Mechanism of action of 15-deoxyspergualin. I. Suppressive effect on the induction of alloreactive secondary cytotoxic T lymphocytes in vivo and in vitro.

The effects of 15-deoxyspergualin (DSG) and its derivative deoxymethylspergualin (MeDSG), which is more stable than DSG in solution, on the induction of alloreactive secondary cytotoxic T lymphocytes (CTL) in mice in vivo and in vitro were determined. Secondary CTL were generated in vivo or in vitro by injecting mice with mitomycin C (MMC)-treated allogenic tumour cells twice with a 1-week interval or by culturing spleen cells of alloantigen-primed mice with MMC-treated allogenic mouse spleen cells, respectively. DSG preferentially suppressed the generation of secondary CTL at the differentiation but not at the effector phase. DSG also suppressed the responsiveness of spleen cells to secondary antigen in a non-specific manner. MeDSG suppressed the in vitro induction of secondary CTL in a dose-dependent manner; however, it did not suppress the activity of CTL already induced. The inhibitory effect of MeDSG on CTL induction was totally abolished by exogenous recombinant murine interferon-gamma (IFN-gamma) and partly by interleukin-2 (IL-2), suggesting that suppressive mechanisms of MeDSG (DSG) may be mainly mediated by the inability of lymphocytes to produce IFN-gamma or by impairment of an unknown cascade(s) reconstituted by IFN-gamma.     

Immunosuppressant deoxyspergualin induces apoptotic cell death in dividing cells.

Deoxyspergualin (DSG) has been found to have an antitumour and immunosuppressive activity. However, the precise mechanism of action of DSG has not been clarified. We have used its analogue, methyldeoxyspergualin (MeDSG) for in vitro culture studies of DSG since it shows good stability in aqueous solution and retains strong immunosuppressive activity. In the present study, we found that MeDSG inhibited proliferation of rapidly dividing murine T-cell hybridomas, resulting in cell death. The cell death was accompanied by chromatin condensation and DNA cleavage at the linker regions between nucleosomes. Furthermore, MeDSG induced a reduction in mitochondrial transmembrane potential. When murine thymocytes were treated with MeDSG for 48 hr, a slight increase of DNA fragmentation was constantly observed, and selective depletion of CD4- CD8- cells was noticed. In contrast, CD4+ CD8+ cells were hardly affected. Moreover, splenic T-cells are resistant to MeDSG-induced apoptosis, as evaluated by measuring DNA cleavage. Our findings may account for the immunosuppressive and antitumour properties of DSG which were described in a number of previous studies.     

Impact of 15-deoxyspergualin on effector cells in experimental autoimmune diseases of the nervous system in the Lewis rat.

The influence of the immunosuppressive antibiotic agent 15-deoxyspergualin (DSG) on macrophages and autoreactive T helper lymphocytes from Lewis rats was analysed in vitro and in vivo. DSG did not inhibit antigen- or mitogen-induced proliferation of encephalitogenic or neuritogenic T helper cell lines in vitro. However, the presence of DSG during in vitro activation of the T cells strongly suppressed or completely abrogated their capacity to induce encephalitis (EAE) or neuritis (EAN) after adoptive transfer to naive rats, although expression of activation markers or adhesion molecules on the T line blasts was not down-regulated by DSG. Like activation-induced T cell proliferation, IL-2-dependent growth of CD4+ T line cells was not affected by DSG. Preincubation of CD4+ T line cells in DSG during IL-2-driven proliferation for 48 h, however, inhibited the subsequent antigen- but not mitogen-induced activation of these T cells, although neither density of T cell receptors nor other surface molecules involved in antigen recognition were lowered on the cells exposed to DSG. Similar to its effect in vitro, in vivo administration of DSG for 10 days even at a concentration with cumulative toxicity did not suppress in vitro proliferation of spleen cells induced by mitogen or a mitogenic combination of anti-CD2 antibodies. Furthermore, spleen cell and peripheral blood lymphocyte (PBL) surface antigens, particularly MHC molecules, were not altered by long-term treatment with DSG for 30 days. While there was a slight reduction in the number of polymorphonuclear cells in both populations, the proportion of the different leucocyte subpopulations remained unchanged. In contrast to the strong functional impact of DSG on autoreactive T helper cells, the drug did not inhibit the oxidative burst of macrophages or their MHC antigen expression. This study demonstrates a clear inhibitory effect of DSG on CD4+ T lymphocytes, but not macrophages. It provides an explanation for recent observations of a strong immunosuppressive in vivo effect of DSG on transplantation rejection and experimental autoimmune diseases, despite a normal mitogen response of T cells exposed to DSG in vivo and in vitro.     

