Antigen-binding receptors on T cells from long-term MLR. evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (VH) determinants strongly inhibited vesicle binding by both primary and long-term MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesicle-binding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectivelly blocked with the anti-VH reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.     

Receptor specificity of Ia-restricted T lymphoblasts activated against trinitrobenzene sulfonate-coupled spleen cells: recognition of distinct trinitrophenyl and Ia moieties

The receptor specificity of H-2-restricted T lymphoblasts activated against trinitrobenzene sulfonate (TNBS)-coupled spleen cells was examined using an antigen binding assay. A population of Lyt-1+,2-T lymphoblasts acquired syngeneic Ia determinants during 4 days of primary culture with hapten-coupled stimulator cells. Syngeneic Ia was not reexpressed after trypsin treatment of the T cells, but was found after incubation with soluble Ia shed from lipopolysaccharide-activated blasts. Self-Ia binding was specific in that Lyt-1+,2- but not Lyt-1-,2+ cells acquired the antigen, and in that self-Ia bound more effectively than allogeneic Ia material. To determine the relationship of self-Ia binding to the recognition of foreign antigen, the binding of trinitrophenyl (TNP)-coupled plasma membrane vesicles by TNP-specific T cells was studied. TNP-vesicle binding occurred via TNP and H-2(Ia) molecules on the vesicles in that binding was inhibited with antibodies against TNP or H-2(Ia) molecules but not non-major histocompatibility complex (e.g., Ly-6.2) molecules on the vesicles. Complete inhibition of TNP-vesicle binding by an Iak-restricted TNP-specific T-cell line occurred with soluble TNP-lysine, but not an unrelated hapten, N-iodoacetyl-N-(5-sulfonic-1-naphthyl)ethylenediamine (I-AED)-cysteine. Conversely, I-AED-cysteine, but not TNP-lysine, inhibited binding of I-AED-coupled B6 vesicles by B6 anti-I-AED T cells. Significant, but weak inhibition of TNP-vesicle binding by the anti-TNP line was observed with glycoprotein preparations containing partially purified self-Ia molecules. However, inhibition was specific for I-Ak molecules, in that inhibition was lost after removal of I-Ak molecules from the glycoprotein preparation, and very little inhibition occurred with soluble glycoproteins prepared from thymocytes which contained very little Ia material or from LPS blasts of an unrelated H-2 haplotype. These results suggest a recognition model in which TNP and Ia determinants are recognized by neighboring receptor combining sites.     

The syngeneic mixed leukocyte reaction: the genetic requirements for the recognition of self resemble the requirements for the recognition of antigen in association with self

We have used cells from inbred strain 2 and strain 13 guinea pigs in order to define further the role of Ia antigens in the syngeneic mixed leukocyte reaction (MLR). The guinea pig syngeneic MLR resembled the autologous MLR in man in that it demonstrated both memory and specificity. The Ia antigens appeared to be the proliferative stimuli in that the primary stimulator cell was an Ia-positive adherent peritoneal exudate cell (PEC) and the reaction could be specifically inhibited by anti-Ia sera directed to the stimulator cell. We also demonstrated the existence of two (2 x 13)F1 T cell populations that were capable of reacting to one or the other parental PEC in the absence of any known exogenous antigen. These results suggest that the syngeneic MLR may represent T cell activation mediated through a receptor for self Ia.     

Mouse T lymphocytes proliferative responses specific for human MHC products in mouse anti-human xenogeneic MLR

It has been reported that human T cells recognize the polymorphism of murine Ia antigens in the human anti-mouse xenogeneic mixed lymphocyte reactions (MLR). In this study, murine T cell recognition of human Class II antigens of the major histocompatibility complex (MHC) was analyzed in mouse anti-human xenogeneic MLR responses. The xenoreactive murine T cell proliferative response was blocked by adding anti-HLA-DR monoclonal antibody to the xenogeneic MLR culture. The specificity of xenoreactive murine T cells was examined with regard to the secondary and tertiary xenogeneic MLR system. The xenoreactive murine T cells were restimulated by distinct human stimulator cells that had no shared HLA antigens with the stimulator used in the primary MLR. The data presented here show that the murine xenoreactive T cells recognize the shared determinant(s) of HLA-DR antigen on non-T, non-B stimulator cells. The xenoreactive murine T cell proliferative responses were mediated by Thy-1+, Lyt-1+, and Lyt-2- cells. Furthermore, the xenoreactive T cell responses required Ia+ cells, and Ia antigen on accessory cells plays a crucial role in eliciting the xenoreactive responses against human stimulator cells, while Ia+ accessory cells in the responding cell population are not essential for the elicitation of allogeneic MLR responses, as reported previously.     

