T-suppressor Factor Research Articles

From Mysterious Supernatant Entity to miRNA-150 in Antigen-Specific Exosomes: a History of Hapten-Specific T Suppressor Factor.

Soon after the discovery of T suppressor cells by Gershon in 1970, it was demonstrated that one subpopulation of these lymphocytes induced by i.v. hapten injection suppresses contact sensitivity response mediated by effector CD4+ or CD8+ T cells in mice through the release of soluble T suppressor factor (TsF) that acts antigen specifically. Our experiments showed that biologically active TsF is a complex entity consisting of two subfactors, one antigen specific and other non-specific, produced by differently induced populations of cells. In following years, we found that the antigen-specific subfactor is a light chain of IgM antibody that is produced by B1a lymphocytes. However, the exact nature of non-specific part remained a mystery for about 30 years. Our current studies characterized TsF as regulatory miRNA-150 carried by T suppressor cell-derived exosomes that are antigen specific due to a surface coat of IgM antibody light chains produced by B1a cells. The present communication briefly summarizes our studies on TsF that led to discovery of regulating miRNA that acts antigen specifically to suppress immune response.

Biochemical Basis of Antigen-Specific Suppressor T Cell Factors: Controversies and Possible Answers

The chapter reviews previous observations made on T-suppressor factor (TsF) and provides interpretations for controversial issues on this factor. Antigen-specific T cell-mediated immunosuppression could be transferred from one animal to other syngeneic animals either with intact suppressor cells or with soluble factors derived from the cells. The chapter also discusses the experiments that provide the evidence that OVA-specific glycosylation inhibiting factor (GIF), which is an effector type TsF, consists of the 55-kDa TCRa chain-GIF conjugate and the TCRP chain. After the identification of T cell receptors (TCR) on helper and cytotoxic T cells, however, fundamental questions can be raised on T cell-mediated suppression with the failure to identify TCR and TCR gene rearrangement in representative Ts hybridomas. In addition, the ability of Ts cells and antigen-specific TsF to bind specifically to nominal antigen, independently of major histocompatibility complex (MHC) gene products, created skepticism on the nature of Ts cells and the origin of TsF.

Cryptococcal capsular polysaccharide utilizes an antigen-presenting cell to induce a T-suppressor cell to secrete TsF.

A T-T hybridoma (F6.6.2) which secretes a T-suppressor factor (TsF) specific for cryptococcal capsular polysaccharide (glucuronoxylomannan, GXM) was tested to determine if antigen-presenting cells (APC) were necessary for activation of the hybridoma to secrete TsF. Normal, syngeneic spleen cells were required along with GXM before TsF could be detected in culture supernatants. Ts cells did not secrete TsF unless the APC were obtained from mice which were identical at the "so-called' I-J sublocus as defined by the difference between B10.A(3R) and B10.A(5R) mice. The APC was adherent and could be depleted from spleen cell suspensions by treatment with anti-I-J and complement but not anti-I-A and complement. Additionally, treatment with anti-T cell serum or anti-immunoglobulin and complement did not remove the APC function of the spleen cell population. A role for I-E antigens in the function of the APC was determined by blocking antigen presentation to the suppressor cell with anti-I-E antibodies. The polysaccharide was associated with splenic adherent cells as extensive washing of the APC after incubation with GXM did not eliminate the antigen presenting function of the population.

Bromocriptine Immunomodulation of Experimental SLE and Primary Anti-phospholipid Syndrome via Induction of Nonspecific T Suppressor Cells

