Generation of suppressor cells by concanavalin A: a new perspective

Significantly lower mitogenic responses of fresh cells co-cultured with Con A-stimulated cells were found when compared with the responses of fresh cells co-cultured with preincubated control cells. We do not agree with the interpretation that this effect represents the generation of suppressor cells by Con A, since the responses of fresh cells cultured alone were also significantly less than when co-cultured with control cells and the same as when co-cultured with the Con A-stimulated cells. Treatment with mitomycin C was sufficient to prevent the preincubated cells from contributing to the mitogenic response of the fresh cells. The increased responses of fresh cells when co-cultured with preincubated cells seems analagous to the increased mitogenic responses of cells aged in vitro by preincubation without mitogen. This effect seems to be transferable to fresh cells in the absence of cell division. Although preincubation in the presence of Con A abrogates this effect, we do not interpret this as the generation of suppressor cells.     

Role of the autologous rosette-forming T cells in the concanavalin A-induced suppressor cell function

It was recently reported that the human autologous rosette-forming T cells (Tar cells) are devoid of Fc receptors for IgM and IgG but that they give rise in vitro to Tμ and Tγ cells and that these cells participate actively in feedback inhibition. We now investigated whether Tar cells participate in the concanavalin A-induced suppressor cell function, using two indicator systems, namely, mitogen- or alloantigen-induced DNA synthesis and mitogen-driven polyclonal immunoglobulin synthesis. When Tar cells were removed from peripheral blood T cells (T-Tar) there was no generation of suppression determined on both DNA synthesis and immunoglobulin production. When Tar cells were readded to T-Tar cells suppressor activity was restored. When purified Tar cells were activated by concanavalin A they showed the highest degree of suppression as compared to that exerted by total T cells or T-Tar cells. Tar cells lose their capacity to generate suppression when treated with mitomycin C before their activation; however, they are no longer sensitive to mitomycin C treatment once they have completed the period of activation. These results together with our previous findings showing that Tar cells give rise in vitro to Tμ and Tγ cells after their activation by Con-A suggest that Tar cells participate in the Con-A-induced suppressor cell function as precursors of the suppressor effector T cells.     

Suppressor T cell growth and differentiation: evidence for induced receptors on suppressor T cells that bind a suppressor T cell differentiation factor

T suppressor cell differentiation factor (TsDF) induces the differentiation of alloantigen-primed suppressor T cells (MLR-Ts) to expression of their effector function, i.e., to active TsF production. The initial activation stimulus to Ts is provided by alloantigen binding; after this binding, Ts are functionally responsive only for a period of hours to the additional stimulus provided by TsDF. The present studies addressed the possibility that MLR-Ts responsiveness to TsDF reflects the induced and transient display of TsDF-binding receptors. TsDF receptor expression was investigated by determining the capacity of TsDF-responsive MLR-Ts to adsorb TsDF activity and to respond to that TsDF pulse by TsF production. Primed Ts populations that were alloantigen restimulated for 8 hr adsorbed TsDF in a cell dose-dependent fashion and produced TsF in response to that adsorption, whereas alloantigen-stimulated naive cells or primed but nonrestimulated cells neither responded to nor bound TsDF. Primed and restimulated L3T4-Ly-2+ but not L3T4+-Ly-2--enriched T cells bound TsDF. TsDF adsorption was saturable and time and temperature dependent. Glutaraldehyde fixation did not prevent TsDF adsorption by restimulated MLR-Ts, whereas pronase treatment abolished their TsDF-binding capacity. Kinetic analyses demonstrated that the capacity to bind TsDF developed rapidly after alloantigen reexposure, with maximal binding within 8 hr, followed by rapid decay with loss of TsDF binding by 36 hr. The kinetics of TsDF-induced TsF production correlated precisely with those of TsDF binding. These observations provide strong evidence that TsDF affects primed alloantigen-reactive Ts by interaction with antigen-induced and transiently expressed cell surface receptors. TsDF-receptor binding is then the stimulus for expression of Ts effector function.     

Suppressor T cells and suppressor macrophages induced by Corynebacterium parvum

Corynebacterium parvum, injected intravenously into C57B1/6 mice (H-2^b) previously alloimmunized with P815 (H-2^d) mastocytoma cells, generated splenic suppressor cells that inhibited the development of primary cytotoxic lymphocytes in vitro. These suppressor cells differed from those generated by intravenous C. parvum injection of naive C57B1/6 mice. The former suppressor cells were effectively induced by administration of 700 μg of C. parvum whereas the latter suppressor cells were dependent upon higher doses (1400 μg) of adjuvant for their activation. Furthermore, suppressor cells generated in alloimmunized mice could only suppress C57B1/6 anti-P815 in vitro cytotoxic responses whereas suppressor cells generated in naive mice could suppress C57B1/6 anti-CBA (H-2^k) responses as well. Suppressor cells were not H-2 restricted in their action. Fractionation of spleens from alloimmunized, C. parvum-treated mice revealed the presence of suppressor T cells and suppressor macrophages. We were unable, however, to determine which cell was responsible for “antigen specificity” of suppression since the fractionation procedures seemed to trigger both suppressor cell types prior to adding them to the primary culture.     