Inhibition of secondary mouse mixed lymphocyte reaction by anti-Ia sera

Secondary mixed lymphocyte reaction (MLR-II) was studied in A.TH anti A.TL and A.TL anti-A.TH combinations in which stimulation was mainly due toH-2I-region differences. In both cases the MLR-II was specifically inhibited by the responder anti-stimulator Ia serum. The level of inhibition was dependent on the ratio of the amount of immune serum to the number of stimulating cells. The inhibitory activity and Ia antibodies were specifically absorbed and eluted together. The results confirm that the lymphocyte-activating determinants of the MLR-II (1) are carried by the Ia molecules and (2) are identical to the serologically defined Ia determinants. —Anti-Ia sera directed against private and public specificities of the stimulating cell induced a higher level of inhibition than anti-Ia sera directed only against public specificities, indicating that both private and public Ia specificities are involved in restimulation during MLR-II. — These results, in connection with others, suggest that the receptor of the proliferating T cell recognizes the same Ia determinant as the combining site of the Ia-recognizing antibody.Abbreviations used in this paper Lad lymphocyte-activating determinant - MLR mixed lymphocyte reaction - MLR-I, MLR-II primary, secondary MLR - PRC primed responder cells - LCT dye exclusion lymphocytotoxicity microtechnique - RR relative response     

Blocking of MLC stimulation by anti-Ia sera: studies with the virus plaque assay.

The development of congenic mouse strains identical at the H-2K and H-2D loci but differing by I-region associated (Ia) determinants has permitted an association to be established between Ia determinants and stimulation in mixed lymphocyte culture reactions (MLR). The present experiments were undertaken to establish whether the Ir-coded control of MLR operated at the level of recognition or of stimulation. Reciprocal MLR were established between A.TH and A.TL mouse spleen cells in the presence or absence of anti-Ia sera directed either at determinants of the stimulating or responding cells. The number of T cells responding was assessed by the virus plaque assay. Anti-Ia sera directed against the responding cells were no more inhibitory of the MLR than normal mouse serum. In contrast, anti-Ia sera directed against determinants of the mitomycin-treated stimulating cells markedly inhibited activation of T cells in the MLR.     

Unique T cell Ia antigen expressed on a hybrid cell line producing antigen-specific augmenting T cell factor

Hybrid cell lines were established by fusion between keyhole limpet hemocyanin(KLH) binding T cells of A/J mice and an AKR T cell tumor line, BW5147. Hybrids were selected for the presence of Ia antigen and KLH-specific augmenting activity of their extracts in the secondary antibody response. The detailed phenotypic and functional analysis of 1 of these clones, FL10, is reported here. The hybrid was positive for both Thy1.1 and Thy1.2 antigens and possessed the Lyt-1+,2-,3- phenotype. Both VH and Ia determinants were detected on their cell surface. The IA locus was mapped in the I-A subregion, but the Ia specificities were serologically distinct from those of B cell Ia antigen. This was demonstrated by the fact that anti-Ia antiserum preabsorbed with B cells could react with the hybrid cells, whereas none of the monoclonal anti-Ia specific for private and public determinations of Iak could. The extract from the cell line specifically augmented the in vitro secondary antibody response against dinitrophenylated KLH, and this activity was removed by absorption with antigen and conventional anti-Ia antisera. The results indicate that the cell line, FL10, carries Ia antigen unique to the T cell, which is associated with the antigen-specific augmenting molecule.     

