Inhibitory Effects of Anti-HLA-A, B, C Heavy Chain and Anti-β2 Microglobulin Monoclonal Antibodies on Alloantigen and Microbial Antigen-Induced Immune Responses in Vitro

The role of HLA class I subunits in class II-restricted immune responses was investigated by means of a panel of monoclonal antibodies (MoAb) recognizing HLA-A,B,C heavy chain and different beta 2 microglobulin (beta 2m) epitopes. MoAb against either class I subunit strongly inhibited mixed lymphocyte cultures, generation of cytotoxic T lymphocyte cultures, generation of cytotoxic T lymphocytes or natural killer-like activity, and lymphoproliferation in response to soluble or particulate microbial antigens derived from Candida albicans. In general, anti-beta 2m MoAb were more efficient inhibitors than anti-HLA-A,B,C heavy chain MoAb. The inhibitory effects were specific, in that the parental myeloma ascitic fluid or a low-affinity MoAb against beta 2m, or MoAb directed against non-HLA surface structures did not affect any of the immune responses studied. The MoAb-induced inhibition could not be attributed to nonspecific toxic effects, since PHA-induced blastogenesis and IL-2-dependent proliferation of mixed lymphocyte culture (MLC) blasts were not inhibited. Furthermore, exogenous IL-2 did not reverse the block of MLC and microbial antigen-induced proliferative responses by MoAb. Taken together, these data suggest an involvement of both subunits of class I antigens in class II-restricted immune responses.     

Interleukin 10 inhibits allogeneic proliferative and cytotoxic T cell responses generated in primary mixed lymphocyte cultures.

The effects of IL-10 on the generation of alloreactivity in primary mixed lymphocyte cultures (MLCs) were investigated. IL-10 inhibited in a dose-dependent fashion the alloantigen-induced proliferative responses. The suppressive effect was maximal when IL-10 was added at the beginning of the cultures, suggesting that it acts on the early stages of T cell activation. The proliferative responses were enhanced in the presence of a neutralizing anti-IL-10 mAb, indicating that endogenously produced IL-10 suppresses proliferation in primary MLC. The inhibitory effects of IL-10 were observed irrespective of whether irradiated allogeneic peripheral blood mononuclear cells, purified monocytes or freshly isolated B cells were used as stimulator cells. The proliferation of both the CD4+ and CD8+ T cell subsets was inhibited to a similar extent. The reduced proliferative responses were only minimally restored by high concentrations of exogenous IL-2, indicating that the effects of IL-10 are not exclusively due to inhibition of IL-2 synthesis. Furthermore, the production of IL-2, interferon (IFN)-gamma, IL-6, granulocyte macrophage colony stimulating factor, and tumor necrosis factor-alpha in primary MLCs was diminished by IL-10 and enhanced in the presence of anti-IL-10 mAb. The strongest effects were observed on the production of IFN-gamma. Although IL-10 reduces the proliferative responses, the ratios of CD3+CD4+ and CD3+CD8+ T cells remained the same in IL-10 treated and control cultures, yet the percentages of activated CD3+ T cells, as judged by CD25 and HLA-DR expression, were consistently reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

TGF beta down-regulates TLiSA1 expression and inhibits the differentiation of precursor lymphocytes into CTL and LAK cells.

This study analysed the regulatory effects of transforming growth factor beta (TGF beta) on the expression of a 70,000 MW cell surface activation antigen, TLiSA1, involved in the differentiation of cytotoxic T lymphocytes (CTL) and lymphokine-activated killer (LAK) cells from their precursor(s), and also examined the role of TGF beta in the generation of these functional cells. TGF beta was shown to suppress the expression of TLiSA1 and to inhibit, in a dose-dependent manner, the generation of both CTL and LAK cells when present from the beginning of mixed lymphocyte culture; the same inhibitory effect upon the development of cytotoxic effector cells was observed with a monoclonal antibody and with monospecific rabbit antibodies against the TLiSA1 protein. Antibody to TGF beta reversed the inhibitory effect of the cytokine on differentiation and on TLiSA1 expression. Exogenous IL-2 or, to a lesser extent, tumour necrosis factor alpha (TNF alpha) added to mixed lymphocyte cultures (MLC) augmented both TLiSA1 antigen expression and cytotoxic function by the resulting blast cells; the co-addition of TGF beta inhibited both of these cytokine-mediated effects. Similarly, it was shown that phytohaemagglutini (PHA)-induced lymphoblasts up-regulate their surface expression of TLiSA1 and exhibit increased LAK activity in response to IL-2, and TGF beta inhibited both of these events; this IL-2-induced increase in LAK cell function was also inhibited by antibodies to TLiSA1. It is suggested that TLiSA1 antigen expression is intimately linked to the differentiation of cytotoxic effector cells and that such differentiation may be a distinct process from IL-2-induced proliferation, although both events can be regulated by TGF-beta.     

