Production Of Suppressor Factor Research Articles

Maternal anti-placental cell-mediated reactivity and spontaneous abortions.

Does Th1/Th2 balance determine pregnancy outcome, and if so, what determines Th1/Th2 balance in pregnancy? Review and synthesis of existing data. A bias toward Th1 is strongly correlated with pregnancy failure in mice and humans. Pregnancy usually shifts the balance toward Th2 and placental factors/progesterone and progesterone-stimulated CD8+ T cell production of suppressor factor; TGFbeta2 and IL4/10 may be responsible. The bias toward a Th1 response may result from intracellular parasitic infection and other as yet undefined factors. The Th1/Th2 balance thesis appears to be valid.

Transfection of TCR alpha-chains into suppressor and T helper cell hybridomas. Production of suppressor factors with predicted antigen specificity.

Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.

HUMAN SUPPRESSOR T CELLS INDUCED IN VITRO WITH AN AUTOLOGOUS RENAL ALLOGRAFT-DERIVED T CELL LINE

Human suppressor T cells induced by autologous mixed lymphocyte reaction (AMLR) using fresh responder PBL from a renal transplant recipient and an autologous irradiated antidonor CTL line (EE-1) established from a biopsy of the patient's own allograft were studied for the production of suppressor factors. The suppressor cell lines propagated (designated TsEE) were capable of inhibiting the in vitro generation of proliferative and cytotoxic responses of responder cells from the recipient or other individuals who shared HLA-B7 with TsEE cells, regardless of the stimulatory cell phenotype. Coculture of TsEE cells with the autologous irradiated EE-1 inducer cell line in vitro yielded a soluble factor (designated TsEEF) capable of inhibiting the generation of MLR and CTL responses, as well as mitogen-induced proliferative responses to PHA and PWM in an HLA-unrestricted manner. TsEEF also inhibited the replication of lymphoblastoid T cell lines (Molt-4 and HSB) but not B cell lines (SB and JC-EBV) or PBL stimulated with the B cell mitogen LPS. Control supernatants obtained from each of the cells used to generate TsEE in AMLR (i.e., EE-PBL and the EE-1 line) cultured alone or together for 48 hr demonstrated no suppressive activity in any of these test systems. TsEEF was nontoxic for lymphoid cells, was nondialyzable (greater than 12kDa), did not act by interfering with IL-1 or IL-2 utilization, and was negative for TNF and IFN-gamma activity. Functionally, the suppressive activity of TsEEF was dose-dependent, did not shift MLR kinetics, and could be absorbed by T cells. T cells incubated with TsEEF for 4 hr were unresponsive to subsequent mitogen or MLR stimulation. These findings indicate that, whereas T suppressor cell lines propagated from the circulation of a stable renal transplant recipient demonstrate class I HLA restriction, the activity of their soluble products is not HLA-restricted, and functionally inhibits T cell proliferation.

Production of suppressor factors induced by ultraviolet irradiation or cis-urocanic acid requires Lyt-2+ lymphocytes.

Both ultraviolet (UV) irradiation and cis-urocanic acid (UCA) are reported to be associated with the suppression of contact hypersensitivity (CHS) and the induction of a soluble factor which suppresses leukocyte adherence inhibition (LAI) assays in vitro. The cellular origin of the suppressor factor (SF) has now been investigated in vivo and in vitro, using monoclonal antibodies against lymphocyte surface markers to deplete certain cell populations, namely T lymphocytes bearing the L3T4 (helper) and Lyt-2 (suppressor) markers. Depletion of Lyt-2+ cells from irradiated mice in vivo and from spleen cell cultures in vitro led to the elimination of detectable levels of SF. Depletion of L3T4+ cells had no such effect. Similarly, Lyt-2+ cells (but not L3T4+ cells) were shown to be necessary for the production of SF by normal spleen cells cultured with cis-UCA. These data suggest that the production of SF following UV irradiation may be related to the action of cis-UCA on Lyg-2+ lymphocytes.

Suppression of cytotoxic responses by a supernatant factor derived from Mycoplasma hyorhinis-infected mammalian cell lines.

A class of interleukin-2-dependent T-cell clones, isolated from a murine fetal thymus, was previously shown to suppress the induction of cytotoxic responses to alloantigens (H.-S. Teh, M. Ho, and W. R. McMaster, J. Immunol. 135:1582-1588, 1985). In that article, the immunosuppressive properties of these T-cell clones were shown to be a direct consequence of infection by Mycoplasma hyorhinis. Suppression of cytotoxic responses was mediated by both the mycoplasmas and a 200-kilodalton factor present in supernatants of infected cultures. This factor was sensitive to proteases but was resistant to heating to 60 degrees C for 1 h and to incubation on ice at pH 2 or pH 14 for 4 h. The production of suppressor factor in infected cultures was independent of the viability or the protein synthesis capability of the mammalian cells, suggesting that it was produced by M. hyorhinis. The factor was most suppressive when it was added during the early stages of the cytotoxic response. Its suppressive effects on cytotoxic responses were not reversed by the addition of an excess of recombinant interleukin-2. This factor also suppressed mitogenic responses to lipopolysaccharide. However, it is not a growth inhibitor since it did not affect the proliferation of tumor cell lines. A simple method for detecting M. hyorhinis in the infected T-cell clones and for eliminating it is described.