Human anergic CD4+ T cells can act as suppressor cells by affecting autologous dendritic cell conditioning and survival.

T cell suppression exerted by regulatory T cells represents a well-established phenomenon, but the mechanisms involved are still a matter of debate. Recent data suggest that anergic T cells can suppress responder T cell activation by inhibiting Ag presentation by dendritic cells (DC). In this study, we focused our attention on the mechanisms that regulate the susceptibility of DC to suppressive signals and analyzed the fate of DC and responder T cells. To address this issue, we have cocultured human alloreactive or Ag-specific CD4+ T cell clones, rendered anergic by incubation with immobilized anti-CD3 Ab, with autologous DC and responder T cells. We show that anergic T cells affect either Ag-presenting functions or survival of DC, depending whether immature or mature DC are used as APC. Indeed, MHC and costimulatory molecule expression on immature DC activated by responder T cells is inhibited, while apoptotic programs are induced in mature DC and in turn in responder T cells. Ligation of CD95 by CD95L expressed on anergic T cells in the absence of CD40-CD40L (CD154) interaction are critical parameters in eliciting apoptosis in both DC and responder T cells. In conclusion, these findings indicate that the defective activation of CD40 on DC by CD95L+ CD154-defective anergic T cells could be the primary event in determining T cell suppression and support the role of CD40 signaling in regulating both conditioning and survival of DC.     

Human placental Hofbauer cells promote mitogen induced T cell proliferation

Hofbauer cells are a major cell type of the human placental villous core particularly numerous at the beginning of pregnancy. In the present study we have investigated whether or not Hofbauer cells could subserve the function of accessory cells for colony formation by phytohemagglutinin stimulated allogeneic T cells. Results showed that Hofbauer cells are capable to play an accessory role on T cell proliferation probably due to the release of interleukin 1-like soluble factors.     

In vitro effects of 4-hydroperoxycyclophosphamide on concanavalin A-induced human suppressor T cells

Summary Treatment in vitro of human peripheral blood lymphocytes (PBL) with ConA induced the generation of suppressor cells which inhibited T cell blastogenic response to ConA and of allogeneic response in the mixed lymphocyte reaction (MLR). Treatment of PBL with 4-hydroperoxycyclophosphamide (4-HPCy) before incubation with ConA markedly decreased the generation of suppressor cells by ConA. The effect of 4-HPCy on generation of suppressor cells was more pronounced in the test of ConA stimulation than in the MLR. Treatment with 4-HPCy had no effect on suppressor cells already induced as shown by incubation of PBL with 4-HPCy after incubation with ConA.     

Cellular immunity in pyelonephritis: identification of suppressor cell activity of spleen cells in response to concanavalin A and inhibition of lymphocyte-mediated L cell cytotoxicity.

A population of fast sedimenting spleen cells was identified as the cause of impaired in vitro response of spleen cells to concanavalin A (Con A) in acute pyelonephritis in rats. These fast-sedimenting cells responded to Con A by suppressing the DNA synthetic response of normal spleen cells to Con A. In addition, spleen cells from acute pyelonephritis rats were significantly less able to mediate in vitro cell destruction in xenogeneic aggressor lymphocyte: target L cell mixtures. On the basis of these findings, the hypothesis is proposed that a suppressor cell can protect the pyelonephritic kidney against immunologically mediated tissue damage.     

Suppressor T cell mechanisms in contact sensitivity. II. Afferent blockade by alloinduced suppressor T cells.

We investigated the mechanism(s) by which MHC-restricted suppressor T cells (Ts) induced by i.v. injection of allogeneic DNP-modified lymphoid cells (alloinduced Ts) suppress the DNFB contact sensitivity response. It was shown that alloinduced Ts acted only during the early phases (afferent limb) of sensitization. They were incapable of suppressing previously sensitized recipients or of inhibiting the expression of DNFB-immune LN cells when co-transferred into normal recipients. The target of alloinduced Ts seems to be cell proliferation, i.e., inhibition of antigen-induced cell proliferation (DNA synthesis) in Ts recipient mice. The failure of recipients of alloinduced Ts to generate DNFB-immune LN cells capable of transferring contact sensitivity to normal recipients also suggests that these Ts act by preventing the development of an expanded clone of mature immune T cells. The suppressive effects of alloinduced Ts also were inhibited by prior in vitro treatment with anti-TNP serum. The data are discussed in terms of current models of suppression, and are compared to mechanisms of suppression in other contact sensitivity models.     

