Modulation of suppressor T cell induction with gamma-interferon

The ability of antigen-coupled splenic adherent cells to induce suppressor T cells (Ts) is dependent on the presence of I-J determinants on antigen-presenting cells. After 4 days of in vitro culture, antigen-coupled adherent cells lose the capacity to induce Ts. Supernatants from Con A-stimulated lymphocyte cultures and purified interferon-gamma can sustain accessory function for the induction of Ts. Furthermore, after in vitro culture of splenic adherent cells, there is an apparent correlation between the loss of I-A determinants and the decrease in I-J-restricted Ts induction. Stimulation of Ia expression with interferon-gamma results in a simultaneous increase in the ability to induce Ts. Finally, elimination of I-A-bearing splenic adherent cells with antibody + C eliminates I-J-restricted Ts induction. The combined data imply a co-regulation of I-A and I-J on the antigen-presenting cells involved in the induction of both the Ts1 and Ts3 suppressor T cell subsets.     

Modulation of immune responses by suppressor T cells.

The activity of suppressor T cells has been demonstrated in almost every phase of the immune response. These regulatory cells modulate both humoral and cell-mediated immunity utilizing antigen-specific and nonspecific mechanisms. For comparative purposes two murine models are described, the nonspecific suppressor T cell stimulated by the mitogen concanavalin A and the antigen-specific suppressor T cell stimulated by injection of the synthetic terpolymer acid 60-L-alanine30-L-tyrosine10 (GAT) in nonresponder mice. These two T cells are similar to expression of Ly alloantigens, ability to inhibit antibody responses, and the mediation of suppression, at least in part, by soluble products. However, differences in radio-resistance and antigenic specificity of the suppressor T cells, as well as differences in molecular characteristics of the soluble factors and their targets suggest that these T cells regulate the immune response by different mechanisms. The relationship of these two suppressor T cells to other nonspecific and antigen-specific suppressor T cells is discussed.     

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.     

Induction of specific suppressor T cells in vitro.

We describe conditions for generating sheep red blood cell-specific suppressor T cells in Mishell-Dutton cultures. The production of specific suppressor cells is favored by increasing antigen dose in the initial culture but can be produced by transferring more cells when lower doses of antigen are used. Transfer of small numbers of cells cultured with low doses of antigen leads to a specific helper effect. Transfer of large numbers of educated cells leads to nonspecific suppression. Suppression can be effected by the effluent cells from nylon wool columns which do not make detectable PFC. A fraction of these cells become resistant to treatment with anti-T cell sera and complement after culture. The suppressor cells are radiation sensitive and must be able to synthesize protein to suppress. They take 2 to 3 days of education to reach maximum suppressive efficiency and will not suppress cultures if added 2 to 3 days after culture initiation. Their production is favored by the absence of mercaptoethanol, suggesting that the observed suppression is not "too much help". The ability to generate specific suppressor cells in vitro should be of great benefit in determining the factors that regulate their appearance in vivo.     

Activation of idiotype-specific suppressor T cells by injection of antibody carrying a recurrent idiotype

Intravenous injection of spleen cells (SC) coated with an antitrinitrophenyl (anti-TNP) IgM monoclonal antibody, Sp6 (Sp6-SC), which carries a recurrent idiotype, resulted in activation of a Lyt-2-positive population which did adhere to Sp6-coated plates. No effect of Sp6-SC injection could be observed in vivo on an anti-TNP B-cell response when mice were primed with an immunogenic dose of TNP-horse red blood cells (HRBC), but an anti-TNP response was observed when Sp6-SC-injected mice were primed with a subimmunogenic dose of TNP-HRBC. Furthermore, after intravenous (iv) injection of Sp6-SC, it was no longer possible to suppress a primary anti-TNP response by iv injection of TNP-haptenized thymocytes. In vitro analysis showed that the Sp6-induced suppressor T cell (Ts) population had no measurable influence on TNP-specific naive B cells, nor did it suppress TNP-specific helper T cells (THTNP), but it did lead to counterregulation of TNP-specific suppressor T cells (TsTNP). Hence, iv injection of antibody carrying a recurrent idiotype resulted in activation of a Ts population which functioned as inhibitor of suppression, thus displaying a helper effect.     

