin (Sigma, St Louis, MO.), a protease inhibitor, at 2 p.g/ml. Five millilitres of ascites were passed through a 30-ml bed volume of Sephadex G-50 (Pharmacia, Dorval, Quebec). Twenty-five millilitres of run-through were collected. The protein concentration of ASHF was estimated by using the value of 1.40 OD2gQ as 1 mg protein/ml. ASHF was usually used at a concentration of 100-300 \ig/rrd (equivalent to dilutions of 1 in 30 to 1 in 100). e. C T L helper assay. This assay was performed as described by Pilarski (97). C B A thymocytes (3 x 105) from 4-5-week-old mice were cultured with 10^ irradiated (2000 rads) B6 or D2 spleen cells in the presence or absence of clone 9 cells in a volume of 0.20 ml/culture in conical-bottomed microtitre wells, six to eight cultures per group. On day 5, the cultures were assayed for cytolytic activity against the appropriate target cells using a 4-hr -^Cr-release assay as described elsewhere (291). The target cells used were spleen cell blasts obtained by stimulating spleen cells with Con A. The spontaneous release for all experiments ranged from 15 to 25% of the maximum counts released. All the assays were performed at least twice and checked for statistical significance by the Student's T-tesL Helper activity in clone 25 ASHF was determined by pulsing 107 irradiated stimulator spleen cells with 100-300 |ig/ml of G-50 passed clone 25 ASHF for 3-4 hr at room temperature. The spleen cells were then washed twice and used as stimulators in the thymocyte assay. f. Analysis of CD4 phenotype. The rat monoclonal IgG25 antibody* GK1.5, developed by Dialynas et al (186), was obtained from the American Type Culture Collection. This mAb is specific for the CD4 cell surface molecule, a marker for helper T cells (186). The CD4 phenotype of clone 9 and its hybrid was analysed by using the antibody-dependent complement-mediated cytotoxicity assay as described previously (291). 58 g. Generation of mAbs against ASHF. Mabs against ASHF were produced by first immunizing allogeneic B6 mice with eight weekly i.p. injections of 10^ clone 9 cells. The mice were given a further injection of 5 x 10^ cells i.v., and 3 days later the spleen cells were fused with the 8-azaguanine-resistant myeloma fusion partner, NS1, and the hybrid cells selected in hypoxanthine, aminopterin and thymidine (HAT) medium as previously described (294). The culture supernatants from the hybrids were tested for their ability to block the helper action of clone 25 ASHF as follows: 1 ml of spent culture medium was preincubated with minimal concentration of clone 25 ASHF (3jig/ml) for 2 hr at room temperature before incubating wit 1 x 10^ irradiated B6 spleen cells for a further 4 hr at room temperature. The cells were then washed and used as stimulator cells in the thymocyte assay. Using this approach, one hybridoma clone (clone 30), which secretes an IgM antibody that is capable of inhibiting the helper action of clone 25 ASHF, was isolated. h. Affinity purification of ASHF. Clone 30 IgM mAb was generated as ascites in pristane-treated and irradiated (650 rads) mice of any strain. The ascitic fluids were cleared by centrif ugation and precipitated with a 100% saturated ammonium sulphate solution to a final concentration of 33% ammonium sulphate. The precipitated IgM was passed over Sephadex G-200 and the void volume fractions were collected. The G-200-purified mAb was coupled to Affigel 10 beads (BioRad, Richmond, CA) at 1 mg/ml of beads. The Affigel column was then used to purify clone 25 ASHF. Five millilitres of leupeptin-treated, G-50-passed ASHF were loaded onto 3 ml of beads. The column was then washed extensively with 1 litre of phosphate-buffered saline (PBS), pH 7.2, and eluted with 15 ml of 0.1 M glycine HCl, pH 2.3, containing 0.15 M NaCl. Eluates were neutralized with 1 M NaOH, and half of the eluate (7.5 ml) was used to assay for biological activity at dilutions equivalent to the non-affinity purified ASHF. The other half of the eluate was dialysed against distilled H 2 O to remove the glycine and salt and then lyophilized. This lyophilized fraction was resuspended in 100 [il sample buffer, boiled for 5 min at 100° C and then 25 (il were analysed on a 7.5% reduced 59 SDS-PAGE. Individual protein bands on the SDS gels were visualized by silver staining (ICN Biochemicals, Irvine, CA). A second Affigel column, to which an equivalent amount of irrelevant IgM mAb had been bound, was used as a control column. For the identification of the protein that was associated with the D^ binding property of clone 25 ASHF, 1 ml of G-50-passed clone 25 ASHF was absorbed with 108 B6 or D2 spleen cells for 4 hr at room temperature and the absorbed ASHF was loaded onto 3 ml of an irrelevant IgM antibody column. The run-through (6 ml) from this column was loaded onto a 3 ml clone 30 column. This column was washed with 1 litre of PBS and eluted with 15 ml of 0.1 M glycine HCl, pH 2.3, containing 0.15 M NaCl. The eluate was extensively dialysed against distilled H 2 O to remove the glycine and salt After dialysis, the eluate was divided into two tubes and then lyophilized. One lyophilized fraction was resuspended with 0.5 ml complete medium and assayed for helper activity at dilutions equivalent to the G-50-passed starting material. The other lyophilized fraction was resuspended in 50 jil sample buffer, boiled for 5 min and 25 |il were analysed on a 7.5% reduced SDS-PAGE gel. Individual protein bands in the SDS gels were visualized by silver staining (ICN Biochemicals). This experiment was repeated at least once except that in the subsequent experiments the ASHF was absorbed with B10. A (2R) or B10. A spleen cells. Hybridoma cells producing the F23.1 (IgG2a) mAb were developed by Staerz et al. (292) and provided to us by Dr. Doug Waterfield, Dept. of Oral Biology, University of British Columbia. This mAb is specific for a determinant on the variable region of the 6 chain of the T-cell receptor (293). F23.1 mAbs were generated as ascitic fluids in pristane-treated and irradiated (650 rads) (B6 x D2) F j mice. The ascites were cleared by centrifugation, precipitated with 50% (w/v) ammonium sulphate and purified over protein-A column (Pharmacia). The purified F23.1 mAb was coupled to Affigel at 1 mg/ml of beads. G-50-passed clone 25 ASHF was loaded onto this immunoadsorbent and eluted with glycine HCl. pH 2.3. Eluates were neutralized and tested for helper activity as well as analysed on a 7.5% reduced SDS-PAGE gel. 60 i. Recombinant IL-2. Human recombinant IL-2 (rIL-2) (lot no. LP-315 from E.coli) was provided by Cetus Corporation, Emeryville, C A (298). The r-IL-2 was 99% pure as assayed by SDS-PAGE and contained less than 0.01 ng endotoxin per 1.5 x 10^ units. One unit of IL-2 was defined as the amount of r-IL-2 required to cause 50% maximal proliferation of an IL-2 dependent T-cell cultured at 1 x 10 4 cells/0.2 ml for 2 days. 3. Results a. Generation of D^-specific T hybridoma cell lines. In the previous chapter, it was shown that supernatant factors derived from clone 9 cells contained a D^-specific helper factor (291). However, since clone 9 cells produced only small amounts of ASHF and grew slowly in culture (doubling time of 36 hr), clone 9 cells were consequently fused with A K R thymoma, BW5147, in order to obtain T hybridoma lines that will produce larger amounts of ASHF. Culture supernatants from the hybridoma lines were screened for helper activity by determining their ability to augment the C T L responses of B6 responder cells to alloantigens as previously described (291). Only one in 48 hybridoma supernatants initially screened contained a significant amount of helper activity. Clone 25 was derived by cloning the hybridoma cells from the well that produced high levels of helper activity. This clone has a doubling time of 14 hr and expresses the Thy-1.2 and H-2 d molecules found on clone 9 cells (data not shown). Using the antibody-dependent complement-mediated cytolysis assay, it was found that clone 9 cells were 95% positive for the T helper cell-associated marker, CD4; clone 25 hybridoma cells were negative for the CD4 molecule. Since clone 25 was functionally active, this implies that the helper function of this clone does not