Immune responses in vitro. I. Cellular requirements for the immune response by nonprimed and primed spleen cells in vitro.

A cell suspension culture system combined with a procedure which separates most macrophages from lymphoid cells was used to investigate some of the cellular requirements for direct and indirect plaque-forming cell responses by nonprimed and primed mouse spleen cells in vitro. The plaque-forming cell response to heterologous erythrocytes in cultures of nonprimed spleen cells required both macrophages and lymphoid cells for its development. A significant indirect plaque-forming cell response did not develop in cultures of nonprimed spleen cells. In contrast, cultures of separated or macrophage-poor lymphoid cells from primed mice exhibited increasing responses relative to the response of unseparated spleen cells as the interval after priming increased. The cultures of separated lymphoid cells were not entirely free of phagocytic cells. Despite some evidence which suggests that these phagocytic cells had little function in the response, one cannot ascertain whether the lymphoid cells were responding directly to a second contact with antigen or whether the few contaminating phagocytic cells were performing a function essential to the response by the lymphoid cells. Physiologically different populations of cells appear to develop after priming and are able to respond in vitro in a macrophage-poor culture. Some of the properties of these populations suggest that they are "memory cell" pools containing precursors of direct and indirect plaque-forming cells highly susceptible to a second antigenic stimulus.

Immune responses in vitro. II. Suppression of the immune response in vitro by specific antibody.

The effects of hyperimmune anti-sheep erythrocyte (SRBC) antibody on the plaque-forming cell (PFC) response to SRBC by mouse spleen cells in vitro were studied. Anti-SRBC antibody specifically suppressed the PFC response against SRBC. The degree of suppression was directly related to the amount of antibody added and was overcome by large amounts of antigen. Suppressive activity was absorbed from the sera by SRBC and could be partially eluted from the antigen by heat. The PFC response in cultures stimulated with antigen-antibody complexes prepared with high concentrations of antibody were suppressed; however, some complexes prepared at lower antibody concentrations stimulated greater responses than SRBC alone. Antibodies collected after four immunizations had greater suppressive ability than those collected after two immunizations. The degree of suppression was as great whether antibody was added at the initiation of the cultures or 24 hr later, suggesting that during the first 24 hr the culture system was antigen-dependent. Incubation of separated lymphoid cells with antibody did not impair their ability to develop a PFC response in vitro. However, if macrophages were incubated with antibody either before or after incubation with SRBC, the subsequent PFC response by lymphoid cells was suppressed. The data are consistent with the conclusion that antibody suppresses the PFC response in vitro by neutralizing the antigenic stimulus at the macrophage-dependent phase of the response.

Antigen-specific suppressor T-cell activity in genetically restricted immune spleen cells.

Virgin spleen cells develop comparable primary antibody responses in vitro to syngeneic or allogeneic macrophages (Mphi) bearing the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT), whereas immune spleen cells primed with syngeneic or allogeneic GAT-Mphi develop secondary responses preferentially when stimulated with GAT-Mphi syngeneic to the GAT-Mphi used for priming in vivo. These restrictions are mediated by products of the I-A subregion of the H-2 complex and are operative at the level of the GAT-Mphi-immune helper T-cell interactions. To investigate why these immune spleen cells fail to develop a significant antibody response to GAT-Mphi other than those used for in vivo immunization and determine the mechanism by which the restriction is maintained, spleen cells from virgin and syngeneic or allogeneic GAT-Mphi-primed mice were co-cultured in the presence of GAT-Mphi of various haplotypes. Antibody responses to GAT developed only in the presence of GAT-Mphi syngeneic to the Mphi used for in vivo priming; responses in cultures with GAT-Mphi allogeneic to the priming Mphi, whether these Mphi were syngeneic or allogeneic with respect to the responding spleen cells, were suppressed. The suppression was mediated by GAT-specific radiosensitive T cells. Thus, development of GAT-specific suppressor T cells appears to be a natural consequence of the immune response to GAT in responder as well as nonresponder mice. The implications of stimulation of genetically restricted immune helper T cells, and antigen-specific, but unrestricted, suppressor T cells after immunization with GAT-Mphi in vivo are discussed in the context of regulatory mechanisms in antibody responses.