Modulation of suppressor activity by thymosin

Modulation of suppressor cell activity by thymosin was evaluated in vivo in a murine tumor system, and in vitro on human lymphocytes. We showed that splenocytes from tumor bearing mice were able to enhance tumor growth in a syngeneic system. This enhancement was T dependent and disappeared by Thymosin treatment. A decrease of Tumor size associated with injection of bone marrow cells treated by thymosin was observed. In the human system we showed that ConA stimulated lymphocytes were able to suppress the response of normal lymphocytes to PHA, PWM and ConA, and in MLC. This effect was significantly blocked in presence of thymosin fraction 5.     

Modulation of autoimmunity in NZB mice by cyclophosphamide-induced, nonspecific suppressor cells

A group of NZB mice received six biweekly injections of cyclophosphamide-induced nonspecific suppressor cells, with treatment commencing at 2 mo of age. Mice were evaluated for Coombs and natural thymocytotoxic antibody at 6-wk intervals thereafter, and for anti-DNA autoantibodies, total IgM and IgG levels, and renal histology at selected time points. The administration of suppressor cells resulted in marked and prolonged suppression of both Coombs and natural thymocytotoxic antibody reactivity in the majority of animals while not measurably affecting the levels of anti-DNA autoantibodies, the total IgM and IgG levels, or the life span of the mice.     

Experimental autoimmune encephalomyelitis induction in naive mice by dendritic cells presenting a self-peptide

Self-reactive T cells escape deletion in the thymus and are found in the peripheral repertoire. Because bone-marrow-derived dendritic cells (BM-DC) are potent activators of antigen-specific T cells, these cells could theoretically activate self-reactive T cells leading to autoimmunity. We investigated whether BM-DC could induce the autoimmune disease experimental autoimmune encephalomyelitis (EAE). Our results show that transfer of BM-DC presenting a self-peptide from the myelin oligodendrocyte glycoprotein (MOG35-55) into naive mice induced EAE 7-14 days later. MOG35-55-specific T cells of the Th1 phenotype were present in the lymph nodes and spleens of mice that received live peptide-pulsed BM-DC. Heat-killed or formaldehyde-fixed BM-DC presenting MOG35-55 could induce neither clinical signs of EAE nor a measurable T-cell response in vitro. These data show that live BM-DC presenting a self-antigen can induce the organ-specific autoimmune disorder EAE in a non-transgenic system. Therefore, this new EAE model could be used as a more clinically relevant model for the human disease multiple sclerosis. These findings could also have implications for the use of DC immunotherapy in a clinical setting.     

Human suppressor T cells induced by concanavalin A: suppressor T cells belong to distinctive T cell subclasses.

Human peripheral blood lymphocytes were stimulated by concanavalin A (Con A) and then evaluated by their suppressive activity for thymus-derived (T) cell- and bone marrow-derived (B) cell-proliferative responses to mitogen and allogeneic cells. Con A-activated T cells markedly suppressed these responses, but Con A-activated B cells failed to demonstrate suppressor activity. Discontinuous bovine serum albumin (BSA) density gradient separation of T cells which had been activated by Con A demonstrated that a fraction containing blast cells as well as fractions containing unproliferated cells manifest the same degree of suppressor capabilities. However, when density gradient separation of T cells followed by subsequent incubation with Con A was performed, fractions of proliferating cells of low density exhibited no suppression; a fraction containing high density T cells produced marked suppression, but this fraction incorporated only little thymidine in response to Con A. Thus, these studies indicate that Con A-induced suppressor T cells belong to a distinctive subpopulation which has already been programmed to express this function before exposure to Con A and that cell proliferation may not be a prerequisite for the development of such suppressor T cells.     