correlate with the expression of the CD4 molecule. b. Helper assay for clone 9 cells and clone 25 ASHF. In the previous chapter, clone 9 cells or their factors were found to be required in the induction of C T L responses to alloantigens in cultures containing low density of D b responder spleen cells. One problem encountered with the use of this assay system for detecting clone 9 helper activity was that the enhancing effect 61 of clone 9 cells or their factors was small. By switching to the thymocyte assay system that was developed by Pilarski (97), more consistent helper activity of clone 9 cells or their factors could be routinely detected. In this assay system, C B A (H-2k) thymocytes were used as responders and irradiated B6 (H-2*5) spleen cells were used as stimulator cells. Thymocytes were used as responder cells since they are known to be deficient in Th cell population (97). By contrast to the previous assay system, the target cell of clone 9 (B6) resided in the stimulator instead of the responder population. The larger number of target cells available for clone 9 (10^ B6 stimulator cells versus 3 x 103 B6 responder cells) likely contributed towards the larger helper effects observed in this assay system. In the absence of clone 9 helper cells, no C T L response to B6 was observed; in the presence of clone 9 cells, the CBA anti-B6 C T L response was induced by the helper cells in a dose-dependent fashion (Fig.1, p.69). By contrast, the C B A anti-D2 response was not helped by clone 9 helper cells (Fig.1, p.69). The failure of clone 9 cells to induce the C B A anti-D2 response was not due to the inability of CBA to respond to D2 since the addition of 10 U/ml of r-IL-2 to these cultures resulted in the generation of a significant C T L response to D2 (51% lysis). These results indicate that the helper action of clone 9 cells was specific for B6 stimulator cells. Clone 9 therefore could help C T L responses either by directly interacting with the B6 responder population and consequently augmenting the C T L responses of B6 responder cells to alloantigens ( see Table HI, p.43), or clone 9 could interact with the stimulator population and could therefore enhance the C T L responses of non-D^ responder cells (eg. C B A thymocytes) to B6 alloantigens. A model which encompasses the mode of action of clone 9 in these two assay systems will be discussed in the next chapter. Similar results were obtained when clone 25 ASHF was used as a source of help in the thymocyte assay system. Optimal helper effects were observed when clone 25 ASHF was preincubated with B6 spleen cells and the ASHF-pulsed and irradiated B6 spleen cells were then used to stimulate C B A thymocytes. Figure 2 (p.71) indicates that clone 25 ASHF could induce a CBA anti-B6, but not a C B A anti-D2, C T L response in a dose-dependent fashion. It was necessary to preincubate B6 stimulator cells with clone 25 ASHF as the helper factor preparation contained non-antigen-specific inhibitory molecules, which could be removed by 62 washing the helper factor-pulsed B6 stimulator spleen cells. When B6 spleen cells were pulsed with BW5147 ascites, which were processed in the same manner as clone 25 ASHF, and used to stimulate C B A thymocytes, no C B A anti-B6 C T L response was observed (data not shown). The finding that pulsing of B6, but not D2, spleen cells with clone 25 ASHF led to the induction of an anti-B6 cytotoxic response suggests that clone 25 ascites contains an ASHF that binds to B6, but not D2, spleen cells. c. Binding of clone 25 ASHF to D^. In order to demonstrate more direcdy that clone 25 ASHF binds specifically to antigens, different concentrations of clone 25 ASHF were preabsorbed with 108 BIO.A (2R) or BIO.A spleen cells for 4 hr at room temperature before they were tested in the thymocyte assay. As can be seen in Figure 3 (p.73), the helper activity of the clone 25 ASHF was absorbed out by BIO.A (2R), but not BIO.A, spleen cells. Similar results were obtained when a constant amount of clone 25 ASHF was absorbed with increasing numbers (2 x 107 to 8 x 107) of B6 or D2 spleen cells and then tested in the thymocyte assay. At a cell dose of 8 x 107 spleen cells, the helper activity of clone 25 ASHF was completely absorbed out by B6, but not by D2 spleen cells (data not shown). Since BIO.A (2R) and BIO.A are congenic strains of mice differing only in the D locus, these results suggest that clone 25 ASHF binds specifically to D*5. d. Production of a mAb specific for clone 25 ASHF. A mAb against clone 25 ASHF was produced by hyperimmunizing B6 mice with clone 9 cells and subsequently fusing the immune lymphocytes with NS1 myeloma cells. B6 mice were used for immunization since clone 9 cells might be continously stimulated by the D*5 alloantigen present in B6 mice and thus produce more D^-specific helper factor. Culture supernatants from hybridoma cells were screened for antibodies capable of neutralizing the helper activity of clone 25 ASHF. This was done by incubating test supernatants with clone 25 ASHF and these mixtures were then used to pulse B6 stimulator spleen cells. Out of 100 macrocultures tested, only the supernatant from one well was able to neutralize the helper function of clone 25 ASHF consistently. Cells from this well were subsequently expanded, cloned and subcloned. A representative result of the 63 neutralization assay used to screen for antibodies to clone 25 ASHF is shown in Table X V (p.75). Only clone 30 IgM supernatant inhibited the helper action of clone 25 ASHF (compare lines 2 and 6). Preincubation of clone 25 ASHF with a control IgM mAb (clone 20), produced from the same fusion did not inhibit the helper activity of clone 25 ASHF (compare lines 2 and 4). The inhibitory effect of clone 30 IgM was specific in the sense that this mAb did not inhibit the rIL-2-induced C B A anti-B6 C T L response (compare lines 7 and 9). e. Binding of clone 25 ASHF to clone 30 and F23.1 mAbs. Clone 30 IgM was bound to agarose beads and used as an immunoadsorbent for clone 25 ASHF. Clone 20 IgM similarly bound to agarose beads was used as a control. The data in Table XVI (p.76) indicate that only acid eluates from clone 30, but not clone 20, affinity columns could help the C B A anti-B6 C T L response. Furthermore, this helper activity was specific for B6 in that clone 30 column eluates did not help the CBA anti-D2 C T L response. A higher B6-specific helper activity was observed with clone 30 eluates when compared to unpassed material (compare lines 2 and 3 with lines 6 and 7). This could be due to non-specific inhibitory molecules that were present in the unpassed material. Clone 25 ASHF also bound to an affinity column made with the F23.1 mAb (Table XVI, p.76, lines 8 and 9). This latter observation suggests that clone 25 ASHF bears an antigenic determinant that is similar, if not identical, to that found on the B-chain of the T-cell receptor (293). f. Identification of a 50.000 molecular weight molecule that binds to B6. In order to identify the DD-binding molecule that is present in clone 25 ASHF, the helper factor was preabsorbed with 10 8 B6 or D2 spleen cells for 4 hr at room temperature. After absorption, the ASHF was loaded onto a clone 30 column. Eluates from this column were tested for helper activity as well as analysed on a 7.5% reduced SDS-PAGE gel. Table XVII (p.77) shows that eluates from the column loaded with supernatants that were either unabsorbed or absorbed with D2 spleen cells still contained helper activity. However, the fraction that was preabsorbed with B6 spleen cells did not contain helper activity, thus indicating that clone 25 ASHF was absorbed out by B6 spleen cells. The gel profiles of the unabsorbed, B6- or D2-absorbed fractions are shown in 64 Figure 4 (p.78). In the unabsorbed fraction (lane 3), there were four major bands with molecular weights of approximately 90,000,68,000,50,000 and 43,000, and three minor bands with molecular weights of approximately 60,000, 35,000 and 30,000. The gel profile of the fraction that was absorbed with D2 spleen cells (lane 1) was essentially the same as the unabsorbed fraction. However, after absorption with B6 spleen cells (lane 2), a 50,000 M W band was selectively