Analysis of T cell hybridomas. I. Characterization of H-2 and Igh- restricted monoclonal suppressor factors

Five hybridoma T cell lines were prepared by fusion of second order suppressor T cells (Ts2) with the BW5147 thymoma. The culture supernates from these T cell hybrids contained a factor, TsF2, which specifically suppressed 4-hydroxy-3-nitrophenyl acetyl hapten (NP)-induced cutaneous sensitivity responses. TsF2 activity was observed when the factor was administered during the effector phases of the immune response. TsF2 bears I-J determinants and has binding specificity for NPb idiotypic determinants. TsF2 suppressor activity could be absorbed on antigen-primed H-2-incompatible T cells but cannot suppress H-2-incompatible mice. In addition to this H-2 restriction, which maps to the I-J subregion, monoclonal TsF2 also has an Igh genetic restriction. The present results are combined with previous data to describe the cellular interactions leading to immune suppression.     

Immunosuppressive factor(s) specific for L-glutamic acid(50)-L- tyrosine(50) (GT). II. Presence of I-J determinants on the GT-suppressive factor

The responses to the synthetic antigens, L-glutamic acid(60)-L- alanine(30)-L-tyrosine(10) (GAT) and L-glutamic acid(50)-L-tyrosine(50) (GT) are controlled by genes in the I region of the mouse H-2 complex (1-3). Preimmunization of the mice bearing the H-2(p,q,s) nonresponder haplotypes with GAT stimulates the development of suppressor T cells that inhibit in vivo or in vitro antibody responses to GAT complexed to the immunogenic carrier, methylated bovine serum albumin (GAT-MBSA) (4). The copolymer GT is not immunogenic in any inbred mouse strain tested, and has a suppressive effect on the antibody responses to GT-MBSA in mouse strains bearing the H-2(d,f,k,s) haplotypes; suppressor T cells have been demonstrated to be responsible for specific GT suppression (3). We have obtained specific suppressive extracts from thymus and spleen cells of GAT-or GT-primed suppressor strains (5,6). The specific suppressive T-cell factors in the active extracts have been characterized (6,7) and appear similar to the carrier-specific suppressor factor described by Tada and Taniguchi (8). These products belong to a family of newly identified molecules coded for by the I region of the H-2 complex with affinity for antigen and helper (9,10) or suppressive (5-8) regulatory activity on the immune response. Recently, Tada et al. have reported that the keyhole limpet hemocyanin (KLH)-specific suppressor factor is coded for by the I-J subregion of the H-2 complex (11). We now demonstrate also that a GT-specific suppressor factor extracted from the spleens and thymuses of B10.BR (H-2(k)) mice bears determinants controlled by the I-J subregion of the H-2 complex.     

Regulation of immune responses by I-J gene products. I. Production and characterization of anti-I-J monoclonal antibodies

Using a novel, two-step functional screening procedure, we have isolated hybridoma B cell lines secreting monoclonal antibodies directed against gene products of the I-Jb and I-Jk subregions of the mouse H-2 complex. These monoclonal antibodies act in vitro by allowing nonresponder spleen cells to respond to normally suppressive quantities of poly(Glu50Tyr50) (GT) (WF8 series of anti-I-Jk monoclonal antibodies) or to suboptimal concentration of poly(Glu60Ala30Tyr10) (WF9 series of anti-I-Jb monoclonal antibodies). Some of the culture supernates that show augmenting activity bind GT-specific T cell- derived suppressor factor (GT-TsF), indicating that some monoclonal antiantibodies display a nonspecific enhancing effect, or, more likely, that anti-I-J monoclonal antibodies have been produced against I-J determinants not found on TsF. It is this last possibility that is most intriguing and that might serve as a means for exploring the heterogeneity of the I-J subregion. It is also possible that some of our monoclonal anti-I-J antibodies might detect antigenic determinants selectively expressed on suppressor T cells, helper T cells, and/or macrophages. In addition, we have demonstrated that monoclonal anti-I-J antibodies should be useful in the biochemical characterization and purification of a monoclonal GT-TsF. These haplotype-specific anti-I-J monoclonal antibodies should prove to be powerful tools for future studies exploring the role of I-J gene products in the regulation of specific immune responses.     

