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.     

Analysis of hapten-specific T suppressor factors: genetic restriction of TsF1 activity analyzed with synthetic hybrid suppressor molecules

We have analyzed the first-order suppressor factor secreted by an azobenzenearsonate (ABA)-specific T suppressor cell (Ts) hybridoma. Treatment of the factor with 5 mM dithiothreitol (DTT) yields two fragments with distinct phenotypes and functional capabilities. One fragment is bound by a monoclonal anti-I-J antibody, the other is not. Further, although neither molecular fragment by itself is sufficient to suppress an ABA response, a mixture of the two reconstitutes the suppressive activity. The I-J- portion of the first-order suppressor factor (TsF1) presumably guides the antigen specificity; activity of the ABA-specific Ts I-J- TsF1 factor can be reconstituted with an I-J+ subunit of a TsF molecule of either sheep red blood cell (SRBC) or ABA specificity. The genetic restriction for Igh-linked determinants of the ABA/SRBC hybrid TsF molecules is influenced by the I-J+ portion, regardless of the original antigen specificity of that molecule. The data support a two-subunit TsF model. Polyclonal ABA-specific TsF1 molecules appear to resemble the monoclonal factor in structure.     

Information transfer between T cell subsets is directed by I-J+ antigen nonspecific molecules

T cell antigen-specific suppressor factors (TsF) consist of two distinct polypeptide chains: one that binds antigen (ABM) and one that bears I-J region markers (I-J+ chain). We studied the functional role of these two molecules in delivering the biologic message of suppression to its appropriate target cell. Two different biologically active TsF were used in these studies: TsiF, a T suppressor-inducer factor consisting of an ABM secreted by Ly-1 T cells (Ti-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Ji), which initiates the suppressor circuit by inducing an Ly-1,2 T cell; and TseF, a T suppressor-effector factor consisting of an ABM secreted by Ly-2 T cells (Te-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Je), which delivers the biologic message of suppression to the T helper (TH) cell. In both TsF, the ABM and I-J+ chain are noncovalently associated and can be easily separated. Both molecules must be present, however, for biologic activity of the TsF to be manifest. We studied the role of each chain in delivering these biologically active messages by constructing "hybrid" factors made from mixing the ABM from TsiF with I-J+ chains from either TsiF or TseF and determined which of these chains could reconstitute functional TsiF activity. Likewise, we mixed the AMB from TseF with I-J+ chains of TsiF or TseF to determine which I-J+ chain could reconstitute TseF activity. We found that I-J+ chain from TsiF (I-Ji) can reconstitute ABM from TsiF to form a functional TsiF capable of inducing suppression but cannot reconstitute ABM from TseF to form a functional TsiF capable of suppressing the activity of TH cells. Likewise, the addition of I-J+ chain from TseF to ABM from TseF can reconstitute its ability to suppress TH responses, but I-J+ chain from TsiF plus ABM from TseF has no effect on these TH cell responses. We did find, however, that this hybrid TsF composed of the ABM from TseF and the I-J+ chain from TsiF is capable of suppressing the Ly-1,2 Ttrans cell, the cell normally induced by the ABM + I-J+ suppressor inducer complex from T suppressor-inducer cells (TsiF).(ABSTRACT TRUNCATED AT 400 WORDS)     

Alloreactive cloned T cell lines. VII. Comparison of the kinetics of IL 2 release stimulated by alloantigen or Con A

Injection of poly(Glu50Tyr50)(GT) into B10.BR (H-2k) mice induces GT-specific suppressor T cells and a T cell-derived suppressor factor (TsF1), which in turn induces a second-order suppressor T cell (TS2). In the present study, we show that B10.BR GT-TSF1 is composed of separate I-Jk and idiotype-bearing chains linked by disulfide bond(s). Functional suppressive activity requires both chains to be in association. Neither chain alone can induce TS2, indicating that both chains must be seen in association and suggesting a single cellular target for the two chains. Experiments designed to interchange I-J-bearing chains of GT-TSF1 derived from different H-2 haplotypes indicate that only the homologous I-J and idiotype-bearing chains can reassociate into a suppressive moiety. These experiments may imply heterogeneity of I-J region gene products.     

