Idiotypic analysis of antibodies against the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). IV. Induction of CGAT idiotype following immunization with various synthetic polymers containing glutamic acid and tyrosine.

The immune responses of all inbred strains of mice specific to the synthetic terpolymer poly(LGlu60LAla30LTyr10), referred to as GAT10, are characterized by the presence of anti-GAT antibodies which share a common (CGAT) idiotype. In this report, we describe the ability of the synthetic polymers, LGlu33LAla33LTyr33, LGlu51-LAla34LTyr15 and poly-L(Tyr, Glu)-DLAla--LLys [(T,G)-A--L] to induce antibodies with CGAT idiotypic specificities. All of these polymers contain "GT"-related determinants. Following immunization with these polymers, antisera from responder mice bind the corresponding 125I-labeled antigen and 125I-labeled poly(LGlu50LTyr50) or GAT10. These antisera shared the CGAT idiotype which is associated with the antibody fraction with binding specificity for GAT10. Collectively, the present results indicate that GT-related determinants are required for the induction of the CGAT idiotype. Moreover, since the immune responses to these synthetic polymers are under distinct H-2-linked immune response (Ir) gene control, a mouse strain can be nonresponder to one polymer and responder to another; in this case, only the latter polymer induces CGAT idiotype. Thus, although the immune responses of inbred strains of mice to different polymers are under distinct Ir gene control, the antibody responses can be idiotypically related.     

Induction by monoclonal anti-idiotypic antibodies of an anti-poly(Glu60 Ala30 Tyr10) (GAT) immune response in GAT-responder and GAT-nonresponder mice

Two different monoclonal anti-idiotypic (Id) antibodies, HP-Id20 and HP-Id22, recognizing two discrete idiotopes characteristic of the anti-poly(Glu60 Ala30 Tyr10) (GAT) response were used to immunize BALB/c (GAT-responder) and DBA/1 (GAT-nonresponder) mice. The monoclonals were injected either copolymerized with keyhole limpet haemocyanin or polymerized with glutaraldehyde. The specific response was studied by two assays: (a) inhibition of binding of monoclonal anti-GAT antibody G5Bb2-2 to HP-Id20 and HP-Id22 and (b) GAT binding assays. In BALB/c GAT-responder mice, HP-Id20 and HP-Id22 immunization led to the preferential stimulation of immunoglobulin idiotypically related to anti-GAT antibodies (Ab1') and expressing anti-GAT activity. The results obtained with BALB/c nu/nu mice indicated that this response is T-cell-dependent. By means of the same experimental protocol GAT-nonresponder animals could be induced to produce anti-GAT antibodies after HP-Id immunization. This last result indicates that anti-Id immunization can bypass Ir gene control and does not preferentially stimulate the induction of GAT-specific T suppressor cells.     

Public and individual idiotopes in the anti-poly(Glu60, Ala30, Tyr10) response: analysis by monoclonal antibodies

From BALB/c mice immunized with anti-GAT monoclonal antibody (mAb) G5, we have obtained anti-idiotypic mAb against individual (or private) idiotopes, expressed by G5 as well as anti-GAT mAb, that are heteroclitic because they recognize poly-(Glu50, Tyr50) (GT) better than poly(Glu60, Ala30, Tyr10) (GAT). From BALB/c mice immunized with BALB/c polyclonal anti-GAT antibodies, anti-idiotypic mAb directed against public idiotopes expressed following GAT immunization in all individuals of all mouse strains tested have been obtained. Nine anti-idiotypic mAb were studied in detail. One of these mAb recognizes only polyclonal anti-GAT antibodies; the other eight recognize polyclonal anti-GAT antibodies and anti-GAT mAb. The distribution of the structures recognized by the different anti-idiotypic mAb on a battery of 20 anti-GAT mAb allows definition of two families of public idiotopes.     

Antigen presentation in the murine T lymphocyte proliferative response. II. Ir-GAT-controlled T lymphocyte responses require antigen-presenting cells from a high responder donor.

