Antigen-specific and antibody-mediated growth inhibition of suppressor T cell hybridomas. Roles of H-2 and the CD3-T cell receptor-alpha/beta complex

Eight different Ts cell hybridomas (including inducer (Ts1) and effector (Ts3) suppressor cells) specific for the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were tested for their ability to respond to Ag or anti-CD3 antibody in a growth-inhibition assay. Results suggest that the expression of the TCR-CD3 complex on Ts hybridomas is required for the Ag or anti-CD3-mediated growth inhibition. One of the CD3+, Ts hybridomas (CKB-Ts3-9.H3) was tested in detail; this CD4- effector suppressor cell hybridoma showed specific inhibition of growth in the presence of NP or NIP-coupled protein conjugates but not in the presence of other irrelevant hapten-protein conjugates. In addition, growth of this hybridoma was specifically inhibited by anti-CD3 and anti-TCR-alpha/beta antibodies but not by control hamster antibodies. In order to study the role of MHC molecules in Ag-mediated growth inhibition, Ts cell hybridomas were incubated with Ag (NP-keyhole limpet hemocyanin) in the presence of spleen cells from various H-2 congenic strains. The results suggest that the Ts hybridomas that express donor Ts-derived TCR beta-chain recognize Ag in an MHC-restricted manner, whereas the two Ts3 hybridomas that utilize BW5147-derived TCR-beta recognize Ag in H-2 unrestricted way. Co-incubation of anti-CD3 and anti-TCR-alpha/beta antibodies with specific Ag enhanced the Ag-mediated growth inhibition, whereas anti-LFA-1 antibody completely blocked the Ag-mediated effect. The combined data suggest that, like Th hybridomas, expression of CD3-associated-TCR complex is essential for the Ag responsiveness of Ts cell hybridomas.     

A monoclonal antibody raised to lipomodulin recognizes T suppressor factors in two independent hapten-specific suppressor networks

Five different Ag-binding suppressor factors from two types of hapten-specific Ts cell hybridomas (TsF1 inducer and TsF3 effector factors) were bound by an anti-lipomodulin mAb (141-B9), that crossreacts with rodent glycosylation inhibition factor (GIF). The Ag-specific suppressor activity in these hybridoma supernatants was bound by anti-lipomodulin columns and could be recovered by elution at acid pH. Additional evidence for the expression of lipomodulin/GIF activity on these TsF molecules was demonstrated by the ability of the eluted fractions to inhibit the glycosylation of IgE-binding peptides during their biosynthesis. The same biologic activity is associated with GIF and lipomodulin. The relationship between TsF and lipomodulin/GIF was confirmed in a serologic assay, which showed that TsF1 and TsF3 molecules, whether purified over Ag, anti-IJ or anti-TsF columns, are recognized by the mAb. 141-B9. The combined results indicate that Ag-binding Ts factors share a common antigenic determinant with phospholipase inhibitory proteins such as lipomodulin and GIF. In addition, the demonstration of glycosylation regulatory activity carried on these TsF molecules suggests a possible mode for their bioactivity.     

Purification and analysis of an antigen-specific suppressor factor from a T cell hybridoma specific for phenyltrimethylamino hapten

The synthetic monovalent antigen L-tyrosine-p-azophenyltri-methylammonium (tyr (TMA)) induces in A/J mice, a cascade of regulatory T cells in the absence of any detectable effector function (e.g., CTL, delayed-type hypersensitivity, etc.). An important component of the activated T cells is a first order suppressor T cell or Ts1 that is Ly-1+2-, functions only at the afferent limb of the anti-TMA response, binds the TMA ligand and bears cross-reactive idiotypes associated with anti-TMA antibodies. This Ts1 produces a suppressor factor (TsF1) that binds the TMA ligand, bears the cross-reactive idiotypes and I-J determinants and functions to induce an idiotype-specific Ts2 population. To study the biochemistry of this TsF, use was made of T cell hybridomas that constitutively produce TMA-TsF1 (8A.1 and 8A.3). The TsF1 was purified from culture supernatant or cell extracts by (NH4)2SO4 precipitation, reverse phase HPLC and either affinity chromatography or by preparative IEF. The TsF1 has an isoelectric point of 6.5 and a m.w. of 26,000 or 62,000 as analyzed by SDS-PAGE or high performance molecular sieve chromatography. Its precipitation in 30 to 40% (NH4)2SO4; elution pattern from reverse phase high performance columns; its capacity to bind to a mAb specific for L-glutamic acid 60L-alanine30-L-tyrosine10 (GAT)-TsF1 strongly suggest that this protein belongs to the same family of proteins as do the GAT-TsF1 described previously. Most noteworthy is that although these TsF1 proteins show remarkable similarities, they are absolutely specific in their biologic activity; TMA-TsF1 will not suppress the response to GAT-BA-TNP and GAT-TsF1 will not suppress the response to TMA-BA-TNP. Thus the TMA-TsF1 represents a second example of a unique group of Ag-specific proteins whose function is to induce or activate other suppressor T cells in the primary immune response to Ag.     