A genetically restricted suppressor factor that requires interaction with two distinct targets

We have previously described a genetically restricted suppressor factor (TsF3) that suppresses the terminal phases of the contact sensitivity response. The activity of TsF3 is restricted by genes in the H-2 (I-J) and Igh complexes. This report analyzes the mechanisms responsible for these genetic restrictions. One cellular target of TsF3 is an I-J-bearing antigen-presenting cell population that is sensitive to low doses of cyclophosphamide. To elicit suppression I-J homology is required between this antigen-presenting cell population and the TsF3 donor. In contrast, the Igh-linked genetic restriction exists between TsF3 and an unprimed cell population present in the recipient. These findings suggest that under these experimental conditions TsF3 acts by bridging the APC with cells of the host. Finally, we demonstrated that nonspecific bystander or cognate suppression can be mediated by TsF3, provided specific antigen is present in the site of the ongoing T cell response.     

Mechanism controlling the genetic restrictions of an NP-specific suppressor factor that inhibits B cell responses

The mechanism of B cell suppression by a T cell hybridoma-derived monoclonal effector suppressor factor (TsF3) was studied in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. The NP-specific effector suppressor cells that produce TsF3 are Lyt-1-, 2+, I-J+, NP-binding T cells and are induced by immunization with NP conjugates. Monoclonal TsF3 inhibits both T cell activity as measured by suppression of contact sensitivity responses and B cell function as measured by suppression of antibody production to both T-independent and T-dependent antigens. The present studies were designed to specifically investigate the mechanisms and genetic restrictions that govern the interactions between TsF3 and its target cells in the plaque-forming cell (PFC) response. The results show that the target of TsF3 is a splenic adherent cell. Suppression will occur only if the restriction specificity of the TsF3 matches the H-2 genotype of the adherent population. Once this TsF3-adherent cell interaction has occurred, suppression of NP-specific B cells can occur across an H-2 barrier. The data also demonstrate that Igh-linked gene products do not appear to play a part in the TsF3-mediated suppression of in vitro PFC responses, which contrasts with the requirements for regulation of T cell-mediated contact sensitivity responses.     

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.     

T suppressor efferent circuit which affects contact sensitivity to picryl chloride: the late-acting, second nonspecific T suppressor factor bears I-A determinants which are responsible for the I-A genetic restriction in its interaction with its target cell

The T suppressor efferent circuit in the picryl (TNP) system, which inhibits the passive transfer of contact sensitivity, involves at least two antigen-nonspecific factors. The second nonspecific T suppressor factor (ns-2) bears I-A determinants of both the alpha and the beta chain as shown by affinity chromatography on immobilized anti-I-A monoclonal antibodies. Sequential absorption shows that the determinants of the alpha and beta chain occur on the same molecular complex. No absorption was obtained with anti-I-E antibody. There are two genetic restrictions associated with ns-2--the first is in its release from the second T suppressor efferent cell (on exposure to antigen) and the second is in its inhibitory interaction with its target cell. Both are MHC restricted and matching in I-A (but not I-E, or I-J) is sufficient. The question was asked whether the I-A of the ns-2 was directly responsible for the I-A genetic restriction in its action. F1 TsF was made in (H-2k X H-2b)F1 mice by injecting picrylated parental cells intravenously and triggering the release of ns-2 with the corresponding picrylated parental cells. Both I-Ak- and I-Ab-positive ns-2 were produced and were separated by affinity chromatography on immobilized anti-I-A monoclonal antibody. The I-A phenotype of these separated ns-2 of F1 origin determines the genetic restriction in their action; i.e., I-Ak+ ns-2 only inhibits passive transfer by H-2k cells and I-Ab+ ns-2 only acts on H-2b cells. In contrast, the I-A haplotype of the picrylated cell used to induce the Ts cell which makes ns-2 is unimportant. It was concluded that the I-A on the ns-2, and not a possible recognition site for I-A, serves as a restriction element. This finding suggests that ns-2 may act directly on the I-A-restricted T cell which mediates contact sensitivity.     

The molecular basis of granuloma formation in schistosomiasis. IV. T cell-derived suppressor-inducer and suppressor-effector factor reactivities are regulated by a TCR beta chain analog

In Schistosomiasis mansoni, granulomatous modulation is mediated by antigenically and genetically restricted T suppressor-inducer and suppressor-effector cells and the soluble factors which they produce. The T suppressor-inducer factor (TsiF) is produced by an L3T4+, 14-30+ T cell. TsiF does not suppress directly, but induces the production of T-cell-derived suppressor-effector factor (TseF). TseF directly suppresses granuloma formation in vitro and in vivo. This study describes the molecular properties of TsiF. The factor is a nonimmunoglobulin heterodimer which can be separated into two component chains by dithiothreitol (DTT) reduction. The alpha chain imparts antigenic specificity and bears both the AgR and the epitope recognized by mAb 14-30 which characterizes T cells and factors of the Tsi phenotype. The beta chain imparts genetic restriction and bears both the I-J phenotypic marker and a T-cell receptor for Ag (TCR) V beta 8 determinant. These two chains can complement each other in vitro to reconstitute functional activity. The beta chain also determines the functional activity of T cell-derived suppressor factor (TsF). A beta chain, derived from TsiF, can complement the alpha chain derived from TsiF or TseF to reconstitute TsiF, but not TseF functional activity. Conversely the beta chain of TseF can reconstitute only TseF activity. These findings suggest that TsiF bears structural homologies to the TCR borne by Tsi cells and that the beta chain mediates the mode of functional interactions between TsFs and their target cells.     

