The role of class II MHC molecules in the activation of class I-reactive T cell hybridomas

To examine the role of Ia molecules in T cell responses to allo-class I major histocompatibility antigens, a series of allo-class I-reactive T cell hybridomas was established. Of 134 T cell hybridomas obtained from the fusion of C3H/HeJm or B10.HTT T cells stimulated with C57BL/6 splenocytes, nine T cell hybridomas were reactive to class I antigens and 126 T cell hybridomas were reactive to class II antigens. Six of the nine IL 2-producing T cell hybridomas were further analyzed: five mapped to H-2Kb and the other mapped to H-2Db. Three of these T cell hybridomas, HTB-157.7, HTB-176.10, and HTB-177.2, could react to the EL-4 cell line that expresses H-2Kb and H-2Db class I antigens but lacks class II I-Ab molecules. Furthermore, the activation of these three T cell hybridomas with C57BL/6-derived splenocytes was not blocked by either anti-I-A or anti-L3T4 antibody. In contrast, the other three T cell hybridomas, CB-127.6, CB-221.7, and HTB-102.7, failed to react with EL-4 but reacted with the LB cell line which expresses class I (H-2Kb, H-2Db) and class II (I-Ab) molecules. Although class II molecules were required for activation of the latter clones, there was no apparent I-A allele specificity, suggesting that a relatively nonpolymorphic Ia determinant was involved. The activation of the three latter T cell hybridoma clones with C57BL/6 splenocytes could be blocked completely by either anti-I-A or anti-L3T4 antibody. The data are interpreted in terms of possible T cell receptor models for recognition of class I with nonpolymorphic class II determinants.     

Role of the L3T4-antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells

We have used two monoclonal antibodies (Mab) to the L3T4 antigen to reexplore the role of this molecule in the process of T cell activation. Both Mab (Gk1.5 and 2B6) were capable of inhibiting Con A-induced IL 2 production by a number of antigen-specific T cell hybridomas in an assay system that was free of major histocompatibility complex (MHC) class II antigen-bearing cells. The inhibition produced by the anti-L3T4 Mab was specific, because other Mab to cell surface antigens expressed on the hybridomas were without inhibitory effects. These studies rule out the possibility that the mechanism of inhibition by anti-L3T4 in this model is mediated by blocking interaction of L3T4 with MHC class II products. Taken together, these results and those of other groups of investigators, are most compatible with a dual function for L3T4 in T cell activation. L3T4 might first interact with MHC class II molecules or other molecules on target or accessory cells; L3T4 would subsequently transmit a signal that would regulate the activation process. Mab to L3T4 might exert inhibitory effects at one or both of these steps.     

Characterization of cell surface molecules involved in the recognition of antigen-presenting cells by cloned helper T-cell lines

The recognition of antigen-presenting cells (APCs) by T helper (TH) cells occurs in an antigen (Ag)-specific, MHC-restricted manner. Recent evidence, however, suggests that other interaction molecules may also be involved in TH:APC interaction in addition to the T-cell receptor (Ti) and class II or la antigens. We chose, therefore, to examine the role of various interaction molecules (Ia, Ti, L3T4, and LFA-1) in Ag presentation using several TH clones with distinct recognition patterns (self-Ia, self-Ia/Ag, and allogenic Ia). We describe here the use of a rapid clustering assay to study the initial binding events that occur between TH cells and APCs of various types. In all combinations of TH cells and APCs, conjugate formation was both Ag-specific and MHC-restricted. Moreover, with one exception cell clustering was prevented by the addition of monoclonal antibodies (mAb) against either the T-cell receptor or class II MHC molecules. In contrast, mAb to L3T4 and LFA-1 generally failed to inhibit cluster formation even though T-cell proliferation was profoundly inhibited. The relative importance of these interaction molecules in conjugate formation appeared to depend on the APC type as well as on the T-cell clone used. The implications of these findings for the mechanisms of Ag presentation and T-cell activation are discussed.     