removed (compare lanes 1 and 3 with lane 2). This result therefore implies that the 50,000 M W band could participate in binding to B6. Several non-specific high M W bands were also observed after absorption with B6, but not D2, spleen cells (compare lanes 1 and 2). A 50,000 M W band was also present in acid eluates of clone 25 affinity purified over F23.1 and clone 30 IgM mAb columns (Figure 5, p.79, lanes 2 and 3, respectively), but not the control clone 20 column (lane 1). This suggests that the 50,000 M W molecule that participates in binding to D b may also bind to the F23.1 mAb. 4.Discussion. Antigen-specific helper factors have been shown to play an essential role in the induction of antibody (31,199) and cytotoxic (280,290,29l,296)responses. Studies with conventional and hybridoma-derived ASHF, which are specific for protein antigens or synthetic polypeptides and are required for the induction of antibody responses, have demonstrated that ASHF possess the following properties: (i) they are antigen-specific in their mediation of help; (ii) they bind to their respective antigen in either a MHC-restricted (43) or unrestricted manner (31); (iii) they are glycoproteins with molecular weights of 50,000 to 70,000 that may be composed of two non-covalently bound chains; and (iv) they carry class IJ-MHC and immunoglobulin V J J determinants on separate polypeptide chains. In this chapter, a helper cell-deficient thymocyte assay system (97) was used to show that monoclonal antigen-specific Th cells or factors are required for the induction of cytotoxic responses to alloantigens. The helper clone (clone 9) was previously shown to induce the cytotoxic responses of B6 ( K D A D E b o b ) m d B 1 0 A ( 2 R ) ( K k A k E k D b ) responder cells to alloantigens but did not help the cytotoxic responses of BIO.A ( K k A k E k D d ) or B10.A (3R) ( K b A b E k D d ) responder cells to alloantigens (291). In this chapter, clone 9 cells were shown to induce the cytotoxic responses of C B A (H-2k) thymocytes to B6 (H-2b), but not D2 (H-2d), alloantigens. One important observation that came out from these studies was that clone 9 appeared to help via two different modes of interaction. Clone 9 could either help C T L responses of B6 responder spleen cells to any alloantigens by directly interacting with the B6 responder cells, or clone 9 could interact with B6 stimulator cells and as a result, augmented C T L responses of non-B6 responder cells to B6 stimulator cells via associative linked recognition. More details on clone 9 help via associative linked recognition will be presented in the next chapter. A model that encompasses the two modes of action of clone 9 will also be discussed in the next chapter. The hybridoma-derived ASHF had the same antigen and MHC-restriction specificity as clone 9 cells. Thus, clone 25 ASHF could induce C B A thymocytes to respond to B6, but not to D2, alloantigens. Furthermore, clone 25 ASHF was specifically absorbed out by B6 or BIO.A (2R), but not by D2 or BIO.A spleen cells. These data suggest that clone 9 and clone 25 ASHF are specific for D b . Recognition of D b also appears to be independent of class n molecules since the D b molecule is recognized in the context of both Ia b and Ia k molecules. As clone 9 cells are of H-2 d origin and they can help C B A (H-2k) thymocytes (Figure 1, p.69) or B6 spleen cells (291) to respond to alloantigens, this suggests that clone 9 cells are not MHC-restricted in their helper action. Clone 25 cells were obtained by fusing clone 9 cells (H-2d) with BW5147 (H-2k). Thus, the ability of clone 25 ASHF to help C B A (H-2k) thymocytes cannot be taken as evidence for the MHC-unrestricted helper action of clone 25 ASHF since BW5147 may contribute class U M H C molecules that may alter the reactivity of clone 9 ASHF. However, as clone 25 ASHF can also help B6 spleen cells or thymocytes to respond to any alloantigen (data not shown), it is likely that clone 25 ASHF is also MHC-unrestricted in its mode of action. The existence of class H-unrestricted allo-class I-specific Th cells such as clone 9 has also been reported by others (297,298). Furthermore, allo-class I-specific helper factors were also obtained from the draining lymph nodes of mice 66 recently implanted with an allogeneic skin graft (280). The frequency of such helper cells in the T-cell repertoire has not been determined. The physiological role of such helper cells and how they may be selected during T-cell ontogeny also remain to be defined. In studies on the C T L repertoire, it has been proposed that all CTL-P within an individual animal may in fact be completely resticted to self-MHC; the existence of allo-MHC-restricted CTL-P can be explained on the basis of cross-reactivity with self-MHC-restricted CTL-P (299). Thus, if Th cells are selected in the same manner during ontogeny as CTL, then the allo-class I specificity of clone 9 may be due to a fortuitous cross-reaction between a self-class II molecule (Iad) plus conventional antigen. It has been generally assumed that ASHF may be a secreted form of the T-cell receptor. The possible relationship of clone 25 ASHF to the T-cell receptor was investigated by determining whether it expressed a determinant that may be recognized by the F23.1 mAb, which is specific for a determinant on the B-chain of the T-cell receptor (293). Recent studies have suggested that the T-cell receptor comprises an aB heterodimer in association with the CD3 complex (180). Studies involving a-loss or B-loss T-cell hybridoma variants (155) as well as the transfer of a and B genes from clones of one MHC-restricted antigen specificity to another clone of different MHC-restricted antigen specificity (156,157) have demonstrated that the aB hetrodimer is responsible for MHC-restricted recognition of antigen. Consequently, the presence of B- chain determinants on ASHF can be taken as strong suggestive evidence that ASHF may be a secreted form of the T-cell receptor. The results reported here also show that affinity purification of clone 25 ASHF over the F23.1 mAb column yielded a helper factor that was specific for the C B A anti-B6 cytotoxic responses (Table XVI, p.76). Preabsorption of clone 25 ASHF with D^-expressing cells followed by affinity purification over the clone 30 IgM mAb column, which is specific for this ASHF, allowed the identification of a 50,000 MW molecule that appeared to bind to D b (Fig.4, p.78). A 50,000 M W molecule was also present in acid eluates of clone 25 ASHF affinity purified over F23.1 and clone 30 mAb columns, but not in acid eluates of an irrelevant mAb column (Fig. 5, p.79). One possible interpretation of these data is that the 50,000 M W that is involved in the binding of the D ° molecule may be 67 closely related, if not identical, to the B-chain of the T-cell receptor. However, until the amino acid sequence of this 50,000 M W molecule is partially or completely defined, it is difficult to deterrnine the precise relationship of this ASHF to the T-cell receptor. The existence of determinants on ASHF that are recognized by mAb to the T-cell receptor may also explain why ASHF is also recognized by immunoglobulin Vjj-specific antibodies since the structure of the T-cell receptor is highly homologous to that of immunoglobulin molecules (140). These studies also lend more direct support to the notion that ASHF may be a secreted form of the T-cell receptor. It remains to be determined whether clone 25 ASHF expresses class II M H C determinants and whether binding to requires one or more polypeptide chains since all the SDS gels were ran under reduced conditions. It is pertinent to note that the antigen-specific suppressor factor for K L H comprises two disulfide-bonded polypeptide chains (300). Thus, the possibility exists that the 50,000 M W molecule that binds Db may be disulfide-bonded to another molecule, particularly to the a-chain of the T-cell receptor. 