Soluble factors in tolerance and contact sensitivity to DNFB in mice. VI. Cellular and lymphokine requirements for stimulating suppressor factor production in vitro.

Coculture of spleen cells from mice tolerized with 2,4-dinitrobenzenesulfonate (DNBS) and DNP-labeled spleen cells (DNP-SC) activates Lyt-2+ T suppressor cells (Ts) to synthesize and release a suppressor factor (SSF) into the supernatant, which suppresses the transfer of contact sensitivity to DNFB. The purpose of the present study was to examine in greater detail the signals required to activate DNBS-primed Ts to produce SSF. The supernatant from cultures of tolerant cells and glutaraldehyde-fixed DNP-SC did not have SSF. In contrast, the soluble cell lysate from these cultures did contain the suppressive activity. Pretreatment of glutaraldehyde-fixed DNP-SC with either anti-DNP or anti-class I, but not anti-class II MHC, antibodies blocked SSF synthesis. The addition of IL 1 to cultures of DNBS-tolerant cells and glutaraldehyde fixed DNP-SC restored the ability of the Ts to release the synthesized factor. These results indicate that Ts recognition of the hapten/class I MHC determinant stimulates the synthesis of SSF, and a costimulator is required to induce the release of the factor. The supernatants from cultures of either L3T4-depleted tolerant cells and DNP-SC or tolerant cells and anti-I-A antibody-treated DNP-SC had no SSF activity. The addition of a costimulator (IL 1) also restored the ability of the Ts to release the synthesized factor in cultures of L3T4-depleted tolerant cells and DNP-SC. These results suggest that an L3T4 cell in the DNBS-primed cell population interacts with I-A determinants on a cell in the DNP-stimulator population to initiate the generation of the mediator required for SSF release. This further suggests that the Ts is unable to induce the costimulator from the hapten-presenting cell during interaction with the DNP/class I MHC ligand. Therefore, the production of SSF is regulated not only by the presentation of the appropriate hapten/MHC determinant but also by the interactions of cells that function in generating the costimulator needed to induce release of the suppressor factor.

Analysis of lymphocyte populations extracted from chronically inflamed human periodontal tissues

The current study was undertaken to analyze in vitro the functional activity of cells extracted from diseased periodontal tissues. Tissue was removed from 33 patients undergoing periodontal surgery. All patients had received repeated oral hygiene instruction and root planing prior to the surgery. The excised tissue was cut finely, digested in collagenase for 90 min, and then forced through a stainless steel grid to obtain a single cell suspension. The responses to various doses of PHA, Con A, and PWM were assessed in 2, 3, and 4‐day cultures using peripheral blood lymphocytes as controls. Blastogenesis was measured by the uptake of 3H‐thymidine.The extraction procedure resulted in a mean of 3.29 ± 0.29 × 105 lymphocytes per 100 mg of tissue. These cells had a mean viability of 76.8 ± 2.2%. Peripheral blood lymphocytes gave typical dose response curves for each of the three mitogens with a peak response occurring at 3 days. However, the lymphocytes extracted from the periodontal tissues failed to respond to any mitogen at any time. The failure to respond was not due to the effect of local anesthetic nor to the effect of collagenase pretreatment. These results may therefore indicate that the cells are maximally stimulated in vivo prior to extraction and/or there is production of suppressor factors in vitro.

Network regulation of the immune response: modulation of suppressor lymphocytes by alternative signals including contrasuppression.

In the preceding paper we demonstrated that comparison of alternative designs for the immune network can be used to examine the functional significance of specified interactions in normal immune responses. In this paper we examine mathematically the functional significance of three interactions affecting the production of suppressor lymphocytes involved in regulation of normal immune responses. The interactions examined in detail are 1) antigenic stimulation of the production of suppressor lymphocytes, 2) idiotypic stimulation of the production of suppressor lymphocytes, and 3) antigenic inhibition of the production of suppressor lymphocytes (i.e., contrasuppression). The results of our analysis suggest that an immune system with only antigenic stimulation of suppressor production is less effective than a system with both antigenic and idiotypic stimulation of suppressor production on the basis of all of the criteria examined in this study. In turn, the latter system is less effective than a system with only idiotypic stimulation of suppressor production. Furthermore, a system with both idiotypic stimulation and antigenic inhibition of suppressor production can be equal or superior to a system with only idiotypic stimulation of suppressor production on the basis of the same criteria. Similar conclusions hold for the comparison of systems in which regulation by the suppressor lymphocytes of interest is exerted upon production of effector molecules rather than upon production of effector lymphocytes, and also for the comparison of systems in which interactions affecting the production of suppressor factors are of interest.