TMA-specific first-order T-suppressor hybridoma. I. Characterization of the hybridoma-derived single-chain inducer suppressor factor, TsF1

Experiments described in this report will characterize a monoclonal phenyltrimethylammonium (TMA) specific, first-order T-suppressor factor (TsF1) produced by a T-cell hybridoma, 8A.3. The hybridoma expressed the Thy-1, Lyt-1, Lyt-2 antigens as well as cross-reactive idiotypic (CRI) determinants but did not express I-J encoded epitopes. It was also found to bear determinants recognized by a monoclonal antibody raised against single-chain GAT-specific TsF1. The hybridoma-derived factor was capable of suppressing primary in vitro trinitrophenol (TNP)-specific responses induced with the Brucella abortus antigen, conjugated with TMA and TNP haptens (TMA-BA-TNP). In addition, in vivo administration of 8A.3 culture supernatant resulted in the specific suppression of TMA-specific delayed-type hypersensitivity (DTH) responses. Analysis of this factor revealed it to be an induction-phase, antigen-binding, CRI+, and I-J+ single chain polypeptide. Our results represent only the second such described single chain, antigen binding, I-J+ suppressor factor derived from a monoclonal T-cell hybridoma.     

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.     

Structural analyses of a monoclonal heterodimeric suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10

A GAT-specific, MHC-restricted "second-order" suppressor T cell factor (TsF2) from the hybridoma 762 B3.7 was biosynthetically radiolabeled with 35S-methionine and was isolated from cell extracts. The isolation procedure involved two-dimensional nonreducing/reducing SDS-PAGE and electroelution of the reduced off-diagonal polypeptide chains from the gel. Biochemical characterization studies revealed that TsF2 is a disulfide-linked heterodimer composed of a basic and an acidic polypeptide chain, both having m.w. of 30,000. Both chains are glycosylated and contain sialic acid residues. The basic polypeptide reacts with anti-I-J antisera, whereas the acidic chain contains the antigen-binding capacity. Monoclonal antibodies induced by immunizing rats with TsF2 purified from hybridoma supernatants were selected for the ability to block immunosuppression mediated by TsF2 in vitro. These antibodies, but not irrelevant antibodies, immunoprecipitated the 35S-methionine-labeled protein that migrates off the diagonal in two-dimensional gels. Thus, we have verified that the immunosuppressive protein that migrates off the diagonal in two-dimensional gels binds to antibodies that are known to inhibit the biologic activity of unpurified TsF2.     

Novel mechanisms of specific suppression of anti-hapten antibody response mediated by monoclonal anti-carrier antibody

The cellular mechanisms of the antibody-induced suppression of immune responses were analyzed in the keyhole limpet hemocyanin (KLH) system. Some of the monoclonal anti-KLH antibodies, like KLH-specific suppressor T cell factor (KLH-TsF), were demonstrated to suppress the anti-2,4-dinitrophenyl IgG but not IgM plaque-forming cell responses in a KLH-specific and H-2-restricted manner. The anti-KLH antibodies with suppressive activity reacted with, and in turn, stimulated the suppressor hybridoma (34S-281) with the anti-idiotypic receptor complementary to the idiotypic KLH-TsF of the inducer type. Moreover, because the suppressive activity of the anti-KLH antibody was completely abolished by the treatment of responding spleen cells with anti-Lyt-2 and complement, it was apparent that the suppressive antibody activated suppressor T cell pathways. The isotype or affinity of antibodies is not related to the suppressive activity, because suppressive and nonsuppressive antibodies possess a similar affinity belonging to the same Ig isotypes. It also has been demonstrated that the Fc portion is not the functional site, because the F(ab')2 fragment still has the activity. The antibody specificity is found to be important for determining whether the antibody is suppressive or not. In fact, anti-KLH 26, but not other antibodies without activity, recognizes the particular KLH epitope seen by KLH-TsF, and exclusively interacts with the anti-idiotypic suppressor T cells. Thus, the anti-idiotypic suppressor T cell receives signals both from the suppressive anti-KLH antibody and from KLH-TsF, and transmits the antibody-induced suppressor signals to the effector-suppressor pathway. The size of the repertoire of anti-idiotypic suppressor T cells involved in the suppression seems to be very limited, because only four out of 120 monoclonal anti-KLH antibodies were found to have suppressor activity. The possible mechanisms of the cell interaction mediated by the suppressive antibody are discussed.     