The activation of T lymphocytes from poly (Glu60Ala30Tyr10)n (GAT)-primed donors by GAT-pulsed nonimmune spleen cells was shown to require identity at the I-1 subregion of the major histocompatibility complex. However, GAT-primed T lymphocytes from (responder x nonresponder) F1 hybrids could only be stimulated to proliferate by GAT bound to high responder or F1 spleen cells but not by GAT bound to spleen cells from the low responder parent. The failure of spleen cells from low responder parental strains to present GAT was shown not to be due to the presence of suppressor cells in either the antigen-presenting or the responding cell populations. These results indicate that control of antigen-presenting cell-T lymphocyte interactions is one site of Ir gene expression.     

The role of suppression in immunoregulation: in vivo analysis using a monoclonal antibody to T suppressor factors

We have used a monoclonal antibody (mAb)-specific for murine T suppressor (Ts) cells (mAb 14-12) to study the role of T cells in tolerance and immunoregulation. We demonstrate that mAb 14-12 can block in vivo Ts cell activity in a variety of experimental systems. It prevents the induction of Ts cells induced by i.v. injection of the water-soluble hapten 2,4,6-trinitrobenzene sulfonic acid, and the protein antigen bovine serum albumin. When 14-12 mAb is given prior to the i.v. injection of trinitrophenyl-conjugated spleen cells (TNP-SC) it blocks the induction of Ts cells and sufficiently overcomes suppression so that TNP-SC is able to induce immunity. mAb 14-12 can convert nonresponder mice into responders for the Ir gene-controlled response to the random terpolymer L-glutamic acid60-L-alanine30-L-tyrosine 10 (GAT), and can substitute for cyclophosphamide in overcoming a suppressor barrier in the adoptive transfer of contact sensitivity. Administration of 14-12 mAb just prior to immunization results in the augmentation of contact sensitivity, antibody and plaque-forming cell responses. These results demonstrate the versatility of this reagent for the study of Ts cell activity.     

Establishment of antigen-specific and MHC-restricted B cell hybridoma clones: a model for studying antigen presentation to T cell lines and clones

Presentation of GAT by GAT-specific hybridoma B cell clones to GAT-specific T cell lines and clones was investigated. B cell clones expressed surface IgG2b, Ia and Fc receptors, and did not secrete immunoglobulins. The clones formed rosettes with GAT-SRBC which could be inhibited by monoclonal antibodies against GAT idiotype. Presentation of GAT by the clones was MHC-restricted and highly efficient, i.e., a few hundred B cells or very low concentration of GAT stimulated proliferation of GAT-specific T cell lines. The fusion partner, M12.4.5.2, clone presented GAT much less efficiently. Although GAT-specific B cell clones presented also heterologous antigen beef insulin to insulin-reactive T cell lines, they were not able to present insulin in the absence of soluble antigen. Presentation of GAT was inhibited by pretreatment of the clones with monoclonal antibodies against I-E, mouse Ig and GAT-idiotype, as well as related antigens GA and GT. The rate of antigen uptake was much shorter compared to resting B cells for cells pulsed with GAT for 4 hours presented the antigen well. The studies suggest that a membrane-associated Il-1 may be required in the presence of antigen presentation by B cells. The results also revealed that antigen-specific hybridomas can present specific antigen in part with association of surface immunoglobulins which may be involved in antigen uptake and focusing to T cells.     

Immune response to the p-azobenzenearsonate-L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) conjugate. III. Mechanisms of Ir gene-controlled phenotype conversion

Immunization of GT (random copolymer of L-glutamic acid51-L-tyrosine49) nonresponder animals with p-azobenzenearsonate (ABA) GT conjugates elicits an antibody response to both ABA and GT epitopes which is induced by ABA-specific T helper cells. Expression of these hapten-specific helpers is under the control of an I region gene which also regulates the proliferative T cell response to ABA. Conversion of the unresponsive phenotype to GT is, therefore, dependent on the ABA Ir gene and escapes the influence of the GT-specific I region-controlled suppressive pathway. Studies on the influence of ABA/polymer coupling ratio on T and B cell responses suggest that ABA-specific T cells, like conventional carrier-specific helpers, require linked interactions with B lymphocytes to provide helper signals. GAT (terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10) nonresponder animals immunized with ABA-GAT conjugates also develop an antibody response to ABA which is induced by ABA-specific T helper cells. Comparison of antibody affinity, specificity, isotypes and idiotypes in different mouse strains demonstrates that hapten-specific helper cells stimulate antibody responses to ABA which are qualitatively similar to those induced by GAT-specific helpers. However, ABA-specific helper cells do not permit the conversion of the I region gene-controlled nonresponder phenotype to GAT. The data suggests that high ABA density, which is required for optimal ABA help expression, extinguishes the immunogenicity of GAT determinants at both T and B cell levels.     