Suppressor T-cell activity in responder X nonresponder (C57BL/10 X DBA/1)F1 spleen cells responsive to L-glutamic acid60-L-alanine30-L-tyrosine10.

The ability of spleen cells from (responder X nonresponder)F(1) mice immunized with various GAT-Mphi, GAT-MBSA, and soluble GAT to develop IgG GAT-specific PFC responses in vitro after stimulation with responder and nonresponder parental and F(1) GAT-Mphi, was investigated. F(1) spleen cells from mice immunized with F(1) GAT-Mphi or GAT-MBSA developed secondary responses to responder and nonresponder parental and F(1) GAT- Mphi, but not to unrelated third party GAT-Mphi. Spleen cells from F(1) mice immunized with either parental GAT-Mphi developed secondary responses to F(1) GAT-Mphi and only the parental GAT-Mphi used for immunization in vivo. Soluble GAT-primed F(1) spleen cells responded to F(1) and responder parental, but not nonresponder parental, GAT-Mphi. Simultaneous immunization in vivo with the various GAT-Mphi or GAT-MBSA plus soluble GAT modulated the response pattern of these F(1) spleen cells such that they developed secondary responses only to F(1) and parental responder GAT-Mphi regardless of the response pattern observed after immunization with the various GAT-Mphi or GAT-MBSA alone. These observations demonstrate the critical importance of the physical state of the GAT used for immunization in determining the subsequent response pattern of immune F(1) spleen cells to the parental and F(1) GAT-Mphi. Further, suppressor T cells, capable of inhibiting primary responses to GAT by virgin F(1) spleen cells stimulated by nonresponder parental GAT-Mphi, were demonstrated in spleens of F(1) mice immunized with soluble GAT, but not those primed with F(1) GAT-Mphi. Because responder parental mice develop both helper and suppressor T cells after immunization with GAT-Mphi, and soluble GAT preferentially stimulates suppressor T cells whereas GAT-Mphi stimulate helper T cells in nonresponder parental mice, these observations suggest that distinct subsets of T cells exist in F(1) mice which behave phenotypically as responder and nonresponder parental T cells after immunization with soluble GAT and GAT- Mphi.

Genetic control of immune responses in vitro. VI. Experimental conditions for the development of helper T-cell activity specific for the terpolymer L-glutamic aicd60-L-alanine30-L-tyrosine10 (GAT) in nonresponder mice.

Mice which are genetic nonresponders to the random terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) not only fail to develop GAT-specific antibody responses when stimulated with soluble GAT either in vivo or in vitro, but develop GAT-specific T cells which suppress the GAT-specific plaque-forming cell response of normal nonresponder mice stimulated with GAT complexed to methylated bovine serum albumin (MBSA).Thus, both responder and nonresponder mice have T cells which recognize GAT. However, nonresponder mice can develop GAT-specific helper T cells if immunized with GAT bound to MBSA or to macrophages. The relevance of Ir gene-controlled responses is discussed.

Genetic control of immune responses in vitro. 3. Tolerogenic properties of the terpolymer L-glutamic acid 60-L-alanine30-L-tyrosine10 (GAT) for spleen cells from nonresponder (H-2s and H-2q) mice.

Although nonresponder, H-2(s) and H-2(q), mice fail to develop GAT-specific PFC responses to GAT, they do develop GAT-specific PFC responses when stimulated by GAT complexed to an immunogenic carrier such as methylated bovine serum albumin. The studies described in this paper show that injection of nonresponder mice with GAT specifically decreases their ability to develop anti-GAT PFC responses to a subsequent challenge with GAT-MBSA. Addition of GAT to cultures of spleen cells from nonresponder mice also prevents development of the GAT-specific PFC responses stimulated by GAT-MBSA. Thus, interaction of nonresponder spleen cells with GAT leads to the induction of unresponsiveness in vivo and in vitro. Various parameters of the tolerance induction have been investigated and described. A comparison of the effects of GAT on B cells indicates that nonresponder B cells are more readily rendered unresponsive by soluble GAT than are responder B cells. The significance of these data for our understanding of Ir gene regulation of the immune response is discussed.