Acquisition of syngeneic I-A determinants by T cells proliferating in response to poly (Glu60Ala30Tyr10)

The T cell proliferative response in mice to the synthetic polymer GAT is under Ir gene control, mapping to the I-A subregion of the H-2 major histocompatibility complex (MHC). Antigen-dependent proliferation in vitro of in vivo GAT-primed lymph node cells can be inhibited by a monoclonal antibody to Ia-17, an I-A public determinant. Using this antibody for direct immunofluorescent analysis, T cells in GAT-stimulated proliferative culture are identified that express syngeneic I-A during culture. This expression is strictly antigen dependent, requires restimulation in vitro, and requires the presence of I-A-positive adherent antigen-presenting cells. T cells bearing I-A can be enriched by a simple affinity procedure, and I-A-positive cells separated on a FACS are shown to retain antigen-specific reactivity. The acquisition of I-A determinants by T cells under these culture conditions is not nonspecific. The Ia determinants borne by T cell blasts appear to be dictated by the I subregion to which the relevant Ir gene maps, and which codes for the Ia molecule involved in presentation of the antigen. Thus, (B6A)F1 (H-2b X H-2a)F1 LNC express I-Ak antigens when proliferating to GAT but not when stimulated by GLPhe, the response to which is under I-E subregion control. The relation of Ir gene function to Ia-restricted antigen presentation and self-Ia recognition is discussed.     

T lymphocyte-enriched murine peritoneal exudate cells. III. Inhibition of antigen-induced T lymphocyte Proliferation with anti-Ia antisera.

The recent development of a reliable murine T lymphocyte proliferation assay has facilitated the study of T lymphocyte function in vitro. In this paper, the effect of anti-histocompatibility antisera on the proliferative response was investigated. The continuous presence of anti-Ia antisera in the cultures was found to inhibit the responses to the antigens poly (Glu58 Lys38 Tyr4) [GLT], poly (Tyr, Glu) ploy D,L Ala-poly Lys [(T,G)-A--L], poly (Phe, Glu)-poly D,L Ala-poly Lys [(phi, G)-A--L], lactate dehydrogenase H4, staphylococcal nuclease, and the IgA myeloma protein, TEPC 15. The T lymphocyte proliferative responses to all of these antigens have previously been shown to be under the genetic control of major histocompatibility-linked immune response genes. The anti-Ia antisera were also capable of inhibiting proliferative responses to antigens such as PPD, to which all strains respond. In contrast, antisera directed solely against H-2K or H-2D antigens did not give significant inhibition. Anti-Ia antisera capable of reacting with antigens coded for by genetically defined subregions of the I locus were capable of completely inhibiting the proliferative response. In the two cases studied, GLT and (T,G)-A--L, an Ir gene controlling the T lymphocyte proliferative response to the antigen had been previously mapped to the same subregion as that which coded for the Ia antigens recognized by the blocking antisera. Finally, in F1 hybrids between responder and nonresponder strains, the anti-Ia antisera showed haplotype-specific inhibition. That is, anti-Ia antisera directed against the responder haplotype could completely block the antigen response controlled by Ir genes of that haplotype; anti-Ia antisera directed against Ia antigens of the nonresponder haplotype gave only partial or no inhibition. Since this selective inhibition was reciprocal depending on which antigen was used, it suggested that the mechanism of anti-Ia antisera inhibition was not cell killing or a nonspecific turning off of the cell but rather a blockade of antigen stimulation at the cell surface. Furthermore, the selective inhibition demonstrates a phenotypic linkage between Ir gene products and Ia antigens at the cell surface. These results, coupled with the known genetic linkage of Ir genes and the genes coding for Ia antigens, suggest that Ia antigens are determinants on Ir gene products.     

Role of nominal antigen and Ia antigen in the binding of antigen-specific T lymphocytes to macrophages.

We have previously demonstrated that when primed T lymphocytes were repeatedly incubated on monolayers of antigen-pulsed macrophages (M phi), the cells that failed to adhere to the monolayer demonstrated a marked depletion of their proliferative response that was specific both for the antigen used for pulsing the M phi and for Ia determinants on the M phi. In order to further analyze the contribution of the nominal antigen and Ia antigens to the physical binding of T lymphocytes to M phi, we have attempted to block the absorption of T lymphocytes to M phi with a large excess of soluble antigen and with anti-Ia sera. Our results demonstrate that anti-Ia sera inhibit but that soluble antigen augments the binding of specific T lymphocytes to M phi. The implications of these findings for "dual recognition" and "linked recognition" models of T lymphocyte receptors are discussed.     