Expression of functional alpha beta T cell receptor gene rearrangements in suppressor T cell hybridomas correlates with antigen binding, but not with suppressor cell function

We have examined the expression of TCR genes in 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific Ts cell hybridomas. Each of three independently isolated hybridomas expressed in-frame TCR alpha-chain rearrangements derived from the original suppressor Ts cell. Different V alpha and J alpha gene segments were rearranged and expressed in each Ts cell line. The only TCR beta-chain expressed in these cells was derived from the BW5147 fusion partner. Expression of the BW5147 beta-chain was found to correlate with cell surface Ag binding, inasmuch as subclones derived from one of the original Ts lines expressed greatly reduced levels of beta-chain mRNA and no longer bound to NP-coupled RBC. Subclones that continued to express beta-chain mRNA did bind to NP-coupled RBC. This suggests that the Ag receptor on Ts hybridomas is a TCR-alpha beta dimer composed of a unique alpha-chain and the BW5147 beta-chain. Ag binding could be modulated by preincubation of Ts hybridoma cells with anti-TCR-alpha beta antibody, thereby supporting this conclusion. Suppressor factor activity was measured in the conditioned media of Ts subclones that differed by 250-fold in levels of beta-chain mRNA expression. No difference in suppressor factor activity was found; conditioned media from these subclones suppressed both plaque-forming cell responses and delayed-type hypersensitivity responses at approximately equivalent dilutions. Suppressor factor activity in the conditioned media of both a beta-chain negative subclone and a beta-chain positive subclone could be absorbed with an antibody that recognizes the TCR alpha-chain, but not with an antibody that recognizes the TCR beta-chain. We conclude that suppressor factor activity in the conditioned media of these Ts hybridomas is not derived from surface TCR-alpha beta receptors, although it does share TCR alpha-chain determinants.     

Monoclonal antibodies specific for single chain or two chain GAT-specific suppressor factors: production and analysis of in vitro modulating properties

Fusion of spleen cells from rats hyperimmunized with T cell hybridoma derived GAT-specific TsF1 or TsF2 suppressor T cell factors has resulted in the generation of hybridomas secreting monoclonal antibodies reactive with the appropriate GAT-TsF used for immunization, and in several cases, reactive with other GAT-TsF1 and TsF2. The monoclonal anti-TsF1 antibodies are capable of modulating in vitro GAT-specific PFC response in a GAT-specific manner; some suppress responses to GAT directly, whereas others reverse GAT-TsF1-mediated suppression of responses. The monoclonal anti-TsF2 antibodies all reverse suppression but are reactive with combinatorial determinants, I-J+ chains or antigen-binding chains of the GAT-TsF2. The data are discussed in terms of the nature of the determinants recognized by these antibodies as well as the potential uses of these reagents for studying the suppressor T cell pathway and potential relationships between Ts1, Ts2, and T helper cells.     

Cloned suppressor T cells derived from mice tolerized with conjugates of antigen and monomethoxypolyethylene glycol. Relationship between monoclonal T suppressor factor and the T cell receptor

