T cell subsets in (responder x nonresponder)F1 mice regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine (GAT)

Immune responses to GAT are controlled by H-2-linked Ir genes; soluble GAT stimulates antibody responses in responder mice (H-2b) but not in nonresponder mice (H-2q). In nonresponder mice, soluble GAT stimulates suppressor T cells that preempt function of helper T cells. After immunization with soluble GAT, spleen cells from (responder x nonresponder: H-2b X H-2q)F1 mice develop antibody responses to responder H-2b GAT-M phi but not to nonresponder H-2q GAT-M phi. This failure of immune F1 spleen cells to respond is due to an active suppressor T cell mechanism that is activated by H-2q, but not H-2b, GAT-M phi and involves two regulatory T cell subsets. Suppressor-inducer T cells are immune radiosensitive Lyt-1 +2-, I-A-, I-J+, Qa-1+ cells. Suppressor-effector T cells can be derived from virgin or immune spleens and are radiosensitive Lyt-1-2+, I-A-, I-J+, Qa-1+ cells. This suppressor mechanism can suppress responses of virgin or immune F1 helper T cells and B cells. Helper T cells specific for H-2b GAT-M phi are easily detected in F1 mice after immunization with soluble GAT; helper T cells specific for H-2q GAT-M phi are demonstrated after elimination of the suppressor-inducer and -effector cells. These helper T cells are radioresistant Lyt-1+2-, I-A+, I-J-, Qa-1- cells. These data indicate that the Ir gene defect in responses to GAT is not due to a failure of nonresponder M phi to present GAT and most likely is not due to a defective T cell repertoire, because the relevant helper T cells are primed in F1 mice by soluble GAT and can be demonstrated when suppressor cells are removed. These data are discussed in the context of mechanisms for expression of Ir gene function in responses to GAT, especially the balance between stimulation of helper vs suppressor T cells.     

T cell subsets regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in virgin and immunized nonresponder mice

T cell subsets from virgin and immunized mice, which are Ir gene controlled nonresponders to GAT, which regulate antibody responses to GAT have been characterized. Virgin nonresponder B10.Q B cells develop GAT-specific antibody responses to GAT, B10.Q GAT-M phi, and GAT-MBSA when cultured with virgin or GAT-primed Lyt-1+, I-J-, Qa1- B10.Q helper T cells. Virgin T cells are radiosensitive, whereas immune T cells are radioresistant (750 R); qualitatively identical helper activity is obtained with T cells from mice immunized with soluble GAT, B10.Q GAT-M phi, and GAT-MBSA. Responses to GAT and GAT-M phi are not observed when virgin or GAT-primed Lyt-1+, I-J+, Qal+ T cells are added to culture of virgin or GAT-primed Lyt-1+, I-J-, Qa1- helper T cells and virgin B cells; the GAT-specific response to GAT-MBSA is intact. The Lyt-1+, I-J+, Qa1+ T cells from mice primed with GAT, GAT-M phi, and GAT-MBSA were qualitatively identical in mediating this suppression. Virgin Lyt-2+ T cells have no suppressive activity alone or with virgin Lyt-1+, I-J+, Qa1+ T cells, whereas responses to GAT, GAT-M phi, and GAT-MBSA are suppressed in cultures of GAT-primed helper T cells containing GAT-primed Lyt-2+ T cells (with or without GAT-primed Lyt-1+, I-J+, Qa1+ T cells). Suppression of responses to GAT-MBSA in cultures of GAT-M phi-primed helper T cells requires both GAT-M phi-primed Lyt-1+, I-J+, Qa1+ T cells and Lyt-2+ T cells; the Lyt-1+, I-J+, Qa1+ T cells appear to function as inducer cells in this case. In cultures containing GAT-MBSA-primed helper T cells, either GAT-MBSA-primed Lyt-1+, I-J+, Qa1+ or Lyt-2+ T cells suppress responses to GAT and GAT-M phi; under no circumstances are responses to GAT-MBSA suppressed by GAT-MBSA-primed regulatory T cells. This regulation of antibody responses to GAT by suppressor T cells is discussed in the context of the involvement of suppressor T cells in responses to antigens under Ir control, and of the evidence that nonresponsiveness to GAT is not due to a defect in the T cell repertoire, but rather is due to an imbalance in the activation of suppressor vs helper T cells.     