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XI. Pseudogenetic restrictions of hybridoma suppressor factors

Suppressor factor derived from three different murine T cell hybridomas were characterized . They specifically inhibited 4-hydroxy-3-nitrophenyl acetyl cutaneous sensitivity responses. The factors bind antigen and bear I-J and idiotypic determinants, but lack conventional immunoglobulin constant-region determinants. The factors function during the induction phase of the immune response, by inducing a second population of suppressor cells (Ts(e)). Suppressor factor can inhibit both cellular and plaque-forming cell responses in appropriate strains of mice. These hybridoma suppressor factors directly suppress strains of mice that are Igh-V homologous with the strain producing the factor. Thus, there is an apparent Igh-V restriction in the activity of these factors. However, this is a pseudogenetic restriction because these factors generate second order suppressor cells (Ts(e)) in Igh-incompatible mice, but in order to express the suppressive activity, the cells must be adoptively transferred into recipients that are Igh compatible with the strain producing the suppressor factor. Finally, it was shown that the factor-induced Ts(e) population is under an apparent dual genetic restriction. Thus, Igh and H-2 homology is required in order for the Ts(e) population to express its suppressive activity.     

Mechanisms of suppression in the transfer of contact sensitivity. Analysis of an I-J+ molecule required for Ly2 suppressor cell activity

The passive transfer of contact sensitivity (CS) by immune cells can be inhibited with an antigen-specific T suppressor factor. This factor is composed of two subfactors: an antigen-specific subfactor made by an Ly1+ cell (PC1-F) and a antigen nonspecific subfactor made by an Ly2+ T cell (TNBSA-F). The suppressive activity of the complete factor can be eliminated by depleting the assay population of Ly2+ cells, even though it is the Ly1+ cell in the population that transfers the adoptive immunity. This suggests that the Ly2+ cell in the assay population is needed to transduce the suppressive signal to the Ly1+ effector cell of DTH. We found that an Ly2+ cell from immune animals could be induced to produce a cell free subfactor that overcame the requirement for this Ttrans cell in the suppression of CS by TsF. The induction required only PC1-F, TNP-coupled spleen cells, and resulted in the production of an antigen-nonspecific I-J+ subfactor by immune Ly2+, I-J+ cells. The need for the Ly2+ transducer cell could also be overcome by addition of an I-J+ molecule secreted by Ly1 T cells hyperimmunized to SRBC. A suppressor complex made from mixing the I-J+ molecule with TNBSA-F could directly suppress the functional activity of immune T cells not only to transfer CS, but also to deliver help to B cells in an in vitro PFC response. This suppressive complex is antigen-nonspecific and does not require Ly2+ T cells in the assay population for suppressive activity. These results indicate that effector factors of the suppressor circuit require two molecules; one that contains the functional suppressor material and one that serves as a "schlepper," a molecule needed to deliver the suppression to the appropriate target cell. The ability to construct a functional suppressor complex from two subfactors raised against different antigens, using different immunization procedures, which were isolated from factors exhibiting different functional activities suggests that certain cells of the immune system may play a universal role in "transducing" the suppressive signal.     