Increase in proliferation and cytotoxic cell development in human mixed lymphocyte cultures in the presence of very low concentrations of LPS: Role of IL-1 and prostaglandin E2

Lipopolysaccharide (LPS) added to human allogeneic mixed lymphocyte cultures (MLC), even at very low concentrations, increased the level of specific cytotoxicity that developed. Proliferation was also increased by LPS in MLC but no increase was detectable when the allogeneic stimulus was absent. LPS enhanced only low cytotoxic responses while having little effect on naturally high responses. Significant enhancement in cytotoxic response was found within the picogram-nanogram per milliliter range of concentrations of LPS and only when it was added at the initiation of cultures. This early action of low concentrations of LPS suggested that IL-1 was involved. Indeed, a supernatant from silica-treated human mononuclear cells containing IL-1 activity also enhanced cytotoxic and proliferative responses. Aside from increasing IL-1 secretion we also found that LPS significantly increased synthesis and secretion of PGE₂ which had a selective inhibitory effect. Namely, addition of indomethacin or flurbiprofen to MLC further enhanced the cytotoxicity of LPS-treated but not that of untreated cultures without increasing the proliferative response. These results suggest a key role for macrophage-derived IL-1 and PGE₂ in the regulation of proliferative and cytotoxic responses of T cells. They also suggest that very low amount of LPS may reach the immune system and contribute to the expression of cell-mediated immune responses.     

Human colostrum contains an activity that inhibits the production of IL-2.

The effect of human colostrum on T cell immune function was investigated. Colostrum inhibited the proliferation of human T cells activated by allogeneic, concanavalin A (Con A) or phytohaemagglutinin (PHA) stimulation. Colostrum also inhibited the production of IL-2 by Con A-activated human peripheral blood T cells and by Con A-activated Jurkat cells, a human T lymphoma line. Similarly, human colostrum inhibited the production of IL-2 by EL4 cells, a murine thymoma line, when stimulated with phorbol myristate acetate. The inhibitory activity was not cytotoxic and could not be neutralized by antibody to transforming human growth factor beta.     

Rheumatoid arthritis synovial fluid enhances T cell effector functions.

Rheumatoid arthritis is a chronic autoimmune joint disease of unknown etiology. T cells are believed to be important in the pathogenesis of rheumatoid arthritis since they infiltrate the joints and express several activation markers, such as MHC class II and IL-2R. In this study we have elucidated the effect on freshly isolated T cells of rheumatoid arthritis synovial fluid (RA-SF), which contains in vivo produced cytokines and enzymes. The mouse mixed lymphocyte culture (MLC) has been used as a model and specific cytotoxicity was evaluated against 51Cr-labelled sensitive target cells. Studies have shown that RA-SF contains a B cell differentiation activity that can cross-react between the human and murine species. Here we have shown that the addition of RA-SF strongly potentiates cytotoxic activity as well as lymphokine production by allogeneic activated effector T cells. The enhanced cytotoxicity induced by RA-SF was found to be due to a combined effect of increased cytotoxic T lymphocyte (CTL) precursor frequency, measured by limiting dilution analysis, and a more efficient killing on a per cell basis. Kinetic studies show that RA-SF must be added within 48 h after initiation of the MLC, otherwise the effect is lost. The target cell specificity of RA-SF was studied, using enriched CD4+ or CD8+ responder cells in the MLC. It was found that RA-SF could act directly on the CD8+ cells and potentiate their development to cytotoxic effector cells: this activity was not found when CD4+ responder cells were used instead. RA-SF could, on the other hand, greatly enhance IL-2 production by CD4+ responder cells. We suggest that B and T cell activity in RA-SF is important in the propagation of chronic inflammation in the joints of patients with rheumatoid arthritis.     