Inhibition of an allospecific T cell hybridoma by soluble class I proteins and peptides: estimation of the affinity of a T cell receptor for MHC

To investigate the molecular basis of the interaction between the T cell receptor and the MHC class I antigen in an allogeneic response, a soluble counterpart of the murine class I molecule, H-2Kb, was genetically engineered. Cells secreting this soluble molecule, H-2Kb/Q10b, inhibited stimulation of an H-2Kb-reactive T cell hybridoma by cells transfected with H-2Kbm10, a weak stimulus, but not by H-2Kb- or H-2Kbm6-transfected cells. Soluble purified H-2Kb/Q10b protein also blocked T cell stimulation. In addition, a peptide from the wild-type H-2Kb molecule spanning the region of the bm10 mutation specifically inhibited activation of the T cell hybridoma by H-2Kbm10 cells, thus suggesting that amino acid residues 163-174 of H-2Kb define a region important for T cell receptor binding. An estimate for the Kd of the T cell receptor for soluble H-2Kb/Q10b was 10(-7) M, while the Kd for soluble peptide 163-174 was 10(-4) M.     

Contribution of antigen processing to the recognition of a synthetic peptide antigen by specific T cell hybridomas

Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the alpha-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the alpha-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.     

The role of Ia molecules in the activation of T lymphocytes. I. The activation of an IL 1-dependent IL 2-producing T cell hybridoma by Con A requires an interaction, which is not H-2-restricted, with an Ia-bearing accessory cell

A model of accessory cell-dependent lectin-mediated T cell activation was investigated by utilizing a mitogen-inducible T cell hybridoma. A continuous MHC-restricted antigen-specific T cell line was fused with the azaguanine-resistant AKR thymoma BW5147. A hybrid, RF1.16B, was identified that is minimally inducible by Con A stimulation alone but is stimulated by Con A in the presence of T cell-depleted accessory cells to produce interleukin 2. The accessory cell function can be replaced by the monokine interleukin 1. Thus the lectin is a sufficient trigger for the hybrid in the absence of MHC restriction elements. The accessory cell function from splenocytes is provided by a non-B, non-T, predominantly Ia-bearing radioresistant cell. The interaction between the RF1.16B hybrid and the accessory cell population is not H-2-restricted. Control experiments, including the use of a cloned source of accessory cells, ruled out contaminating T cells or direct lectin effects as an explanation for the lack of H-2 restriction. The finding that an Ia-bearing cell is required for activation in an MHC-nonrestricted manner is discussed, and a hypothesis is raised that Ia antigens may play a role in addition to that of being a restriction element.     

Role of L3T4 and Ia in the heteroclitic response of T cells to cytochrome c

The activation of helper T lymphocytes has been proposed to result from the sum of low-affinity interactions between the specific immune receptor, as well as nonpolymorphic receptors such as L3T4 on the T cell surface, and nominal antigen and Ia displayed in a multivalent array on the antigen-presenting cell surface. The present work takes advantage of a T cell hybridoma specific for pigeon cytochrome c in the context of I-Ek, which responds to tobacco hornworm moth cytochrome c at one hundredth the concentration of the homologous antigen, to determine if the T cell's requirement for L3T4 and Ia is directly related to its functional affinity for antigen. The results demonstrate that the T cell's activation by pigeon cytochrome c was blocked by antibodies directed to L3T4 and to I-Ek, even at antigen concentrations twofold to fourfold above those required for maximal responses. In contrast, the response to tobacco hornworm moth cytochrome c was not as affected by these antibodies under equivalent superoptimal conditions. The same phenomenon was observed for the T cell's activation by the carboxyl-terminal peptide fragments of the two cytochromes c, which do not require processing, indicating that the differences were not due to the relative efficiency of processing and/or presentation of the antigens. Although both I-Ek- and L3T4-specific antibodies blocked the T cell response to pigeon cytochrome, antibodies to I-Ak had no effect, even though I-Ak had been considered to be a ligand for L3T4. Thus, either Ia does not bind L3T4 or, if it does, I-Ek must be a sufficient ligand for L3T4 for T cells that recognize their antigen in the context of I-Ek. These studies provide more definitive evidence that the T cell's requirement for the functions of Ia and of L3T4 is dependent on the T cell's functional affinity for its antigenic determinant. This data is consistent with a model of T cell activation in which, given a high enough affinity of the T cell receptor for the processed antigen, the requirement for other components of a stimulatory complex, such as Ia and L3T4, may diminish to undetectable levels.     

