Suppression of lymphocyte proliferative responses: characterization of the suppressor and kinetics of suppression

Culturing spleen cells for 2 or more days in the absence of mitogenic stimulation results in the generation of suppressor cells that can effectively inhibit the proliferative responses of freshly prepared spleen cells to mitogen or alloantigen stimulation. Suppression does not appear to be mediated by prostaglandins nor other soluble factors produced during the preculture period. The suppressor cell is described as a plastic-adherent Thy-1.2-, IgM-, FcR+ macrophage-like cell. Significant suppression of Con A responses can be detected at suppressor to target ratios as low as 1:100. The plastic-adherent suppressor is capable of terminating Con A-induced proliferation of spleen cells whether added at the onset of the Con A response or added as late as 48 hr after mitogenic stimulation. The suppressed spleen cell population displays an absence of large blast cells and a decrease in surface density of Thy-1.2 determinants.     

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.     

Virus-specific proliferative T-cell responses: Parameters and specificity

The genetic requirements for inducing virus-specific T-cell proliferation were investigated by taking spleen cells from animals primed with vaccinia virus in vivo, then culturing the cells in vitro with vaccinia virus-infected syngeneic peritoneal macrophages, and finally restimulating these cells a second time in vitro with vaccinia virus-infected macrophages from several strains of mice. Under these conditions, T cells proliferated in the tertiary response to virus-specific stimulation, whereas background proliferation caused by allogeneic differences between stimulator and responder cells was minimal. Compatibility between T cells and infected stimulator cells at the K or I regions alone or at I-A or I-A + I-B regions of the major histocompatibility complex (MHC) produced strong proliferative responses, whereas compatibility at D alone often resulted in somewhat weaker responses. However, these responses were rarely as great as in combinations of completely syngeneic stimulator and responder cells. Homology between responding and virus-infected stimulating cells in more than one of the H-2K, D, or I regions resulted in an additive, but not potentiating, effect. Genes coded outside the H-2 region did not seem to play a role in this system. In some rare cases, a weak response occurred across allogeneic barriers, but in general, virus-specific T-cell proliferation was strongly H-2 restricted.     

Effect of L-ornithine on proliferative and cytotoxic T-cell responses in allogeneic and syngeneic mixed leukocyte cultures

The effect of L-ornithine on cytotoxic and proliferative responses in mixed leukocyte cultures has been analyzed. The activation of cytotoxic T lymphocytes (CTL) was strongly inhibited when 9 X 10(-3) M L-ornithine was added at the initiation of the cultures. The CTL precursor cells were not completely and irreversibly inactivated, however, since the cells generated normal cytotoxic activity if resuspended after 6 days in fresh culture medium together with a fresh set of stimulator cells. Experiments in microcultures with nylon-wool-nonadherent T-cell-enriched spleen cells as responder cells and "plastic adherent cells" as stimulator cells revealed that the cytotoxic responses were almost completely suppressed if ornithine was added within the first 20 hr but were only partially suppressed if ornithine was added after 48 hr. Also, ornithine had only a mild suppressive effect on proliferative responses in allogeneic and syngeneic mixed leukocyte cultures. The strong suppressive effect of the cytotoxic response was therefore not explained by a general toxic effect of L-ornithine on the responding cells in the culture. The addition of interleukin 2 (IL-2)-containing EL-4 supernatants did not prevent but rather enhanced the suppressive effect of L-ornithine. This indicated that the inhibitory effect was not (exclusively) expressed at the level of the IL-2-producing helper T cells. Since activated macrophages have been reported to secrete arginase, it appears that L-ornithine may be part of a regulatory circuit that selectively regulates the development of cytotoxic effector T cells.     