68 5. Summary A Th clone (clone 9), isolated from a H-2 d anti-H-2b mixed lymphocyte culture, was previously found to produce an ASHF that could be specifically absorbed out with B10.A (2R) ( K k A k E k D b ) , but not BIO.A ( K k A k E k D d ) , spleen cells. In order to characterize this ASHF further, T-cell hybridoma lines were constructed by fusing clone 9 cells with the A K R thymoma, BW5147. One of these hybridoma clones, referred to as clone 25, produced an ASHF that was specific for the D b alloantigen. Immunization of allogeneic B6 mice with clone 9 cells and subsequent fusion of these immune spleen cells with non-secreting myeloma cells led to the isolation of a mAb (clone 30 IgM) that was capable of neutralizing the helper activity of clone 25 ASHF. Clone 30 IgM affinity column was found to retain clone 25 ASHF; clone 30 IgM column eluates augmented the cytotoxic responses of CBA/J thymocytes to B6 but not D2 (H-2d), alloantigens. Preabsorption of clone 25 ASHF with Db-bearing spleen cells prior to affinity purification over a clone 30 IgM column resulted in the abrogation of Db-specific helper activity as well as the loss of a 50,000 M W band in SDS-PAGE run under reducing conditions. Clone 25 ASHF was also retained by immunoadsorbents made with an IgG2a mAb (F23.1), the reactivity of which is against the B-chain of the T-cell receptor. Furthermore, affinity purification of clone 25 ASHF over a F23.1 affinity column, but not an irrelevant mAb column, also yielded a 50,000 M W molecule. These findings suggest that this particular ASHF may be intimately related to the T-cell antigen receptor. 69 Figure 1. Helper activity of clone 9 cells in the thymocyte assay. 3 x IO 3 CBA thymocytes and 10^ irradiated B6 or D2 spleen cells were cultured with the indicated number of irradiated clone 9 cells in V-bottomed wells. On Day 5, the cultures were assayed for C T L activity in a 5^Cr-release assay. Each point represents the mean of eight cultures ± standard error. o CBA ANTI - B6 o Figure 1 71 Figure 2. Helper activity of clone 25 ASHF in the thymocyte assay. 10' B6 or D2 spleen cells were pulsed with 1 ml of the indicated dilution of G-50-passed clone 25 ASHF for 3 hr at room temperature. These cells were washed, irradiated and cultured at 1 x lofywell with 3 x 105 C B A responder thymocytes. On Day 5, cultures were assayed for C T L activity in a *^Cr-release assay. Each point represents the mean of eight cultures ± standard error. o CBA ANTI-B6 • CBA ANTI-D2 RECIPROCAL OF DILUTION OF HELPER FACTOR Figure 2 73 Figure 3. Binding of clone 25 ASHF to D D . One ml. of the indicated dilution of ASHF was either unabsorbed or absorbed with BIO.A or BIO.A (2R) spleen cells for 3 hours at room temperature. The absorbed and unabsorbed ASHFs were then tested in the helper assay. Each point represents the mean of six cultures ± standard error. • unabsorbed A B10.A absorbed • 2R absorbed RECIPROCAL DILUTION OF ASHF Figure 3 Table X V . Neutralization of the helper action of clone 25 ASHF by a mAb a RESPONDER STIMU- CULTURE SN rIL-2 % SPECIFIC LYSIS LATOR - 5056 (V/V) ASHF lOU/ml (M ± S.E.) CBA B6 - - - 2.2 ± 0.4 CBA B6 - • 17.3 ± 3.1 CBA B6 Clone 20 b - - 3.1 ± 0.5 CBA B6 Clone 20 + - 16.1 ± 3.6 CBA B6 Clone 30 b - - -1.0 ± 1.0 CBA B6 Clone 30 + - 5.2 ± 0.8 CBA B6 - • 58.7 ± 3.9 CBA B6 Clone 20 - + 50.2 ± 7.1 CBA B6 Clone 30 - + 52.2 ± 5.9 a. Clone 25 ASHF (3vg/ml) was preincubated with 1 ml of spent culture medium from clone 20 or clone 30 for 2 hours at room temperature. This mixture was then used to pulse 10 B6 stimulator spleen cells for 3 hours at room temperature. After pulsing, the spleen cells were washed 6 5 and cultured at 1x10 /well with 3 x 10 CBA responder thymocytes. On day 5, cultures were assayed for CTL activity against B6 Con A 51 blasts in a 4 hour Cr-release assay. b. Clone 20 and clone 30 hybridomaa were obtained from fusions between NS1 myeloma cells and C57BL/6 spleen c e l l s hyperimmunized with clone 9 c e l l s . Table XVI. Clone 30 and F23.1 mAb columns can retain clone 25 A S H F a HELPER FACTOR DILUTION % S P E C I F I C L Y S I S (MBAN ± S . E . ) CBA ANTI-B6 CBA ANTI-D2 1.4 ± 3.0 0.1 ± 0.5 Unpassed Unpassed 1/30 1/100 13.1 ± 1.5 13.7 ± 2.7 2.4 + 0.6 5.7 ± 2.4 Clone 20 eluate 1/30 Clone 20 eluate 1/100 0.6 ± 3,1 -1.9 ± 2.0 2.8 ± 1.3 1.5 ± 2.2 Clone 30 eluate 1/30 Clone 30 eluate 1/100 39.3 ± 6.2 23.7 ± 6.9 0.1 + 0.1 2.4 ± 1.5 P23.1 eluate P23-1 eluate 1/30 1/100 26.1 i 8.2 25.3 ± 6.8 0.1 ± 0.9 3.5 ± 0.9 rIL-2 20 U/ml 35.3 ± 4.9 48.1 ± 1.8 a. 5 ml of G-50-passed ASHF were loaded onto the appropriate affinity column. The column was washed extensively with 1 l i t r e PBS and eluted with 15 ml of 0.1 H glycine HCl, pH 2.3, containing 0.15 M NaCl. Eluates were neutralized with 1 M NaOH and assayed for biological activity at equivalent dilutions to the starting unpassed material. 77 Table XVII. Biological activity of absorbed ASHF purified over clone 30 mAb column* ASHF TREATMENT PURIFICATION OVER CLONE 30 COLUMN ASHF DILUTION % SPBCIFIC LYSIS (MEAN ± S.E.) G-50 passed B6 absorbed D2 absorbed Unabsorbed No No No No No 1/30 1/30 1/30 1/30 6.5 + 1.8 26.2 + 5.9 5.7 t 2.3 32.2 ± 6.3 19.8 ± 2.7 B6 absorbed D2 absorbed Unabsorbed Ye 8 Yes Yes 1/30 1/30 1/30 5.9 ± 0.9 21.5 ± 3.9 19.8 ± 6.2 a. 1 ml ASHF was either absorbed with 10 B6 or D2 spleen cells for 4 hours at room temperature. The preabsorbed ASHFs were passed over clone 30 mAb column. Both the aff i n i t y - p u r i f i e d ASHF as well as the non-affinity purified ASHP were tested for helper activity in the thymocyte assay. 78 200 • 97 > 68 • 43 • 25 • D2 ABSORBED B6 ABSORBED UNABSORBED Figure 4. Gel profile of affinity-purified ASHF that had been preabsorbed with B6 or D2 spleen cells. One ml. of G-50-passed clone 25 ASHF was absorbed with 108 B6 or D2 spleen cells for 4 hr at room temperature. The unabsorbed and absorbed ASHF were individually passed over an irrelevant IgM mAb column. Each run-through from this column was loaded onto a clone 30 mAb column. Acid eluates from clone 30 mAb column were dialysed, lyophilized and run on a 7.5% reduced SDS-PAGE gel. Individual proteins were visualized by silver staining. 79 1 2 3 Figure 5. Gel profile of ASHF purified over different mAb columns. Five mis. of G-50-passed clone 25 ASHF were loaded onto clone 20 Qanel), F23.1 (Lane 2) or clone 30 (lane 3) mAb columns. The columns were washed extensively and then eluted with 0.1 M glycine HCl , pH 2.3, containing ).5 M NaCl. The eluates were dialysed, lyophilized and then run on a 7.5% reduced SDS-PAGE gel. Individual proteins were visualized by silver staining. 80 Chapter 4. Mechanism of action of a D^specific helper clone and factor in cytotoxic responses to alloantigens 1. Introduction The production of C T L in mixed lymphocyte cultures involves interactions between accessory cells, T helper cells and CTL-P (49). Stimulation of Th cells with specific antigen or mitogen leads to the production of non-antigen-specific helper factors that contribute towards a cytotoxic response. The best characterized non-antigenic-specific factor, which is required for the growth and differentiation of antigen-stimulated CTL-P, is IL-2 (211). Under certain culture conditions, IL-2 alone can induce the growth and maturation of antigen- (242) or mitogen- (241) activated Lyt-2 + cells into effector CTL. In other experimental systems, in addition to IL-2, other non-antigen-specific factors that have early (237,286) or late (238,239,288) effects were also reported to be required for cytotoxic responses. The requirement for ASHF in cytotoxic responses can only be demonstrated in a limited number of assay systems. These include C T L responses to tumour-associated antigens (290), C T L Tesponses to alloantigens using helper-deficient thymocytes as responder cells (296) and C T L responses to alloantigens using a low density of responder spleen cells (291). The above studies suggest that CTL-P can be activated by more than one mechanism. The different activation requirements for CTL-P may be due to one or more of the following considerations: (i) CTL-P are not a homogeneous population, and individual subsets of CTL-P require different activation signals, (ii) the requirement for non-specific helper factors other than IL-2 may be determined by the effective concentration of IL-2 present in the cultures, (iii) the number of high-affinity IL-2 receptors that are required for the growth of T cells (300,301) on antigen-activated CTL-P may be determined by the affinity of interaction between CTL-P and the specific antigen; in cases where the affinity of CTL-P for antigen is low, early-acting (237,286) factors may also be required for the induction of IL-2 receptors on CTL-P, and (iv) ASHF may mediate their effects by increasing the production of non-specific helper factors such as IL-2 or via hitherto undefined mechanisms. 