Properties of the antigen-specific suppressive T-cell factor in the regulation of antibody response of the mouse. IV. Special subregion assignment of the gene(s) that codes for the suppressive T-cell factor in the H-2 histocompatibility complex

The locus of the gene that codes for the antigen-specific suppressive T-cell factor was determined to be in a new subregion "I-J" which locates between I-B and I-C subregions in the H-2 histocompatibility complex. This was shown by two different lines of evidence: (a) The absorbing capacity for the suppressive T-cell factor of several alloantisera against restricted I subregions did not correlate with their specificity for previously known Ia molecules which are coded for by genes in I-A and I-C subregions, but was associated with the specificity for the products of genes putatively present between I-B and I-C subregions. By the occurrence of special recombinant strains, i.e. B10.A(5R), B10.A(3R), B10.S(9R), and B10.HTT, which differ with respect to the I-J subregion, we were able to produce alloantisera which distinguish I-J subregion gene products. The absorption studies using these special alloantisera directed to I-J subregion clearly indicated that the suppressive T-cell factor is a product of I-J subregion gene(s), and that the molecule is distinct from known Ia molecules expressed on splenic B cells. (b) Taking advantage of the fact that there is a strict histocompatibility requirement for the effective suppression between the donor and recipient strains of the suppressive T-cell factor, we were able to determine the required identities of the genes in the H-2 complex existing among those present between I-B and I-C. Again, utilizing the T-cell factors obtained from special recombinant strains, i.e. B10.A(4R) and B10.A(5R), we were able to locate the gene that codes for the suppressive T-cell factor reactive only with relevant haplotype strains between I-B and I-C subregions. These results are most reasonably explained by the presence of a new subregion I-J which is specialized in coding for the suppressive T-cell factor as a different molecule from previously known Ia molecules.     

Haplotype-specific suppression of antibody responses in vitro. II. Suppressor factor produced by T cells and T cell hybridomas from mice treated as neonates with semiallogeneic spleen cells

Culture supernatant fluids from spleen cells from C57BL/10 or BALB/c mice neonatally treated with semiallogeneic (B 10.D2 x B10)F1 cells to induce haplotype-specific suppressor T cells and restimulated with macrophages syngeneic at I-A with the allogeneic haplotype encountered as neonates contain a soluble factor capable of suppressing primary in vitro antibody responses of normal syngeneic spleen cells in a non- antigen-specific manner. This haplotype-specific suppressor factor, TsF- H, has also been recovered in culture fluids of a T cell hybridoma produced by fusion of the AKR thymoma BW5147 and the haplotype-specific suppressor T cells. TsF-H is inactivated by low pH (3.5) trypsin, for 30 min at 50 degrees C, and has a molecular weight in the range of 45,000 to 68,000. Studies with specific immunoabsorbents demonstrate the presence of determinants encoded by the I-A subregion of the haplotype of the T cell producing TsF-H but not I-J subregion or immunoglobulin constant-region determinants on the TsF-H. Suppression is restricted to primary in vitro antibody responses, and not secondary antibody, mixed lymphocyte, or cytotoxic lymphocyte responses by spleen cells syngeneic at the I-A subregion of H-2 with the T cell producing the factor. The properties and activities of TsF-H and the haplotype- specific suppressor T cell are compared and contrasted with antigen- specific and genetically restricted suppressor T cells and their factors.     

Mechanisms of regulation of cell-mediated immunity. III. The characterization of azobenzenearsonate-specific suppressor T-cell- derived-suppressor factors