Functional analysis of cloned macrophage hybridomas. V. Induction of suppressor T cell responses

It has been suggested that macrophage-like accessory cells are involved in suppressor T cell (Ts) induction. To further analyze this issue, we obtained several cloned macrophage hybridoma cell lines by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serological and functional characteristics of macrophages. We evaluated the ability of these hybridomas to induce third order or effector Ts (Ts3) to suppress the contact sensitivity response against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). In contrast to the parental P388D1 and two other macrophage hybridomas, one macrophage hybridoma clone, termed 63, when conjugated with NP, induced Ts3, which suppressed contact sensitivity responses against NP but not DNFB, showing that the Ts3 were antigen specific. Macrophage hybridoma 63 could specifically induce Ts3 activity in either H-2k, H-2d, or H-2k/H-2d heterozygous hosts. Thus, macrophage hybridoma 63 functionally expressed major histocompatibility complex-related restricting determinants, and the fusion with cells from a H-2k macrophage donor caused a functional complementation of H-2d-related, Ts-inducing elements. The genetic restriction governing induction of Ts3 was controlled by genes that mapped to I-J region. Furthermore, NP-conjugated macrophage hybridoma 63 could serve as a target for elicitation of suppressor responses after administration of I-Jk, but not I-Jb, restricted suppressor factor. The data suggest that macrophage hybridomas represent a means to dissect heterogeneity within the macrophage population. The data also imply that the I-J determinants expressed on macrophages represent a ligand for the antigen receptor of Ts.     

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.     

A novel suppressive activity: complementation between a T cell induced with first-order T-suppressor factor and an I-J-restricted antigen-nonspecific T cell

Previous studies demonstrated that the first-order T-suppressor factor (TsF1) requires the presence of antigen to induce idiotype-specific Ts cells which readily suppress phenyltrimethylamino (TMA) hapten-specific delayed-type hypersensitivity (DTH) responses when transferred into already immune recipients. In this study we show that TsF1 in the absence of antigen induces a splenic population which limits DTH in recipient mice only when an additional accessory lymphoid population was also cotransferred. Neither of these populations alone was sufficient to mediate suppression and depletion of T cells in either population's abrogated suppression, indicating the T-cell dependency of the complementing cell types. Moreover, suppression was seen only when TMA-TsF1-induced and not normal spleen cell lysate-induced cells were cotransferred with the antigen-induced population, suggesting the requirement for a specific signal to induce the factor-induced population. Further experiments showed that the antigen-induced lymphoid population could be replaced by either heterologous antigen-induced or adjuvant alone-induced splenic populations, indicating the lack of specificity of this secondary population. Further analysis showed that the cell complementation between TMA-TsF1-induced and the nonspecific accessory lymphoid population resulted in antigen-specific and genetically restricted immune suppression. The TsF1-induced lymphoid population was not responsible for the genetic restriction, and furthermore, there was no restriction observed between the two complementing populations. However, matching of the nonspecific accessory cell with the recipient host at the I-J subregion of the H-2 complex was essential for immune suppression. Finally, the activity of complementing cells was found to be independent of cyclophosphamide-sensitive Ts populations of the recipient mice. The ramifications of these findings with reference to the existing suppressor pathways are discussed.     

Expression of cell surface antigens by suppressor T cell hybridomas. I. Comparison of phenotype and function