Monocyte-dependent regulation of T lymphocyte activation through CD98

CD98 is a 125 kDa heterodimer, which is strongly expressed on the surface of activated and proliferating cells. Its expression is strikingly regulated during T cell differentiation and activation, but the role of CD98 during T lymphocyte responses is not yet understood. We report here that proliferation of resting peripheral blood mononuclear cells (PBMC) induced by lectin, superantigen (SAg) or conventional antigens was blocked by anti-CD98 heavy chain (CD98hc) mAb. In contrast, anti-CD98hc did not block responses of T cell clones or lines. Anti-CD98hc inhibited IL-2 receptor expression and progression of T cells from G1 to S phase, but did not reduce expression of the IL-2 gene. Anti-CD98hc mAb did not regulate the initial activation events involving the TCR and co-receptor structures, but instead inhibited T lymphocyte responses even when added 18 h or more after the activation stimulus. Further experiments demonstrated that anti-CD98 was not directly affecting T cells in this system, but was instead acting on accessory cells. This was supported using a novel xenogeneic system that takes advantage of the lack of xenoreactivity of purified human T cells against mouse splenocytes. Despite absence of a direct xenoresponse to murine spleen cells, human T cells were activated by SAg presented by murine splenic antigen-presenting cells (APC). Murine anti-human CD98hc did not block T cell proliferation in this system. Furthermore, responses using monocyte-depleted PBMC as APC were not blocked by anti-CD98hc. Taken together, the present data suggests that triggering of human monocyte CD98 can suppress T cell proliferation by a process that halts progression through the cell cycle of recently activated T lymphocytes. This may represent a novel pathway for monocyte regulation of T cell activation.      

间充质干细胞对混合淋巴细胞反应体系中T淋巴细胞的免疫调节作用

目的:探讨间充质干细胞(MSC)在体外对T淋巴细胞免疫调节作用的特点。方法:通过Ficoll梯度密度离心法分离出正常人骨髓单个核细胞,体外培养扩增MSC,获取第3代细胞。将其按照不同的比例加入双向混合淋巴细胞培养(MLC)体系中,在第3、5天,采用MTT比色法检测各组MLC中的T淋巴细胞的增殖情况,再用流式细胞术分析其与MSC共孵育前后细胞表面标记的变化情况。结果:加入了MSC的MLC体系中,MSC对T淋巴细胞的增殖抑制具有剂量依赖性,且随着时间延长,抑制程度增强;其中,CD4 T细胞亚群受抑不如CD8 T细胞亚群显著;另外,T淋巴细胞表面CD25的表达虽然较对照组有所下降,但是CD4、CD25共表达的细胞却较对照组有明显的上升趋势。T淋巴细胞表面活化抗原HLA-DR的表达较对照组有轻度减低。结论:MSC在体外能够明显抑制T淋巴细胞的增殖,其主要是针对CD8 T细胞(CTL)。另外,它还能下调活化T淋巴细胞上一些较特异的表面标记CD25和HLA-DR的表达。     

Production of interferon in the murine mixed lymphocyte culture. II. Interferon production is a T cell-dependent function, independent of proliferation.

Interferon production occurs after two days of culture in murine mixed lymphocyte cultures (MLC). This was demonstrated in various combinations of mouse spleen cells differing at the major histocompatibility (H-2) locus. Interferon production could be demonstrated in one-way MLC when F1/parent combinations were used and in reactions in which one partner was treated by puromycin. After treatment of both cell populations with mitomycin C, interferon production occurred in the absence of lymphoproliferation. Interferon production in response to alloantigen did not occur in spleen cell cultures of nude mice and in cultures treated by anti-theta antiserum plus complement indicating that interferon production is a T cell-dependent function.     

Interaction of IL-1 beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) in human T cells activated by murine antigens.

We used a mixed leucocyte culture between human T cells and irradiated murine splenocytes which allowed us to distinguish between cytokine production from the responder and stimulator cells by the use of species-specific assays for mRNA up-regulation. Using this model of T cell activation by antigen, we studied the effects of human antigen-presenting cell-derived cytokines IL-1 beta, IL-6 and TNF-alpha on the activation of human T cell subsets. We show in this system that exogenously added IL-1 beta, IL-6 and TNF-alpha induces IL-2 receptor (R) up-regulation and IL-2 production, and proliferation by both CD4+ and CD8+ cells. The addition of IL-1 beta induces IL-6 mRNA, and anti-IL-1 antibodies or an IL-1R antagonist protein completely suppresses IL-6 and TNF-alpha supported proliferation. Similarly, addition of IL-6 or TNF-alpha induces up-regulation of IL-1 beta mRNA. However, anti-IL-6 and anti-IL-6R antibodies only partially block proliferation supported by IL-1 beta. These findings suggest that IL-6 and TNF-alpha will induce IL-2R up-regulation/IL-2 secretion via the induction of IL-1 beta production.     