The immunobiology of T cell responses to Mls-locus-disparate stimulator cells. II. Effects of Mls-locus-disparate stimulator cells on cloned, protein antigen-specific, Ia-restricted T cell lines

Cloned, protein antigen-specific, Ia-restricted T cell lines frequently (approximately 20%) also respond strongly to stimulator cells from strains expressing stimulatory alleles at the chromosome 1-encoded Mls-locus. Furthermore, such responses are blocked by monoclonal antibodies specific for Ia antigens expressed by the stimulator rather than the responder cells. However, such responses show no specificity for polymorphic determinants on Ia molecules, although in such responses, as in primary and secondary T cell responses to stimulating Mls-locus alleles, I-E molecules appear to play a central role. These results, combined with the unique immunobiology of the primary T cell proliferative response to Mls-locus-disparate stimulator cells, suggest to us that this response involves the interaction of the receptor on T cells for antigen:self Ia with a relatively nonpolymorphic region of Ia glycoproteins. This hypothesis is supported by the observation that a monoclonal antibody to the T cell receptor will inhibit both responses, although the response to Mls-locus-disparate stimulators appears to be more sensitive to these antibodies. We propose that the interaction of the T cell receptor with Ia is stabilized by a cell interaction molecule encoded or regulated by the Mls-locus gene product permitting the T cell receptor:Ia glycoprotein interaction to lead to T cell activation.     

Inhibition of secondary mouse mixed lymphocyte reaction by anti-Ia sera

Secondary mixed lymphocyte reaction (MLR-II) was studied in A.TH anti A.TL and A.TL anti-A.TH combinations in which stimulation was mainly due to H-21-region differences. In both cases of MLR-II was specifically inhibited by the responder anti-stimulator Ia serum. The level of inhibition was dependent on the ratio of the amount of immune serum to the number of stimulating cells. The inhibitory activity and Ia antibodies were specifically absorbed and eluted together. The results confirm that the lymphocyte-activating determinants of the MLR-II (1) are carried by the Ia molecules and (2) are identical to the serologically defined Ia determinants. - Anti-Ia sera directed against private and public specificities of the stimulating cell induced a higher level of inhibition than anti-Ia sera directed only against public specificities, indicating that both private and public Ia specificities are involved in re-stimulation during MLR-II. - These results, in connection with others, suggest that the receptor of the proliferating T cell recognizes the same Ia determinant as the combining site of the Ia-recognizing antibody.     

Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: Evidence for two classes of Ia antigens

Previously published data suggest that both xenogeneic and allogeneic anti-Ia sera can recognize carbohydrate-defined antigenic determinants on the surface of lymphocytes. There is also evidence, based on studies with allogeneic anti-Ia sera, that protein-defined Ia antigens exist. In this paper the relationship between these two types of Ia antigen was examined. It was found that in capping studies, the allogeneic anti-Ia serum could cap off the antigens recognized by the xenogeneic antiserum, whereas the xenogeneic antibodies could, at least partially, clear the surface of lymphocytes of Ia antigens detected by the allogeneic antibodies. On the other hand, when immunoprecipitates of radioiodinated cell-surface antigens were examined by SDS-polyacrylamide-gel electrophoresis, it was found that the xenogeneic anti-Ia serum did not immunoprecipitate any labeled material. In contrast, the allogeneic antiserum immunoprecipitated a labeled molecule which corresponded to the protein-defined Ia antigen described by others. Finally, it was shown that serum Ia antigens could be bound by either mouse or rabbit anti-Ia antibody, and this binding blocked any further reactivity with either serum. These results were interpreted as suggesting that two separate classes of Ia antigen molecule appear on the lymphocyte surface-one class has carbohydrate-defined antigenic specificities and the other has protein-defined determinants. Allogeneic anti-Ia sera contain antibodies against both these antigenic systems, whereas xenogeneic sera recognize only the carbohydratedefined series. The genetic implications of this interpretation are discussed.     