Cloned Ts cells specific for the Ag, human monoclonal (myeloma) IgG, were derived from spleen cells of mice that had been immunosuppressed by treatment with a tolerogenic conjugate of HIgG and monomethoxypolyethylene glycol. The cloned Ts cells (clone 23.32) suppressed in vitro antibody responses in an Ag-specific and MHC-restricted manner. By FMF with appropriate antibody reagents, these cells were shown to be Thy-1+, CD4-, CD5-, and CD8+ and to express CD3 and the alpha beta-TCR. These results are consistent with the view that Ts cells use Ag recognition structures similar to those reported for Th cells and CTL. A soluble factor (TsF) extracted from the cloned Ts cells also suppressed in vitro antibody responses in an Ag-specific and H-2Kd-restricted manner, i.e., restricted to MHC class I molecules. The suppressive activity of this TsF could be abrogated by addition of mAb H28-710 that reacts with a determinant on the alpha-chain of TCR. Moreover, the TsF bound to and could be recovered from an immunosorbent consisting of the anti-alpha-TCR mAb H28-710 coupled to Sepharose 4B. In contrast, the TsF was not bound by immunosorbents consisting of mAb to the beta-chain of TCR (H57-597) or to V beta 8 (F23.1). It was, therefore, concluded that the TsF of clone 23.32 is serologically related to the alpha-chain of the TCR; however, it is not identical to TCR, because it lacks the determinants expressed on the TCR beta-chain that are recognized by the two anti-beta mAbs used in this study.     

A monoclonal antibody raised to tumor-specific T cell-derived suppressor factors also recognizes T suppressor inducer factors of the 4-hydroxy-3-nitrophenyl acetyl hapten suppressor network

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.     

In vitro generation of suppressor T cells. Induction of CD3+, IgH-restricted suppressor cells

Previous studies of the immune response of C57BL/6 mice to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten determined that challenge with antigenic forms of hapten induces both immunity and suppression. The anti-NP plaque-forming cell response can be down regulated by an Ag-induced cascade consisting of three suppressor T cell subsets. These three populations, termed Ts1, Ts2, and Ts3 have been characterized to have inducer, transducer and effector functions, respectively. Although the functions of each of these subsets have been examined in vivo, the cellular requirements for in vitro Ts induction have only been investigated for the Ts3 population. The present study characterizes the cellular events that lead to the induction of the Ts2, suppressor transducer population. Culture of naive C57BL/6 spleen cells with Ts1-derived suppressor factor in the absence of exogenous Ag leads to the generation of Ts2 cells that mediate Ag-specific suppression of NP plaque-forming cell responses. Phenotypic analyses demonstrate that a CD3+, CD4-, CD5+, CD8+, and I-J+ precursor population is stimulated by TsF1 to become mature Ts2 cells that express CD3, CD8, and I-J but not CD5. Although previous studies have reported an essential role for B cells in the induction of other Ts populations, depletion of B cells from Ts2 induction cultures had no effect on Ts2 generation. Despite the absence of B cells in these cultures, the mature Ts2 cells were functionally IgH restricted. Studies with IgH congenic B.C-8 mice suggest that this restriction specificity was imposed by the idiotype-related determinants expressed on the TsF1, not the T cell genotype.     

Manipulation of anti-LDH-B response by T suppressor factors

Hybridomas produced by fusion between the BW5147 thymoma and an LDH-B-specific B10.A(2R) suppressor T cell line secrete two T suppressor factors (TsF). One factor (TsF-A) shares Mhc determinants with the A alpha A beta molecule and suppresses proliferating Th cells; the other (TsF-E) shares determinants with the E alpha E beta molecule and it inhibits the maturation of the T suppressor (Ts) cells. Here we demonstrate that the two factors can be used to alter the immune response status of cultured T lymphocytes or of an animal. When added to a culture of LDH-B-primed cells or injected into mice, the TsF-A turns responders into nonresponders, presumably by blocking the proliferation of the Th cells. The TsF-E converts nonresponder cultures or mice into responders, presumably by preventing the differentiation of Ts cells. As there are good prospects for obtaining TsF in large quantities and in a highly purified form, this manipulation of the immune response by the deployment of specific factors promises to become an efficient new method of immunotherapy.     