The phenotypic expression of cell surface markers by T cell hybridomas that elaborate suppressor factors specific for the polymers L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) or L-glutamic acid50-L-tyrosine50 (GT) has been analyzed. We found that determinants encoded by the I-J subregion of the H-2 complex were borne on the surface of these hybrid cells and on the factors they secrete, whereas I-J determinants were not expressed by the AKR thymoma fusion parent, BW5147. The level of expression of I-J determinants fluctuated widely depending upon culture conditions, but I-J products and other cell surface markers of normal T cells could be quantitatively increased, or induced to appear, by treatment of the hybridomas with chemical agents, such as dimethyl sulfoxide (DMSO) or phorbol myristate acetate (PMA). In contrast, the surface expression of the viral product gp70 was decreased by the same treatment. Using chemical induction, we typed BW5147, a group of antigen-specific suppressor T cell hybridomas, and two control hybridomas for expression of I-J, Thy-1, Lyt, and H-2K alloantigens. Also, a haplotype-specific hybridoma that produces an antigen-nonspecific factor was analyzed. The results demonstrated that BW5147 failed to express I-J or Lyt alloantigens but expressed Thy-1.1 and H-2Kk gene products. The pattern of expression of these antigens by T cell hybridomas was very complex, but three conclusions could be drawn: 1) Good correlation exists between the expression of certain I-J determinants and the ability of T cell hybridomas to produce suppressor factor. 2) The expression of Thy-1, Lyt, or H-2Kk determinants is variable, and no correlation was found between expression of these antigens and the ability to produce active suppressor factors. 3) I-Jk products contributed by the AKR thymoma fusion partner are expressed by T cell hybridomas.     

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.     

Mechanism of action of a T suppressor factor (TsF) in contact sensitivity: the T cell target for TsF activity in adoptive transfer of immunity is not effector cell

The passive transfer of contact sensitivity (CS) by immune cells into normal animals requires the interaction of two distinct Ly-1+ T cells, one which is Vicia villosa lectin (VV)-nonadherent, the other which adheres to VV. Functional deletion of either cell type abrogates the adoptive transfer of CS into normal animals, whereas VV-nonadherent cells alone can transfer CS into animals pretreated with cyclophosphamide (Cy). An antigen-specific T suppressor factor, designated TNP-TsF, inhibits the transfer of CS into normal adoptive recipients. TNP-TsF mediates its suppressive activity by inducing an I-J+ subfactor (designated I-J2) from the assay population by the interaction of PC1-F (a TNP-binding subfactor of TNP-TsF) with antigen-primed Ly-2+ T cells. This I-J+ subfactor then complements TNBS-F (an antigen-nonbinding subfactor of TNP-TsF) to form an antigen-nonspecific effector molecule which suppresses DTH responses in an antigen-nonspecific fashion. We report here that TNP-TsF suppresses the adoptive transfer of CS into normal animals but not into animals pretreated with Cy. TNBS-F + I-J2, the effector complex of TNP-TsF, also suppresses the transfer of CS into normal but not Cy-treated animals. When the Ly-1 immune cells were separated into VV-adherent and -nonadherent populations, the TNBS-F + I-J2 suppressor complex suppressed the functional activity of the VV-adherent cell population, but not the VV-nonadherent cells. This suppressive activity correlates with the need for VV-adherent cells in the transfer of CS into normal but not Cy-treated recipients. When an I-J+ molecule (I-J1) from an SRBC-specific TsF was used in place of I-J2 to form a suppressor complex with TNBS-F, this TNBS-F + I-J1 TsF suppressed the transfer of CS into both normal and Cy-treated recipients. This difference in functional suppressive activity correlated with a difference in target cell specificity: TNBS-F + I-J1 suppressed the VV-nonadherent TDTH cell, whereas TNBS-F + I-J2 suppressed the VV-adherent T cell of CS. Immune cells which are transferred under conditions which do not require the VV-adherent cell for transfer are not suppressed by TNBS-F + I-J2 or TNP-TsF, but are suppressed by the TNBS-F + I-J1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional equivalence of cryptococcal and haptene-specific T suppressor factor (TsF). I. Picryl and oxazolone-specific TsF, which inhibit transfer of contact sensitivity, also inhibit phagocytosis by a subset of macrophages.

Monoclonal and conventional cryptococcal-specific T suppressor factors (TsF) (also called TsFmp) depress phagocytosis by a subset of macrophages, while picryl- and oxazolone-specific TsF depress the passive transfer of contact sensitivity. This paper shows that these haptene-specific TsF also inhibit phagocytosis by a subset of macrophages and, using this assay, that the anti-haptene TsF resemble the anti-cryptococcal TsF in five respects: (i) the need for reexposure to specific antigen to trigger the release of TsF; (ii) genetic restriction in action; (iii) possession of an antigen-binding site; (iv) expression of I-J determinants; and (v) inactivation by reduction and alkylation. Purification of the anti-picryl TsF by sequential affinity chromatography indicates that the inhibition of phagocytosis is due to the TsF itself and not to a TsF-antigen complex. The TsF inhibits phagocytosis by a direct action as macrophages treated with TsF and exposed to antigen do not release a second factor which inhibits phagocytosis. These results and those of the accompanying paper indicate that the anti-cryptococcal and anti-haptene TsF are functionally equivalent, antigen-specific suppressor factors.