Antigen-specific tachycardia and hypotension in rodents

Tachycardia and hypotension, two cardiovascular responses to anaphylaxis, were specifically induced by antigen in mice and rats, respectively. Intravenous injection of poly (Glu60Ala30Tyr10) (GAT) elicited tachycardia within 30-40 sec in GAT-primed B6 mice. Moreover, a minute amount of GAT (0.2 micrograms) was enough to sensitize the mice to subsequent GAT-induced tachycardia. Challenging doses ranging from 100 ng to 500 micrograms. could elicit tachycardia. The kinetics of tachycardia induction was different from that of antibody production or delayed-type hypersensitivity. Tachycardia was induced from day 6 after immunization, while delayed-type hypersensitivity developed as early as day 4, and anti-GAT antibodies were undetectable on day 6 and would not reach a maximum until day 8. Specific antigen-induced hypotension was also observed in rats. Furthermore, cardiovascular changes in both species could be passively transferred by heat-treated (56 degrees C, 30 min) sera from immunized animals. These benchmarks of antigen-induced cardiovascular changes in mice or rats could be used as models to study the immune control of cardiovascular changes in anaphylactic responses.     

Stimulation of specific suppressor T cells in newborn responder mice by the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT).

The effects of immunization with the terpolymer of L-glutamic acid60-L-alanine40-L-tyrosine10 (GAT), the copolymers of L-glutamic acid60-L-alanine40 (GA) and of L-glutamic acid50-L-tyrosine50 (GT), were compared in adult and newborn BALB/c and BALB.B mice. as expected, BALB/c (H-2d) and BALB.B (H-2b) adult mice were responders to GAT and GA and nonresponders to GT, which induced suppressor T cells in BALB/c but not in BALB.B mice. in contrast, newborn mice expressed different phenotypes. Two-week-old mice developed responses to GAT, GA and GT-complexed methylated bovine serum albumin, but immunization at birth with these copolymers induced a cross-reactive tolerance in both strains. Neonatal GAT tolerance could be transferred in adult and involved suppressor T cells in the two inbred strains, whereas the GT-specific immune suppression was not demonstrable in newborn BALB/c mice. The significance of these data to our understanding of the regulation of specific immune response and tolerance is discussed.     

The avidity of IgM antibody in high and low responder rats.

This study examined IgM antibody produced by highly responding ACI and poorly responding F344 rats follwing immunization with poly(Glu52Lys33Tyr15) or poly(Glu52Lys33Tyr15) aggregated with methylated bovine serum albunim (MeBSA). The ACI rats produced both IgM and IgG plaque-forming cells (PFC) following immunization with either form of antigen. The F344 rats did not respond to unaggregated poly(Glu52Lys33Tyr15), but they produced significant amounts of IgG PFC and extremely small amounts of IgM PFC after immunization with poly(Glu52Lys33Tyr15)/MeBSA. Both high and low responder rats had similar kinetic profiles of IgM antibody production, and this antibody had nearly identical avidity in both strains with no evidence for any maturation in avidity. thus, one of the genetic defects in the antibody response to poly(Glu52Lys33Tyr15) is an inability of the F344 strain to produce large amounts of IgM in response to this antigen.     

Poly(Glu60,Ala30,Tyr10) (GAT)-specific T cells do not express B cell public idiotopes but can be primed by monoclonal anti-idiotypic antibodies

Eight monoclonal anti-idiotypic antibodies directed against public idiotopes have been further characterized: (a) they bind to public idiotopes with a high affinity; (b) they recognize all anti-poly(Glu60,Ala30,Tyr10) (GAT) antibodies as measured by inhibition of the anti-GAT plaque-forming cell response. This has been verified in three strains of mice. These reagents were not able to detect idiotope expression on eight GAT-specific helper T cell lines and clones. This result was obtained by two techniques: (a) idiotope expression at the T cell surface was measured by indirect immunofluorescence using a cell sorter with surface antigens H-2D, Thy-1.2, Lyt-1 and L3T4 as positive controls; (b) after immunoadsorption of [35S] methionine-labeled cellular extracts from two lines, no unique molecule was retained by the HP-idp22 monoclonal anti-idiotypic antibody coupled to Sepharose. Despite these negative results, this antibody was found to prime lymph node cells in vivo, which were able to proliferate specifically in response to GAT. Two T cell lines derived from this lymphocyte population do not express any of the idiotopes tested. These results suggest that monoclonal anti-idiotypic antibodies may be influencing T lymphocyte activity indirectly.     