Genetic restrictions in the development of antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 by nude mice implanted with semiallogeneic thymus glands

Athymic nude mice implanted with F1 thymus glands were used to investigate genetic restrictions regulating T cell-macrophage (M phi) interactions in the development of antibody responses to GAT. Spleen cells from conventional mice developed comparable primary plaque-forming cell (PFC) responses when stimulated by syngeneic and allogeneic GAT-M phi. However, spleen cells from strain A nude mice implanted with (A X B)F1 thymus glands were tolerant of strain B alloantigens and developed GAT-specific PFC responses to strain A GAT-M phi and allogeneic strain C GAT-M phi, but failed to respond to strain B GAT-M phi. The lack of primary GAT-specific PFC responses by spleen cells from (A X B)thy----A nude mice stimulated by strain B GAT-M phi was not due to detectable suppressor mechanisms. However, an allogeneic effect stimulated by H-2- or non-H-2-disparate GAT-pulsed or unpulsed M phi was able to overcome the inability of spleen cells from (A X B)F1 thy----A nude mice to respond to strain B GAT-M phi. Furthermore, the inability to respond to strain B GAT-M phi was overcome by the addition of supernatant fluids from independent cultures of H-2-disparate cells. These results 1) demonstrate that T cells from A nude mice implanted with (A X B)F1 thymus glands did not recognize nominal antigen in the context of B MHC antigens, and 2) suggested that the T cell repertoire was altered in strain A nude mice implanted with (A X B)F1 thymus glands, such that T cells that could recognize GAT in association with strain B MHC antigens were functionally deleted.     

Identification of suppressor T cells in virgin non-responder spleen cells responsible for primary unresponsiveness to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

T cell subsets that regulate antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in mice that are Ir gene non-responders have been further characterized. We previously defined several T cell subsets in GAT-primed non-responder mice. The Lyt-2+ suppressor-effector T cells suppress responses to GAT and GAT complexed to methylated BSA (GAT-MBSA). The Lyt-1+ cell population is complex and can be separated into I-J- Th cells, which support responses to GAT and GAT-MBSA. After priming, the Lyt-1+, I-J+ cell population contains suppressor-inducer cells that activate precursors of suppressor-effector cells to suppress responses to GAT and GAT-MBSA as well as Ts cells that directly inhibit responses to GAT but not GAT-MBSA. By contrast, the Lyt-1+ cells from virgin mice contain only cells that directly suppress responses to GAT but not GAT-MBSA. The major question addressed in the present studies was whether the Lyt-1+, I-J+ Ts cells in virgin and primed mice and the suppressor-inducer cells in GAT-primed mice were functionally and serologically distinct subsets. The studies used mAb and panning procedures to separate cell populations and inhibition of PFC cell responses to functionally define the activity of the cell populations. We used the following two mAb that were raised by immunizing rats with GAT-specific suppressor factors: 1248A4.10 (known to react with suppressor-inducer cells) and 1248A4.3, another reagent from the same fusion. Lyt-1+ cells from virgin spleens contained Ts cells that were A4.10-, A4.3+ and no suppressor-inducer T cells, whereas Lyt-1+ cells from GAT-primed spleens contained Ts cells that were A4.10-, A4.3+ as well as A4.10+, A4.3- suppressor-inducer cells. Thus, the Lyt1+, I-J+ cell subset can be divided into two functionally and serologically distinct subsets, direct Ts cells (1248A4.3+), which suppress responses to GAT but not GAT-MBSA, and GAT-primed suppressor-inducer T cells (1248A4.10+).     

Insulin-specific suppressor T cell factors

Murine antibody responses to heterologous insulins are controlled by MHC-linked immune response genes. Although nonresponder mice fail to make antibody when injected with nonimmunogenic variants of insulin, we have recently shown that nonimmunogenic variants stimulate radioresistant, Lyt- 1+2- helper T cells that support secondary antibody responses. However, the helper activity can not be detected unless dominant, radiosensitive Lyt-1-2+, I-J+ suppressor T cells are removed. In this paper we report that extracts of primed Lyt-2+ suppressor T cells contain insulin-specific suppressor factors (TsF) that are capable of replacing the activity of suppressor T cells in vitro. The activity of these factors is restricted by MHC-linked genes that map to the I-J region, and immunoadsorption studies indicated that they bind antigen and bear I-J-encoded determinants. Insulin-specific TsF consists of at least two chains, one-bearing I-J and the other the antigen-binding site. Furthermore, mixing of isolated chains from different strains of mice indicates that the antigenic specificity is determined by the antigen-binding chain and the MHC restriction by the H-2 haplotype of the source of the non-antigen-binding, I-J+ chain. Moreover, mixtures containing antigen-binding chain from allogeneic cell donors and I-J+ chain from responder cell donors have activity in cultures containing responder lymphocytes. This suggests that preferential activation of suppressor T cells, rather than differential sensitivity to suppression, results in the nonresponder phenotype to insulin.     