Existence of T cells manifesting self-reactivity indistinguishable from alloreactivity

The studies reported here were designed to analyze the phenotypic characteristics of self-reactive T lymphocytes induced in culture by allogeneic effect factor (AEF), as well as the control of their functional activities by the major histocompatibility complex (MHC). Unprimed T cells cultured with AEF in the absence of exogenous stimulating target cells become activated against self-antigens, as evidenced by their ability to manifest two distinct activities. First, such cells could lyse syngeneic target cells. This cytolytic activity was directed against H-2K antigens and was mediated by Lyt-2+ T cells. Second, the AEF-activated T cells could be stimulated in a secondary culture to high levels of proliferative activity by irradiated syngeneic spleen cells. The stimulator cells in this syngeneic mixed lymphocyte reaction (MLR) were found to be Thy-1-negative, Ia-positive splenic adherent cells. Stimulation in the secondary syngeneic MLR was provided by I-region specificities, and the majority of the proliferating cells were Lyt-1+ cells. Finally, AEF-induced T cells were effective in serving as effectors of graft-vs-host reactions in vivo in syngeneic recipients. These results prove that, under appropriate conditions, murine T lymphocytes can display aggressive patterns of self-reactivity that are similar in both quantity and quality to the classical patterns of alloreactivity and may have great significance for our understanding of MHC recognition processes.     

Immunologic effects of whole-body ultraviolet (uv) irradiation. III Defective splenic adherent cell function in alloantigen-stimulated T-cell proliferation

The daily exposure of a mouse to ultraviolet (uv) radiation causes a selective depletion of Ia-bearing adherent cells in that animal's spleen. This depletion manifests itself in functional deficiencies in the presentation of protein antigens and haptens to T cells. The present studies demonstrate a defect in splenic adherent cells (SAC) from uv-irradiated mice resulting in defective alloantigen presentation. We show that unfractionated splenocytes and SAC from uv-irradiated mice show decreased stimulatory activity in allogeneic MLR. We then utilize this phenomenon induced by uv radiation to characterize the stimulator cell in the M locus (Mls) determinant-driven MLR. We show that the stimulator cell in Mls determinant-driven MLR is an adherent cell and demonstrate that this stimulator cell bears Ia determinants by showing that whole spleen cells and SAC from mice treated with uv radiation are inefficient stimulators of the Mls determinant-driven MLR. The importance of the Ia determinant on the stimulator cells in Mls determinant-driven MLR is corroborated by the demonstration that a monoclonal antibody directed at this determinant fully blocks the Mls determinant-driven MLR. The significance of these studies to the problem of alloreactions in vivo is discussed.     

Characterization and function of autoreactive T-lymphocyte clones isolated from normal, unprimed mice

Self-Ia-reactive cloned T-cell lines, designated PK, were established by long-term culture of T cells from normal DBA/2 mice with irradiated syngeneic splenic adherent cells (SAC), rich in macrophages and dendritic cells. The cell lines were Thy 1+, Lyt 1+, Lyt 2-, produced IL-2 following stimulation with syngeneic spleen cells, and did not exhibit alloreactivity when screened against six different H-2 haplotypes. Of the five cloned PK cell lines tested, four were I-Ed restricted while one was I-Ad restricted as determined by genetic mapping and blocking studies carried out with monoclonal anti-Ia sera. Extensive specificity studies suggested that the PK cells reacted to syngeneic Ia molecules alone and not to foreign antigens such as fetal calf serum (FCS) used in the culture medium, in association with self-Ia. SAC pulsed with FCS or other protein antigens such as turkey gamma-globulin (TGG) were tested for their ability to induce proliferation of autoreactive T cells and other antigen-specific T cells using culture conditions consisting of serumless medium and interleukin 2 (IL-2). The data showed that the autoreactive T cells proliferated better in response to antigen-unpulsed SAC, while FCS-specific and TGG-specific cell lines, developed independently, proliferated only in response to FCS- or TGG-pulsed SAC, respectively, but not to antigen-unpulsed SAC. These results clearly distinguished the autoreactive T-cell clones from the antigen-specific T-cell clones. Preliminary studies carried out to investigate the functions of autoreactive T cells suggested that these cells helped in the in vitro differentiation of alloantigen-specific cytotoxic T lymphocytes (CTL) from CTL precursors obtained from the thymus and augmented syngeneic, allogeneic, and antigen-specific immune responses in vitro. The autoreactive T cells were also capable of inducing both proliferation and differentiation of antigen-specific populations of B cells in the absence of antigen. The present investigation suggests that autoreactive, non-antigen-reactive T cells can be cloned from normal, unimmunized mice and that such cell lines may provide a powerful tool for analyzing the role of the syngeneic mixed lymphocyte reaction in induction and maintenance of both T-and B-cell immune responses.     