The role of antigen recognition and suppressor cells in mice with oral tolerance to ovalbumin.

The induction of tolerance by feeding proteins may prevent potentially harmful delayed-type hypersensitivity (DTH) reactions to food antigens. Suppressor T cells (Ts) are present in mice with tolerance of systemic DTH after feeding ovalbumin (OVA) but, as other immunoregulatory mechanisms have also been described, the exact role of Ts in maintaining tolerance is not known. In this study, we have used the ability of native and denaturated OVA to cross-react at the level of helper/effector T cells, but not Ts, to re-examine the role of Ts in oral tolerance to OVA. Mice immunized with native OVA (nOVA) or denatured OVA (dOVA) in adjuvant had fully cross-reacting DTH to either nOVA or dOVA, but intravenous administration of antigen induced Ts which were specific for the appropriate form. Mice fed nOVA or dOVA had identical tolerance of systemic DTH to both forms of OVA, and feeding nOVA induced splenic Ts which suppressed the DTH response to both nOVA and dOVA. Splenic Ts could not be detected in mice fed dOVA. The results support the hypothesis that tolerance of systemic DTH in mice fed native proteins is due to Ts. Although, for the moment, there is no complementary evidence for a role for Ts in oral tolerance to denatured proteins, this study is consistent with the idea that Ts are the mechanism which normally prevent enteropathy due to DTH against dietary proteins. In addition, our study underlines the differences between orally and parenterally induced Ts and reinforces the view that fed proteins induce Ts after processing by the gut or its lymphoid accessory cells.     

Tolerance to thymus-independent antigens. Characteristics of induction of tolerance to thymus-independent synthetic polypeptides

The conditions have been studied for induction of tolerance in mice to two thymus-independent multichain synthetic polypeptides, poly(DTyr,DGlu)-polyDPro--polyDLys, and poly(DTyr,DGlu)-polyLPro--polyLLys. The antigens are of very similar structure, differing only in the optical configuration of proline and lysine residues.The tolerance thresholds were established by giving a single injection of antigen in solution in saline, followed later by challenge of antigen in adjuvant. For both antigens it was possible to induce high dose tolerance, which was preceded by a zone of priming at lower doses. In neither case was low zone tolerance observed. The induction of high zone tolerance was accompanied by a passing phase of antibody production in the case of poly(DTyr,DGlu)-polyDPro--polyDLys, but not in the case of poly(DTyr,DGlu)-polyLPro--polyLLys. Furthermore, the threshold doses for high zone tolerance for the two antigens differed by two orders of magnitude, being at about 100 μg per mouse for the former and at 1 μg per mouse for the latter. It appears that thymus-independent antigens, even if very similar structurally, can differ very markedly in the characteristics of their interaction with B cells.     

Presentation of cryptococcal capsular polysaccharide (GXM) on activated antigen-presenting cells inhibits the T-suppressor response and enhances delayed-type hypersensitivity and survival.

A hallmark of infection with Cryptococcus neoformans is depression of the immune system characterized by poor inflammatory responses and loss of delayed-type hypersensitivity (DTH) and antibody responses. T-suppressor cell (Ts) responses, elicited by the capsular polysaccharide (GXM) of the organism, are known to develop during infection. This study was undertaken to develop a method to inhibit the anti-GXM Ts response and thereby study the influence of the Ts response on immune responsiveness and survival in cryptococcosis. Antigen-presenting cells (APC), elicited with complete Freund's adjuvant (CFA), were treated in vitro with GXM (GXM-APC). The GXM-APC were injected intravenously into normal mice. These mice were resistant to induction of anti-GXM Ts cells when soluble GXM was administered in tolerogenic doses or when animals were infected with C. neoformans. Inhibition of the anti-GXM Ts response was specific to GXM as levan-APC did not inhibit induction of anti-GXM Ts cells. Inhibition of the anti-GXM Ts response could not be attributed to increased clearance of GXM due to induction of anti-GXM antibodies or other mechanisms. Anti-cryptococcal DTH responses were lost in mice by the second week of infection. However, treatment with GXM-APC, but not levan-APC, allowed mice to maintain their DTH response. GXM-APC pretreatment enhanced survival of infected mice compared with mice pretreated with levan-APC. These results show that GXM-APC induces immune responses that inhibit the induction of Ts responses and enhances DTH responses in infected mice. These responses correlate with enhanced survival after cryptococcal infection.     