81 In the Chapter 3, it was shown that a Db-specific helper clone or factor is required for the induction of C T L responses to alloantigens using the helper cell-deficient thymocyte assay system (97,296). It was also shown that the Db-specific helper factor may be a secreted form of the T-cell antigen receptor since it is bound by a mAb (F23.1) (292) that is specific for a determinant on the B-chain of the T-cell receptor (293). In this chapter, the mechanism by which this Db-specific helper clone or factor activates CTL-P to alloantigens is described. A model describing how antigen-specific and non-antigen-specific helper factors may function in cytotoxic responses is proposed. 2. Materials and methods a. Mice. CBA, B6,D2 and (B6xD2)Fj (BDFj) mice were purchased from the Jackson Laboratory, Bar Harbor, Maine. b. Helper cell lines. Clone 9, a Db-specific T helper clone, was isolated from a H-2 d anti-H-2b mixed lymphocyte culture and maintained as previously described (291, see chapter 2). Clone 25 is a T-cell hybridoma that produces a Db-specific helper factor and was obtained by fusing clone 9 cells with the A K R lymphoma BW 5147, as described in Chapter 3. c. Sources of IL-2. Human recombinant IL-2 (rIL-2) was kindly provided by the Cetus Corporation, Emeryville, C A , and the details for this lot of rIL-2 are specified in the preceding chapter. IL-2-containing supernatants were also produced by stimulating EL4.JJL-2 cells with PMA, and the JJL-2 activity in these supernatants (EL4.PMA) was determined as previously described (265). The batch of EL4.PMA used in this study had an IL-2 activity of 600 U/ml. d. Helper assay. Details for the production of the Db-specific helper factor from clone 25 cells and the C T L helper assay are described in Chapter 3. Total nonspecific C T L activity was determined by incubating cultures with 3 x 103 -^Cr-labelled P815 cells with lectin Phytohemaglutinin-P (Sigma)(268) during the 51Cr release assay. 82 3. Results a. Helper function of clone 9 cells and clone 25 ASHF is dependent on responder cell dose. In Chapter 3, it was shown that clone 9 cells or clone 25 ASHF can induce CBA thymocytes to form C T L against B6 alloantigens. The mechanism by which this antigen-specific helper clone or factor, referred to as ASHF from now on, induces cytotoxic responses in mixed lymphocyte cultures was determined by first examining the effect of varying the responder (CBA thymocytes) cell dose on the helper function of clone 9 cells or ASHF. In cultures supplemented with optimal numbers of clone 9 cells, it can be seen that the C B A anti-B6 C T L response was helped by clone 9 cells at all responder doses tested (0.75 to 6 x 10 )^ (Figure 6, p.91). By contrast, optimal amounts of ASHF exerted helper effects only at high responder cell doses (3 or 6 x 105) but not at a low responder cell dose (0.75 x 10 )^. Furthermore, the level of help observed with ASHF was lower than that observed with clone 9 cells. This implies that clone 9 cells may be able to exert helper effects additional to those observed with ASHF. At responder cell doses of less than 0.75 x 10-\ clone 9 cells were also incapable of helping the C B A anti-B6 C T L response (data not shown). Since B6-specific CTL-P were not limiting in these cultures, this result implies that both clone 9 and ASHF may not act directly on CTL-P, but rather exert their helper function through an intermediary cell that is present in limiting numbers at low responder cell doses. b. Clone 9 or ASHF can help a bystander response. One explanation for the ability of clone 9 or ASHF to help specifically the C B A anti-B6 response is that clone 9 or ASHF, which is specific for D b , can bind B6 stimulator cells and as a result of this, is brought into close proximity with the B6-specific C B A responder cells which are also bound to the B6 stimulator cells. This form of help has been referred to as linked recognition (97). The experiments in Table XVHI (p.93) were designed to determine whether there was an obligatory requirement 83 for clone 9 or ASHF to be in close proximity with the CBA responder cell in order to mediate their helper function. As shown in Chapter 3, only the CBA anti-B6, but not the C B A anti-D2, response was helped by clone 9 or ASHF (also see Table XVITJ, p.93, lines 1-4 and 9-12). However, cytotoxic responses to D2 were also helped by clone 9 or ASHF when BDFj spleen cells were used as stimulators and when a 1:1 mixture of B6 or D2 spleen cells was used as stimulators (lines 5-8 and 13-16). The latter result clearly shows that clone 9 or ASHF need not be in close proximity with the C B A responder cell to exert their helper effects. The most likely interpretation of these results is that following interaction of B6 stimulator with clone 9 or ASHF, long-ranged, non-antigen-specific helper factors that can induce cytotoxic responses are produced. In Table XVHI (p.93), it can be seen that the helper effects of clone 9 cells on the C B A anti-B6 or the 'bystander' C B A anti-D2 response was much higher than that observed with ASHF (compare lines 2,6 and 8 with lines 10,14 and 16). This again implies that clone 9 may mediate more helper effects than ASHF. 'Bystander' help also suggests that the immediate target cell of clone 9 or ASHF is not the CTL-P since the D2-specific CTL-P and clone 9 or ASHF are not in close proximity during bystander help. c. Enhancement of IL-2 production by clone 9 or ASHF. The simplest explanation for bystander help is that clone 9 or ASHF can induce the production of long-ranged acting factors such as IL-2 in B6-stimulated cultures. The ability of clone 9 or ASHF to induce IL-2 production by B6 stimulator cells or in CBA anti-B6 mixed lymphocyte cultures was therefore determined. The data in Table XLX (p.94) show that clone 9 cells could induce a 4.7 fold increase in IL-2 production by irradiated B6 spleen cells (compare lines 7 and 9) but did not induce IL-2 production by irradiated D2 spleen cells (compare lines 10 and 12). By contrast, ASHF could not induce B6 spleen cells to produce JJL-2 (compare lines 7 and 8). Since clone 9 cells were incapable of producing IL-2 following stimulation by antigen or Con A (data not shown), it was concluded that clone 9, but not ASHF, can induce irradiated B6 spleen cells to produce IL-2. In C B A anti-B6 cultures, clone 9 or ASHF increased the amount of IL-2 produced by 5.7- and 3.8-fold, respectively (lines 1-3); this increase in EL-2 production was not observed in C B A anti-D2 cultures (lines 4-6). The data in Table XIX (p.94) also indicate 84 that, following clone 9 stimulation, C B A anti-B6 cultures produced more JJL-2 than B6 spleen cells (compare lines 3 and 9). This suggests that clone 9 cells can also stimulate the production of IL-2 by C B A responder cells in B6-stimulated cultures. Since ASHF could not induce B6 spleen cells to produce IL-2, it is likely that ASHF can induce B6-stimulated C B A responder cells to produce IL-2 via linked recognition. The greater amount of IL-2 produced in clone 9 stimulated cultures may partially or completely account for the more potent helper effects of clone 9 when compared to ASHF. Since ASHF could not induce B6 spleen cells to produce IL-2, this implies that binding of ASHF to B6 is insufficient to trigger EL -2 release. As clone 9 can induce B6 spleen cells to release IL-2, it is possible that, in addition to the allogeneic effects induced by clone 9 on B6 stimulator cells, clone 9 cells may also deliver additional helper signals to B6. d. Production of antigen-specific and non-antigen-specific helper factors following stimulation  of clone 9 cells with antigen or Con A. It has been shown that clone 9 cells and ASHF differ quantitatively in their capacity to help cytotoxic responses. One reason for this quantitative difference is that clone 9 cells are more effective