Bromocriptine (BRC) is a dopamine agonist that suppresses the secretion of prolactin by the pituitary gland and is known to have immunomodulating properties. In the present study, we investigated the effect of BRC on the development of two autoimmune experimental models: (i) systemic lupus erythematosus (SLE), induced by injection of a human anti-dsDNA monoclonal antibody (MIV-7); (ii) primary anti-phospholipid syndrome (PAPS), induced by injection of a mouse anti-cardiolipin monoclonal antibody (CAM). BRC had a suppressive effect on in vivo autoantibody production, as well as on the appearance of other manifestations of the respective disease. The BRC activity seems to be nonspecific, since the same effect was demonstrated in mice with lupus and with PAPS. These data were supported by the in vitro nonspecific effect of CD8 cells induced in vivo by bromocriptine, on specific lymph node cell proliferation in the presence of the pathogenic monoclonal antibodies (MIV-7 or CAM, respectively), and on the response to an irrelevant autoantigen, myelin basic protein. These data were complemented by the nonspecific suppressive effect by the T-suppressor factor, produced by the CD8 cells, on specific thymidine uptake by lymph node cells from experimental SLE or PAPS in the presence of the immunizing mAb. Injection of CD8 cells from BRC-treated mice with SLE or PAPS abolished the disease development in the lupus and PAPS experimental models. The data suggest a possible novel role of bromocriptine in downregulating autoimmune phenomena through induction of natural nonspecific CD8 1 suppressor cells.

A nonspecific inhibitor of contact sensitivity elaborated by macrophages: Genetic restriction in its production but not in its action

It is known that macrophages armed with haptene-specific T suppressor factor (TsF) and then exposed to antigen (haptenized spleen cells) liberate a nonspecific inhibitor of the transfer of contact sensitivity (CS). This is called macrophage suppressor factor (MSF). This paper shows that MSF is only released when the source of the TsF and the haptenized spleen cells share the same I-J subregion. This is based on the comparison of B10.A(3R) and B10.A(5R) mice. In contrast, the action of MSF is antigen nonspecific and genetically unrestricted. In these respects it resembles the antigen-nonspecific inhibitor (nsTsF-1) made by the T acceptor cell when armed with TsF. However, it differs from nsTsF-1 in acting directly on the I-A − population which transfers contact sensitivity and not indirectly via I-A + T cells. In vitro, MSF fails to inhibit the proliferative response of lymph node cells to specific antigen and their production of IL-3 activity, IFN- t , and IL-2. This indicates that MSF is not a global inhibitor of T cell activity. The finding that MSF inhibits systemic passive transfer of contact sensitivity, but has no effect on local passive transfer strongly supports the view that MSF affects the arrival of certain cells critical for the development of the reaction to the skin challenge site.

Functional equivalence of cryptococcal and haptene-specific T suppressor factor (TsF): 1. 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 antihaptene 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 TsFare functionally equivalent, antigen-specific suppressor factors.

Functional equivalence of cryptococcal and haptene-specific T suppressor factor (TsF): II. Monoclonal Anti-cryptococcal TsF Inhibits both Phagocytosis by a Subset of Macrophages and Transfer of Contact Sensitivity

Monoclonal anti-cryptococcal TsF (which inhibits phagocytosis by macrophages) and antipicryl TsF use the same two circuits to block the transfer of contact sensitivity (CS). Both arm macrophages which then release a macrophage suppressor factor (MSF) when exposed to antigen. This MSF depresses the transfer of CS. The evidence suggests that a single molecular species of TsF (MW ca. 70 kDa), which bears an antigen-binding site and I-J determinant, is responsible for MSF production and inhibition of phagocytosis. Anti-cryptococcal TsF also arms the T acceptor cell which then releases nsTsF-1 after triggering with a specific antigen (SCPA). This nsTsF-1, which depresses the transfer of contact sensitivity, was authentic, as shown by its I-J positivity (in contrast to MSF) and its role in the production of nsTsF-2. As anti-picryl TsF also inhibits phagocytosis, it was concluded that anti-cryptococcal TsF, originally detected by the inhibition of phagocytosis, and anti-picryl TsF, originally detected by inhibition of CS, are functionally equivalent.

Serologic, biologic, and western blot analysis of a T suppressor factor with specificity for the hapten 4-hydroxy-3-nitrophenyl acetyl derived from serum-free medium.

A T cell hybridoma producing a T suppressor factor (TsF) with specificity for the hapten nitrophenyl was converted to long term growth in serum-free medium and its product tested by serology, bioactivity, and Western blot analysis. Results indicated that Ag-specific suppressive activity was present in serum-free medium and this TsF could exhibit the characteristics ascribed to it by various groups: it could bind nominal Ag with specificity, it was bound by anti-TsF mAb, and it could mediate Ag-specific suppression both in vivo and in vitro. Western blot and SDS-PAGE analysis of this purified TsF revealed a 43-kDa single chain protein.