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.

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.

Haplotype-specific suppression of antibody responses in vitro. I. Generation of genetically restricted suppressor T cells by neonatal treatment with semiallogeneic spleen cells.

C57BL/10 mice were injected with semiallogeneic (B10.D2 X C57BL/10)F(1) spleen cells via the anterior facial vein within 24 h of birth to induce tolerance to B10.D2 (H-2(d)) alloantigens. Spleen cells from these mice as adults developed reduced, but significant, mixed lymphocyte and cytotoxic lymphocyte responses in vitro to H-2(d) stimulator cells and these treated mice rejected first-set B10.D2 skin grafts within a normal time-course, indicating that at best only a state of partial tolerance had been induced. Spleen cells from these mice failed to develop antibody responses to a variety of antigens in vitro when H-2(d) macrophages were in the cultures. Partially purified T cells from these neonatally treated mice suppressed primary antibody responses by normal syngeneic spleen cells in the presence of H-2(d) but not other allogeneic macrophages. These radiosensitive, haplotype-specific suppressor T (Ts) cells inhibited primary antibody responses by blocking initiation of the response, but failed to suppress secondary antibody responses and mixed lymphocyte or cytotoxic lymphocyte responses by appropriate responding spleen cells. To activate H-2(d) haplotype-specific Ts cells, stimulation with IA(d) subregion antigen(s) was necessary and sufficient; syngenicity at the I-A subregion of H-2 between the activated Ts cells and target responding spleen cell populations was also necessary and sufficient to achieve suppression. Comparable results have been obtained with spleen cells from BALB/c mice injected as neonates with (B10.D2 x C57BL/10)F(1) spleen cells where IA(b) antigens activate the haplotype-specific Ts cells. Implications for the significance of this population of haplotype-specific Ts cells in immune regulation are discussed and the properties of these Ts cells are compared and contrasted with other antigen-specific and nonspecific Ts cells whose activity is restricted by I- region products.

Immune responses in vitro. IV. Suppression of primary M, G, and A plaque-forming cell responses in mouse spleen cell cultures by class-specific antibody to mouse immunoglobulins.

The suppressive effects of monospecific goat anti-mouse globulins on primary immunoglobulin class-specific plaque-forming cell responses in mouse spleen cell cultures were investigated. Anti-micro suppressed responses in all immunoglobulin classes, whereas anti-gamma(1) and anti-gamma(2) suppressed the gamma(1) and gamma(2) responses but not gammaM or gammaA responses, and anti-gammaA suppressed only gammaA responses. The mechanism of action of the anti-micro was studied in detail because of its suppression of responses in all immunoglobulin classes. The anti-micro was specific for micro-chain determinants; its activity was dose dependent, but was not mediated by killing cells with surface micro-chain determinants. Free gammaM but not gammaG myeloma proteins in solution effectively competed with micro-bearing cells for the anti-micro. An excess of anti-micro was necessary in the cultures for 48 hr to insure complete suppression of 5-day responses. However, after removal of excess anti-micro at 48 hr, responses could be stimulated by newly added antigen in cultures where incubation was prolonged to 7 days. Anti-micro was most effective when added at the initiation of cultures and had no suppressive effect when added at 48 hr. Excess antigen did not effectively compete with anti-micro for antigen receptors. Precursors of antibody-forming cells were shown to be the cell population where the suppressive activity of anti-micro was mediated. The experiments suggest that anti-micro combines with micro-chain determinants in antigen-specific receptors on the surfaces of antibody-forming cell precursors, prevents effective stimulation by antigen and subsequent antibody production. To explain suppression of responses in all Ig classes by anti-micro, several models were proposed. It is not possible to determine from the data whether stimulation of precursor cells with gammaG or gammaA receptors requires concommitant stimulation of separate cells with only gammaM receptors, or whether cells bearing gammaM receptors are precommitted to or differentiate into cells capable of synthesis of other Ig classes, or whether receptors of gammaM and another Ig class are present on some virgin precursors or the second Ig receptor appears after antigenic stimulation.