than ASHF in inducing IL-2 production by both the stimulator and responder cells (Table XTX, p.94). In addition to inducing IL-2 release, it is possible that antigen-stimulated clone 9 cells can produce additional helper factors. This possibility was investigated by stimulating clone 9 cells with Con A or antigen and examining the supernatants from these cultures for IL-2 and non-IL-2 helper activities. The data in Table X X (p.95) indicate that supernatants from Con A, B D F j or B6-stimulated clone 9 cells were highly potent in inducing the C B A anti-B6 C T L response (lines 3,5 and 9). Supernatants from D2-stimulated clone 9 cells did not contain such an activity (lines 7 and 11); this indicates that only supernatants from H-2b-stimulated clone 9 cultures produced helper factors). The CTL-enhancing activity found in these supernatants was not due to IL-2 since the amount of IL-2 in all supernatants were less than 0.2 U/ml. Such a low level of IL-2 was insufficient to induce a C T L response (data not shown). In Table XIX (p.94), clone 9 cells were shown to be able to induce a low level of IL-2 production by B6 spleen cells (1.4 U/ml). The lower B6 or BDFj spleen cell doses used in Table X X (p.95) could account for the low amounts of IL-2 in 85 these supernatants. The helper activity in these supernatants was not due to Con A since an excess of alpha methyl mannoside was added to neutralize the effects of Con A (see line 1). The above experiment demonstrated that, upon stimulation by Con A or antigen, clone 9 cells produce a non-JL-2 helper factor that helps the C B A anti-B6 C T L response. Clone 9 cells were previously shown to produce Db-specific helper factors upon antigenic stimulation (291). The presence of H-2b-specific helper factors in these supernatants was therefore investigated. The data in Table XXI (p.96) indicate that clone 9 cells produced B6-specific helper factors upon stimulation with Con A. Thus, supernatants from ConA-stimulated clone 9 cells only helped C B A or D2 thymocytes to respond to B6 and did not help CBA thymocytes to respond to D2. The failure of C B A thymocytes to respond to D2 was overcome by adding 11^2-containing supernatants, indicating that these cultures were not limiting in CTL-P (line 5). Similar results were also observed with supernatants from clone 9 cells that were stimulated with BDF1 spleen cells (data not shown). Thus, one of the helper factors produced by clone 9 cells following mitogen or antigen stimulation is the H-2b-specific helper factor. The supernatant of Con A-stimulated clone 9 cells was also examined for the existence of other CTL-inducing factors. In a recent study, it was found that antigen-stimulated clone 9 cells could synergize with rIL-2 in the activation of CTL-P (H.-S. Teh and P.C. Kwong, unpublished observations). Thus, the supernatants of Con A-stimulated clone 9 cells were examined for the existence of factors) that could synergize with rIL-2 in the polyclonal activation of CTL-P. This was determined by culturing BDFj or D2 spleen cells with supernatants from Con A-stimulated clone 9 cells in the presence or absence of excess rIL-2. Five days later, C T L of all specificities were assayed by incubating 51Cr-labelledP815 cells with effector cells in the presence of PHA (302). The data in Table X X n (p.97) indicate that Con A-stimulated clone 9 supernatant or rIL-2 by itself was incapable of inducing C T L formation. Only in cultures that were supplemented with both supernatants from Con A-stimulated clone 9 cells and rIL-2 did one observe significant cytotoxic activity (lines 4 and 8). The amount of residual Con A was effectively neutralized by alpha methyl mannoside since cultures containing an equivalent amount of Con A did not lead to the production of C T L (lines 2 and 6). By contrast to the ASHF, this activity in supernatants of Con A-stimulated clone 9 cells was not specific for B6 since both BDFj and D2 spleen cells were helped by this factor (lines 4 and 8). Culture supernatants from antigen-stimulated clone 9 cells also contained this non-antigen-specific helper activity (data not shown). In summary, the data in Tables X X - X X U (pp.85-87) suggest that clone 9 cells can produce a H-2D-specific helper factor as well as a non-antigen-specific helper factor upon stimulation with antigen or mitogen. This non-antigen-specific helper factor can synergize with rIL-2 in the activation of CTL-P in the absence of stimulation with specific antigen. For discussion purposes, this non-antigen-specific helper factor will be referred to as IL-X. 4. Discussion The mechanism by which C T L to alloantigens is activated was investigated using helper cell-deficient thymocytes as responder cells and irradiated allogeneic spleen cells as stimulator cells. Help was added in the form of a Th clone (clone 9) or an ASHF. Both clone 9 and ASHF were previously shown to be specific for the D D class I molecule (291). The helper properties of clone 9 can be summarized as follows: (i) it helped C B A anti-B6, but not C B A anti-D2, C T L responses; in clone 9 supplemented cultures that were stimulated with both B6 and D2 spleen cells, C T L responses to D2 were also enhanced; (ii) the helper function of clone 9 cells was dependent on the responder cell dose, and clone 9 cells were insufficient to induce a C T L response at low responder cell doses; (iii) clone 9 cells increased the production of IL-2 by B6 spleen cells and in CBA anti-B6 cultures; (iv) upon antigen or Con A stimulation, clone 9 cells produced an ASHF (B6-specific) and a non-antigen-specific helper factor (TL-X) that was distinct from DL-2; (v) IL-X synergized with excess rIL-2 in activating cytotoxic precursors in the absence of intentional stimulation with antigen or mitogen. The properties of the D^-specific ASHF were similar to that of clone 9 in that: (i) it also stimulated a bystander response; (ii) it was only effective at high responder cell doses, and (iii) it increased IL-2 production in C B A anti-B6 cultures. It differs from clone 9 cells in the following aspects: (i) it was less efficient than clone 9 in mediating help; (ii) it did not induce IL-2 production by B6 87 spleen cells, and (iii) it did not possess any IL-X activity (data not shown). On the basis of the above observations, the following model for C T L activation, involving helper cells, ASHF, IL-2 and IL-X, is proposed. This model is illustrated in Figure 7 (p.98). In C B A anti-B6 cultures, clone 9 cells are stimulated by the D D alloantigen on B6 antigen-presenting cells. This results in the production of IL-X and ASHF by clone 9 cells. ASHF can directly stimulate the production of IL-2 by activated B6 T cells or stimulate the production of IL-2 by activated C B A T cells via associative linked recognition. The IL-2 produced induces the proliferation and differentiation of C B A CTL-P that have been activated by H-2 b antigens. IL-X may synergize with IL-2 in inducing the proliferation and/or differentiation of these antigen-activated CTL-P. The production of IL-X and IL-2 in these cultures also provides a simple explanation for the bystander C B A anti-D2 C T L response since these factors are non-antigen-specific in their mode of action and can therefore help C B A CTL-P that are specific for H-2 d . In C B A cultures stimulated with only D2 spleen cells, clone 9 cells are not activated. No ASHF or IL-X are produced in these cultures and the level of IL-2 produced will also be reduced since its production can be augmented by ASHF. The net result is that no C T L response can be detected in these cultures. The data in Table XXII (p.97) suggest that when high concentrations of IL-2 and IL-X were added to BDFj spleen cells, C T L were produced despite the absence of intentional antigen stimulation. This implies that the supra-optimal concentrations of IL-2 and IL-X can synergize in the activation of CTL-P. However, since it was previously shown that the C T L produced in clone 9-supplemented cultures were highly specific for the stimulating antigen (291), it would appear that in the presence of the stimulating antigen, IL-X may not work optimally in the non-specific induction of CTL-P. Therefore, it is possible