Immune amplification of murine CD8 suppressor T cells induced via an immune-privileged site: quantifying suppressor T cells functionally

Background

CD8⁺ suppressor T cells exert antigen-specific suppression of the expression of hypersensitivity by activated T cells. Therefore, CD8⁺ suppressor T cells serve a major regulatory role for the control of active immunity. Accordingly, the number and/or activity of CD8⁺ suppressor T cells should be influenced by an immune response to the antigen. To test this hypothesis we used an adoptive transfer assay that measures the suppression of the expression of delayed-type hypersensitivity (DTH) by CD8⁺ suppressor T cells to quantify the antigen-specific suppression of DTH by these suppressor T cells.

Methods

Suppressor T cells were induced in the spleens of mice by the injection of antigen into the anterior chamber of an eye. Following this injection, the mice were immunized by the same antigen injected into the anterior chamber. Spleen cells recovered from these mice (AC-SPL cells) were titrated in an adoptive transfer assay to determine the number of AC-SPL cells required to effect a 50% reduction of antigen-induced swelling (Sw50) in the footpad of immunized mice challenged by antigen.

Results

Suppression of the expression of DTH is proportional to the number of AC-SPL cells injected into the site challenged by antigen. The number of AC-SPL cells required for a 50% reduction in DTH-induced swelling is reduced by injecting a cell population enriched for CD8⁺ AC-SPL cells. Immunizing the mice receiving intracameral antigen to the same antigen decreases the RSw50 of AC-SPL cells required to inhibit the expression of DTH.

Conclusions

The results provide the first quantitative demonstration that the numbers of antigen-specific splenic CD8⁺ suppressor T cells are specifically amplified by antigen during an immune response.     

Effects of concanavalin A-induced cells on the proliferative response of T cells. Concanavalin A-induced suppressor and amplifier cells to the proliferative response of human T cells to trinitrophenyl-modified autologous lymphocytes.

Effects of Con A-induced human mononuclear cells on the proliferative response of peripheral T cells were examined by using TNP-modified autologous lymphocytes as stimulator cells. Cells induced by incubation with Con A contained both suppressor cells and amplifier cells. The former were induced from nylon wool-nonadherent T cells and these precursor cells were sensitive to mitomycin treatment. On the other hand, amplifier precursor cells were nylon wool-nonadherent T cells and were resistant to mitomycin treatment. Cell proliferation was required for the induction of suppressor cells but not for the induction of amplifier cells. Con A-induced suppressor effector cells were both nylon wool-adherent and nonadherent cells, on the contrary, Con A-induced amplifier effector cells were nonadherent cells. A small number of macrophages enhanced the suppressive activity of nonadherent T cells when added at the induction phase of suppressor T cells.     

Hapten-specific responses to the phenyltrimethylamino hapten. IV. Occurrence of mechanistically distinct idiotypic suppressor T cells before the appearance of anti-idiotypic suppressor T cells induced by the monovalent antigen L-tyrosine-p-azophenyltrimethylammonium

We have previously shown that a single i.p. injection of the monovalent synthetic antigen, L-tyrosine-p-azophenyltrimethylammonium [tyr(TMA)] in complete Freund's adjuvant induces an anti-idiotypic T suppressor cell (Ts2) population that can be detected 6 wk later by its ability to shut down delayed-type hypersensitivity (DTH) specific for the TMA hapten. In this paper we present evidence that 2 wk after tyr(TMA) administration, a subset of Ts, termed Ts1, appears that is both functionally and phenotypically distinct from the late appearing Ts2 population. The early occurring Ts1 act only at the induction phase of the DTH response and can also suppress this response intrinsically. This latter point is in marked contrast to our previous observation that the tyr(TMA)-induced anti-idiotypic Ts2 fail to function intrinsically and can only be detected upon adoptive transfer into naive mice. Ts1 bear idiotypic receptors and are Ly-1+,2- in contrast to the anti-idiotypic Ly-1-,2+ Ts2 population. In addition, unlike the Ts2 population, Ts1 are comparatively nylon wool-adherent. Adsorption of Ts1 on either antigen- or idiotype-coated petri dishes indicate that the suppressor activity can be transferred only by antigen-binding cells. Cellfree factors prepared from spleens containing the Ts1 population can suppress DTH only if administered at the induction phase of the response, in contrast to the factors derived from the Ts2 population that act both at induction as well as effector phases, suggesting that Ts1 and Ts2 can function via soluble mediators. Finally, we show that when Ts1-bearing mice are primed and boosted for anti-TMA antibody formation, the resulting response was overall reduced with respect to the idiotype-positive and negative plaque-forming cells that differs from the Ts2-bearing hosts wherein the idiotypic component is preferentially suppressed. The appearance of Ts1 before the detection of Ts2 in the same experimental animals is discussed with reference to a normal physiologic sequence of events involved in suppressor pathways.     