Mechanism of hapten-specific T-suppressor factor inhibition of hapten-modified proliferation of human and mouse leukemia cells

Mechanism of hapten-specific T-suppressor factor inhibition of hapten-modified proliferation of human and mouse leukemia cells

Preadministration of a T-suppressor factor enhances tumor immunity in DBA/2 mice.

Previously we have described the isolation and characterization of a T-suppressor factor (TsF) from a T cell hybridoma (A10F), which has a degree of specificity for the DBA/2 mastocytoma P815. Administration of A10F intravenously at the time of tumor cell injection resulted in an accelerated rate of tumor growth, decreased cytotoxic T lymphocyte antitumor activity, and reduced survival time. In the work reported here, we have shown that administration of affinity-enriched A10F 7-14 days prior to tumor cell injection causes what appear to be reverse effects, in that an enhanced resistance to the P815 tumor is observed in vivo, an effect which we can correlate with the demonstration of antitumor cytotoxic T lymphocyte activity in vitro. These effects are dose-dependent since only doses of TsF at 20 micrograms or greater are effective. A similar effect was found when A10F was administered to DBA/2 mice 10 days prior to challenge with two unrelated tumors (L1210 and M-1). However, when another TsF (Fd11F) with apparent specificity for a nominal antigen was tested in this system, it had no effect on tumor growth.

Isolation and Characterization of A T-Suppressor Factor Specific for the Lupus-Associated Autoantigen RNP-Sm

We have previously described a monoclonal antibody, B16G, which has been found to be specific for T-cell derived suppressor factors (TsF). B16G has been shown to react with T-suppressor cells, TsF in the spleen of normal or tumor-bearing mice, the TsF produced by tumour-specific, or hapten-specific T-cell hybridomas, and with polyclonal whole human TsF isolated from tonsillar tissue. This pan-reactivity inherent to the B16G antibody suggests that it recognizes some common, shared epitope of the TsF molecule. In this study, we have used B16G as a probe to isolate a TsF-producing T-cell hybridoma, S-50, from CBA mice (H-2k) that is specific for the Lupus-associated antigen, RNP-Sm. The TsF bound specifically to RNP-Sm and inhibited the production of anti-RNP-Sm antibody cell cultures from MRL-lpr mice. SDS-PAGE analysis of purified S-50 TsF revealed a B16G-reactive band with a molecular weight of 43 kd.

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

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

Infectious and noninfectious tolerance are blocked by a monoclonal antibody to T-suppressor factor

Two forms of hapten-specific unresponsiveness have been demonstrated following intravenous (iv) injection of hapten-conjugated syngeneic spleen cell based on the nature of the antigen-presenting cell (APC): I-J +, I-A − APC have been shown to induce T-suppressor cells (Ts cells) which are demonstrated upon adoptive transfer, while I-J −, I-A + APC induce a nontransferable tolerance. In this paper we report that a monoclonal antibody specific for T-suppressor effector cells and factors (14-12) can block the Ts cells induced by I-J +, I-A − APCs and the tolerance induced by I-J −, I-A + APCs. In addition, it sufficiently overcomes suppression such that injection of TNP-spl iv induces immunity rather than suppression. We show that the I-A +, I-J − TNP-spl, which induce nontransferable tolerance upon iv injection, are the cells which induce immunity in 14-12-treated recipients. These results demonstrate that injection of I-J −, I-A + APC does not lead to clonal deletion and the tolerance induced by the iv injection of both I-J +, I-A − and I-J −, I-A + APC Operate via Ts Cells.

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

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

Isolation of an antigen-specific T suppressor factor that suppresses the in vivo response of DBA/2 mice to ferredoxin.