白血病细胞相关的免疫抑制活性对正常人IL－2及CD抗原的作用

６５例白血病患者的细胞制备物（白细胞培养上清与溶解物）测定对正常人ＰＢＬ的ＩＬ－２产生、ＩＬ－２反应性及ＣＤ４、ＣＤ８、ＣＤ２５抗原表达的影响，并探讨与抑制淋转和ＭＬＲ的关系。结果表明制备物对ＩＬ－２产生及／或ＩＬ－２反应性的抑制是抑制淋转与ＭＬＲ的主要因素。抑制ＩＬ－２产生及／或ＩＬ－２反应者对正常人ＰＤＬ的ＣＤ４表达有明显抑制，但对ＣＤ８、ＣＤ２５表达未见影响，提示来自白血病细胞的免疫抑制是通过抑制淋巴细胞ＣＤ４表达、ＩＬ－２产生及／或ＩＬ－２反应起作用的。     

Immunosuppression of triptolide and its effect on skin allograft survival.

In this study the immunosuppressive properties of triptolide were evaluated. Triptolide was found to inhibit skin allograft rejection in a dose-dependent manner. This inhibitory effect was time dependent. Triptolide at 0.1 mg/kg/day significantly prolonged the graft survival when triptolide was given for 9 days after transplantation, but not before transplantation. In vitro studies showed that triptolide markedly suppressed cytotoxic T-lymphocyte (CTL) induction and mixed lymphocyte reaction (MLR) at concentrations ranging from 0.08 to 10 ng/ml. The inhibition on MLR was also significant when triptolide was added to the cultures at 36 h after initial incubation. Furthermore, exogenous IL-2 did not reverse this inhibitory effect of triptolide. Our results suggest that triptolide inhibits lymphocyte activation at a relatively late stage, and its effect on immune response is not exerted through altering IL-2 production.     

Deoxyspergualin preferentially inhibits the growth and maturation of anti-CD40-activated surface IgD+ B lymphocytes

Deoxyspergualin (DSG), an analogue of spermidin, is a potent immunosuppressive drug with an action quite distinct from that of cyclosporin, rapamycin, or FK506. In this study we investigated the effect of DSG and methyldeoxyspergualin (MeDSG) on the proliferation and differentiation of human B cells stimulated with anti-CD40 MoAb. Highly purified B cells obtained from tonsillar samples were used as target cells. Both agents inhibited the proliferative response of anti-CD40-stimulated B cells in the absence and presence of IL-4, IL-2 or IL-10 in a dose-dependent manner. This inhibitory effect differed markedly among cell populations based on surface IgD expression: strong inhibition of sIgD⁺ B cells but little inhibition of sIgD⁻ B cells. The drugs also suppressed the production of IgG, IgM and IgA by unfractionated B cells, which suggests that DSG acts against post-switch (sIgD⁻) B cells. Although the drugs suppressed immunoglobulin synthesis by both sIgD⁺ and sIgD⁻ B cells, the effect was more marked in the sIgD⁺ B cells. Analysis of the subclass of IgG secreted by slgD⁺ B cells revealed a decline in IgG1 and IgG3 in the presence of DSG. These results suggest that DSG preferentially inhibits the growth and maturation of sIgD⁺ naive B cells.     

A soluble 'anchorminus' interleukin 2 receptor suppresses in vitro interleukin 2-mediated immune responses

The immunosuppressive effects of a recombinant soluble IL-2 receptor L chain (s-IL-2R) were analyzed. S-IL-2R protein was obtained from the conditioned medium of L cells transfected with a mutant cDNA clone encoding the extracytoplasmic portion of the IL-2 receptor (IL-2R) and was purified to homogeneity by an IL-2-coupled sepharose column, following by reverse phase chromatography (HPLC). Soluble IL-2R protein thus prepared retained the ability to bind IL-2 specifically and suppressed the in vitro IL-2-mediated immune responses, including proliferation of IL-2-dependent cell line (CTLL-2), induction of secondary cytotoxic T lymphocytes (CTL) and the mixed lymphocyte reaction (MLR), but did not suppress the growth of IL-3-dependent cell line. Kinetic studies revealed that s-IL-2R exhibited the suppressive effects on the proliferative responses of alloantigen stimulated human tonsillar cells, only when added at an early stage, namely 0-48 h after culture onset, whereas cyclosporin A (CsA) exhibited an inhibitory effect only when added at between 0 and 24 h. This implies that s-IL-2R exerts its effect on an early stage of lymphocyte activation. The observed immunosuppressive effects of s-IL-2R suggest the possibility that s-IL-2R might be useful for the protection of rejection crisis in organ transplantation.     