Delayed type hypersensitivity to the hapten azobenzenearsonate (ABA) can be induced and suppressed by the administration of hapten-coupled syngeneic spleen cells by the appropriate route. Suppressor T cells stimulated by the intravenous administration of ABA-coupled spleen cells have been shown to produce a discrete subcellular factor(s) which is capable of suppressing delayed type hypersensitivity to azobenzenearsonate in the mouse. Such suppressor factors may be produced by the mechanical disruption of suppressor cells or by placing such suppressor cells in culture for 24 h. The suppressor factor(s) (SF) derived from ABA-specific suppressor cells exhibit biological specificity for the suppression of ABA delayed type hypersensitivity (DTH), but not trinitro-phenyl DTH, as well as the capacity to bind to ABA immunoadsorbents. Passage of suppressor factor(s) over reverse immunoadsorbents utilizing a rabbit anti-mouse F(ab')2 antiserum demonstrated that the antigen-specific T-cell derived SF does not bear conventional immunoglobulin markers. The suppressor factor(s) are not immunoglobulin molecules was further demonstrated by the inability of anti-ABA antibodies to suppress ABA DTH. Gel filtration of ABA suppressor factor(s) showed that the majority of the suppressive activity was present in a fraction with molecular weight ranging between 6.8 x 10(4) and 3.3 x 10(4) daltons. We also analyzed for the presence of determinants encoded by the H-2 major histocompatibility complex (MHC) and found that immunoadsorbents prepared utilizing antisera capable of interacting with gene products of the whole or selected gene regions of H-2 MHC, i.e., B10.D2 anti-B10.A and B10 anti-B10.A immunoadsorbents, retained the suppressive activity of ABA-SF. Elution of such columns with glycine HCl buffers (pH 2.8) permitted recovery of specific suppressive activity. Taken collectively such data supports the notion that suppressor T-cell-derived ABA suppressor factors have antigen-binding specificity as well as determinants controlled by the K end of the H-2 MHC. The distribution of strains capable of making SF has also been analyzed. The relationship of the antigen-binding specificity to VH gene products is discussed in this and the companion paper.     

A mechanism responsible for the induction of H-2 restricted second order suppressor T cells

The mechanism by which I-J restrictions were imposed on second-order suppressor cells (Ts2) was analyzed. The induction of Ts2 cells requires presentation of an inducer suppressor factor by a specialized population of factor-presenting cells. The I-J phenotype of this factor-presenting population controls the H-2 restriction of the Ts2 cells. The splenic cells responsible for presenting inducer factor appear to be of macrophage or dendritic cell lineage. Several homologies exist between the mechanism responsible for the induction of H-2-restricted suppressor and helper T cells. Thus, the I region products on specialized presenting cells determine the specificity and genetic restrictions of the T cell. In an H-2 heterozygous F1 animal, two distinct populations of cells can be induced, one specific for each parental H-2 heplotype. Furthermore, the data suggest that the suppressor cells also bear receptors for self H-2 products. The ramifications of these observations for the suppressor cell cascade are discussed.     

Analysis of the interactions between two molecules that are required for the expression of Ly-2 suppressor cell activity. Three different types of focusing events may be needed to deliver the suppressive signal

We described a T suppressor factor made by an I-J- Ly-2 T cell (Ly-2 TsF) that expresses biological activity only when its acceptor cell shares H-2-linked polymorphic genes with the cells that made the Ly-2 TsF (or when the producer cell had differentiated in a thymic environment where the gene products of the acceptor cell were expressed). The Ly-2 TsF requires the presence of I-J+ Ly-1 cells in the assay culture to express its suppressive activity, although removal of the I-J+ Ly-1 cells in the assay cultures with an I-J+ soluble factor derived from them. This I-J+ molecule not only fails to bind antigen but is also antigen nonspecific in that it can come from Ly-1 cells making factors of irrelevant specificities. For the I-J+ molecule to replace the activity of the I-J+ Ly-1 cell in the assay population, in restoring suppressive function in cultures depleted of I-J+ Ly-1 cells, it must share genetic polymorphisms linked to the I-J subregion with the Ly-2 TsF and genetic polymorphisms linked to Igh-V with the target cell. These results indicate that an I-J+ antigen-nonspecific molecule combines with an antigen-specific Ly-2 TsF via an I-J- anti-I- J "type" of interaction. The resultant molecular complex is focused on a cell surface receptor of the acceptor cell. This focusing event is controlled by the antigen-nonspecific I-J+ molecule, and the precise interaction with the receptor on the acceptor cell is controlled by Igh- V-linked polymorphic gene products. The antigenic specificity of the interaction is controlled by a receptor for antigen on the I-J- component of the complex. Thus, three focusing events are required for Ly-2 TsF to express biologic activity: (a) the Ly-2 TsF must be focused on an acceptor cell that has the same antigenic specificity (most likely via an antigen bridge); (b) it must also be focused onto an I-J+ antigen-nonspecific molecule that we refer to as a "schlepper" molecule (most likely via an I-J anti-I-J bridge); and (c) the schlepper molecule must focus the molecular complex on an Igh-V-controlled receptor on the antigen-specific target cell.     