Immune responses in vitro. V. Suppression of M, G, and A plaque-forming cell responses in cultures of primed mouse spleen cells by class-specific antibody to mouse immunoglobulins.

Suppression of Ig class-specific PFC responses by class-specific antibody to mouse immunoglobulin was studied in cultures of spleen cells from immunized mice. In contrast to cultures from normal mice where anti-micro suppressed responses in all Ig classes, anti-micro had progressively less suppressive effect on gamma(1) and gamma(2) responses in cultures from immunized mice with time after immunization. This was most pronounced at 10 days after immunization when anti-micro suppressed gammaM and gammaA responses, but had no or slight effect on gamma(1) or gamma(2) responses which were still suppressed with anti-gamma(1) and anti-gamma(2). These changes in precursor cell susceptibility to anti-micro were antigen specific.

Genetic control of immune responses in vitro. I. Development of primary and secondary plaque-forming cell responses to the random terpolymer 1-glutamic acid 60-1-alanine30-1-tyrosine10 (GAT) by mouse spleen cells in vitro.

In vivo, the antibody response in mice to the random terpolymer L-glutamic acid(50)-L-alanine(30)-L-tyrosine(10) (GAT) is controlled by a histocompatibility-linked immune response gene(s). We have studied antibody responses by spleen cells from responder and nonresponder mice to GAT and GAT complexed to methylated bovine serum albumin (GAT-MBSA) in vitro. Cells producing antibodies specific for GAT were enumerated in a modified Jerne plaque assay using GAT coupled to sheep erythrocytes as indicator cells. Soluble GAT stimulated development of IgG GAT-specific plaque-forming cell (PFC) responses in cultures of spleen cells from responder mice, C57Bl/6 (H-2(b)), F(1) (C57 x SJL) (H-2(b/s)), and A/J (H-2(a)). Soluble GAT did not stimulate development of GAT-specific PFC responses in cultures of spleen cells from nonresponder mice, SJL (H-2(s)), B10.S (H-2(s)), and A.SW (H-2(s)). GAT-MBSA stimulated development of IgG GAT-specific PFC responses in cultures of spleen cells from both responder and nonresponder strains of mice. These data correlate precisely with data obtained by measuring the in vivo responses of responder and nonresponder strains of mice to GAT and GAT-MBSA by serological techniques. Therefore, this in vitro system can effectively be used as a model to study the cellular events regulated by histocompatibility-linked immune response genes.

Genetic control of immune responses in vitro. II. Cellular requirements for the development of primary plaque-forming cell responses to the random terpolymer 1-glutamic acid 60-1-alanine30-1-tyrosine10 (GAT) by mouse spleen cells in vitro.

The cellular requirements for the development of primary IgG GAT-specific PFC responses in cultures of spleen cells from responder, C57Bl/6, mice stimulated with GAT and GAT-MBSA and in cultures of spleen cells from nonresponder, SJL and B10.S, mice stimulated with GAT-MBSA were investigated. Macrophages were required for development of responses to GAT and GAT-MBSA in cultures of spleen cells from responder mice and for responses to GAT-MBSA in cultures of spleen cells from nonresponder mice. Macrophages from nonresponder mice supported the development of responses to GAT by nonadherent responder spleen cells, indicating that the failure of nonresponder mice to respond to GAT is not due to a macrophage defect. Furthermore, responder macrophages supported the responses of nonadherent, nonresponder spleen cells to SRBC and GAT-MBSA, but not to GAT. This indicates that the capacity to respond to GAT is a function of the nonadherent population which is composed of thymus-derived (T) helper cells and precursors of antibody-producing cells. Treatment of spleen cells with anti-theta serum and complement before culture initiation abolished PFC responses to GAT and GAT-MBSA thus establishing the requirement for T cells in the development of PFC responses to these antigens. Since precursors of antibody-producing cells in nonresponder mice are capable of synthesizing antibody specific for GAT after stimulation with GAT-MBSA and since the response to GAT is thymus-dependent, it appears that nonresponder mice lack GAT-specific helper T cell function.