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.     

T lymphocyte heterogeneity in the rat. III. Autoreactive T cells are activated by B cells

Evidence has been presented to show that CD4+ autoreactive T cell lines (ATs)2 in the rat require periodic stimulation with syngeneic spleen cells for in vitro proliferation. This proliferation can be blocked by treatment of the stimulator (spleen) cells with mAb to Ia antigens. Although ATs are Ia+ and can activate the allogeneic MLR, they fail to be autostimulatory. Fractionation of the spleen cells revealed that ATs can be stimulated with B cells and not by macrophages, although the latter were efficient in several accessory cell functions, including antigen presentation, lectin-dependent T cell activation and allogenic MLR response. Moreover, B cells proliferated and differentiated in response to AT cells. These data are compatible with a model in which ATs respond to hitherto undetermined B cell membrane antigen(s) in association with MHC class II antigens. These results may have important implications in understanding autoimmune responses.     

Murine class II (Ia) molecules associate with human invariant chain

Polymorphic class II (Ia) major histocompatibility complex (MHC) gene products associate intracytoplasmically with a third nonpolymorphic class II molecule, the invariant chain (Ii), which is encoded by gene(s) unlinked to the MHC. Although the role of the Ii chain in the expression of cell surface Ia molecules is unclear, it has been suggested that the Ii chain helps in the assembly and intracellular transport of class II antigens. In this study, we demonstrate that the murine polymorphic class II antigens of an interspecies mouse-human hybrid, which has segregated the murine invariant chain gene, associates with the human invariant chain gene intracytoplasmically. The murine Ia antigens are expressed on the cell surface and can function as restriction elements in antigen presentation to T cells. The biochemical analysis demonstrates that the regions of the Ii gene that are critical to its interaction with Ia molecules are conserved between species.     

Accessory cell function in the Con A response: role of Ia-positive and Ia-negative accessory cells

We have examined the role of Ia-positive and Ia-negative accessory cells (AC) and soluble factors in Con A-stimulated murine T cell activation. Supernatant fluids containing interleukin 1 (IL 1) derived from the P388D1 macrophage cell line and from a lipopolysaccharide (LPS)-stimulated macrophage hybridoma provided only partial reconstitution of the response of purified T cells (18 to 27%). The complete reconstitution obtained with gamma-irradiated spleen cells or LPS-activated B cells was inhibited by approximately 60 to 77% when anti-Ia antibody was included in the culture. Despite this apparent involvement of Ia+ spleen AC, Ia-negative L cell AC could also reconstitute the response of both Class I-restricted Lyt-2+ T cells and Class II-restricted L3T4+ T cells. When the Ia-negative AC were employed, the L3T4 antigen on L3T4+ T cells played a critical role because addition of anti-L3T4 antibody to the culture inhibited the response by 85 to 90%. In contrast, anti-L3T4 did not inhibit the response in the presence of spleen AC. These results suggest that the molecules involved in T cell-AC interactions may vary depending on the AC source. Moreover, at least one of the putative target ligands for L3T4 presumably is not Ia, because anti-L3T4 inhibited T cell stimulation when Ia-negative AC were used.     