IFN-alpha-induced modulations of the events in human mixed lymphocyte cultures

Autologous mixed lymphocyte cultures (AMC) with T-enriched subset of human blood lymphocytes as responders and B-cells or plastic adherent cells as stimulators and allogeneic mixed lymphocyte cultures (MLC) were assayed for blastogenesis and generation of cytotoxic potential. The activated cells lyzed K562 and Daudi, autologous and allogeneic PHA-blasts. The AMC population affected the autologous and allogeneic blasts at a similar strength and there was no indication for selective effects. B-Blasts induced with Staphylococcus aureus were not lyzed. The MLC populations had a stimulation-specific cytotoxic component. This was revealed by the stronger effect against the stimulator PHA-blasts and by the lysis of the stimulator B-blasts. Short-time interferon (IFN) treatment prior to the lytic assay enhanced the anti-Daudi and anti-K562 lytic activity of the AMC and MLC populations. With AMC the lytic efficiency against the autologous and allogeneic PHA-blasts were not changed while with MLC they were also elevated. This increase was confined to the non-specific component of the cytoxicity. The proliferation of lymphocytes was suppressed when interferon was added at the initiation of the mixed cultures. On a per-cell basis the cytotoxic potential of these cultures were stronger. In the MLC the stimulation-specific component increased more substantially than the effect against the non-specific targets. It is possible that the IFN-induced modification of the culture conditions such as suppression of the initial proliferation favored the growth of the specific clone. Re-exposure of these cells to another dose of interferon prior to the lytic assay had no effect on the lytic potential.     

Involvement of T11 molecules in antigen receptor-mediated T lymphocyte functions: effect of anti-T11 monoclonal antibody on functional capabilities of alloreactive T cell clones

As shown by previous studies, the sheep erythrocyte-binding T11 molecule is involved in T cell activation, as well as in mechanisms of specific allogeneic target cell lysis. In this study, we utilized two anti-T11 monoclonal antibodies (mAb) that inhibited the specific cytolytic activity of mixed lymphocyte culture (MLC)-activated T cells to analyze, at the clonal level, the involvement of T11 molecules in (a) antigen-specific vs. nonspecific mechanisms of target cell lysis, and (b) antigen-driven T cell proliferation and interleukin 2 (IL 2) production vs. IL 2-induced cell proliferation. In contrast to anti-T3 or anti-T8 mAb, antibodies to T11 molecules inhibited the cytolytic activity of MLC-derived allospecific clones in a uniform manner. In addition, anti-T11 antibodies inhibited the specific activity of cytotoxic T lymphocyte clones resistant to anti-T3 antibodies, even after antibody-induced modulation of T3 molecules (while anti-T3 mAb had no effect). Similarly, anti-T11 antibodies inhibited the alloantigen-induced proliferation and IL 2 release of alloreactive clones independent of their T4+ or T8+ phenotype. The inhibitory activity of anti-T11 antibodies appears to be confined to antigen-specific T cell functions since neither natural killer-like activity of cytotoxic T lymphocyte clones nor the IL 2-induced clonal proliferation was affected. Thus, our results indicate that T11 molecules are functionally involved in antigen recognition by T cell regardless of their function and T4/T8 phenotype. The possible mechanisms of anti-T11 antibody-mediated inhibition are discussed.     

Involvement of fibronectin and its receptor in human lymphocyte proliferation.

Adhesion between lymphocytes and antigen-presenting cells is necessary for the development of certain immune reactions. We have previously shown that fibronectin (FN) added to mixed lymphocyte cultures (MLC) can restore a decreased lymphocyte proliferation in immunocompromised individuals. Using highly purified cell populations from peripheral blood for depletion and adding back experiments we show here that exogenous FN enhanced proliferation only when allogeneic monocytes were co-cultured with responder lymphocytes. Although lymphocyte proliferation in MLC was augmented by FN, there was no preferential proliferation of any particular major lymphocyte subpopulation in cultures supplemented with FN as compared to control cultures lacking its addition. Antibody against the FN receptor (FN-R) of the beta 1 integrin family, as well as Arg-Gly-Asp containing peptide, could inhibit alloantigen-induced lymphocyte proliferation in a concentration-dependent manner. Anti-CD3-induced proliferation was inhibited by anti-FN-R antibody but not Arg-Gly-Asp peptide whereas no inhibition was seen with the phytohemagglutinin (PHA)-induced lymphocyte proliferation. This study presents further evidence that FN and its receptor (alpha 5 beta 1) are involved in the augmentation of T-cell responsiveness to proliferative stimuli.     