Biological expressions of lymphocyte activation. II. Generation of a population of thymus-derived suppressor lymphocytes.

A population of thymus-derived lymphocytes has been identified that, upon activation by the nonspecific plant mitogen concanavalin A, suppresses the development of plaque-forming cell responses in fresh or 48-h antigen-stimulated cultures of mouse spleen cells. Suppressor cells can inhibit both primary and secondary IgM and IgG responses in vitro. X-irradiation before activation of peripheral thymus-derived cells by concanavalin A abrogates generation of suppressor cells. After a 48 h activation period, however, the function of concanavalin A-activated suppressor cells is radioresistant. As yet uncertain is whether these suppressor cells are a population of cells distinct from thymus-derived "helper" cells. In certain important regards, the cells mediating these two opposing functions share similar characteristics; the effect observed may be determined by the circumstances of activation or the numbers of activated cells, and may consequently represent different functions of a single thymus-derived regulator cell population.

Functional maturation of thymic lymphocyte populations in vitro.

Mouse thymocytes were cultured for short periods of time either alone or with one of two supporting cell populations, splenic adherent cells or thymic epithelial cells. The thymus-derived (T) cell activity of thymocytes cultured on supporting cell populations increased dramatically during 2 days of culture, as assayed in the mixed lymphocyte interaction (MLI), response to phytomitogens, and helper cell activity in the in vitro antibody response. The level of activity attained was equal to that of spleen and lymph node lymphocytes and greater than that of steroid-resistant thymocytes. The cultured thymocytes had surface antigens characteristic of mature T lymphocytes with regard to theta and H-2. The appearance of functionally active lymphocytes in vitro depended upon cell division. Most of the active cultured cells arose from cells already undergoing maturation, i.e., from cells with reduced theta determinants and increased H-2 determinants. We therefore have generated a population of thymocytes indistinguishable from peripheral T lymphocytes using simple in vitro techniques. The extent to which the production of these active lymphocytes depends upon in vitro differentiation is discussed.

Cell-mediated immune responses in vitro. I. Suppression of the generation of cytotoxic lymphocytes by concanavalin A and concanavalin A-activated spleen cells.

The effects of soluble concanavalin A (Con A) or Con A-activated spleen cells on the generation of cytotoxic lymphocytes (CL) in mixed leukocyte cultures (MLC) were examined. Mitogenic concentrations of soluble Con A or small numbers of Con A-activated spleen cells substantially inhibited CL responses. The suppression was partial rather than absolute and was critically dependent upon the concentration and time of addition of soluble Con A or Con A-activated spleen cells to the MLC. Suppressive effects of Con-A activated spleen cells were mediated by T cells since suppressor cell activity was abrogated by treatment of spleen cells with anti-theta serum and complement before or after Con A activation. X irradiation of spleen cells before Con A treatment also abrogated generation of suppressor cell activity. After activation by Con A, however, the function of suppressor cells was radioresistant. Although the precise mechanism(s) of suppression is, as yet, unknown, the precursors of CL must be exposed to Con A-activated cells during the early phases of the immune response for suppression to occur. Kinetic studies revealed that suppression of CL responses was not due to a failure to initiate an immune response, but represented a response which developed initially, but subsequently aborted. The relevance of these observations to the concepts of T-cell-T-cell interaction and regulatory control of immune responses by T cells is discussed.

Biological expressions of lymphocyte activation : I. Effects of phytomitogens on antibody synthesis in vitro.

The effects of nonspecific phytomitogens on primary plaque-forming cell (PFC) responses of mouse spleen cells to heterologous erythrocytes in vitro were studied. Spleen cell cultures treated with concanavalin A or phytohemagglutinin in vitro or established with spleen cells derived from mice injected with concanavalin A 24 h previously were similarly affected. In both cases, submitogenic doses resulted in substantial enhancement of PFC responses, whereas 10-fold larger doses were profoundly inhibitory. In contrast to the suppressive effects of mitogenic doses of phytomitogens added at culture initiation, addition of these same doses to cultures 48 h later resulted in increased PFC responses. This enhancement could be observed within 1 h after treatment and consequently could not be ascribed only to mitotic expansion of the antibody-synthesizing clone. Activation of spleen cells with specific antigen before mitogen treatment was not required for expression of the enhancing or suppressing effects on PFC responses. IgM and IgG PFC responses were similarly affected. Studies of cell interactions revealed that as few as 10(5) spleen cells obtained from mice treated with concanavalin A in vivo synergistically enhanced the PFC responses of 10(7) normal spleen cells. This enhancement was mediated by mitogen-activated T lymphocytes which were resistant to 2000 R irradiation 24 h after activation. The relevance of these observations to emerging concepts of helper and suppressor T cell activity is discussed.