Direct stimulation of T lymphocytes by antigen-conjugated beads

To examine T lymphocyte recognition of foreign antigen, specific responses to the photoreactive antigen N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) were determined by using an HSAB/I-Ad-reactive murine T cell hybridoma. It was found that covalent coupling of HSAB to aminoethyl polyacrylamide beads at particular densities directly activated the T cells for IL 2 production, and beads conjugated at higher or lower doses of HSAB were nonstimulatory. This stimulation was specific for the phenyl ring composition of HSAB and for HSAB-reactive T cells. In addition, T cell activation by HSAB-coupled beads was specifically inhibited by soluble monomeric HSAB-glycine. These results indicate that HSAB-specific T cells may be directly stimulated by insolubilized HSAB in the absence of Ia antigens, suggesting direct T cell binding of foreign antigen.     

The fine specificity of antigen and Ia determinant recognition by T cell hybridoma clones specific for pigeon cytochrome c

The activation of proliferative T lymphocytes normally involves the simultaneous recognition of a particular foreign antigen and a particular Ia molecule on the surface of antigen-presenting cells, the phenomenon of major histocompatibility complex (MHC) restriction. An analysis of T cell clones specific for pigeon cytochrome c, from B10.A and B10.S(9R) strains of mice, revealed the unusual finding that several of the clones could respond to antigen in association with Ia molecules from either strain. Using these cross-reactive clones, we performed experiments which demonstrated that both the Ia molecule and the T cell receptor contribute to the specificity of antigen recognition; however, MHC-linked low responsiveness to tuna cytochrome c (an immune response gene defect) could not be attributed solely to the efficacy with which the Ia molecules associated with the antigen. These results imply that antigen and Ia molecules are not recognized independently, but must interact at least during the process of T cell activation.     

Coreceptor function of CD4 in response to MHC class I molecule

Specificity of T cell receptor (TCR) and its interaction with coreceptor molecules play decisive role in successful passing of T lymphocytes via check-points during their development and finally determine the efficiency of adaptive immunity. Genes encoding alpha- and beta-chains of TCR hybridoma 1D1 have been cloned. The hybridoma 1D1 was established by the fusion of BWZ.36CD8alpha cell line with CD8+ memory cells specific to MHC class I H-2Kb molecule. Exploiting retroviral transduction of thymoma 4G4 cells with TCR genes and coreceptors CD4 and CD8, variants of this cell line expressing on the surface CD3/TCR complex and coreceptors, separately or simultaneously have been obtained. The main function of CD4 is stabilization of interaction between TCR and MHC class II molecule. Nevertheless, we have found that CD4 could successfully participate in the activation of transfectants via TCR specific to MHC class I molecule H-2Kb. Moreover, coreceptor CD4 dominates CDS, because the response of transfectants CD4+CD8+ is blocked by antibodies to CD4 and MHC Class II Ab molecule but not to coreceptor CD8. The response of CD4+ cells was not due to cross-reaction between TCR 1D1 with MHC class II molecules, because transfectants do not respond to splenocytes of H-2b knockouted mice with impaired assembly of TCR/beta2-microglobulin/peptide complexes resulting in their absence on the cell surphace. The effect of domination was not due to sequestration of kinase p56lck, because truncated CD4 with the loss of binding motif for p56lck remained functional in 4G4 cells. Results obtained can explain the number of features of intrathymic selection and represent experimental basis for development of new methods of cancer gene therapy.     

Cutting edge: a single MHC class Ia is sufficient for CD8 memory T cell differentiation

Recent studies have suggested a role for MHC class Ib molecules in providing signals for memory T cell differentiation during the early phases of acute infection. To test this hypothesis, we assessed the development of effector and memory CD8 T cells in transgenic mice expressing a single chain H-2D(d)/beta2-microglobulin (beta2M) fusion protein on a beta2M-deficient background. These mice thus express a single MHC class Ia in the absence of all other beta2M-dependent class Ia and Ib molecules. Following infection with a recombinant vaccinia virus expressing a known D(d)-restricted epitope from HIV-1 gp160, the development of effector and memory cells CD8 T cells was comparable to control mice. Furthermore, these memory cells responded rapidly and robustly to antigenic restimulation. Therefore, we conclude that full CD8 memory differentiation requires only a single MHC class Ia chain, ruling out a requirement for MHC class Ib molecules in this process.     