Production of T-T hybrids from T cell clones. Direct comparison between cloned T cells and T hybridoma cells derived from them

It has been assumed, without direct evidence, that T cell hybridomas and non-transformed T cell clones are both good models of normal Ag-specific T cells. To compare directly the difference in activation of cloned normal T cells and T hybridoma cells with the same TCR, cloned T hybridoma cells were obtained by fusing pre-established, myoglobin-specific, Iad-restricted T cell clones (14.5 and 9.27) with BW5147 cells. T cell clones were pre-activated with IL-2 as well as specific Ag before fusion. Cloned T hybridoma A3.4C6 was derived from Lys 140-specific and I-Ed-restricted clone 14.5. The other cloned T hybridoma, C7R14, was a fusion product of Glu 109-specific and I-Ad-restricted clone 9.27. Both T hybridomas showed the same Ag specificity and Ia restriction as the parental cloned T cells. However, C7R14 showed higher apparent affinity and broader cross-reactivity than 9.27. Clone 14.5, but not hybridoma A3.4C6, appeared to stimulate splenic cells to secrete cytokines inhibiting HT-2A cell proliferation. The most striking difference between the clones and hybridomas was that both clones, but neither of the matched hybridomas, were induced to synthesize IL-1 on stimulation with Ag. Finally, both cloned T cells and T hybridomas killed Ag-pulsed Iad-bearing B lymphoma target cells. This evidence suggests that killing function can be inherited from clones to hybridomas. However, the clones were much more efficient at killing than the hybridomas, and the hybridomas were more efficient at IL-2 production than the clones. Thus, matched pairs of clones and hybridomas differ in their capacity to mediate the two functions or may tend to be selected differently during cloning. Thus, although our results generally support the validity of T cell hybridomas as faithful models of the corresponding T cell clones, a number of subtle and not-so-subtle differences indicate that caution must be used in such an extrapolation.     

Antigen-specific suppressor T cell interactions. I. Induction of an MHC-restricted suppressor factor specific for L-glutamic acid50-L-tyrosine50

The synthetic polymers L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) and L-glutamic acid50-L-tyrosine50 (GT) stimulate specific suppressor T cells in certain strains of mice. Extracts from these T cells contain factors (TsF) that inhibit GAT- or GT-specific antibody responses by normal spleen cells or proliferative responses by primed T cells. We constructed T cell hybridomas that constitutively produce GAT-TsF or GT-TsF, which functionally and serologically are identical to factors extracted from suppressor T cells. In this report we demonstrate that monoclonal GT-TsF can induce specific unresponsiveness in vivo or in vitro and that this unresponsiveness is due to development of second-order antigen-specific suppressor T cells. T cell hybridomas were constructed by fusion of BW5147 with GT-TsF1 induced second-order suppressor T cells and clones that produced suppressor factor (GT-TsF2) were isolated and characterized. GT-TsF2 differs from the GT-TsF1 used to induce it in that GT-TsF1 acts across allogeneic barriers whereas GT-TsF2 does not. This restriction is controlled by genes in the H-2 gene complex and maps to the I-J subregion. GT-TsF2 is antigen-specific in suppressive activity and also in its antigen-binding site(s). Thus, GT-TsF2 closely resembles the carrier-specific, I-J+, genetically restricted factor described by Tada and his colleagues. Because GT-TsF2 was induced by GT-TsF1, we suggest cells producing GT-TsF1 are an early cell in the pathway of suppression, and that this cell is required for the activation of antigen-specific, MHC-restricted TsF.     

Age-related changes within a suppressor T cell circuit

The effects of aging on cellular and molecular components of the 4-hydroxy-3-nitrophenyl acetyl-specific suppressor T (Ts) cell circuit were analyzed in vitro using inducer (Ts1), transducer (Ts2), and effector (Ts3) cells and activating factors (TsF1 and TsF2) derived from young or old mice. The activation of Ts2 cells by TsF1 and of Ts3 cells by TsF2 was found age-restricted, suggesting a loss of Ts2 and Ts3 cell subsets in old mice. However, the activation of Ts3 cells by small amounts of TsF2 is more efficient when both are derived from old rather than from young mice while the same level of maximum suppression is attained. Higher affinity of the interactions involved in Ts cell activation may compensate for loss of Ts cell subsets in old mice. No age restriction was found for antigen presentation to Ts1 cells and for the interaction between Ts3 cells and target B cells. Thus, the effects of aging on immunosuppression result from changes within the Ts cell circuit.     

