Suppression of murine cytotoxic T-lymphocyte induction following exposure to 7,12-dimethylbenz[A]anthracene: dysfunction of antigen recognition

Exposure of murine lymphocytes to the carcinogenic polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA) results in significant suppression of a variety of immunological parameters, including the induction of cytotoxic T-lymphocytes (CTL). This CTL suppression may be reversed in vitro by the addition of exogenous cellular activation products, including IL-2. The current study demonstrated that DMBA-induced CTL suppression occurs throughout the induction process, with the most pronounced effect occurring within 24-48 h of initiation. IL-2-mediated restoration of suppression is effective only within this critical window. These findings suggest an effect by DMBA on both the T-helper (Th) and CTL-precursor (CTLp) subsets. Alloantigen-specific CTL generated from murine thymocytes in the presence of DMBA and Th-derived factors (thus circumventing the Th) displayed an almost identical degree of CTL suppression to that of splenocytes, indicating a direct effect of DMBA on the CTLp. Antigen nonspecific, polyclonal CTL activation by the lectin leucoagglutinin or by IL-2 (LAK cells) was unaffected by chemical exposure, indicating that DMBA preferentially affects antigen-specific activation rather than the cytolytic process itself. Conversely, polyclonal CTL induced with monoclonal antibodies directed against the T-cell receptor-associated subunit CD3, in the presence of conditioned medium, was suppressed in a manner similar to antigen-mediated CTL. The CD3 receptor subunit acts as a transmembrane activation signal following binding of antigen to the specific receptor, further implicating dysfunction of antigen recognition or signal processing following DMBA exposure. This was confirmed by the observation that DMBA exposure prevents the anamnestic response of CTL memory cells following re-exposure to eliciting tumor antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Target cell specificity of immunosuppression in murine lymphocytes exposed to 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD)

Exposure of murine lymphocytes to the highly-substituted anthraquinone 1,4-bis[(2-amino-ethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD) results in a profound suppression of cytotoxic T-lymphocyte function. This immunosuppression is confined to CTL induction and cannot be restored by addition of exogenous interleukin-2 (IL-2), suggesting a defect at the level of the cytotoxic T-lymphocyte precursor (CTL_p) cell. Production of both IL-1 and IL-2 were normal, and responsiveness to IL-1 was unaltered, although lymphocytes displayed a reduced responsiveness to IL-2. Cell surface marker analysis demonstrated no changes in relative numbers of lymphoid cell subsets, implying functional rather than population changes. The ability to activate CTL_p with lectins was markedly reduced, as was CTL induction in thymocytes lacking functional T-helper cells. These data suggest that CTL suppression following in vivo or in vitro exposure to AEAD is due primarily to a direct effect on the CTL_p cell.     

Secondary cytotoxic allograft response in vitro. I. Antigenic requirements

The antigenic requirementsfor in vitro induction of secondary murine cytotoxic allograft responses were tested. The proliferative responses were assayed by the [³H]thymidine uptake technique; the generation of cytotoxic T lymphocytes (CTL) was tested in a ⁵¹Cr-cytotoxicity assay. Spleen cells from normal or alloantigen preimmunized CBA mice (H-2^k) were used as responder cells. Allogeneic x-irradiated splenic lymphocytes (normal stimulator cells) were UV light treated, heat treated or glutaradehyde fixed and subsequently tested for their capacity to induce CTL in a primary or secondary mixed lymphocyte culture (MLC). In addition allogeneic fibroblasts were tested as stimulator cells. The results obtained suggest that although they fail to trigger significant proliferative and cytotoxic T cell responses, in a primary MLC certain allogeneic stimulator cells, are able to induce strong cytotoxic T cell activity in a secondary MLC. The generation of these secondary CTL is preceded by only marginal cell proliferation.     

Trophoblast Does Not Cause the Cytotoxic T Lymphocyte Generation Due to the Lack of Ability to Stimulate lnterleukin-2 Production

ABSTRACT: Trophoblast was demonstrated to be unable to cause the production of interleukin-2 (IL-2) by allogeneic splenocytes in vitro in two ways: 1) The addition of lymphocyte-trophoblast culture-supernatant (LTC-SN) did not stimulate the proliferation of IL-2 dependent cytotoxic T lymphocyte (CTL-L cells; 2) When responder cells were cultured with heat-treated splenocytes (usually no CTL generation) an increase of CTL formation could be seen in the presence of mixed lymphocyte culture-supernatant (MLC-SN) but not of SN from the cultures in which trophoblast cells served as stimulators. In parallel, the trophoblast cells were found to be very poor stimulators of alloreactive CTL. The addition of interleukin-2-enriched media resulted in a significant amplification of trophoblast-induced CTL generation. The resulting killing lymphocytes were capable of destroying the only specific targets, did not lyse syngeneic target cells, and could not be generated in the absence of allogeneic trophoblast. The incubation of these lymphocytes with anti-Thy 1.2 monoclonal antibody in the presence of complement eliminated their killing effect. Lack of class II antigenic determinants on the surface of trophoblast and/or local immunosuppression of IL-2 production by trophoblast cells seem to be responsible for nondelivery of helper factor, which is essential for CTL production.     