that the role of IL-X may be relevant only in cases where antigen cannot optimally interact with the CTL-P and IL-X may be required as an alternative mechanism to stimulate CTL-P. This model can also be extended to account for the helper effects of clone 9 in low density B6 responder spleen cell cultures (see Chapter 2, Results section). In that system, clone 9 cells were stimulated by the D D alloantigen present on the responder B6 antigen-presenting cells. As a result of this specific interaction, clone 9 cells produce ASHF 88 and IL-X. ASHF can induce IL-2 production in alloantigen-stimulated B6 T cells. The IL-2 produced can then induce the proliferation of alloantigen-activated B6 CTL-P. IL-X may also synergize with JJL-2 in the activation of CTL-P. This model, together with the current three signal model for C T L activation (242), can be used to provide an explanation for the observation that clone 9 supernatant factors could synergize with EL-2-containing medium in augmenting C T L responses to alloantigens (See Table XII, p.52). In this case, ASHF probably synergized with other factors besides IL-2 in the augmentation of C T L responses. If purified ASHF were to be tested for synergy with other factors in the augmentation of C T L responses, ASHF is expected to synergize with other factors such as CTDF or RTF but not with IL-2 since the final effect of ASHF is to induce JJL-2 production. By contrast, one would expect IL-X to synergize with JJL-2 as well as with other factors such as CTDF and ASHF in the augmentation of C T L responses. Many aspects of this model remain to be tested. For instance, more direct experiments are required for the identification of the target cell for clone 9, ASHF and IL-X. The relationship of IL-X to other factors that have been implicated in C T L responses is presently unknown. Nevertheless, this model provides new insight as to how antigen-specific and non-specific factors can collaborate in activating CTL-P during a mixed lymphocyte reaction. As mentioned in the introductory section of this chapter, the requirements for activation of C T L may depend on variables such as the affinity of CTL-P for the antigen and the effective concentration of lymphokines such as IL-2. ASHF and factors such as IL-X may be more relevant under physiological conditions where only limited amounts of IL-2 are produced and where CTL-P with widely differing affinities for antigen may be activated. In these studies, the role of ASHF in inducing a C T L response was best demonstrated using helper cell-deficient thymocytes as responder cells. This mdirectly suggests that ASHF is required for C T L induction only in cases where help is deficient. If the thymocyte cultures were supplemented with excess IL-2, a C T L response readily took place in the absence of clone 9 cells or ASHF (see Chapter 3). However, activation of C T L by excess IL-2 may not be physiologically relevant since such high effective concentration of JJL-2 may be difficult to achieve under in vivo conditions. 89 The studies reported in this chapter also demonstrate that cloned antigen-specific helper cells can produce more than one type of factor that can influence C T L differentiation. Mosmann et al. (193) have performed a systematic study on the lymphokines that are produced by antigen-specific helper cells and found that Th cells can be classified into two broad categories on the basis of the types of lymphokine activities produced by each clone. Thl cells produce IL-2, EL-3 and y-IFN, whereas Th2 cells produce EL-3, BSF-1 and other lymphokine activities that can be attributed to BSF-1 (193,220). Whether or not clone 9 cells fall into the Th2 category remains to be determined. Since BSF-1 has been shown to have T-cell activating properties (220), it is important to determine if the IL-X activity in clone 9 supernatant is also due to BSF-1. The existence of two classes of helper cells, namely an EL-2-producing Thl cell and an EL-X and ASHF-producing Th2 cell (e.g. clone 9) would allow the C T L response to be more finely regulated. This form of regulation may be important in C T L responses to tumour-associated or conventional antigens where the frequency of helper cells to these antigens is expected to be low. 90 5. Summary The mechanism by which clone 9 and clone 25 ASHF induced the C T L responses to alloantigens was investigated. Clone 9 or ASHF helped C B A thymocytes to produce C T L against B6 (H-2b), but not D2 (H-2d), alloantigens. However, when BDFj (H-2 b/ d) spleen cells or an equal mixture of B6 or D2 spleen cells were used as stimulator cells, C T L responses to both B6 and D2 were induced. This suggested that clone 9 cells or ASHF could induce the production of long-ranged, non-antigen-specific helper factor(s) in B6-stimulated cultures. One of these long-ranged factors produced in B6-stimulated cultures was found to be IL-2. Thus, clone 9, which is a non-IL-2 producer, increased the production of IL-2 by irradiated B6 spleen cells and by C B A anti-B6 cultures by 4.7- and 5.7-fold, respectively. ASHF did not increase the amount of IL-2 produced by irradiated B6 spleen cells but increased the amount of IL-2 produced in C B A anti-B6 cultures by 3.8-fold. Clone 9 cells or ASHF did not increase IL-2 production in D2-stimulated cultures. Upon stimulation with Con A or antigen, clone 9 cells also produced a non-antigen-specific helper factor. This factor (TL-X) synergized with excess rIL-2 in the polyclonal activation of BDFj or D2 CTL-P. A model that involves the participation of ASHF, clone 9 cells, IL-2 and IL-X in the induction of a cytotoxic response is proposed. 91 Figure 6. Effect of varying the responder cell dose on the helper effect of clone 9 cells and ASHF. The indicated number of C B A thymocytes were cultured with 10^ irradiated (2000 rads) B6 spleen cells (o), 10 6 irradiated B6 spleen cells that had been pulsed with 300 |ig/ml ASHF (A), or 10 6 irradiated B6 spleen cells plus 10 4 irradiated (2000 rads) clone 9 cells (°) in V-bottomed microtitre wells in a volume of 0.20 ml. After 5 days, the cultures were assayed for cytotoxic activity using ^ Icr-labelled B6 Con A blasts as target cells. Each point represents the mean of eight cultures ± standard error. C B A Thymocytes(x 10"5) Table X V I I I . Clone 9 and A S H F can help bystander responses1 % Specific Lysis  Responder _ Stimulator Help** B6 Targets D2 Targets Thymocytes Spleen Cells (3xl0 5) (10 6) CBA B6 - .0.0 ± 1.2 CBA B6 ASHP 9.2 ± 2.4 CBA D2 _ -2.6 ± 1.9 CBA D2 ASHP 3.9 ± 0.9 CBA BDPi — 2.4 ± 0.8 -4.3 ± 1.2 CBA BDPX ASHF 20.1 ± 3.2 13.2 ± 3.7 CBA B6+D2 _ 5.2 t 1.2 -2.8 ± 1.5 CBA B6+D2 ASHP 20.4 ± 6.0 11.8 ± 3.3 CBA B6 -0.5 ± 1.8 CBA B6 C1.9 49.6 ± 7.3 CBA D2 _ 0.8 ± 2.3 CBA D2 C1.9 -4.1 ± 0.9 CBA BDPi _ 1.6 ± 1.2 0.1 ± 1.2 CBA BDPX C1.9 26.3 ± 5.9 36.2 ± 6.0 CBA B6+D2 _ 6.6 ± 3.8 8.0 ± 2.4 CBA B6+D2 C1.9 30.6 ± 4.9 48.4 ± 7.6 aCBA thymocytes (3xl0 5) were cultured with 10 irradiated (2000R) B6, D2, or BDFj spleen cells or with a 1:1 mixture of Irradiated B6 and D2 spleen cells ln a volume of 0.20 ml ln V-bottomed microtiter plates. The cultures were assayed for CTL acti v i t y after 5 days against the indicated target c e l l s . bHelp was added ln the form of 10* Irradiated (2000R) clone 9 cells or stimulator spleen cells which had been pulsed with 300 vg/ml ASHP. In cultures containing a 1:1 mixture of B6 and D2 stimulator c e l l s , only the B6 cells were pulsed with ASHP. Table XLX. Induction of IL-2 production by clone 9 or A S H F a CBA Spleen Cells Help 0 Amount of IL-2 produced Thymocytes (2000R) (Dnlts/ml) 3xl0 5 10 6 B6 0.6 3xl0 5 10 6 B6 ASHP ~ 2.3 3xl0 5 10 6 B6 Clone 9 3.4 3xl0 5 10 6 D2 1.9 3xl0 5 10 6 D2 ASHP 1.8 3xl0 5 10 6 D2 Clone 9 2.1 10 6 B6 0.3 IO 6 B6 ASHP 0.4 10 6 B6 Clone 9 1.4 10 6 D2 0.6 10* D2 . ASHP 0.3 10 6 D2 Clone 9 0.7 Clone 9 0.3 Unirradiated CBA thymocytes were cultured with irradiated B6 or D2 spleen c e l l s i n the presence or absence of help i n V-bottom wells, 0.2 ml/well, for 2 days. The culture supernatants were then harvested and i t s IL-2 a c t i v i t y determined. bHelp was added l n the form of 10* i r r a d i a t e d (2000R) clone 9 c e l l s or spleen c e l l s which had been pulsed with 300 yg ASHP/ml. 95 Table X X . Supernatants from B6-stimulated clone 9 cultures contain CTL-inducing activity Culture Combinations Used to Generate Supernatants* CBA Anti-B6 CTL Response0 % Specific Lysie (H+SB) . Spleen Cells Clone 9 ConA • 5.8 ± 0.9 - 6.6 ± 1.2 — 56.5 ± 1.6 BDP1 , , , 8.9 ± 1.2 BDF1 • 40.0 ± 1.5 D2 - 16.1 ± 1.5 D2 + 14.3 ± 1.9 B6 (2000R) _ — 10.8 ± 0.9 B6 (2000R) • 49.4 ± 1.9 D2 (2000R) - 9.2 ± 1.3 D2 (2000R) + 7.3 ± 0.3 a. IO 4 irradiated clone 9 cells were stimulated with Con A (2 (ig/ml), unirradiated BDF1 or D2 spleen cells (4 x ltVfywell), or with irradiated B6 or D2 spleen cells (2 x 105/well) in V bottomed wells in a total of 0.2 ml/well. On day 1, the culture supernatants were collected and tested for CTL-inducing activity and for IL-2 activity. b. The CTL-inducing activity was determined by testing the ability of the supernatants to help a C B A anti-B6 C T L response. Culture conditions were the same as in Table XVIII. When testing supernatants containing Con A , 50 mM alpha methyl mannoside was included to neutralize the effects of Con A. Culture supernatants were used at a final dilution of 20% (v/v). 