I-J restriction of T cells that suppress in vivo antibody responses to Thy-1

The contact-sensitizing haptens dinitrophenyl (DNP) and oxazalone (Ox) act as helper determinants for antibody responses to Thy-1 when conjugated to donor thymus cells. The helper effect is transferrable from primed to naive mice with spleen cells, producing specific augmentation of in vivo PFC responses to Thy-1. The helper cells are hapten-specific and require associative recognition of hapten and Thy-1, excluding a role for nonspecific B cell activation. The phenotype of the helper cells is Thy-1+ and Lyt-1+2-. Antigen-specific suppression could be readily generated by using an inoculum of DNP-modified syngeneic RBC. T cells from these suppressed donors (Ts) were shown to abolish the helper effects of TH in adoptive transfer experiments in vivo. These Ts were characterized as Thy-1+ and Lyt-1-2+. A requirement for MHC compatibility at the I-J subregion was necessary between the Ts and the recipient to obtain a transfer of suppression.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.     

T suppressor factor activity is due to two separate molecules. The Lyt-1(-)2+I-J+ cells of mice primed with antigen make an antigen binding molecule which is only active when complemented by cofactor made by Lyt-1+2(-)I-J+ cells

Mice primed with picrylsulfonic acid (PSA) and then painted on the skin with picryl chloride produce antigen-specific T suppressor factor (TsF). In contrast unpainted primed mice fail to produce active TsF. This is not due to the absence of the antigen binding part of TsF but to the absence of a cofactor. This cofactor is (a) antigen nonspecific and occurs in potassium chloride extract of normal spleen cells. It also occurs in the 24 hr supernatant of normal cells modified by haptenisation with picryl or the unrelated NP antigen (4-hydroxy-3-nitrophenylacetyl), and in preparations of conventional TsF (PSA/PCl) from painted PSA-primed mice; (b) bears I-J determinants; and (c) is produced by Lyt-1+2(-)I-J+ cells. The antigen binding molecule occurs alone in the supernatant of PSA-primed mice. It lacks I-J determinants and has a molecular weight around 35,000 and 75,000. It is produced by Lyt-1(-)2+I-J+ cells and is only active when complemented by cofactor. However, the complementation is genetically restricted and the restriction maps to the I-J subregion of the MHC.     

Immunoregulatory pathways in adult responder mice. II. Regulation of delayed-type hypersensitivity responses by GAT-specific suppressor factors present in GAT-tolerant adult responder mice

We studied the effects of T cell extracts from adult responder BALB/c mice tolerized with poly(Glu60Ala30Tyr10) (GAT)-coupled syngeneic spleen cells (GAT-SP) on delayed-type hypersensitivity (DTH), T cell-proliferative (Tprlf), and plaque-forming cell (PFC) responses. Adult responder mice injected i.v. with GAT-SP develop Lyt-1-2+ suppressor T cells (Ts), which suppress the induction of GAT-specific DTH and PFC, but not Tprlf responses. Sonicates from these Ts contain an afferent-acting, soluble factor(s) (GAT-TsFdh) that specifically suppresses the same responses as the intact Ts (i.e., DTH and PFC, but not Tprlf). Immunosorbent chromatography studies were employed to determine the molecular nature of the suppressive material active on both cellular and humoral responses. In both assay systems, GAT-TsFdh was found to bear determinants encoded by the I subregion of the H-2 complex and a receptor(s) for GAT. BALB/c-derived GAT-TsFdh suppressed the induction of GAT DTH in syngeneic BALB/c and H-2-compatible B10.D2, but not in allogeneic C57BL/6 or CBA/Cum, suggesting a possible H-2 restriction in the suppression. It was also shown that one target of functional regulation by GAT-TsFdh is the T helper cell for DTH responses (DTH-Th). The results suggest that similar Ts and TsF regulate humoral and cell-mediated responses, perhaps by affecting a target common to both pathways (e.g., the T helper cell). The resistance of Tprlf responses to suppression by GAT-TsFdh indicates that the effector DTH-Th target is not a major component of the proliferative response. These data are discussed with respect to GAT-specific TsF-regulating PFC responses, which have been identified in nonresponders and in responders tolerized as neonates with GAT.     