Antigen-specific suppression in genetic responder mice to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Characterization of conventional and hybridoma-derived factors produced by suppressor T cells from mice injected as neonates with syngeneic GAT macrophages

Spleen cells from C57BL/10 mice injected with syngeneic B10 L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-pulsed macrophages (GAT-M phi) within 18 h of birth were unable to respond to soluble GAT, GAT-methylated bovine serum albumin, or B10 GAT-M phi as adults. Spleen cells from these neonatally treated mice responded at control levels to GAT presented in allogeneic M phi and to sheep erythrocytes. Partially purified T cells from these neonatally treated mice suppressed responses by syngeneic virgin, but not primed, spleen cells in an antigen-specific manner and acted during the early phases of the response. These responder GAT-specific suppressor T cells (GAT-TSR) were sensitive to anti-Thy-1 + C and 500-rad irradiation and have the phenotype Ly-1-2+, I-J+; GAT-TSR cells can only suppress responses by spleen cells syngeneic with the GAT-TSR cells at the I-J subregion of H-2. Restimulation of these Ts cells with syngeneic GAT-M phi induces an antigen-specific suppressor factor within the supernatant fluid. The factor, GAT-TsFR, is a glycoprotein with a molecular weight between 48,000 and 63,000, as determined by gel filtration chromatography using isotonic buffers; it bears serologically detectable determinants encoded by the I-J subregion of the H-2 complex, has an antigen-binding site for GAT and L-glutamic acid50-L-tyrosine50, and shares idiotypic determinants with anti-GAT antibodies. The presence of GAT-TsFR in the first 36 h of in vitro culture is required for significant suppression. Furthermore, only responses by spleen cell syngeneic with the cells producing GAT-TsFR at the I-J subregion are suppressed. The fusion of GAT-TsFR-producing cells with BW5147 resulted in generation of two hybridomas with properties and characteristics identical to those of the conventional GAT-TsFR with one exception: conventional and hybridoma 372.D6.5 GAT-TsFR only suppress responses by spleen cells of the I-Jb haplotype, whereas suppression mediated by the second hybridoma GAT-TsFR (372.B3.5) is genetically unrestricted. These hybridoma GAT-TsFR are compared with nonresponder GAT-Ts factor (GAT-TsF) and these responder and nonresponder GAT-TsF are considered in the context of suppressor pathways.     

Analysis of T cell hybridomas. II. Comparisons among three distinct types of monoclonal suppressor factors

Five hybridoma T cell lines were prepared by fusion of Ts3 cells with the BW 5147 thymoma. The culture supernatants from these T cell hybrids contained a factor, TsF3, which specifically suppressed 4-hydroxy-3-nitrophenyl acetyl hapten (NP(-hapten cutaneous sensitivity responses. The properties of this new series of hybridoma factors was compared with those of two previously characterized types of NP-specific suppressor factors (TsF1 and TsF2). TsF3 activity was only observed if the factor was administered during the effector phases of the immune response. TsF3 bears I-J and C57BL anti-NP antibody idiotypic determinants and has binding specificity for the NP hapten. Furthermore, TsF3 does not suppress H-2 (I-J)-incompatible mice. In addition to this H-2 restriction, the monoclonal TsF3 factors also demonstrated an Igh genetic restriction. Finally, the TsF3 factors could be distinguished by their ability to suppress cyclophosphamide-treated recipients.     