Genetic control of the immune response to the terpolymer L-glutamic acid 60-L-alanine 30-L-tyrosine10 (GAT). III. Restricted heterogeneity of the anti-GAT response from BALB/c responder mice.

The heterogeneity of the anti-GAT [terpolymer poly(Glu60, Ala30, Tyr10)] response of GAT responder mice has been analyzed. Purified anti-GAT antibodies from BALB/c mice belong only to the gamma 1 kappa subclass. The isoelectric focusing pattern obtained indicates that the anti-GAT antibodies are particularly basic and restricted. These results have been confirmed by two-dimensional polyacrylamide gel electrophoresis; by this technique, we have shown that the gamma 1 chain and the corresponding kappa chain of anti-GAT antibodies are restricted. All the anti-GAT antibodies from 10 BALB/c mice bear the cross-reactive GAT idiotype previously defined (J. Thèze and G. Sommé, Eur. J. Immunol. 1979, 9: 924). The present results indicate that the anti-GAT repertoire expressed in BALB/c mice is very limited.     

Accessory molecules and T cell activation. I. Antigen receptor avidity differentially influences T cell sensitivity to inhibition by monoclonal antibodies to LFA-1 and L3T4

A series of BALB/c-derived T hybridoma cells, capable of producing interleukin 2 (IL 2) in response to poly(Glu60, Ala30, Tyr10) (GAT) presented by syngeneic B lymphoma cells in the context of Ad restriction determinants, was used as a model system to evaluate the roles of LFA-1 and L3T4 accessory molecules in antigen-specific T cell activation. Examination of the antigen requirement for optimal IL 2 responses revealed marked differences in the apparent avidity of these cells for GAT/Ad complexes. A relationship was observed between this parameter and the susceptibility of T hybridoma cells to inhibition by monoclonal antibodies directed at 5 distinct epitopes of LFA-1, and at A beta d allodeterminants. In contrast, L3T4a-specific monoclonal antibodies were found to block in a similar fashion the antigen-specific IL 2 responses of T hybridoma cells, regardless of the apparent avidity of their antigen receptors. It was also shown that both L3T4+ and L3T4- T hybridoma cells were capable of recognizing GAT plus Ad with high avidity. Thus, the quality of T cell antigen recognition appears to critically influence the involvement of LFA-1, and only to a marginal extent that of L3T4, in antigen-specific T cell activation. The implications of these findings are discussed in the context of recent data indicating that L3T4 may not only be an Ia-binding protein.     

Effect of irradiation on the precursor, activated and memory suppressor T cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

The relative radiosensitivities of precursor (Tsp), activated (Ts) and memory (Tsm) suppressor T cells for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) were investigated in mice. Spleen cells from CBA mice, primed i.v. with 10(9) SRBC 3-4 days previously, contain specific Ts cells which substantially impair the induction of DTH to SRBC in normal syngeneic recipients. Exposure of mice to 400 rad irradiation 1 day before the priming completely eliminated the subsequent development of Ts cells. In contrast, 3 days after the priming injection, Ts cell activity in mice is resistant to doses higher than 600 rads. Mice primed 40 days previously with 10(9) SRBC contain Ts-cell memory which can be readily recalled by i.p. injection of 10(8) SRBC. The secondary Ts cells which specifically inhibit DTH induction can be demonstrated adoptively in normal recipients. Mice were exposed to various doses of irradiation 40 days after the priming and 1 day before the i.p. injection. Ts memory was significantly reduced by 300 rads and was completely abrogated by 400 rads. The relative radiosensitivities of the three subsets of suppressor T cells are in the order of Tsm = Tsp greater than Ts.     