The suppressor T cell induced by syngeneic splenic cell antigen down-regulates hapten-specific cytotoxic T cells by elaborating a factor inhibitory for IL2-dependent cell replication

An in vitro study has been made of the mechanism by which a suppressor T cell, that is induced in lymph nodes by a syngeneic splenic cell antigen, prevents generation of cytotoxic T cells specific for hapten-altered self antigens. When popliteal lymph node cells exposed in vivo to syngeneic splenic cells were immunized in vitro with heat-treated syngeneic TNP-coupled thymocytes and excess helper factors, the Ts remained inactive. In this condition the exposed popliteal lymph node cells routinely demonstrated approximately twice the CTL response developed by lymph node cells from normal mice. Nevertheless, when triggered in vitro by splenic antigen on either X-irradiated B or T cells, the exposed but not the normal lymph node cells exhibited reduced hapten-altered self-specific CTL responses. Furthermore, T cells within spleen cell-exposed popliteal lymph node cell populations when reexposed to splenic T cells made a factor that was found to be suppressive of CTL generation by normal lymph node cells in vitro. The nondialyzable T-cell suppressor factor (TsF) did not appear to act on lymph node precursor CTLs, nor on helper T cells but instead acted at the level of utilization of helper factors in the development of CTLs. In an examination of the effect of TsF on cellular replication, TsF was found to be nontoxic for CTLL-20, an IL-2-dependent T cell, and it did not hinder the uptake of IL-2 by receptor blockade of this cell. Nevertheless, the replication of CTLL-20 that is IL-2 driven was diminished in the presence of TsF. Similarly, TsF was found to be inhibitory for T-cell proliferation stimulated by mitogen but had no effect on a B myeloma cell proliferative response. Thus, TsF appears to act as an inhibitor of a T cell's capability to replicate despite the presence of the stimulus for replication, namely, IL-2.     

Suppression of interleukin-2 production by human concanavalin A-induced suppressor cells

When PHA-activated normal responder cells (R cells) were cocultured with mononuclear cells (MN cells) which had been preincubated for 48 hr in medium alone (C cells) an enhanced proliferative response was observed. This enhancement was only obtained when the R cells were cultured with allogeneic C cells or when PHA was in the cocultures for the entire culture period. This effect was due to greater production of interleukin 2 (IL-2) by irradiated C cells in the presence of allogeneic or mitogenic stimulation. Con A-treated mononuclear cells (S cells) cultured with PHA-activated allogeneic or autologous responder cells showed reduced [3H]thymidine incorporation and IL-2 production as compared to activated R cells alone. Glutaraldehyde-treated S cells (which retained the ability to absorb IL-2) did not affect the proliferative response or IL-2 production by the R cells, indicating that passive absorption of IL-2 was not entirely responsible for suppression induced by S cells. S cells, pretreated with IL-2, still inhibited R-cell activity. These results show that Con A-treated MN cells suppressed or prevented [3H]thymidine incorporation by actively inhibiting IL-2 production.     

Major histocompatibility complex-restricted augmenting T cells induced by in vitro cultivation of antigen-primed T cells. A new parallelism between suppressor and augmenting circuits

In vitro cultivation of primed T cells with antigen resulted in the induction of a regulatory T cell that nonspecifically augmented the in vitro antibody responses of H-2-compatible T and B cells. This T cell, designated as the augmenting T cell (Ta), was unable to help B cells by itself but enhanced the antibody response of B cells to several multitudes only when conventional helper T (Th) cells or cloned Th cells from the same H-2 haplotype coexisted. Ta was radioresistant and belonged to Lyt-1+, 2-, L3T4+, I-J- T cell lineage. Ta exhibited interesting H-2-restricted activities: when primed T cells from (A X B) F1 were cultured with the antigen in the presence of parent A type antigen-presenting cells, the induced Ta was able to augment the antibody response of (A x B) F1 B cells in the presence of Th cells from F1----A but not from F1----B radiation bone marrow chimeras. This indicates that the induction of Ta in an F1 T cell population is dependent on the H-2 haplotype of antigen-presenting cells during in vitro cultivation. The restriction specificity of the established Ta is, however, not directed to the class II antigen itself but to the restriction specificity of Th cells that recognize class II antigen. In support of this is the fact that the elimination of A-restricted Th cells during cultivation by treatment with anti-I-J mAb, which is known to react with H-2-restricted Th cells, resulted in failure of induction of Ta cells having the augmenting activity for the A-restricted response.