Concanavalin A-induced receptor redistribution on Biomphalaria glabrata hemocytes: Characterization of capping and patching responses

Exposure of rounded, glass-adherent hemocytes from a Schistosoma mansoni-susceptible (PR albino) and S. mansoni-refractory (10-R2) stock of snails, Biomphalaria glabrata, to fluoresceinlabeled concanavalin A induces a redistribution of surface membrane Con A receptors. Receptor redistribution (patching and capping) on hemocytes from both snail stocks can be characterized as (1) rapid, with maximum cap formation occurring within 15 min of lectin treatment at 22°C, (2) sodium azide sensitive, but only at relatively high inhibitor concentrations (100–200 mm^?N₃ for capping and 200 mm^?N₃ for patching inhibition), (3) pronase sensitive (partial), but trypsin resistant, and (4) generally unaffected by exposure of snails to S. mansoni miracidia 60 or 180 min prior to extraction of hemolymph (hemocyte) samples for Con A testing. Although differences in the time course of receptor redistribution are exhibited between PR albino and 10-R2 snail hemocytes, the results of experiments involving sodium azide, proteolytic enzymes, and schistosome exposure strongly suggest that Con A-binding determinants and their associated membrane components on rounded hemocytes are very similar in both susceptible and refractory Biomphalaria stocks. It is concluded that if schistosome recognition in refractory 10-R2 snails is mediated through specific hemocyte membrane components, those components associated with Con A reactivity probably are not directly involved in the recognition process.     

Serological and biological cross-reactivity of class II antigens between mice and humans in antigen-specific T-cell proliferative responses

Many studies have already been reported with regard to the serological cross-reactivities between the polymorphic determinants of murine Ia antigens and human HLA-DR antigens. In this paper, we examined the biological cross-reactivity of the polymorphism of Class II antigens in the xenogeneic antigen-presenting cell (APC)-T-cell interaction. The data indicate that purified protein derivative (PPD)-specific human T cells were not stimulated by PPD-pulsed murine APC from B10.S(9R) which possess I-As and I-Ek molecules serologically cross-reacting with human Class II antigens. On the contrary, B10.S(9R) T cells primed to PPD were stimulated by PPD-pulsed human APC. The failure of the murine APC-human T-cell interaction was not caused by the suppressive effect in culture with ongoing xenogeneic mixed lymphocyte reactions (MLR) or other cell culture conditions. Thus, a hierarchy of antigen-presenting ability in the xenogeneic APC-T-cell interaction was shown to exist.     

Expansion of myeloid suppressor cells in SHIP-deficient mice represses allogeneic T cell responses

Previously we demonstrated that SHIP(-/-) mice accept allogeneic bone marrow transplants (BMT) without significant acute graft-vs-host disease (GvHD). In this study we show that SHIP(-/-) splenocytes and lymph node cells are poor stimulators of allogeneic T cell responses that cause GvHD. Intriguingly, SHIP(-/-) splenocytes prime naive T cell responses to peptide epitopes, but, conversely, are partially impaired for priming T cell responses to whole Ag. However, dendritic cells (DC) purified from SHIP(-/-) splenocytes prime T cell responses to allogeneic targets, peptide epitopes, and whole Ag as effectively as SHIP(+/+) DC. These findings point to an extrinsic effect on SHIP(-/-) DC that impairs priming of allogeneic T cell responses. Consistent with this extrinsic effect, we found that a dramatic expansion of myeloid suppressor cells in SHIP(-/-) mice impairs priming of allogeneic T cells. These findings suggest that SHIP expression or its activity could be targeted to selectively compromise T cell responses that mediate GvHD and graft rejection.     

Antigen-specific suppressor cell activity in man: I. Temporal, antigenic, and proliferative requirements

The temporal, antigenic, and proliferative requirements of antigen-specific suppression of the in vitro plaque-forming cell (PFC) response of human peripheral blood mononuclear cells (PBM) were studied. Suppressor cell activity (SCA) was generated by priming PBM with high doses of the T-cell-dependent antigen ovalbumin (OA) and measured by adding washed primed cells to target PFC cultures. Priming with high doses of OA was shown to induce a population of antigen-specific T lymphocytes which interfered with the anti-OA PFC response of optimally stimulated target cultures. The generation of SCA was demonstrated following as little as 24 hr of high-dose OA priming and could be abrogated by prolonged priming (72 hr) or by pretreatment with mitomycin prior to priming. The expression of optimal SCA required the addition of primed PBM at the initiation of the target culture and could be directly correlated to both the OA concentration used for priming and the number of primed cells added. Higher priming doses of OA (up to 100 μg) generated increasing numbers of cells capable of antigen-specific SCA as measured via a cell dilution protocol. Our data suggest that an antigen-driven and dose-dependent expansion of an antigen-specific T-suppressor cell pool is an early regulative event limiting the in vitro PFC response of human lymphocytes to OA.     