Augmentation of specific immune response against a syngeneic SV40-induced sarcoma in mice by depletion of suppressor T cells with cyclophosphamide.

When cyclophosphamide was administered to mice before immunization with syngeneic SV40-transformed cells, the specific immune response elicited, as was measured by the tumor cell neutralization assay with a syngeneic SV40-induced sarcoma, was stronger and lasted longer as compared to the response generated in non-cyclophosphamide-treated mice. The augmentation effect of the drug was dependent on cyclophosphamide concentration, being optimal at 100 mg/kg, and on the time of drug administration in relation to antigen immunization, being optimal at 2–4 days before antigen administration. Transfer of T cells from normal syngeneic mice to drug-treated animals abolished the cyclophosphamide-induced augmentation of immune response. These results implied that cyclophosphamide-sensitive T cells suppressed the in vivo generation of specific effector T cells against SV40-induced sarcoma.     

Idiotypic analysis of myeloma proteins: anti-DNA activity of monoclonal immunoglobulins bearing an SLE idiotype is more common in IgG than IgM antibodies

The anti-idiotype 3I which recognizes a determinant on kappa-chains of anti-DNA antibodies in SLE patients also recognizes a determinant on kappa-chains of 82/706 myeloma proteins tested. Twenty-nine of these 82 proteins bind to double-stranded DNA, including two monoclonal IgM, one monoclonal IgA, and 26 monoclonal IgG proteins. DNA binding is much more frequent in the IgG than in the IgM myeloma proteins (p less than 0.001), and is also associated with cationic antibody charge. Two-dimensional gel electrophoresis reveals markedly increased charge heterogeneity of both heavy and light chains of the monoclonal IgG as compared with the monoclonal IgM proteins. We postulate that the increased charge heterogeneity of the IgG-associated 3I-reactive kappa-light chains may reflect somatic mutation, and that DNA specificity within the 3I idiotype system arises by somatic mutation of germ-line genes found in normal individuals. DNA binding may be associated with those mutations that give rise to a cationic immunoglobulin charge.     

Modulation of dendritic cell function by naive and regulatory CD4+ T cells

The consequences of interactions between dendric cells (DCs) and either naive CD4+ T cells or regulatory CD4+CD25+ T cells on the expression of proinflammatory IL-6 and anti-inflammatory IL-10 in DC were examined over a period of 12 h, spanning the time frame during which stable T cell-DC interactions shape the development of tolerance and immunity in vivo. We demonstrate that the basal production of IL-6 and IL-10, which is initiated following DC stimulation with LPS, is modified in distinctly different ways by interaction with the two T cell populations. Naive CD4 T cells skew DC cytokine production toward IL-6 and suppress IL-10, whereas CD4+CD25+ T cells have the opposite effect. CD8 T cells or memory CD4 T cells do not influence basal cytokine production by stimulated DC. The effect of CD4+CD25+ T cells is dominant in coculture with naive CD4 T cells as long as inflammatory LPS is absent; the addition of LPS abrogates the suppression of IL-6. However, the modulating influence of CD4+CD25+ T cells remains evident in the enhancement of IL-10 production. Thus, mutual interactions between DC and CD4+ T cell subpopulations following contact with pathogens are likely to influence the strength and quality of incipient immune responses in the local microenvironment.     