Human suppressor T cells induced in vitro with an autologous renal allograft-derived T cell line. I. Suppressor cell induction, function, and specificity

The functional characteristics of T suppressor (Ts) cells generated from the peripheral blood lymphocytes (PBL) of a kidney transplant recipient who had excellent graft function for 1 year were examined. Ts cells were induced by co-culture of PBL with an autologous alloreactive cytotoxic T lymphocyte (CTL) line (EE-1) previously grown from a routine renal allograft biopsy of this patient performed 10 days posttransplant. The EE-1 line included CD3+ T cells of CD8+ and CD4+ phenotypes with cytotoxic specificity for disparate class 1 (HLA-B8) and class II (HLA-DR1 and 3) antigens of the kidney donor (JC). The EE-1 induced Ts cell lines (designated TsEE) were found to significantly suppress (50%-95%) autologous fresh responder EE-PBL stimulation by donor EBV-transformed cells (JC-EBV) in mixed lymphocyte reaction (MLR) assay. TsEE cells were CD3+ (98%) and predominantly CD8+ (68-80%), showed no cytotoxic activity, and were suppressive only at the early phase of MLR stimulation. In three-party cell test MLR assays, TsEE-mediated suppression appeared restricted to responder cells sharing HLA-B7 with the suppressor line, and was not abrogated by the addition of exogenous interleukin-2 (IL-2). TsEE cells also showed restricted suppression of CTL generation but not mature CTL activity. The restricted suppressor activity of TsEE lines was dependent upon their induction and restimulation with the autologous EE-1 line.     

Direct effect of cyclosporin A on CD8+ T lymphocytes in a primary mixed lymphocyte reaction

Cyclosporin A (CsA) blocks the development of cytotoxic T lymphocytes (CTL) in a primary mixed lymphocyte reaction (MLR) when added during the first few days of culture. We found that addition of recombinant IL-2 (rIL-2) or an IL-2-containing supernatant (2 degrees MLR SN) either alone or in combination was unable to reverse the suppression. Purified CD8+ responder cells were inhibited as effectively by CsA as unfractionated cells, suggesting that CsA has a direct, lymphokine-independent effect on CD8+ cells. The frequency of CTL precursors (CTLp) responding in a primary MLR was measured by limiting dilution in the presence and absence of CsA. Using either unfractionated cells or cell-sorter-purified CD8+ responder cells, no suppression was seen at very low cell numbers but increased markedly as the cell number per culture increased. These results imply that either a CD8+ regulatory/suppressor cell is being diluted out at low cell numbers, or that low cell-density culture conditions might override the inhibitory effects of CsA.     

Stromal cells from term fetal membrane are highly suppressive in allogeneic settings in vitro

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have immunosuppressive properties and have been used to treat steroid-refractory acute graft-versus-host disease (GVHD) in stem cell transplant patients. Cells with similar capacities can also be found in term placental tissue. We have isolated stromal cells from term fetal membrane (FMSCs), umbilical cords (UCSCs) and placental villi (PVSCs) as well as from bone marrow and compared their immunoregulatory capacity in allogeneic settings. We found that FMSCs and UCSCs suppressed proliferation significantly in mixed lymphocyte reactions (MLRs), whereas PVSCs showed inconsistent suppressive effects. When added to MLR cultures, FMSCs suppressed the production of interferon (IFN)-γ and interleukin (IL)-17, whereas UCSCs and PVSCs promoted the production of IL-17 instead. Secretion of IL-10 was increased after addition of FMSCs and UCSCs. In this setting, BM-MSCs had no significant effect on secretion of IFN-γ, IL-17 or IL-10 in MLR cultures. When analysing the expression of adhesion markers, we noted that FMSCs expressed the highest levels of CD29 (β1), CD49d (α4) and CD54 (ICAM-1) compared to the other types of stromal cells. Thus, our data indicate that stromal cells isolated from term fetal membrane have great immunosuppressive capacity in terms of proliferation and production of proinflammatory cytokines from alloreactive T cells, and also promote anti-inflammatory IL-10. They express high levels of integrins that may be of importance in homing to inflamed tissues. Fetal membrane may provide a valuable source of cells with immunosuppressive properties and could possibly be used for treatment of acute GVHD and other inflammatory disorders.     