Mechanisms of action of Mycobacterium bovis BCG-induced suppressor cells in mitogen-induced blastogenesis.

Spleen cells from Mycobacterium bovis BCG-infected C57B1/6 mice when cultured in vitro for 72 h elicited soluble suppressor mediators capable of nonspecifically suppressing the mitogen-induced blastogenesis of normal splenocytes. Maximal production of suppressor mediators occurred during the first 24 h in culture, and their production ceased after 72 h. Attempts to isolate the mediators from fresh nonincubated splenocytes failed. After incubation, a strong residual suppressive activity was constantly detected in cell preparations used for production of suppressor factors. Supernatants prepared from cultures of spleen cells of mice infected 14 days earlier possessed higher suppressive activity than did those obtained 28 days after infection. In contrast, the residual cellular suppressive activity increased during the course of the infection. Although the activity of soluble factors was not inhibited, the residual activity of incubated cells was highly depressed by the presence of mouse erythrocytes in the cultures. Thus, the incubated cells appear to act through a direct cell-to-cell contact with the mitogen-responding cells. Finally, the results of depletion experiments suggest that the two populations of BCG-induced suppressor cells, namely, T lymphocytes and macrophage-like cells, are able to elicit suppressor mediators and to retain thereafter suppressive activity.

Suppressor factor from tumor-allosensitized spleen cells—Its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

Abstract C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF) 2 which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.

Induction of Suppressor Cells and Cells Producing Antigen-Specific Suppressor Factors by Haptens Bound to Self Carriers

Injection of TNP-, DNP- or oxazolone-substituted syngeneic cells into mice causes the development of hapten-specific T suppressor cells which prevent the animals from being activity sensitized with homologous hapten. These cells injected together with immunized cells abrogate the latter's ability to transfer passively the contact sensitivity (CS) reaction into normal recipients. T lymphocytes from animals made unresponsive and sensitized with homologous hapten synthesize in vitro antigen-specific suppressor factors (SF) which when incubated with immune lymphocytes prevent them transferring adoptively the CS reaction. The type of cell used to induce suppression or production of suppressor factor (haptenated erythrocytes, thymocytes or macrophages) is not critical suggesting that a hapten-substituted common membrane structure is recognized as a tolerogen. The present work demonstrates that while the specific unresponsiveness induced by cell-bound hapten in vivo is long lasting, cells from tolerized animals are able to suppress the immunized cells in passive transfer or produce in vitro antigen-specific suppressor factors only when tested several days after tolerization.

Physiology of Mixed Leukocyte Reaction Suppressor Factor

Abstract The secretory physiology of the T cell-produced lymphokine, mixed leukocyte rection suppressor factor (MLR-TsF), was characterized with respect to its kinetics of secretion and its sensitivity to a variety of metabolic blocking agents. It was found that spleen cells from alloantigen-immunized mice released active MLR-TsF after freeze-thaw lysis. Upon restimulation with the same priming alloantigen, MLR-TsF was secreted into culture supernatants, and the rate of secretion was determined to be nearly constant. Although colchicine and vinblastine, which bind to microtubules, are known inhibitors of lectin-induced proliferation, it was demonstrated that these drugs had no effect on the secretion of MLR-TsF. However, cytochalasin B, an inhibitor which also binds to some cytoskeletal and membrane-associated proteins, did inhibit the production of MLR-TsF. The above findings indicated that the activation-secretion mechanism of MLR-TsF was much like that described for lymphotoxin and macrophage migration inhibition factor. The dissociation between DNA synthesis and lymphokine secretion was also demonstrated in the MLR-TsF system. DNA synthesis plays no role in the in vitro production of suppressor factor, as determined by resistance to treatment with mitomycin C and γ-irradiation. However, new protein synthesis is required as indicated by the potent inhibitory effects of cycloheximide. Experiments utilizing timed addition and removal of cycloheximide defined a broad period of drug sensitivity, starting from the beginning of culture and lasting for 12 to 16 hr. In addition, experiments measuring the effect of cycloheximide on the MLR-TsF content of cell lysates demonstrated that the cell-associated lymphokine activity is lost when protein synthesis is interrupted. These experiments support the conclusion that MLR-TsF is synthesized de novo in culture. In addition, the secretory process itself may require protein synthesis.