Thymic function and allogeneic T-cell responses in stem-cell transplantation

The thymus is the primary site of T-cell production early in life, and has now been shown to continue to function in both healthy and immunocompromised individuals late into life. Positive and negative selection occurring in the thymus are two of the most important processes that govern the development and specificity of peripheral T cells, including their restriction to self HLA and their ability to respond in an alloreactive manner. In the chimeric state that follows successful allogeneic stem-cell transplants, the specificity of alloreactive cells may be governed by either host- or recipient-derived cellular elements, as well as maturing lymphoid cells, which are, in turn, derived from donor stem cells or host cells surviving transplant conditioning. The ability to measure recent thymic emigrants via the detection of T-cell receptor excision circles has facilitated studies of thymic function in immunodeficient individuals, including HIV-1 infected subjects and recipients of autologous or allogeneic stem-cell transplant (SCT). These studies have now demonstrated that thymic function is likely to play a beneficial role in immune reconstitution in these settings, but have yet to clearly demonstrate what clinical variables are the most important determinants of thymic persistence. It is also not yet clear how much the degree of thymic function following allogeneic SCT influences the alloreactive T-cell repertoire, although studies in animal models and early clinical studies suggest that GvHD results in thymic injury and dysfunction. Future studies will further clarify how thymic function shapes the repertoire of T cells that mediate alloreactivity, as well as protective pathogen-specific immune responses, following SCT. Finally, these studies will also demonstrate whether endogenous mediators of thymic function could be selectively applied to regulate post-SCT thymic function and alloreactivity.     

T cell responses to allogeneic human mesenchymal stem cells: immunogenicity,tolerance, and suppression

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN- and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN- and IL-10, and down-regulation of TNF- production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.     

A 45-kDa human T-cell membrane glycoprotein functions in the regulation of cell proliferative responses

A 45-kDa human T cell surface glycoprotein which is tightly bound in the membrane of the resting T cell is released into the cell medium in soluble form after cell growth activation by phytohemagglutinin or neuraminidase/galactose oxidase treatments. In limited proteolysis by Staphylococcus aureus V8 protease, two major 35-kDa and 27-kDa peptide fragments of the surface-iodinated 45-kDa protein are common to the membrane-bound and the released forms, but a third 18-kDa fragment is observed exclusively with the released protein. The apparent molecular masses of the deglycosylated peptide backbones of the membrane-bound and the released molecule are 30 +/- 1 kDa, although a small size difference cannot be excluded. A polyclonal rabbit anti-(T cell membrane protein) antiserum precipitates the 45-kDa protein. A monoclonal anti-(45-kDa protein) antibody precipitates the membrane-bound 45-kDa protein solubilized with octyl glucoside, but does not precipitate the released protein. In cell culture assays, the monoclonal anti-(45-kDa protein) antibody specifically enhances the cell proliferative responses in phytohemagglutinin-treated and mixed lymphocyte cultures. These observations suggest that the 45-kDa protein has a specific receptor function in the regulation of cell proliferative responses.     

CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer and various pathological conditions. However, the phenotypic and functional heterogeneity of human MDSCs represents a major hurdle for the development of therapeutic strategies targeting or regulating MDSCs in tumor progression, inflammation, and graft-versus-host disease (GVHD). We previously shown that circulating HLA-DR^-CD14⁺ monocytic MDSCs are a major contributor to clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we identified, using high-throughput screening, a set of surface markers that are strongly expressed in HLA-DR^-CD14⁺ monocytic MDSCs isolated from the peripheral blood (PB) of patients receiving allo-HSCT. Subsequent experiments showed the consistent dominant expression of CD1d in monocytic MDSCs of allo-HSCT PB in comparison with granulocytic MDSCs. In addition, CD1d-expressing cells isolated from PB of allo-HSCT patients showed the suppressive activity of T cell proliferation and higher expression of MyD88 and IDO compared with CD1d^? cells. Our results suggest that CD1d could be a valuable marker for further therapeutic evaluation of human monocytic MDSCs for immune-related diseases, including GVHD.     

Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

Cytotoxic and proliferative lymphocyte responses to allogeneic and xenogeneic antigens in vitro.