Relationship between T cell receptors and antigen-binding factors. I. Specificity of functional T cell receptors on mouse T cell hybridomas that produce antigen-binding T cell factors

Upon antigenic stimulation with OVA-pulsed syngeneic macrophages, the mouse T cell hybridoma 231F1 produced glycosylation inhibiting factor (GIF) having affinity for OVA and IgE-suppressive factors, whereas another T cell hybridoma, 12H5, cells produced OVA-binding glycosylation enhancing factor (GEF) and IgE-potentiating factor. The OVA-binding GIF from the 231F1 cells is an Ag-specific Ts cell factor, whereas OVA-binding GEF from the 12H5 cells is an Ag-specific augmenting factor. Both hybridomas express CD3 complex and functional TCR-alpha beta. Cross-linking of TCR-alpha beta or CD3 molecules on the hybridomas by anti-TCR-alpha beta mAb or anti-CD3 mAb and protein A resulted in the formation of the same factors as those obtained by the stimulation of the cells with OVA-pulsed syngeneic macrophages. It was also found that both the 231F1 cells and 12H5 cells formed IgE-binding factors upon incubation with H-2d and H-2b APC, respectively, with a synthetic peptide corresponding to residues 307-317 in the OVA molecules (P307-317). Six other synthetic peptides, including those containing the major immunogenic epitope, i.e., P323-339, failed to stimulate the hybridomas in the presence of APC. Indeed, all of the 10 T cell hybridoma clones, which could produce either OVA-binding GIF or OVA-binding GEF, responded to P307-317 and APC for the formation of IgE-binding factors. In contrast, GIF/GEF derived from six other hybridoma clones, whose TCR recognized P323-339 in the context of a MHC product, failed to bind to OVA-coupled Sepharose. The results indicate the correlation between the fine specificity of TCR and the affinity of GIF/GEF to the nominal Ag. The amino acid sequence of P307-317 suggested that TCR on the cell sources of Ag-binding factors are specific for an external structure of the Ag molecules.     

The AKR thymoma BW5147 is able to produce lymphokines when stimulated with calcium ionophore and phorbol ester

We produced the T cell hybridoma D9C1.12.17 by fusing an IL-4-producing T cell clone D9.1Hi with the AKR thymoma BW5147. The resulting hybridoma produced IL-2 as well as IL-4 even though none of the parental cells produced IL-2 after stimulation with Con A. The production of IL-2 was confirmed at the mRNA level by using an S1 nuclease protection assay. Further analysis indicated that Con A-induced IL-2 production was a common phenomenon among T cell hybridomas derived from this fusion. Although BW5147 does not produce detectable lymphokines after Con A stimulation, this line was able to produce IL-2, granulocyte-macrophage colony stimulating factor, and small amounts of IL-3 and IFN-gamma when stimulated with calcium ionophore and phorbol ester. The latter agents are thought to mimic the activating signal(s) delivered through the Ag:MHC TCR. This observation indicates that BW5147 has the ability to produce lymphokines but may lack component(s) which couple the extracellular signal to lymphokine production, and suggests that in T cell hybridomas, part of the spectrum of lymphokines produced may be contributed by BW5147.     

Hapten-specific responses to the phenyltrimethylamino hapten. V. A single chain antigen-binding I-J+ first-order T suppressor factor requires antigen to induce anti-idiotypic second-order suppressor T cells

We have previously shown that a single i.p. injection of the monovalent antigen, L-tyrosine-p-azophenyltrimethylammonium in complete Freund's adjuvant induces a Ly-1+2-, idiotype-bearing, and antigen-binding first-order T suppressor (Ts1) population. We showed that soluble factors extracted from these cells could suppress delayed-type hypersensitivity responses if administered at the induction phase of the response. In this paper we additionally characterize the suppressor factor, TsF1, with respect to its biologic, serologic, and chemical properties. The studies show that the TsF1 is neither allotype nor H-2 restricted and can induce anti-idiotypic T suppressor cells (Ts2), but it requires the presence of antigen to do so. The factor binds antigen, bears I-J encoded determinants, is resistant to reduction and alkylation, and elutes as a single chain factor after adsorption onto monoclonal anti-I-J antibody-coupled Sepharose beads in the presence of dithiothreitol (DTT). This is in marked contrast to TsF2 (derived from Id-specific Ts2-containing spleen cells), which lost its suppressive activity after reduction and alkylation, and behaves as a two chain factor after adsorption and elution from anti-I-J-coupled beads in the presence of DTT. The TsF1 is discussed with respect to the properties of it and those of TsF1 from other similar idiotype-dominated antigen systems.     