Specific lymphocyte-target cell conjugate formation between tumor-specific helper T-cell hybridomas and IA-bearing RCS tumors and IE-bearing allogeneic cells. I. Role of Ia and both L3T4 and LFA-1 antigens in recognition/binding

The studies reported here describe the feasibility of using single cell techniques with nonadherent target cells for the formation of T helper lymphocyte-target cell conjugates in an Ia recognition system. We have taken advantage of four tumor-specific T cell hybridomas lines, two of which respond only to IA-bearing RCS tumor cells of SJL/J (H-2s) origin, and the other two that respond to both RCS and IA- or IE-bearing allogeneic cells of H-2k,d haplotypes. The conjugate frequency between the T cell hybridomas and target cells was scored microscopically and was facilitated by labeling the lymphocyte with fluorescein. The frequency of conjugate formation ranged from 20 to 40% above background. Conjugate formation was antigen specific and correlated well with the hybridoma specificity determined by IL 2 responses after antigenic stimulation. The cross-reactive hybridomas formed conjugates with RCS and LPS blasts derived from CBA or DBA/2 origin, but not with cells of syngeneic or other allogeneic strains. Conjugate formation with RCS was inhibited greater than 50% with mAb directed against IAs determinants on the RCS tumor cells, and conjugate formation with allogeneic cells was blocked only with mAb directed to either IA/IEk or IA/IEd specificities directed against the alpha or beta polypeptide chain. Blocking of conjugate formation was also achieved by various mAb directed against surface membrane molecules associated with the T cell hybridomas. LFA-1 mAb inhibited significantly the formation of conjugates. However, L3T4 mAb blocked only partially the conjugates. Other antibodies directed against Lyt-1 or Thy-1.2 antigens were without blocking effect. The poor blocking observed with L3T4 mAb did not correlate with the almost complete blocking observed in the IL 2 response by the same hybridomas. These studies of the syngeneic anti-RCS tumor response directed against IA-bearing RCS showed that the conjugate assay permits mapping of tumor-associated Ia epitopes. In addition, the results of these studies demonstrate the feasibility of conjugate formation in determining the antigenic specificity of the T helper system. This assay system can be used to establish the minimal frequency of antigen-reactive cells and can divide the T helper response into multiple steps (i.e., recognition/binding, activation, proliferation, and lymphokine release) and determine the surface membrane molecules involved in recognition.     

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.     

A single monoclonal anti-Ia antibody inhibits antigen-specific T cell proliferation controlled by distinct Ir genes mapping in different H-2 I subregions

A xenogeneic rat anti-mouse Ia monoclonal antibody, M5/114 (gamma 2b, kappa), was studied for its effects in vitro on T cell proliferative responses. Strain distribution studies revealed that M5/114 could inhibit I-A subregion-restricted T cell responses of the H-2b,d,q,u but not the H-2f,k,s haplotypes, indicating that this xenoantibody recognizes a polymorphic determinant on mouse Ia molecules. This same monoclonal antibody was found to inhibit BALB/c (H-2d) T cell proliferation to both G60A30T10 and G58L38 phi 4. The Ir genes regulating responses to these antigens map to either the I-A subregion (GAT), or the I-A and I-E subregions (GL phi), raising the possibility that M5/114 recognizes both I-A and I-E subregion-encoded Ia glycoproteins. It could be shown, using appropriate F1 responding cells, that M5/114 does in fact affect GAT and GL phi responses by interaction with both the I-A and the I-E subregion products, and not by any nonspecific effect resulting from binding to the I-A subregion product alone. These results are consistent with genetic and biochemical studies directly demonstrating that M5/114 recognizes A alpha A beta and E alpha E beta molecular complexes. The existence of a shared epitope on I-A and I-E subregion products suggests the possibility that these molecules arose by gene duplication. Finally, the precise correlation between the Ia molecules recognized by M5/114 and the ability of this antibody to block T cell responses under Ir gene control strengthens the hypothesis that Ia antigens are Ir gene products.     