Regulatory mechanisms in cell-mediated immune responses. VII. Presence of I-C subregion determinants on mixed leukocyte reaction suppressor factor

The presence of H-2 gene products on mixed leukocyte reaction (MLR) supressor factor was investigated by passage of MLR-suppressor factor (SF) over solid immunoadsorbents prepared with various anti-H-2 subregion sera. Antisera with specificity for all or certain I subregion determinants removed or significantly reduced suppressor activity; adsorption was not consistent with K or D region specificity. The single I subregion specificity common to all adsorbing preparations was I-C. Serologic differentiation of I-C products of k and d haplotypes expressed on MLR-SF was established with antisera prepared in I-Cd/I-Ck disparate strain combinations. These sera define allelic T cell restricted Lad determinants encoded by I-C genes. MLR-SF prepared from (BALB/c X CBA)F1 mice and exposed to the I-Cd and I-Ck specific adsorbents demonstrated d and k haplotype specific adsorption respectively. F1 suppressor activity adsorbed on an anti-I-Cd column was eluted by glycine-HCl buffer and suppressed only BALB/c (H-2d) responses. B10.A suppressor activity was removed by anti-I-Cd sera, but was unaffected by anti-I-Ck sera, indicating that B10.A suppressor activity is encoded by an I-C subregion derived from the d haplotype. Antisera with anti-I-Jk specificity did not remove suppressor activity of various H-2k factors. Finally, adsorption with antisera directed against H-2-associated determinants of the allogeneic cell used to stimulate suppressor factor generation demonstrated that sensitizing alloantigens are not components of MLR suppressor factor. Thus among the major histocompatibility complex (MHC)-controlled suppressor factors, MLR suppressor factor is uniquely determined by the I-C subregion.     

Shared idiotypic determinants on antibodies and T-cell-derived suppressor factor specific for the random terpolymer L-glutamic acid60- L-alanine30-L-tyrosine10

T-cell derived suppressor factors (TsF) specific for the random copolymers L-glutamic acid60-L-alanine30-Ltyrosine10 and L-glutamic acid60-L-alanine40, referred to as GAT and GA, respectively, were prepared and partially purified on the approprate antigen immunoadsorbents. GAT-TsF obtained from nonresponder DBA/1 (H-2q) and SJL (H-2s) mice were passed over immunoadsorbents prepared from normal guinea pig serum (NGPS) or guinea pig anti-idiotype antiserum (anti-CGAT) specific for a common cross-reactive idiotype found on most anti-GAT antibodies in all mouse strains tested. Both the directly suppressive activity of the GAT-TsF and the ability of GAT-TsF to induce new suppressor T cells (Ts2) in vitro were adsorbed to and fully recoverable from the guinea pig anti-CGAT-Sepharose immunoadsorbent, while the TsF passed through the control NGPS-Sepharose without appreciable binding. The SJL GAT-TsF specifically eluted from anti-CGAT-immunoadsrobents was shown to still posses I-J determinants. These data provide evidence suggesting a sharing of V region structures between B-cell antibody and T-cell suppressor factor specific for an antigen (GAT) under Ir gene control, in agreement with earlier studies on T and B-cell alloreceptors, T-cell helper factors, and T and B-cell receptors for conventional antigens.     

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. VIII. Suppressor cell pathways in cutaneous sensitivity responses

In the current study, we examine the mechanism of suppression of cutaneous sensitivity (CS) responses to 4-hydroxy-3-nitrophenyl acetyl succinimide ester. Intravenous administration of haptenated syngeneic spleen cells induces a state of hapten-specific tolerance involving I-J bearing suppressor T cells that function at either the induction phase or the effector phase of the CS response. The effective phase suppressor cells (Tse) are genetically restricted by both Igh and H-2 region genes. However, a third cell population is also required in he immune lymphocyte population for immune suppression. This third cell population, termed Ts3, is an I-J+, cyclophosphamide-sensitive T cell, as shown by reconstitution experiments. Further, the Tse-Ts3 interaction is restricted by genes in he H-2 and Igh gene complexes. The results are discussed with respect to the pathway of cellular interactions leading to immuno suppression.     