96 Table X X I . Supernatants from Con A-stimulated clone 9 cells contain B6-specific helper factors Combinations used to % Specific lysis (M t S.B.)b generate supernatants* CBA anti-B6 D2 anti-B6 CBA antl-D2 Medium 4.5 + 1.4 8.6 + 2.4 6.6 ± 2.4 ConA 11.3 ± 1.1 2.3 ± 2.3 -1.0 ± 0.6 Clone 9 7.8 ± 3.0 19.4 ± 5.1 1.6 ± 0.9 Clone 9 + ConA 52.6 ± 1.9 29.8 ± 6.0 0.5 ± 0.6 (65.7 ± 3.9) c (51.0 ± 4.2) (57.9 ± 3.4) a. 1 x 10 4 irradiated clone 9 cells were stimulated wfdith 2 Ug/ml Con A in a volume of 0.2 m] in V-bottomed wells. The supernatant was harvested 24 hr later and tested for ASHF activity. Supernatants from control cultures containing medium, Con A or clone 9 cells were also included in the test for ASHF activity. b. Culture conditions for determining ASHF activity were the same as Table X V m . Each culture contained 3 x 10 5 responder thymocytes and 1 x 10 6 stimulator spleen cells plus 20% (v/v) helper supernatant Where Con A was present in the supernatant, 50 mM alpha methyl mannoside were added to neutralize the effects of Con A. c. The numbers in parenthesis are specific lysis values for cultures which were supplemented wfith 2% (v/v) EL4.PMA. 97 Tabic XXIJ. Presence of a non-antigen-specific helper factor (IL-X) in the supernatants of Con A-stimuIated clone 9 cells Combinations used to Spleen cells % Specific lysis (M ± S.B.b generate supernatants* (1x10*) -rIL-2 +rIL-2 Medium BDP1 5.0 ± 1.0 -0.7 ± 0.4 ConA BDP1 -1.2 + 0.6 2.7 ± 1.4 Clone 9 BDP1 -1.6 ± 0.5 3.0 + 1.7 Clone 9 • ConA BDP1 -1.6 ± 0.5 20.6 ± 7.6 Medium D2 1.0 ± 0./3 -0.2 ± 0 .4 ConA D2 2.0 ± 0.4 3.6 ± 1.1 Clone 9 D2 -1.2 ± 0.5 10.1 ± 5.6 Clone 9 + ConA D2 1.4 ± 0.3 31.3 ± 6.4 a. Supernatants were produced by stimulating clone 9 cells with Con A as described in Table XXI. b. 1 x 104 BDF1 or D2 spleen cells were cultured with 20% (v/v) of the indicated supernatants in the presence or absence of 10 U/ml rIL-2 in a volume of 0.20 ml in V-bottomed wells. Where the supernatant contained Con A, 50 mM alpha methyl mannoside was also included to neutralize the Con A. On day 5, each culture was assayed for total CTL activity by incubating with 3 x 103 51Cr-labelled P815 cells and 10 Ug/ml of the lectin Phytohemaglutinin-P. Refer to materials and methods section for further details. 98 Figure 7. A proposed model for the activation of C B A anti-B6 CTL-P by clone 9 cells, ASHF, IL-2 and IL-X. In CBA anti-B6 cultures, clone 9 cells are stimulated by the D b alloantigen on B6 antigen presenting cells (APC). This results in the production of IL-X and ASHF by clone 9 cells. ASHF can directly stimulate the production of IL-2 by activated B6 T cells or stimulate the production of IL-2 by activated C B A T cells via associative linked recognition. The IL-2 produced induces the proliferation and differentiation of C B A CTL-P that have been activated by H-2 b antigens. IL-X may synergize with IL-2 in inducing the proliferation and/or differentiation of these antigen-activated CTL-P. See Text for more detailed explanation. Symbols: APC, antigen-presenting cells; B6-T or CBA-T, IL-2 producing T cells of B6 or C B A origin; broken arrow, associative linked recognition for the stimulation of C B A - T by ASHF. 100 Chapter 5. Conclusions and future directions The work presented in this thesis has described the isolation of an ASHF-producing Th cell clone, the characterization of this Th cell clone and its soluble products and the study of the mechanism whereby this Th clone and its soluble products participated in the induction of C T L responses. The major conclusions reached from these studies are as follows: 1) Clone 9 is an H-2 d , Thy-1 + , Lyt-1"2", C D 4 + helper cell which specifically enhanced the C T L responses to alloantigens via the action of soluble mediators. 2) One of these soluble mediators is the ASHF. This ASHF contains a 50,000 M W molecule which binds D b antigen in an MHC-unrestricted manner and in so doing, can mediate antigen-specific help in the induction of C T L responses. 3) Due to its reactivity with mAbs to the B-chain of the T-cell receptor, this ASHF may be closely related to the T-cell receptor. 4) One of the mechanisms by which clone 9 or its ASHF mediate their helper function is through the induction of IL-2 production in B6-stimulated cultures. In the case of ASHF, the induction of IL-2 production can occur via an associative linked recognition mechanism. 5) The other factor produced by clone 9 cells is a non-specific T-cell activating factor termed IL-X which has been found to synergize with EL-2 in the nonspecific activation of C T L responses. By inducing IL-2 production, ASHF can therefore act in concert with IL-X in the induction of C T L responses. Apart from the new insights gained with regard to the nature and mechanism of action of Th cells, the work described herein has also provided the ground work for future investigations. Some experiments and suggestions can be considered for future investigations. For example, the biochemical characterization of the ASHF should be further investigated. In particular the subunit structure of ASHF and the biological activity of the individual subunit of the ASHF should be determined. Furthermore, the potential presence of Ia determinants on ASHF should be examined. With the availability of a mAb specific for the ASHF, it is now possible to devise a binding assay for ASHF, which will be more rapid and sensitive than the biological assay. The mAb can also be employed for the isolation and biochemical purification of ASHF. In addition, this mAb can be applied in the screening of cDNA libraries for ASHF-encoding cDNA genes. Since clone 9 cells appear to produce a novel lymphokine upon 101 stimulation by antigen or Con A, it should be of interest to further characterize this novel lymphokine. There are still many unanswered questions with regard to the mode of action of ASHF. The target cell of ASHF, for instance, should be investigated. Preliminary studies suggested that the target cells of ASHF may be a Thy-1 + , C D 4 + , glass-adherent cell and radiation-sensitive cell. This conclusion should be verified. Also, additional effects of ASHF on other cell types, such as the increase in IL-2 receptor expression or the production of other lymphokines, should be analysed. It should also be of interest to determine whether this ASHF possesses helper activity in B cell responses. Finally, the genes encoding ASHF should be characterized and compared to those encoding the T cell antigen receptor. Since T-cell antigen receptors are integral membrane proteins and ASHF is a secreted protein, it will be of interest to determine whether ASHF uses a novel CB gene which is devoid of a transmembrane exon or whether ASHF is a truncated form of the T-cell receptor. The study presented in this thesis provides a rationale for continued interest in Th cells and their products, in particular ASHF. It is believed that with increased effort, patience and faith, the elucidation of the nature of ASHF will be completed in the near future. 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