Characterization of suppressor cells regulating in vitro expression of IgG2A and IgG2B antibody responses

In vitro cooperative responses between hapten-primed anti-Thy-1.2 plus C-treated spleen cells and carrier-primed T cells have different isotypic patterns depending on the source of the T helper cells. T helper cells from primed lymph node induce IgG1, IgG2a, and IgG2b PFC responses, whereas T helper cells from primed spleen induce only an IgG1 type of response. The addition of activated spleen cells to the lymph node cells suppresses their ability to generate IgG2a and IgG2b PFC responses. The suppressor cells involved have been characterized. Functionally, they appear as nonantigen specific and isotype specific, because they never reduce the IgG1 response. They are Lyt-2.2 positive, Lyt-1 negative, and radiosensitive. Their relative resistance to anti-Thy-1.2 plus C treatment indicates that they express low amounts of this antigen or that they are heterogeneous concerning the expression of Thy-1.2.     

Antigen-specific T cell-mediated suppression. II. In vitro induction by I-J-coded L-glutamic acid50-L-tyrosine50 (GT)-specific T cell suppressor factor (GT-T8F) of suppressor T cells (T82) bearing distinct I-J determinants.

The induction of new suppressor T cells (Ts2) by suppressive extracts (TsF) from L-glutamic acid50L-tyrosine50 (GT) nonresponder mice was examined. Incubation of normal spleen cells with allogeneic GT-TsF for 2 days in vitro led to the generation of Ts2 cells able to suppress subsequent responses to the immunogen GT-methylated bovine serum albumin (GT-MBSA) in vivo. This induction occurred efficiently when TsF donor and target cells differed at all of H-2, including the I-J subregion. B10.BR (H-2k) GT-TsF, adsorbed on, then acid eluted from GT-Sepharose and anti-I-Jk [B10.A (3R) anti-B10.A (5R)]-Sepharose in a sequential fashion could induce BALB/c (H-2d) spleen cells to become Ts2 only if nanogram quantities of GT were added to the purified GT-TsF. This indicates a requirement for a molecule or molecular complex possessing both I-J determinants and antigen (GT)-binding specificity, together with GT itself, for Ts2 induction. The induced Ts2 are I-J+, since their function can be eliminated by treatment with anti-I-Jk plus C. These I-J determinants are coded for by the precursor of the Ts2 and do not represent passively adsorbed, I-J coded TsF, since anti-Ijk antiserum [(3R X DBA/2)F1 anti-5R] which cannot recognize the BALB/c (I-Jd) TsF used for induction still eliminates the activity of induced A/J (I-Jk) Ts2. These data provide further evidence for and information about the minimum of two T cells involved in antigen-specific suppressor T cell systems.     

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XIII. Characterization of a third T cell population involved in suppression of in vitro PFC responses

The involvement of a third-order suppressor T cell population (Ts3) in the suppression of in vitro PFC responses was analyzed. It was shown that Ts2 effector-phase suppressor cells, induced by the i.v. injection of NP-coupled syngeneic spleen cells, require a third suppressor T cell population to effect NPb idiotype-specific suppression of an in vitro B cell response. This Ts3 population was shown to be present in NP-primed but not unprimed donors. The Ts3 population specifically binds NP and is Lyt-1-, Lyt-2+, I-J+ and bears NPb idiotypic determinants. The involvement of the Ts3 population in a suppressor pathway that requires recognition of idiotypic determinants is discussed.     

Characterization of L-glutamic acid60-L-alanine30-L-tyrosine10-specific suppressor T cells in responder mice restricted by Igh-C-linked genes