Opposing effect of mesenchymal stem cells on Th1 and Th17 cell polarization according to the state of CD4+ T cell activation

Mesenchymal stem cells (MSCs) are multipotent progenitors with broad immunosuppressive properties. However, their therapeutic use in autoimmune disease models has shown dissimilar effects when applied at different stages of disease. We therefore investigated the effect of the addition of MSCs on the differentiation of Th1, Treg and Th17 cells in vitro, at different states of CD4(+) T cell activation. CD4(+) T lymphocytes purified by negative selection from mouse C57BL/6 splenocytes were cultured under Th1, Th17 and Treg inducing conditions with IL-12, TGF-β+IL-6 or TGF-β, respectively. C57BL/6 bone marrow derived MSCs were added to CD4(+) T cell cultures at day 0 or after 3 days of T cell polarizing activation. Intracellular cytokines for Th1, Th17 and Treg cells were quantitated at day 6 by flow cytometry. While early addition (day 0) of MSCs suppressed all CD4(+) T cell lineages, addition at day 3 only decreased IFN-γ production by Th1 polarized cells by 64% (p<0.05) while markedly increased IL-17 production by Th17 polarized cells by 50% (p<0.05) and left IL-10 production by Treg polarized cells unchanged. MSCs exhibit their typical suppressive phenotype when added early to cell cultures in the presence of CD4(+) T cell polarizing stimuli. However, once T cell activation has occurred, MSCs show an opposite stimulating effect on Th17 cells, while leaving Treg IL-10 producing cells unchanged. These results suggest that the therapeutic use of MSCs in vivo might exert opposing effects on disease activity, according to the time of therapeutic application and the level of effector T cell activation.     

Modulation of the Functions of Dengue Virus-Specific Human CD8+ Cytotoxic T Cell Clone by IL-2, IL-7 and IFNγ

Lymphokines play an important role in immune responses to viruses by modulating functions of T lymphocytes. In the present study, we examined the effects of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), and interferon gamma (IFNγ) on proliferation, cytotoxic activity and lymphokine production of a dengue virus-specific CD8⁺ human cytotoxic T lymphocyte (CTL) clone. IL-2 and IL-7 induced proliferation of the CD8+ CTL clone in the presence or absence of specific antigen, while IFNγ suppressed proliferation of the clone. IL-7 and IFNγ augmented dengue virus-specific cytotoxic activity without inducing non-specific cytotoxic activity, and IL-2 induced non-specific cytotoxic activity. IL-2 induced IFNγ production by the CD8+ CTL clone. IL-4 and IL-6 did not modulate the functions of the CD8+ CTL clone in these experimental conditions.

These results suggest that functions of dengue virus-specific CD8+ CTL are modulated by IL-2, IL-7 and IFNγ, and that IL-7 is a lymphokine useful to induce growth and to maintain specific cytotoxic activity of CD8+ CTL clones in vitro.     

Quantitative measurement of T-lymphocyte activation by an enzyme-linked immunosorbent assay (ELISA) detecting interleukin-2 receptor expression

A monoclonal antibody prepared against the murine interleukin-2 receptor (IL-2R) was employed to develop an ELISA method for measuring the immunological activation of T-cells. The assay detects an increase in IL-2R expression on activated lymphocytes. Stimulated splenic lymphocytes displayed markedly higher IL-2R expression compared to unstimulated controls. A significant increase in IL-2R expression on lymphocytes was detected in mitogen-stimulated responses, in a one-way mixed leukocyte reaction (MLR) and in the antigen-specific responses to conalbumin and purified protein derivative (PPD) in vitro. At a constant cell number, the level of IL-2R expression was found to be dependent on the dose of the stimulant. A comparative study of the kinetics of activation of splenic lymphocytes in response to mitogen, antigen and allogeneic cells as measured by the IL-2R ELISA and the conventional tritiated thymidine (3HTdR) uptake assay revealed remarkable similarity. For both assays, the mitogenic response was detected within 12 h and peaked at 72 h, the MLR was detectable within 2-3 days and peaked at day 6, and the specific antigenic response was detected within 2 days and peaked on day 4-5. Hydroxyurea, an inhibitor of DNA synthesis, had no effect on early IL-2R expression by mitogen-stimulated splenic lymphocytes, however, only 20% of maximum IL-2R expression could be detected at later stages of incubation. In contrast, cycloheximide, an inhibitor of protein synthesis, completely abrogated IL-2R expression and proliferation of stimulated lymphocytes.     