Immunosuppressive factor(s) extracted from lymphoid cells of nonresponder mice primed with L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) II. Cellular source and effect on responder and nonresponder mice.

The synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) fails to stimulate development of GAT-specific antibody responses in nonresponder strains of mice, but does stimulate the development of GAT-specific suppressor T cells that inhibit the development of normal anti-GAT antibody responses to GAT complexed to methylated bovine serum albumin (GAT-MBSA). Furthermore, extracts prepared from lymphoid cells of GAT-primed, but not control, nonresponder mice inhibit the development of antibody responses to GAT-MBSA by normal nonresponder mice. This suppression is specific, dose-dependent, and can be readily analyzed in vitro. The suppressive factor is a T-cell product. An extract from GAT-primed DBA/1 mice inhibits the response to GAT-MBSA by spleen cells from histoincompatible strains of mice that are nonresponders to GAT, but not strains that are responders to GAT.

Regulation by the H-2 gene complex of macrophage-lymphoid cell interactions in secondary antibody responses in vitro.

The ability of antigen-bearing syngeneic and allogeneic peptone-induced peritoneal exudate macrophages to support development of primary and secondary antibody responses by murine lymphoid or spleen cells in vitro has been investigated. The antigen used was the terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Syngeneic and allogeneic macrophages supported development of comparable primary antibody responses to GAT, indicating that genetic restrictions do not limit efficient macrophage-lymphocyte interactions in primary responses. By contrast, immunized spleen or lymphoid cells developed secondary antibody responses preferentially when stimulated in vitro with GAT on macrophages syngeneic to the macrophages used to present GAT during in vivo immunization. Thus, genetic restrictions regulate efficient macrophage-lymphocyte interactions in secondary antibody responses. These restrictions have been demonstrated from 2 to 8 wk after a single immunization with limiting quantities of GAT and are controlled by the H-2 gene complex. The implications that immune lymphocytes selectively recognize and respond to antigen presented in the context of the macrophage membrane-antigen complex which sensitized the lymphocytes initially are considered.

Antigen-specific suppressor T cell interactions. II. Characterization of two different types of suppressor T cell factors specific for L-glutamic acid50-L-tyrosine50 (GT) and L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT).

We have previously reported that two types of suppressor T cell factors (TsF) specific for L-glutamic acid50-L-tyrosine50 (GT) or L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) can be distinguished based upon differences in their ability to suppress responses by allogeneic mice. Injection of GAT or GT induces a suppressor T cell subset that produces an antigen-binding, I-J+, genetically unrestricted, specific suppressor factor (TsF1). Injection of this factor plus small amounts of antigen induces a second-order suppressor T cell that produces an antigen-binding, I-J+, genetically restricted, specific suppressor factor (TsF2). In this report, we demonstrate that these two factors are also biochemically distinct. Monoclonal TsF1 molecules are composed of a single polypeptide chain that bears both the antigen-binding site and I-J determinant, whereas TsF2 molecules are composed of two disulfide-linked polypeptide chains, one of which is antigen-binding and I-J-, and the other, nonantigen-binding, I-J+. The antigen-binding chain must be added at culture initiation to achieve suppression, but the I-J+ chain can be added as late as day 3 with complete suppression observed. However, isolated chains from TsF2-producing hybridomas derived from three different haplotypes were unable to suppress immune responses when chains from heterologous TsF2 were mixed. Indirect evidence is presented that suggests that this restriction is because the chains fail to interact rather than the inability of the target cells to recognize both chains.