A Ts cell subset has been identified in the spleens of responder mice 3 to 6 wk after immunization with an optimally immunogenic dose of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). These Ts were positively selected by panning procedures by using a mAb (1248 A4.10) produced by immunization of rats with semipurified mouse GAT-specific, single polypeptide chain suppressor factor. These Ts cells inhibited the activity of virgin Th cells but not memory Th cells and this activity was genetically restricted by genes which are linked to the Ig H chain (Igh) locus on chromosome 12. Use of the Igh recombination strain, BAB.14, which has a crossover near the VHCH region junction, demonstrated that the genes regulating the Igh restriction map telomeric to the VH genes. The Igh-linked restriction regulated the interaction of A4.10+ Ts cells with virgin T cells and not B cells. However, A4.10+ Ts did not act directly on Lyt-2-Th cells, but required the presence of Lyt-2+ cells for suppression. Suppression by GAT-primed A4.10+-Ts cells also required syngenicity at Igh-linked genes by both Lyt-2- and Lyt-2+ T cells. These results indicated that A4.10+-Ts cells were inducer Ts cells which activated Lyt-2+ effector Ts cells which prevented primary GAT specific Th cell activity. The interaction between A4.10+-Ts inducer and effector Ts cells and/or the interaction of the effector Ts and its target cell were restricted by genes linked to the Igh constant region.     

Regulation of the secondary in vitro antibody response by endogenous natural killer cells: kinetics, isotype preference, and non-identity with T suppressor cells

Our previous studies had demonstrated that depletion of endogenous natural killer (NK) cells resulted in an augmented primary antibody response in vivo and in vitro. We have now examined the effect of NK cell depletion on the in vitro secondary response to antigen. Treatment of primed murine spleen cells with anti-NK-1.1 allo-antibody and complement before culture resulted in a significant increase in the magnitude of the antigen-specific plaque-forming cell (PFC) response. This treatment did not affect the proportions of Lyt-2+, L3T4+, or sIg+ cells in the population, however, indicating that the augmentation in PFC was not due to changes in the ratio of T to B cells. Removal of endogenous NK cells had a greater effect on the IgG (indirect) PFC response (100 to 200% increase) than on the IgM (direct) PFC response (25 to 50% increase). In contrast, removal of Lyt-2+ cells before culture affected the IgM and IgG responses similarly. Moreover, the kinetics of augmentation differed between cultures depleted of Lyt-2+ cells and those depleted of NK-1.1+ cells. NK cells appeared to act earlier in the response than did T suppressor cells. The NK-1.1+ cells involved in antibody regulation were not involved in the generation of the in vitro derived T suppressor cells. The conclusion that the regulation of the antibody response by NK-1.1+ cells is distinct from that involving T suppressor cells was confirmed in experiments in which removal of both regulatory cell populations resulted in an increase in PFC that was greater than in cultures depleted of either NK or T suppressor cells.     

Characterization of an antigen-specific suppressive factor derived from a cloned suppressor effector T cell line

A cloned effector-type suppressor T cell line, 3D10, which is known to suppress the antibody response against dinitrophenylated keyhole limpet hemocyanin (KLH), produced a soluble KLH-specific factor (TsF) that can replace the function of parental T cell clones. High activity of TsF was released spontaneously into the culture supernatant when cultured in interleukin 2 (IL 2)-containing medium, requiring no antigenic stimulation. The culture supernatant of 3D10 was also capable of inhibiting the KLH-induced proliferative response of primed T cells in an antigen-specific manner. The direct target of TsF was found to be Lyt-1+2- T cells undergoing an early stage of antigen-specific proliferation. TsF was antigen binding but lacked any other serologic markers such as I-J and immunoglobulin heavy chain-linked allotypic determinants on T cells. No genetic restriction was found in its action on allogeneic T cells. The production of IL 2 in proliferative T cells by antigenic stimulation was not inhibited by TsF. These results indicate that the TsF described here is the legitimate mediator produced by the effector-type suppressor T cell that suppresses the antigen-specific responses of Lyt-1+2- T cells. The m.w. of TsF was approximately 75,000.     

Characterization of anti-idiotypic suppressor T cells (Tsid) induced after antigen priming

The induction and fine specificity of idiotype-specific suppressor T cells (Tsid) were studied. Spleen cells from C57BL/6 mice, immunized 4 wk previously with NP-KLH, failed to express NPb3 idiotype-bearing PFC when challenged in vitro with NP-Ficoll or NP-Brucella abortus. After treatment of NP-primed responder cultures with anti-Thy-1.2 anti-serum + C, NPb idiotype-bearing B cells could be detected. This B cell subset was preferentially suppressed by the addition of T cells from NP-primed mice. With this reconstitution protocol, it was determined that suppression of the NPb idiotype-bearing portion of the B cell response was mediated by a specifically induced T cell population (Tsid) that directly suppressed NPb-bearing B cells. As with a previously described suppressor population induced with hapten-modified syngeneic spleen cells (Ts2), the Tsid population bound and was lysed by NPb idiotype-bearing serum antibodies. However, the Tsid could be distinguished from the Ts2 population because it lacked I-J determinants and functioned as an effector T cell, not an intermediary suppressor cell. Furthermore, fine specificity studies with monoclonal NP-specific antibodies expressing various levels of serologically detectable NPb idiotypic determinants indicated that unlike the Ts2, the Tsid population reacts with conventional, serologically detected members of the NPb family. The combined idiotype binding studies for the Tsid and Ts2 populations demonstrate that the fine specificity of suppressor T cell populations reflects their independent mechanisms of regulation.     

Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation of this is that antigen-specific host suppressor T cells (Ts) are activated. Alternatively, donor Lyt-2+ T cells, introduced in the priming inoculum, may inactivate host CTL precursors (CTLp) that recognize the priming (donor) alloantigens. Donor cells that act in this way are termed veto T cells. The experiments described here exclude veto T cell participation in transferable alloantigen-specific suppression, and demonstrate the operation of an alloantigen-specific host-derived T suppressor (Ts) cell. The origin of the Ts has been studied directly by using Thy-1-disparate BALB/c mice. The cell responsible for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally non-cross-reactive-third party antigen (H-Y), provided the two antigens were expressed on the same cell membrane. Such third-party suppression is incompatible with the operation of veto T cells. Depletion of Thy-1.2+ or Lyt-2+ cells from the suppression-inducing donor SC inoculum did not abrogate suppression induction in BALB/c mice; instead, suppression was enhanced. The demonstration of veto cell activity in similarly primed mice by other groups of investigators indicates that both types of suppression may operate. However, our results show that only antigen-specific Ts can mediate the transferable suppression of CTL responses to alloantigens.     

Induction of T15-specific suppressor T cells in mice with the CBA/N defect by neonatal injection with anti-T15 idiotype antibody

Mice with the CBA/N defect (xid) are unresponsive to phosphorylcholine (PC), To determine whether idiotype-specific suppressor T cells can also be generated in these defective mice, defective (CBA/N X BALB/c)F1 male and nondefective (CBA/N X BALB/c)F1 female or (BALB/c X CBA/N)F1 male mice were neonatally injected with antibodies specific for the major idiotype of anti-PC antibody, i.e., anti-TEPC-15 idiotype (T15id) antibody. Suppressor cell activity was examined by co-culturing spleen cells from neonatally treated F1 mice with spleen cells of normal nondefective F1 mice in the presence of antigen. Spleen cells from defective (CBA/NM X BALB/c)F1 mice treated with anti-T15id antibody demonstrated a level of suppressor activity (greater than 83% suppression) comparable to that of similarly treated nondefective F1 mice. This suppression was specific for the T15id of anti-PC response, and a Lyt-1-2+-bearing T cell population appeared to be responsible for the active suppression. These suppressor T cells recognized T15 but not PC, based on a functional absorption test. These results indicate that the CBA/N defects, including the deficiency in the anti-PC response by B lymphocytes and a possible T cell defect, do not influence the generation of T15id-specific suppressor T cells by neonatal injection with anti-T15id antibody.     

Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells

Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.     

Role of lipomodulin, a phospholipase inhibitory protein, in immunoregulation by thymocytes

The treatment of murine thymocytes with anti-lipomodulin antibody during Con A stimulation causes selective loss of suppressor activity, but not of helper activity on PFC assay, when co-cultured with T cell-depleted spleen cells. Interaction of the antibody with responder cells in thymocyte culture were necessary in the early stage rather than in the later stage of lymphocyte activation by Con A, which suggests that anti-lipomodulin antibody acts in the stage of suppressor T cells generation. When thymocytes were cultured with purified lipomodulin for 48 hr, suppressor activity was induced. Lipomodulin as detected by radioimmunoassay was found to be released from T cells with the phenotype of I-J+, Lyt-1-, Lyt-2+. The immunoprecipitates from the media of Con A-stimulated thymocyte with anti-I-Kk antibody and anti-lipomodulin antibody were analyzed on SDS-gel electrophoresis. I-J products had m.w. 36,000 and 24,000, whereas lipomodulin had m.w. 36,000, 24,000, and 15,000. Because anti-I-Jk antibody could precipitate 125I-labeled lipomodulin purified from rabbit neutrophils, these results suggest that lipomodulin is a product of I-J genes that induces suppressor T cells.