Effects of interleukin-1 alpha and cyclosporin A in vivo and in vitro on bone and lymphoid tissues in mice

The purpose of this study was to investigate the effects of interleukin-1 alpha (IL-1 alpha) infusion and the ability of cyclosporin A (CYA) to alter IL-1 alpha-induced effects on bone in vivo and in vitro and lymphoid organs in vivo. Mice were administered: IL-1 alpha (2, 4, or 6 days), CYA (6 days), or IL-1 alpha and CYA (6 days). Hypercalcemia was induced in mice treated with IL-1 alpha compared to controls and CYA treated mice, and decreased urinary calcium excretion was present in IL-1 alpha and CYA groups. Osteoclastic bone resorption was increased with a resultant loss of total bone area and bone formation (as measured by mineral apposition rate) was decreased in mice infused with IL-1 alpha. Although CYA-treatment increased bone formation as compared to IL-1 alpha-treatment; CYA in combination with IL-1 alpha did not alter the reduction in mineral apposition rate caused by IL-1 alpha, IL-1 alpha also stimulated bone resorption in vitro which was significantly inhibited by cyclosporin A. IL-1 alpha-induced splenic granulopoiesis, peripheral blood neutrophilia, thymic atrophy, and lymphoid hyperplasia in lymph nodes. CYA-treatment resulted histologically in a severe depletion of lymphocytes in the thymus, a moderate depletion of lymphocytes in lymph nodes but no difference in the histology of the spleen compared to controls. In summary, interleukin-1 alpha was effective in stimulating hypercalcemia and bone resorption both in vivo and in vitro but cyclosporin A was effective in inhibiting IL-1 alpha-mediated bone resorption only in vitro. IL-1 alpha also had marked effects on spleen, thymus, and circulating blood cells; however, most parameters were not affected by the concurrent administration of cyclosporin A.     

Possible mechanisms of immunotherapy for maintaining pregnancy in recurrent spontaneous aborters: analysis of anti-idiotypic antibodies directed against autologous T-cell receptors

We examined whether immunotherapy for recurrent spontaneous abortion (RSA) using paternal lymphocytes induces anti-T-cell receptor (TCR) idiotypic antibodies in RSA patients. The sera of these patients were assessed for inhibitory activity against mixed lymphocyte reactions (MLR) between maternal responder cells and paternal stimulator cells. Sera of four of the five women who maintained pregnancy successfully after immunotherapy showed significant MLR inhibition, whereas none of the five women who had unsuccessful pregnancies showed significant MLR inhibition. These sera inhibited the MLR of autologous responder T-cells, when stimulated with lymphocytes having the same HLA-DR antigens as the patient's husband, but not when stimulated with lymphocytes having unrelated HLA-DR antigens. This MLR inhibitory activity was absorbed by autologous maternal T-lymphoblasts induced by stimulation with lymphocytes having the paternal HLA-DR type but not by those induced by stimulation with lymphocytes having other HLA-DR types. The maternal serum inhibited the proliferation of autologous T-cells, but not of non-autologous T-cells, stimulated with paternal lymphocytes. These results indicate that anti-TCR idiotypic antibodies were induced in RSA patients by immunotherapy. These antibodies may contribute to maintaining pregnancy by negatively regulating maternal T-cells directed against HLA-DR antigens of the fetus.     

Role of IL-2 and interferon in the generation of natural cytotoxic activity in influenza virus-stimulated PBL cultures: analysis with the use of prednisolone.