Murine APC Activation in the Xenogeneic MLC

Purified human T cells proliferate in response to direct and indirect presentation of human alloantigens. However, until recently it was believed that human T cells could respond only indirectly to murine xenoantigens. We recently used the mixed leucocyte culture (M LC) to demonstrate that purified human T cells proliferate in response to direct presentation of murine xenoantigens by murine antigen-presenting cells (APC) in the presence of human cytokines. We suggested that cytokines might function poorly across species barriers. In this study, we demonstrate that although proliferation occurs in the presence of exogenously added cytokines, the precursor frequency of responding human T cells is much lower in a xenogeneic than in an allogeneic MLC. We demonstrate that human T cells also proliferate in response to murine APC in the presence of murine cytokines, and murine cytokines augment the proliferative response seen in a human anti-human MLC, ruling out the possibility that an absolute cytokinc incompatibility exists between these species. We show that exogenously added human IL-1 causes maximal proliferation of human T cells in response to murine xenoantigens only when added early in the culture. We further demonstrate that murine APC preincubatcd in human rIL-1, and washed extensively, prior to use as stimulating cells, cause human T-cell proliferation without the need for exogenously added cytokines. Finally, we noted that during interactions of human T cells and murine APC, little to no IL-1 is produced, whereas after the addition of exogenous IL-1, a marked increase in the production of IL-1 is seen. These data suggest that during interactions between human T cells and murine APC, the murine cells do not receive adequate stimulation to produce sufficient costimulatory signals to allow proliferation of the human T cells.     

Distinct expression and inhibitory function of B and T lymphocyte attenuator on human T cells

B and T lymphocyte attenuator (BTLA) has been recently identified as a new inhibitory receptor of the CD28 superfamily, with similarities to cytotoxic T lymphocyte activation antigen (CTLA)-4 and programmed death (PD)-1. Engagement of BTLA on T lymphocytes can profoundly reduce the T cell receptor (TCR)-mediated activation. In this study, we generated four monoclonal antibodies (mAbs) against human BTLA. Using the produced mAb 8H9, the BTLA molecule was found to distinctly express on many subgroups of immunocytes and show a regulatory expression, which was in accordance with its unique ligand herpes virus entry mediator (HVEM) in the process of T cell activation. In addition, the expression of BTLA was increased in the CD4(+) and CD8(+) T cells of pleural fluid in lung cancer patients. Furthermore, we showed that the BTLA-induced negative signals could be triggered by mAb 7D7. Cross-linking of BTLA with mAb 7D7 suppressed T lymphocyte proliferation, downregulated the expression of T cell activation marker CD25, and inhibited the production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10.     

The glycosphingolipid globoside as a serological marker on cytolytic T lymphocyte precursors and alloantigen-responsive proliferating T lymphocytes in murine spleen

Biochemical analyses of murine lymphocytes have shown that the glycosphingolipid globoside (Glo) is present exclusively on alloantigen-stimulated murine T lymphocytes (Gruner, K. R., Van Eijk, R. V. W. and Mühlradt, P. F., Biochemistry 1981. 20: 4518). An anti-Glo antibody has now been raised in rabbits immunized with purified antigen. Most activity was recovered in the IgM fraction. The specificity of the antibody was ascertained in an enzyme-linked immunosorbent assay with purified glycosphingolipids bound to the solid phase. In antibody-dependent complement lysis experiments the anti-Glo eliminated about 20% of nylon wool-nonadherent splenic T cells of CBA/J mice. To determine the functional identity of these Glo+ cells, the effects of Glo+ cell elimination on mitogen stimulation with concanavalin A and lipopolysaccharide, as well as the effects on the mixed lymphocyte culture (MLC) reaction and cell-mediated lympholysis with mitomycin-treated DBA/2 splenocytes as stimulator cells were studied. Whereas lipopolysaccharide stimulation was not affected by elimination of Glo+ cells, there was a slight inhibitory effect on the concanavalin A stimulation, and a severe inhibition of the MLC reaction and the generation of H-2d-specific cytolytic T lymphocytes. Addition of interleukin 2 increased the MLC reaction, but interleukin 2-saturated cultures were also severely inhibited by anti-Glo and complement treatment. Combined treatment with anti-Glo and anti-Lyt-1 or anti-Lyt-2 antibodies, and determination of cytolytic T lymphocyte precursor frequencies in limiting dilution cultures after Glo+ cell elimination showed that a large proportion of T cells proliferating in a primary MLC are Lyt-1+,2+,3+Glo+, whereas in secondary MLC they are Lyt-1+,2-,3-,Glo+. Fifty % of the cytolytic T lymphocyte precursors in primary as well as secondary MLC are Glo+. The Glo marker is lost upon differentiation to cytolytic T lymphocyte effector cells. It is discussed herein that Glo is a marker for alloantigen-stimulated precursor T lymphocytes of both helper and cytolytic T cells.     