Direct demonstration of specific suppressor T cells in the mice tolerant to type III pneumococcal polysaccharide: two-step requirement for development of detectable suppressor cells

Spleen cells from CAF1 mice made tolerant to type III pneumococcal polysaccharide (S3) with S3 coupled to syngeneic spleen cells (S3-SC) develop S3-specific suppressor T cells (Ts). These Ts could be demonstrated consistently only when spleen cells from tolerant mice were cultured in vitro with the specific antigen and the specific tolerogen. Spleen cells from normal mice cultured under the same conditions did not suppress the antibody response to S3. When different numbers of Ts were transferred to normal CAF1 mice, an unusual dose-effect pattern was observed. Maximal suppression of the S3 response occurred when relatively low numbers of Ts, 3 to 30 x 10(5) per recipient, were transferred, whereas larger numbers of cells, 150 x 10(5) per recipient, were not suppressive. These results indicate that a presumably T-independent antigen, S3, can activate antigen-specific Ts. These Ts exhibit unusual dose effects upon transfer and require both an in vivo induction period and in vitro activation for development of maximal activity. These latter observations suggest that S3 may activate a different population of T cells with suppressor function than do conventional T-dependent antigens. The loss of suppression observed when greater than optimal numbers of cells were transferred suggests that a second type of T cell, which has the ability to 'neutralize' the activity of S3-specific Ts, is also induced in the same spleen cell population.     

Enhancement of T suppressor activity in mice by high doses of BCG

Summary The spleen cells from mice injected 2 weeks previously with high doses of BCG have a lower reactivity as shown when they are engaged in a graft versus host reaction or in a mixed lymphocyte reaction. This suppression is an active and nonspecific phenomenon, at least partly attributable to the T-cell population.This work was supported by CRAMP and Action urgente DGRST 74-7-11-85, by CRL 74.5.015.01 from the Institut National de la Santé et de la Recherche médicale.     

Dynamics of pathogenic and suppressor T cells in autoimmune diabetes development

In the nonobese diabetic (NOD) mouse, pathogenic and suppressor CD4(+) T cells can be distinguished by the constitutive expression of CD25. In this study, we demonstrate that the progression of autoimmune diabetes in NOD mice reflects modifications in both T cell subsets. CD4(+)CD25(+) suppressor T cells from 8-, but not 16-wk-old NOD mice delayed the onset of diabetes transferred by 16-wk-old CD25-depleted spleen cells. These results were paralleled by the inhibition of alloantigen-induced proliferation of CD4(+)CD25(-) cells, indicating an age-dependent decrease in suppressive activity. In addition, CD4(+)CD25(-) pathogenic T cells became progressively less sensitive to immunoregulation by CD4(+)CD25(+) T cells during diabetes development. CD4(+)CD25(-) T cells showed a higher proliferation and produced more IFN-gamma, but less IL-4 and IL-10, whereas CD4(+)CD25(+) T suppressor cells produced significantly lower levels of IL-10 in 16- compared with 8-wk-old NOD mice. Consistent with these findings, a higher frequency of Th1 cells was observed in the pancreas of 16-wk-old compared with 8-wk-old NOD mice. An increased percentage of CD4(+)CD25(-) T cells expressing CD54 was present in 16-wk-old and in diabetic NOD, but not in BALB/c mice. Costimulation via CD54 increased the proliferation of CD4(+)CD25(-) T cells from 16-, but not 8-wk-old NOD mice, and blocking CD54 prevented their proliferation, consistent with the role of CD54 in diabetes development. Thus, the pathogenesis of autoimmune diabetes in NOD mice is correlated with both an enhanced pathogenicity of CD4(+)CD25(-) T cells and a decreased suppressive activity of CD4(+)CD25(+) T cells.     

Deficiencies in suppressor T cell activity seen in patients with active systemic lupus erythematosus are due to the dilution of normally functioning suppressor T cells by nonsuppressor T cells