We have examined the role of interleukin 2, interferon-gamma and interferon-alpha in the generation of natural cytotoxic (NC) activity and cytotoxic T-lymphocyte (CTL) activity in peripheral blood lymphocyte cultures stimulated with influenza virus, using the immunosuppressive effects of prednisolone. In addition to an inhibitory effect on the generation of CTL activity, prednisolone also inhibited the generation of NC activity in a similar dose-and time-dependent manner. Prednisolone suppressed the production of interferon-gamma when it was added on the first day of culture of PBL with influenza virus. Levels of interferon-alpha were not affected. The effects of prednisolone on the generation of NC activity and CTL activity in kinetic terms were not paralleled by the effects on interferon-alpha and interferon-gamma production. The diminished generation of NC activity could be reversed by the addition of interleukin 2 (IL-2), but interferon-gamma had little if any restorative effects. Interferon-alpha had no effect. These findings support the hypothesis that IL-2 is the major inducer of NC activity in CTL generation cultures. The inhibitory effect on CTL generation could only be reversed by IL-2 and not by interferon-alpha- and interferon-gamma. Thus, in the absence of IL-2, interferon-alpha and interferon-gamma cannot support the generation of CTL activity or the concomitantly induced NC activity.     

Modulation of the Functions of Dengue Virus-Specific Human CD8+ Cytotoxic T Cell Clone by IL-2, IL-7 and IFNγ

Lymphokines play an important role in immune responses to viruses by modulating functions of T lymphocytes. In the present study, we examined the effects of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), and interferon gamma (IFNγ) on proliferation, cytotoxic activity and lymphokine production of a dengue virus-specific CD8⁺ human cytotoxic T lymphocyte (CTL) clone. IL-2 and IL-7 induced proliferation of the CD8+ CTL clone in the presence or absence of specific antigen, while IFNγ suppressed proliferation of the clone. IL-7 and IFNγ augmented dengue virus-specific cytotoxic activity without inducing non-specific cytotoxic activity, and IL-2 induced non-specific cytotoxic activity. IL-2 induced IFNγ production by the CD8+ CTL clone. IL-4 and IL-6 did not modulate the functions of the CD8+ CTL clone in these experimental conditions.

These results suggest that functions of dengue virus-specific CD8+ CTL are modulated by IL-2, IL-7 and IFNγ, and that IL-7 is a lymphokine useful to induce growth and to maintain specific cytotoxic activity of CD8+ CTL clones in vitro.     

Transient regulatory T-cells: a state attained by all activated human T-cells

CD4(+)CD25(+)FOXP3(+) regulatory T-cells (T(regs)) form an important arm of the immune system responsible for suppressing untoward immune responses. T(regs) can be thymically derived or peripherally induced, even from CD4(+)CD25(-)FOXP3(-) T-cells. FOXP3 expression and in vitro suppressive activity are considered unique hallmarks of this dedicated and stable lineage of regulatory cells. Here we show that virtually all human CD4(+)CD25(-)FOXP3(-) T-cells and CD8(+)CD25(-)FOXP3(-) T-cells attain a transient FOXP3(+)CD25(+) state during activation. In this state of activation, these cells possess the classic phenotype of T(regs), in that they express similar markers and inhibit in vitro proliferation of autologous CD4(+)CD25(-) T-cells. This state is characterized by suppressed IFN-gamma production and robust TNF-alpha and IL-10 production. Interestingly, the great majority of the activated cells eventually downregulate FOXP3 expression, with a concomitant drop in suppressive ability. Our results show that, in humans, FOXP3 expression and T(reg) functionality are not exclusive features of a stable or unique lineage of T-cells but may also be a transient state attained by almost all T-cells. These results warrant caution in interpreting human studies using FOXP3 and suppressive activity as readouts and suggest that attempts to induce "T(regs)" may paradoxically result in induction of effector T-cells, unless stability is confirmed.     

Cytotoxic T Lymphocytes are the Prime Mediators of Suppression of the Mixed Lymphocyte Reaction by Alloactivated Cells

An alloactivated secondary mixed lymphocyte reaction (MLR) culture (SMC), which was suppressive in the MLR with autologous responder cells, was studied in more detail. In particular, we investigated whether the suppressive activity of this SMC was mediated by cytotoxic T cells or whether bona fide suppressor cells were involved. The SMC suppressed an MLR when the stimulator cells shared Bw57, Bw60, or Dw19 with the original stimulator. Separation of the SMC into CD4+ and CD8+ fractions demonstrated that the CD8+ fraction contained suppressive and cytotoxic activity against Bw57 or Bw60 antigens, while the CD4+ fraction contained both activities against the Dw19 specificity. The CD8+ fraction was also suppressive in the reverse MLR, while the CD4+ fraction was not. Limiting dilution analysis demonstrated that all CD8+-suppressive cultures were also cytotoxic, whereas in the CD4+ fraction both cytotoxic and non-cytotoxic suppressor cultures were found.