Effects of anti-Ia sera on mitogenic responses. III. Mapping the genes controlling the expression of Ia determinants on concanavalin A- reactive cells to the I-J subregion of the H-2 gene complex

We have shown that the Ia determinants expressed on nylon wool-purified T lymphocytes reactive to concanavalin A (Con A) in serum-free media are coded in a single I subregion of the H-2 gene complex. This region, I-J, is defined by two pairs of intra-H-2 recombinant haplotypes: H-2t3, H-2t4 and H-2i3, H-2i5, carried by B10.HTT, B10.S(9R), B10.A(3R), AND B10.A(5R), respectively. No activity against Con A-reactive T cells has been detected in any antiserum that was produced in strain combinations which shared a common I-J region. This suggests that Ia antigens expressed on Con A-reactive T cells are restricted to the I-J subregion.     

Regulatory mechanisms in cell-mediated immune responses. Role of I-J and I-C determinants in the activation of H-2I and H-2K/D alloantigen-specific suppressor T cells

The role of individual H-2I subregion determinants in the activation of H-2I alloantigen-primed mixed leukocyte response suppressor T cells (MLR Ts), as well as their possible expression on stimulator cells required to trigger primed H-2K- or D-specific MLR Ts, was addressed in these studies. Both genetic and serologic studies demonstrated that MLR Ts potentially primed to alloantigens encoded by the entire H-2I region were triggered to MLR Ts factor production only by stimulator cells bearing the priming I-J and/or I-C, but not I-A or I-E alloantigens. The relevant I-J and I-C determinants were demonstrated on a single antigen-presenting cell population that is used in common by independent I-J-specific and I-C-specific MLR Ts. Unexpectedly, the stimulator cell population necessary to trigger MLR Ts primed to class I H-2K or D alloantigens expressed not only the priming class I determinant, but in addition, I-C alloantigens syngeneic with the MLR Ts haplotype. Stimulator populations bearing the appropriate H-2K or D alloantigen but serologically depleted of I-C+ cells or genetically constructed to display MLR Ts-disparate I-C determinants were ineffective stimulators of class I antigen-primed MLR Ts. Thus these data suggest that as allogeneic determinants, I-J- and I-C-encoded molecules are together the major triggering elements for MLR Ts primed to disparate H-2I region determinants. In addition, self-I-C molecule recognition appears to constitute an important feature of the triggering, and by implication, priming process of H-2 class I antigen-specific Ts cells.     

Purification and characterization of an L-glutamic acid60-L-alanine30-L- tyrosine10 (GAT)-specific suppressor factor from genetic responder mice

A hybridoma-derived, GAT-specific suppressor T cell factor (GAT-TsFR) from responder C57BL/10 mice has been purified to apparent chemical homogeneity using reversed phase HPLC techniques. 40 l of starting material yielded approximately 880 micrograms protein with a specific activity of 28.4 X 10(3) S50 U/ng protein representing a purification factor of 4.2 X 10(6). Purified GAT-TsFR is a hydrophobic protein with a minimum molecular weight of 18,000 that is capable of forming biologically active aggregates with molecular weights of 28,000, 64,000 and approximately 84,000 and has a pI of 6.4. GAT-TsFR is a glycoprotein that binds GAT and GT, but not GA, and bears determinants encoded by the I-J subregion of the H-2 complex. This GAT-TsFR derived from an H-2b responder haplotype to GAT is compared with GAT-TsF derived from the nonresponder H-2q haplotype on the basis of biochemical and some serological properties.     