Ligand-receptor relationships in immune regulation

The relationship between ligand, idiotype-bearing ligand-binding T suppressor cells (Ts), and antiidiotypic Ts is discussed. The suppressor pathway involves the activation by ligand of first-order idiotypic Ts (Ts1) which elaborate idiotype-bearing T suppressor factors (TsF). TsF readily induced second-order antiidiotypic TS2 cells. The genetic restrictions imposed on the immune system once perturbed by antigen are evaluated.     

Modulation of experimental allergic encephalomyelitis with anti-T suppressor factor antibodies

mAb reactive with T suppressor factors (TsF) were used to alter the course of myelin basic protein-induced experimental allergic encephalomyelitis in (SJL/J x PL/J)F1 mice. In vivo administration of mAb 14-12, reactive with effector TsF, exacerbated the clinical expression of encephalomyelitis as evidenced by prolonged periods of total limb paralysis in affected animals. This aggravation of disease signs is probably related to the inhibition of effector Ts function by mAb 14-12 thus allowing T cell autoreactivity to proceed unchecked. Disease course was influenced more favorably by i.v. administration of mAb 14-30 reactive with a subset of inducer TsF. Ten days of treatment with this mAb resulted in a reduction in the incidence and severity of disease, noted as the development of minimal limb weakness but no paralysis in the majority of affected animals. Adoptive transfer experiments revealed the presence of Ag-specific Ts in mAb 14-30-treated mice that inhibited recipient Lyt-1+ responses to myelin basic protein, the immunizing autoantigen. Suppression by transferred Ts was revealed only by treatment of the donor population with anti-Lyt-1.2 plus C, however, indicating a role for contrasuppressor activity in the regulation of autoimmune T cell function. Results are considered relevant to the potential for immunotherapeutic management of multiple sclerosis in man.     

Regulation of granulomatous inflammation in murine schistosomiasis. IV. Antigen-induced suppressor T cells down-regulate proliferation and IL-2 production

In murine schistosomiasis mansoni the cell-mediated immune response to the deposited eggs is mediated by CD4+ delayed-type hypersensitivity effector T (TDH) cells that produce vigorous granulomatous responses in the liver and intestines of acutely infected animals. The response is significantly down-modulated in chronically infected mice by Ag-specific Ts cells. The present study was undertaken to establish an in vitro model by which TDH-Ts cell interactions could be analyzed. To this end, Ts cells were induced in vitro by preculture of chronic or acute infection spleen cells with soluble egg Ag (SEA) for 48 h. The induced cells suppressed the SEA-specific proliferation of acute infection spleen cells by 80 to 95%. The induced suppressor cells were Ag specific in both induction and elicitation of function, and were not cytotoxic to the acute infection splenic target cells. Suppression by the induced cells was manifested within the first 24 h of the SEA-induced response as IL-2 produced by acute infection spleen cells was suppressed 62%. Phenotypic analysis by flow cytometry of the induced suppressor cells showed that CD8+ cells from acute infection spleens and CD4+ and CD8+ cells from chronic infection spleens were effector Ts cells. Taken together, CD4+ and CD8+ SEA-specific Ts cells can be induced in vitro to effectively suppress the SEA-specific lymphoproliferation and IL-2 production of acute infection spleen cells. Establishment of this in vitro model will allow us to further analyze the mechanisms of Ts cell-mediated suppression of TDH cells.     

Regulation of the immune response to tumor antigens. X. Activation of third-order suppressor T cells that abrogate anti-tumor immune responses

The experiments described further define the suppressor T cell pathway in the S1509a tumor system. We demonstrated previously that S1509a-induced Ts1, TsF1, and Ts2 specifically suppress in vivo Ly1+2- T cell-dependent responses to S1509a and that Ts1 suppress in vivo Ly-1+2- T cell-mediated proliferative responses to S1509a. We have now shown that in vivo administration of either S1509a-induced TsF1 or TsF2 suppresses both in vivo and in vitro Ly-1+2- T cell-mediated responses to S1509a. Furthermore, we revealed the existence of Ts3, which are activated by S1509a tumor antigen and TsF2, in this murine tumor system. Finally, we demonstrated that cyclophosphamide abrogates the suppressive effect of TsF2 but not that of Ts3. These results are discussed with respect to T cell-mediated suppression in other murine tumor systems and the possible pivotal role for a tumor antigen-presenting cell in activating Ts3 in the S1509a tumor system.