Effect of Pseudomonas aeruginosa exotoxin a on PHA-induced lymphocyte proliferation

P. aeruginosa Exotoxin A induced a dose dependent suppression of PHA-stimulated human lymphocyte proliferation. The suppressor effect was not reversed by interleukin 2 (IL-2) added to the culture medium. Expression of IL-2 receptors (IL-2R) on PHA-blasts a well as their ability for IL-2 binding were decreased by Exotoxin A. Kinetic studies of IL-2 production showed that IL-2 activity was still present in cultures of lymphocytes activated for 72 hours with PHA and Exotoxin A. In contrast, supernatants of lymphocyte cultures activated with PHA and Exotoxin A for 18 hours had low activity of IL-2. Hence, Exotoxin A probably could decrease the utilization of IL-2 due to either inhibition of IL-2R expression or suppression of their ability for IL-2 binding.     

小鼠PTA1/CD226分子参与杀伤性T细胞(CTL)的分化

目的 阐明小鼠VIA1（mFFA1）/mCD226对杀伤性T淋巴细胞（CTL）分化的影响。方法 构建小鼠PTA1-hIgFc真核表达载体，表达并纯化mPTA1融合蛋白，免疫家兔，获得抗mPTA1的多克隆抗体，并用偶联mPTA1-Fc融合蛋白的Sepharose-4B亲和层析柱纯化多克隆抗体。间接免疫荧光染色后经流式细胞术鉴定多克隆抗体的特异性。通过混合淋巴细胞培养（MLC）诱导产生CTL效应细胞，以^51Cr释放试验检测mPTA1多克隆抗体在MLC中对T淋巴细胞分化及杀伤功能的影响。结果 获得了mPTA1-hIgFc融合蛋白和针对mPTA1胞膜外区的多克隆抗体，该抗体能与转染细胞表面天然mPTA1分子结合。mPTA1多克隆抗体对MLC诱导的CTL的杀伤有明显的抑制作用，并呈剂量依赖关系，mPTA1多抗加入的时间愈早，抑制杀伤作用的效果愈明显。结论 抗小鼠PTA1的多克隆抗体可在小鼠混合淋巴细胞培养中明显抑制CTL的分化。     

Effect of Bacillus Calmette-Guérin on the in vitro generation of cytotoxic T lymphocytes. II. Role of interleukin-1-like factors and of soluble suppressor factors.

The injection of BCG vaccine in C57BL/6J mice results in the suppression of the generation of cytotoxic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC) and of mitogenic reactions to concanavalin A (Con A). Suppression is mediated by macrophage-like suppressor cells. Since previous work had indicated that suppression involved the inhibition of the production of interleukin-2 (IL-2), the effects of BCG on interleukin-1 (IL-1), a monokine required for IL-2 production, were investigated. It was found that the release of IL-1-like activity in spleen cell cultures stimulated with LPS or Con A was increased by previous BCG treatment of the cell donors. In MLC, the release of IL-1-like activity was also increased by BCG. However, the detection of IL-1-like activity in MLC supernatants was prevented by the presence of a suppressor factor. In this case, the IL-1-like activity could be separated with gel filtration from the suppressor factor which had higher molecular weight. The production of IL-1-like activity by CBA/J spleen cells, which are not suppressed by BCG, was not significantly different from that of C57BL/6J cells, which are markedly suppressed. Moreover, the addition of IL-1 to the BCG-suppressed cultures not only did not restore normal reactivity, but actually further suppressed CTL formation. It was concluded that BCG-induced suppression cannot be attributed to decreased IL-1 activity. The suppressor factor discovered during these investigations may have a role in this type of suppression.