A T cell hybridoma (Fd11) has been produced from B10.D2 mice that secretes a putative antigen-specific T suppressor factor (TsF). The TsF is isolable from culture supernatants of Fd11 by affinity purification over columns containing either a monoclonal antibody (B16G) shown previously to be capable of binding murine TsF or ferredoxin (Fd), the nominal antigen to which the Fd11 TsF binds. Specificity of the Fd11 TsF for Fd was established by comparing it to another TsF isolated by us (A10 TsF) in a sandwich ELISA, and by demonstrating the specific reactivity to Fd of the hybridoma in calcium flux studies. The Fd11 affinity-purified TsF was shown to contain two major unique components with m.w. in the region of 80,000 and 35,000 when run on reducing polyacrylamide gels in the presence of sodium dodecyl sulphate. Specific immunosuppressive properties of Fd11 were demonstrated when Fd11 TsF (10 micrograms) was injected i.v. into Fd-primed syngeneic mice at the time of antigen boost. Fd11 TsF specifically and significantly diminished the secondary antibody response to Fd in DBA/2 mice.

GENERAL CONSIDERATIONS IN THE INTERPRETATION OF I‐J GENETIC RESTRICTIONS: EVIDENCE THAT THE ANTIGEN‐BINDING CHAIN OF ANTIGEN‐SPECIFIC T‐SUPPRESSOR FACTOR HAS TWO RECOGNITION SITES FOR MEMBERS OF THE I‐J HIERARCHY

(CBA X B10)F1 [(H-2k X H-2b)] mice produce two types of antigen-specific T-suppressor factor (TsF), which can be separated by affinity chromatography on anti-I-J monoclonal antibody. After reduction and alkylation, both chains of F1 TsF are required for biological activity. However, the antigen-binding chain (AgBC) of F1 TgFk (AgBCk) is only complemented by I-Jk and likewise for F1 TsFb. In other words, interchain complementation shows the same genetic restriction in interchain complementation in parental and F1 mice. F1 TsF bearing, for example, I-Jk (TsFk), interacts with haptenized 'antigen-presenting cells' ('APC') and parental I-Jk chains interacts with haptenized 'APC' of both parental haplotypes (dual reactivity). In contrast, the combination of parental AgBCb and F1 I-Jb shows single reactivity and only interacts with haptenized 'APCb'. It was inferred that the antigen-binding chain is responsible for the dual reactivity and, hence, for the interaction with a member of the I-J hierarchy on the 'APC'. It was concluded that the antigen-binding chain of TsF has two recognition sites for members of the I-J hierarchy--one for interchain complementation and the other for interaction with the haptenized 'antigen-presenting cell'. The term member of the I-J hierarchy is used instead of I-J because it is not clear whether the AgBC interacts with I-J or a receptor for I-J, or indeed a receptor for that receptor.

Characterization of an anti-idiotypic T cell hybridoma involved in the regulation of the immune response to the P815 mastocytoma.

We report the isolation and characterization of a T cell hybridoma (A29) which secretes a factor that exhibits anti-idiotypic and immune-modulating characteristics. The A29 cell line is thought to represent the hybrid analog of the Ts2 suppressor cell population in the cascade regulating the immune response to the P815 tumor in DBA/2 mice. The putative TsF2 molecule is reactive with the monoclonal antibody B16G, shown previously by us to bind a public specificity of T suppressor factors (TsF). A29 TsF also exhibits specific binding to a TsF1 secreted by another T cell hybridoma, A10, which shows specificity for antigen from the P815 tumor (this has been described previously). A29 itself does not exhibit binding to P815 antigens. Affinity-purified material from A29 appears to share characteristics with A10 molecules in that the predominant material has an apparent m.w. of 70,000. Studies with calcium flux of A29 cells showed that they respond significantly and specifically on exposure to A10 TsF stimulus. We showed further that affinity-purified A29 TsF molecules can specifically suppress the in vitro generation of syngeneic CTL to the P815 tumor, and that panning of DBA/2 splenocytes over A29-TsF-coated plates renders cell populations capable of generating a higher in vitro CTL response to P815 than appropriately treated controls.

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)

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 (TsF 1) 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 TsF 1 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-TsF 1induced 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-TsF 1-induced and the nonspecific accessory lymphoid population resulted in antigen-specific and genetically restricted immune suppression. The TsF 1-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.

TMA-specific first-order T-suppressor hybridoma: I. Characterization of the hybridoma-derived single-chain inducer suppressor factor, TsF 1

Experiments described in this report will characterize a monoclonal phenyltrimethylammonium (TMA) specific, first-order T-suppressor factor (TsF 1) 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 TsF 1. 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.