In vitro lymphoproliferative responses to foreign histocompatibility antigens are phylogenetically restricted. Responses occur most readily to allogeneic or closely related xenogeneic leucocytes, but not to unrelated xenogeneic cells. Specific cytotoxic T cell responses to foreign histocompatibility antigens show the same phylogenetic restriction. This lack of xenoreactivity is not due to a lack of precursor cells for the xenoantigens; guinea-pig lymphocytes, although normally unresponsive to mouse antigens, have a similar precursor frequency for these antigens as do lymphocytes of allogeneic mouse strains. Specific cytotoxic responses of guinea-pig lymphocytes to mouse antigens can be generated if a factor released from con A stimulated guinea-pig spleen cells is added to the culture medium. The factor produced by con A-activated spleen cells (CS) is also phylogenetically restricted in its action; CS must be obtained from animals homologous with the donor of the responding lymphocytes.     

In vitro generation of suppressor cell activity: suppression of in vitro induction if cell-mediated cytotoxicity.

It was observed that when normal mouse spleen cells were cultured alone in vitro (precultured) for 3 to 7 days, these cells lost the ability to generate cell-mediated cytotoxicity (CML) during subsequent in vitro sensitization with allogeneic spleen cells, trinitrophenyl (TNP)-modified syngeneic spleen cells, or syngeneic tumor cells. These precultured cells, which were themselves unable to generate CML, were also shown in mixing experiments to suppress, actively, the generation of CML by freshly explanted spleen cells. Suppression occurred at the sensitization phase of CML, and not at the effector level; supernatants from suppressive precultured cells were not suppressive. Suppression was totally abrogated by the treatment of spleen cells with a T cell-specific rabbit anti-mouse brain serum and complement (RalphaMB+C) either before or after preculturing, suggesting that a T cell eas essential both to the generation of suppressor activity and to its expression. Suppressor activity was entirely absent in precultured nylon wool column-nonadherent spleen cells, a T cell-enriched population containing most of the RalphaMB+C-sensitive cells in the spleen. Precultured nylon column-adherent cells (T cell-depleted) did have suppressive activity, and a mixture of nylon-adherent and nylon-non-adherent cells was a suppressive after preculture as the precultured unseparated spleen. Moreover, the ability of nylon-adherent spleen cells to generate suppressive activity during preculturing was abrogated by treatment with RalphaMB+C. Thus, the "spontaneous" generation of CML-suppressive activity was dependent upon a limited subpopulation of splenic T cells isolated in the nylon column-adherent fraction. The relationship of these data to a previously described synergy between subpopulations of normal spleen in the generation of CML is discussed, and the findings related to other suppressor systems described in the literature.     

A syngeneic splenic cell antigen induces a suppressor T cell in lymph nodes that controls cytotoxic T-cell and primary antibody responses

The nonspecific suppression of immunological responses that is generated within host popliteal lymph nodes upon exposure to syngeneic normal spleen cells has been examined. The suppression, which had previously been described as being capable of preventing initiation of cytotoxic T lymphocytes (CTLs) to hapten-altered self antigens, arises within 3 to 7 days after injecting the spleen cells. Suppression was shown to be attributable to an induced T cell that was functional when transferred intravenously. Although the cell surface marker(s) on both splenic B and T cells that stimulates appearance of Ts has not yet been identified, the cells possessing the marker were not required to be viable to cause the induction. We have shown here that the Ts is fully functional when it is put in the antigenic site used for CTL immunization. The induced Ts has been identified as bearing the Lyt 2.1 cell surface marker. Furthermore, it has been shown to be insensitive to cyclophosphamide (CY), thus differentiating it from the naturally occurring Ts cell (TS0) that is known to be CY sensitive. In addition to preventing induction of CTLs toward hapten-altered self antigens, exposing popliteal lymph nodes to syngeneic spleen cells induced Ts capable of suppressing the primary IgM antibody response to sheep red blood cells. The Ts cells that suppressed the primary antibody response possessed the same Lyt cell surface markers and CY insensitivity as the Ts that mediated suppression of the CTL response. Thus, evidence that two dissimilar immunological reactions may be down-regulated by the same suppressor mechanism has been provided. Results of a kinetic study showed that the Ts prevented development of both the humoral and the cell-mediated immune responses by affecting their inductive phases. Possible targets for suppression that more than likely would have to be common to the two widely different immune responses have been indicated.