Concanavalin A (Con A)-activated T lymphocytes from patients with active, but not inactive, systemic lupus erythematosus (SLE) failed to express normal suppressor activity, regardless of the phenotype of CD4+ or CD8+. Con A-activated CD4+ or CD8+ T lymphocytes from the SLE patients and from normal controls were further separated into two populations, using the autologous erythrocyte rosette technique. One population very rich in cells capable of forming rosettes with autologous erythrocytes from the active patients showed the same degree of suppressor activity, as did that from normal controls; the CD4+ or CD8+ population poor in autorosetting cells derived from Con A-activated T lymphocytes from both the controls and patients did not express suppressor activity. Moreover, when autorosetting T cells from the active patients and nonrosetting cells from the same patients were mixed at a normal ratio (4:6), normal suppressor activity could be restored. It was notable that the frequency of autorosette-forming cells was markedly reduced in the Con A-activated T lymphocytes from the active, but not inactive, SLE patients, regardless of the phenotype of CD4+ or CD8+. These findings indicate the presence of a normally functioning suppressor T cell population in patients with active SLE. It seems that the lack of suppressor T cell function in patients with active SLE is due to the dilution of a few normal suppressor T cells by large numbers of nonsuppressor T lymphocytes.     

Involvement of protein kinase C in competence induction of macrophages to generate T suppressor cells.

We have previously described an Ia-expressing macrophage hybridoma clone, termed clone 59, which attains the ability to induce Ts cells after activation with murine rIFN-gamma. In this report, we show that a protein kinase C (PKC) activator, PMA (10 ng/ml) can replace IFN-gamma in inducing this form of macrophage competence. IFN-gamma-induced cellular competence was abrogated specifically by a PKC inhibitor but not by inhibitors that have specificity for cyclic nucleotide-dependent protein kinases. Furthermore, PGE2 known to induce protein kinase A in murine macrophages also failed to induce competence. In contrast, the ability to induce Th responses was neither dependent on IFN-gamma nor inhibited by prior treatment with protein kinase inhibitors. Furthermore, PKC depletion of macrophages by treatment with high doses (100 ng/ml) of PMA abrogated their ability to induce Ts cells. In addition, PKC-depleted macrophages failed to regain the ability to stimulate Ts cells after further treatment with IFN-gamma. The ability of IFN-gamma to modulate macrophage-mediated induction of Ts cells does not clearly correlate with an increased Ia expression as inducible expression of Ia was not consistently abrogated by PKC inhibitor treatment. In addition, PKC inhibitors failed to prevent the production of the cytokines IL-1 and IL-6. However, incubation of IFN-gamma or PMA-treated macrophages with antibodies recognizing the putative IJ ligand blocked the ability to induce Ts cells, suggesting the expression of these determinants on accessory cells is responsible for Ts induction.     

Persistent activity of suppressor cells in T cell-mediated immune response.

Tolerance to dinitrochlorobenzene contact sensitivity induced i.v. injection of dinitrobenzenesulfonic acid in guinea pigs is a long-lasting phenomenon (up to 1 year). The tolerogen, however, was traceable in the circulation only up to 3 months after its application. In spite of that, tolerance was adoptively transferred by parabiosis 6 months after being induced. Moreover, active suppressor cells eliminated by cyclophosphamide treatment are able to regenerate in those adoptively tolerized animals. These results indicate that the tolerogenic injection stimulates precursors of suppressor cells to generate active suppressor cells and memory cells of suppression. The further formation of active suppressor cells from memory cells seems to be tolerogen independent, but the existence of specific stimulator cells for suppression may be considered. These cells may bind undetectable small amounts of tolerogen. The recovery of suppression might, however, be also due to recovery of suppressor cells which were temporarily inactivated but not destroyed by cyclophosphamide treatment.     

Studies of the induction of anti-DNA in normal mice

Injection of PBA has previously been demonstrated to induce anti-DNA. In the present study, we found that the combination of neonatal thymectomy and chronic administration of PBA (LPS + poly rI . rC) led to significantly higher anti-DNA levels than either PBA or thymectomy separately. These results suggested that a thymic regulatory process normally serves to suppress anti-DNA after chronic PBA exposure. Indeed, antigen-nonspecific suppressor function was found to be deficient in such thymectomy + PBA-treated mice. In addition, the cells of such mice in vitro interfered with the development of normal suppressor function by control cells.