Regulatory mechanisms in cell-mediated immune responses. VIII. Differential expression of I-region determinants by suppressor cells and their targets in suppression of mixed leukocyte reactions

The phenotypic expression of I-region determinants on cells producing and responding to MLR suppressor factor (MLR-TsF) was established in these studies. Alloantigen-activated MLR suppressor T cells (MLR-Ts), which produce MLR-TsF bearing gene products of the I-C subregion, were exposed to anti-I subregion sera and complement (C) before in vitro culture for MLR-TsF production. Suppressor activity was prevented by removal of cells bearing I-C determinants, whereas elimination of cells expressing I-A/B determinants had no effect. Interestingly, cytotoxic elimination of cells displaying I-J determinants also prevented MLR-TsF production. Admixture of anti-I-J and I-C antiserum-treated cells for MLR-TsF production failed to reconstitute suppressor activity, indicating that I-C and I-J gene products are expressed on a single population of cells critical to MLR suppression, rather than on distinct interacting subpopulations. Anti-I-C serum activity specific for I-C+ MLR-Ts was removed by adsorption with nylon wool-nonadherent splenic T cells and concanavalin A-activated thymocytes; adsorption with splenic B cells from anti-Thy-1,2 serum and C-treated spleen failed to remove relevant anti-I-C activity. These data suggest that regulatory I-C molecules, like I-J molecules, are preferentially expressed on T lymphocytes. Expression of I-C, or other I-region molecules on responder cell targets of MLR-TsF activity was also investigated. Responder cells were pretreated with anti-I subregion-specific sera in blocking or complement-dependent cytotoxic protocols before addition to MLR with MLR-TsF. Neither blocking nor the cytotoxic removal of cells bearing I-C or other I-region determinants from MLR responder populations interfered with MLR-TsF suppression. Because it has previously been demonstrated that MLR-TsF interacts optimally with activated, I-C syngeneic target cells, blocking and cytotoxic studies with anti-I subregion sera were also performed with responder cells activated by 24 h culture in MLR in the absence of MLR-TsF. Brief MLR-TsF pulse after antiserum treatment generated marked suppression regardless of blocking or absence of cells bearing serologically detected I-region determinants. I-C restricted suppression may thus be mediated not by interaction with I-C-bearing cells, but by target cells which exist in requisite association with populations of I-C+ cells.     

Analysis of T cell hybridomas. IV. Characterization of inducible suppressor cell hybridomas

The Ts3 subset of suppressor cells is generated after antigen priming, but, in order to express suppressor activity these cells require an additional activation step involving triggering with specific suppressor factors (TsF2). This report characterizes two cloned hybridoma cell lines (pTs3 hybridomas) that represent this stage of Ts3 cell differentiation. These hybridoma cells could be specifically activated with TsF2 to release another antigen-specific suppressor factor (TsF3) within 6 h. The inducible feature of these cells permitted analysis of the signals necessary for Ts3 activation. Antigen was not required for activation. Only TsF2 factors derived from antiidiotypic second-order suppressor cells could activate pTs3 hybridoma cells. There were stringent genetic restrictions on the ability of Ts2 to activate pTs3 cells. Triggering of pTs3 required corecognition of two determinants on the TsF2 molecular complex, i.e., the I-J and Igh-related idiotypic determinants. Thus, although pTs3 cells could absorb TsF2 from an I-J-mismatched source, these pTs3 were not activated by the allogeneic TsF2. For activation to occur, the H-2 (I-J) and Igh complexes of the TsF2 donor had to match those of the strain from which the pTs3 cells were derived. Mixing two distinct TsF2, one derived from an H-2-matched source and the other from an Igh- matched source, failed to activate pTs3 cells. Once activated, the pTs3 cells released a suppressive material that was indistinguishable from the TsF3 factors previously characterized in this system. Finally, the activation of the pTs3 cells apparently does not induce the de novo synthesis of TsF3 since the suppressive activity could be extracted from nonactivated pTs3 cells. Thus, the inducible pTs3 hybridomas represent a mature stage in the differentiation cycle of Ts3 cells and provide a means for studying the nature of the specific signals required for Ts3 activation.