Serological analysis of antigen-specific helper factors specific for poly-L(Tyr, Glu)-poly-DLAla--poly-LLys [(T, G)-A--L] and L Glu60-LAla30-LTyr10 (GAT).

In vitro prepared antigen-specific helper factors reactive to the synthetic polypeptide antigens poly-L(Tyr, Glu)-poly-DLAla--poly-LLys [(T, G)-A--L] or LGlu60-LAla30-LTyr10 (GAT) and bearing Ia determinants were analyzed serologically to determine the nature of the Ia determinants they expressed. I subregion-specific mouse anti-Ia antisera were used, and showed that (T, G)-A--L-specific helper factor (HF) contains I-A subregion-controlled determinants, whereas GAT-specific HF carries I-J subregion-controlled antigens. This unexptected finding was confirmed in both the H-2k and H-2 b haplotypes, using a variety of anti-I-J antisera. Rabbit anti-Ia antisera also reacted with both HF which raised the possibility that the Ia determinants on HF may be carbohydrate in nature. The fact that HF has a low molecular weight and yet contains Ia determinants, antigen-binding capacity and idiotypic markers is compatible with this interpretation.     

Oral tolerance in protein-deprived mice. II. Evidence of normal 'gut processing' of ovalbumin, but suppressor cell deficiency, in deprived mice.

The induction of oral tolerance for DTH responses to ovalbumin is impaired in protein-deprived mice. This may be via the effects of protein malnutrition on short-lived Ts cells, but an alternative explanation is that the gut handling of antigen is abnormal. We have attempted to transfer tolerance from protein-deprived and control mice to naive recipients by using spleen cells, collected 7 days after an OVA feed at a time when Ts cells should be present, or by using serum, collected 1 hr after feeding, which should contain tolerogenic, 'gut-processed' antigen. Suppression of DTH was transferred with 7-day spleen cells and with 1-hr serum from normal, protein-sufficient mice. Mice that received spleen cells from protein-deprived donors were not tolerant, but suppression was readily transferred with serum from deprived mice, indicating that their capacity for intestinal antigen processing was normal. Furthermore, the quantity of absorbed antigen in the serum 1 hr after feeding was similar in both protein-deprived and normal groups. The results obtained are consistent with the hypothesis that short-term protein deprivation depletes a population of short-lived Ts cells which control DTH oral tolerance.     

Effects of first-order Cryptococcus-specific T-suppressor cells on induction of cells responsible for delayed-type hypersensitivity.

Cell-mediated immunity is an important aspect of host resistance against Cryptococcus neoformans. Using a CBA/J murine model, we demonstrated that injection of cryptococcal antigen (CneF) at dosages sufficient to stimulate the antigenemia observed in cryptococcosis patients induces specific T-cell-mediated suppression of the cryptococcal delayed-type hypersensitivity response. The purpose of this study was to establish whether Lyt 1+, first-order T-suppressor (Ts1) cells block the induction of T cells responsible for delayed-type hypersensitivity (TDH cells) or whether they function by inducing Lyt 2+, efferent suppressor (Ts2) cells. In one set of experiments, suppression was observed when Ts1 cells were adoptively transferred to recipient animals the day before, the day of, or the day after immunization; however, when Ts1 cells were transferred after TDH cells were present, no suppression occurred. In other experiments, putative TDH cells from lymph nodes (LN) or spleens were adoptively transferred from mice after immunization or after a suppressive dose of CneF or adoptive transfer of Ts1 cells and immunization. Delayed-type hypersensitivity could not be transferred with LN or spleen cells from mice receiving the suppressive dose of CneF or the Ts1 cells, even when the LN or spleen cells were treated with anti-Lyt 2.1 antibody and complement to remove any Ts2 cells. Delayed-type hypersensitivity was readily transferred with LN or spleen cells from immunized mice whether the cells were or were not treated with anti-Lyt 2 and complement. Furthermore, the cells in the tolerized LN cell pools responsible for suppression of TDH cell induction were Lyt 1+ 2-, I-J+ cells, which is the phenotype of the Ts1 cells. Taken together, these data indicate that Ts1 cells inhibit the induction of TDH cells. This finding, coupled with the previous demonstration that Ts1 cells or a Ts1 cell-derived soluble factor (TsF1) induces Ts2 cells, establishes that the cryptococcal Ts1 cells are bifunctional in the suppressive pathway.