Properties of anti-idiotypic T cell lines propagated with syngeneic B lymphocytes. I. T cells bind intact idiotypes and discriminate between the somatic idiotypic variants in a manner similar to the anti-idiotopic antibodies

We describe the development of T cell lines possessing a binding specificity for syngeneic T15 idiotopes (Id) expressed on phosphorylcholine (PC)-reactive Ig. The lines were obtained by cultivation of BALB/c splenic T cells with T15 Id+ stimulator cells BCg3R-1d, a BALB/c lymphoma transfected with genomic sequences mu and kappa with S107 (T15) variable regions. Resulting Thyl-2+, L3T4+ cell lines depend on the T15 Id+ BCg3R-1d cells for growth and demonstrate the ability to bind TEPC15, a S107 germline-encoded, PC-specific Ig alpha. The specificity of the 125I-TEPC15 binding was studied in a competitive RIA with various unlabeled Ig. The isolated H and L chains of TEPC15 failed to inhibit the 125I-TEPC15 binding, and the T15-, PC-binding proteins M603 (alpha) and M511 (alpha) inhibited the binding either poorly or not at all. Moreover, the T cell lines had a discriminatory binding specificity for various T15+ Ig that are somatic variants of TEPC15 and that differ from each other in discrete, conformational Id (epitopes) detectable with specific monoclonal anti-Id. The T cell lines could be grouped according to their binding patterns, which were comparable to the recognition patterns of certain monoclonal anti-Id. These data suggest the existence of T cells with specificity for serologically-defined determinants of syngeneic idiotypes.     

Cellular interaction between cytotoxic and suppressor T cells against syngeneic tumors in the mouse.

Splenic T cells from animals bearing growing syngeneic tumors specifically inhibited the effector process of tumor cell lysis by the cytotoxic T cell which had been activated in vitro by mitomycin C treated homologous tumor. The suppression was strictly specific for the individual tumor by which suppressor cells were generated, whereas in some cases cytotoxic T cells generated by two closely related sarcomas showed a certain degree of crossreactivity. This suggests that suppressor and cytotoxic T cells recognize different antigenic moieties on tumor cells; one unique to the individual tumor and the other shared by related tumor cell lines.The suppressor T cell from tumor bearing animals possessed Ia antigen controlled by a gene in I-J subregion of H-2 major histocompatibility complex. Cytotoxic T cells generated by some but not all syngeneic tumors were also killed by anti-Ia and complement; however, the Ia antigen on such cytotoxic T cells was found to be controlled by a locus in I-A subregion. In general, the cytotoxic T cells generated by newly established tumor cell lines had Ia antigen, whereas some old cell lines, which were capable of growing across the H-2 barrier, activated the Ia negative cytotoxic T cell. These results collectively indicate that the immunological resistance against tumors is dependent on the balance of activations of the cytotoxic and suppressor T cells with different specificities and phenotypic expressions.     

Characteristics and functions of Sendai virus-specific T-cell clones

Several murine Sendai virus-specific T-cell clones were characterized in vitro and in vivo. All T-cell clones were phenotypically Thy-1.2+, and most clones were Lyt-1+,2-; one T-cell clone was Lyt-1-,2-. Some of the clones proliferated in response to antigen presented on I region-compatible stimulator cells. Proliferation could be inhibited by monoclonal antibodies directed against class II antigens. Clones which proliferated in response to antigen secreted lymphokines which could be identified as Interleukin 2 and Interleukin 3. All of the clones tested in vivo induced a delayed-type hypersensitivity response in syngeneic mice challenged with antigens. Depending on the experimental conditions chosen, Interleukin 2-producing clones as well as non-Interleukin 2-producing clones mediated help for stimulation of cytolytic T lymphocytes.     

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.