Restriction by the major histocompatibility complex of antigen-induced T lymphocyte proliferation in new inbred guinea pig strains.

Employing new inbred guinea pig strains, JY 1, JY 2 and JY 3, established in this Institute in addition to strains 2 and 13, the authors investigated histocompatibility restriction in macrophage-T lymphocyte interaction. These five strains are known to possess distinct major histocompatibility complex (MHC) gene profiles (1, 2). This fact was supported by our results concerning the mixed leukocyte reaction (MLR) and cytotoxicity test with alloantisera. Using various combinations of T lymphocytes and peritoneal exudated cells (PECs) from these strains, in vitro proliferative responses of T lymphocytes from BCC-immune animals to PPD-pulsed normal PEC were tested. Successful activation of T cell response was observed not only in syngeneic combinations but also in allogeneic combinations among strains JY 1, JY 3 and strain 13 which share common Ia antigens detected by strain 2 anti-strain 13 alloantiserum. Because JY 1 and JY 3 seem to share a common B antigen differing from strain 13, it was suggested that identification in the I region of MHC is sufficient for effective antigen-presentation by the macrophage. Although a part of Ia is shared, no T lymphocyte activation was observed in the combination between JY 2 and JY 1 or JY 3, whereas strong MLR occurred in these allogeneic combinations. At the present stage of the study, it can be said that disparity in the part(s) of Ia antigens which is responsible for strong MLR cannot lead to effective T cell-macrophage interaction. These results support the concept that functional activation of primed, proliferating T lymphocyte requires the participation of gene products of macrophages coded for by the I region in MHC. By employing JY 1, JY 2 and strain 2, which appear to possess distinct B and Ia antigens, it was shown that the T lymphocyte and macrophage interactions essential for mitogen-induced T lymphocyte proliferation are not restricted by histocompatibility.     

An IL 2-secreting T cell hybridoma that responds to a self class I histocompatibility antigen in the H-2D region

A self-reactive T cell hybridoma that secretes IL-2 in response to H-2d haplotype cells resulted from a fusion of BALB/cBy lymph node cells with the AKR thymoma BW5147. The lymph node cells used had been enriched for cells reactive to (TG)-A--L, but neither this antigen nor fetal calf serum were required for stimulation of the hybridoma designated 3DT52.5. The gene product responsible for stimulation mapped to the H-2D region. Allogeneic cells of the b, f, k, q, and s haplotypes failed to stimulate. Not all H-2d haplotype cells were effective stimulators of 3DT52.5. Peritoneal cells and splenic B cells were much more stimulatory than splenic T cells. Most tumor cell lines of H-2d derivation and of B cell or macrophage/monocyte lineage were stimulatory, whereas H-2d T cell lines were not. The capacity to stimulate 3DT52.5 did not correlate with the ability to stimulate I region-restricted hybridomas, or with the ability to be induced to stimulate such hybridomas. Stimulatory cell lines did not apparently produce a soluble factor required for stimulation, and negative cell lines were not inhibitory. The monoclonal antibody 27-11-13, which reacts with H-2D of the b, d, and q haplotypes, inhibited stimulation of 3DT52.5 but did not inhibit stimulation of the sibling hybridoma 3DT18.11, which responds to (TG)-A--L plus I-Ad. Conversely, the monoclonal anti-I-Ad antibody MK-D6 inhibited stimulation of 3DT18.11 but not 3DT52.5. Although it is clear that 3DT52.5 recognizes a class I antigen coded for in the H-2D region, the precise molecular nature of the antigen is unknown. The structure of the antigen receptor on this hybridoma may prove to be of interest when it can be compared with receptors found on T cell hybridomas restricted by class II histocompatibility antigens.