Reduced lysis by CD8+ cytotoxic T cells in mixed lymphocyte reactions induced via CD4+ T cells exposed to chemically modified antigen presenting cells.

The resistance by T lymphocytes to activation by antigen (anergy) is well documented for CD4+ T-helper (Th) cells, although less is known about CD8+ cytotoxic T lymphocytes (CTL). One widely used method of inducing anergy of CD4+Th is presentation of antigen by ECDI (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide)-fixed antigen-presenting cells (APCs). We report here that in murine mixed lymphocyte reactions (MLRs), a marked reduction in detected cytotoxicity (which is mediated predominantly by CD8+ CTL) occurs on day 7 if the bulk cultures are restimulated 2 days previously with ECDI-fixed allogeneic splenocytes. No differences were seen between untreated cultures on days 5 and 7, or on day 7 of cultures to which were added unfixed allogeneic splenocytes, fixed or unfixed syngeneic splenocytes, or 'third-party' allogeneic splenocytes, 2 days previously. The effect is not mediated directly on CD8+ cells, since MLRs depleted of CD4+ cells immediately prior to exposure to fixed allogeneic splenocytes fail to show reduced lysis. On the other hand, reduced lysis did occur if CD4+ cells, purified from the MLRs on day 4, were exposed to ECDI-fixed allogeneic splenocytes and then returned to MLRs previously depleted of CD4+ cells. Moreover the effect is overcome using exogenous interleukin-2 (IL-2). We propose that CD4+ cells, restimulated by a regimen shown previously to induce their anergy, can cause a reduction in CD(8+)-mediated cytotoxicity in MLRs.     

Immunosuppression following 7,12-dimethylbenz[a]anthracene exposure in B6C3F1 mice--II. Altered cell-mediated immunity and tumor resistance

We have previously demonstrated that the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) produce a marked decrease in spleen weight, spleen and bone marrow cellularity and the number of IgM plaque forming cells generated in response to a T-dependent antigen. Exposure to DMBA, but not B[a]P, increased susceptibility to challenge with PYB6 tumor cells and Listeria monocytogenes suggesting that DMBA produces immune impairment involving cell-mediated immunity (CMI) and tumor resistance mechanisms. In this study, female B6C3F1 mice received total doses of 5, 50 and 100 micrograms DMBA/g of body weight in ten subcutaneous injections of 0.5, 5, or 10 micrograms/g over a 2 week period and CMI and tumoricidal functions were examined 3-5 days following the final injection of DMBA. DMBA exposed mice exhibited suppressed splenic cellularity (decreased 62%) and decreased numbers of resident peritoneal cells (down to 47% of control), although the proportion of T cell and T cell subsets, B cells and macrophages in spleens from exposed mice was not altered. Lymphocyte blastogenesis in response to mitogens was suppressed up to 49% with PHA, 48% with Con A and 76% with LPS. The response to alloantigens in unidirectional mixed lymphocyte culture was depressed as much as 73% following exposure to DMBA. Tumor cytolysis mediated by cytotoxic T cells (CTL) was impaired at doses of 50 and 100 micrograms DMBA/g body weight (88-95% suppressed respectively) as was natural killer cell (NK)-mediated tumor cytolysis (24% and 55% suppressed). Antibody-dependent cytotoxicity was significantly depressed in the highest exposure group. Peritoneal macrophage accumulation was decreased in DMBA-treated mice, but the macrophages present were pushed towards activation. The ability of DMBA-exposed mice to eliminate intravenously injected B16F10 tumor cells from the lungs was not impaired. Since NK- and M phi-mediated tumor cytotoxicity are thought to be primarily responsible for pulmonary elimination of B16F10 melanoma cells, the extent of NK suppression observed following DMBA exposure appeared to be insufficient to alter in vivo B16F10 pulmonary elimination. In contrast, the loss of the CTL tumoricidal response correlated with an increased frequency of tumors following challenge with PYB6 tumor cells.     

Persistent suppression of humoral and cell-mediated immunity in mice following exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene

Carcinogen-induced immunosuppression has been implicated as an epigenetic mechanism in promoting the outgrowth and metastasis of neoplastic cells. It has previously been reported that the complete carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) suppresses both humoral immunity (HI) and cell-mediated immunity (CMI) 3-5 days following exposure. Since persistent systemic immunosuppression may be more relevant in tumor outgrowth, assays quantitating HI and CMI, including those functions involved in tumor resistance, were performed 4 and 8 weeks following exposure to tumorigenic doses of DMBA. Adult B6C3F1 female mice were administered DMBA dissolved in corn oil subcutaneously at 5, 50 and 100 micrograms/g body weight in ten equal doses over 2 weeks (corn oil = vehicle control). The number of splenocytes producing IgM antibody to the T-dependent antigen, sheep erythrocytes, was suppressed up to 95% and 98% at 4 and 8 weeks, respectively. The IgG response was similarly depressed 75% and 98% at 4 and 8 weeks, respectively. Lymphoproliferation of splenocytes in response to the mitogens LPS, PHA and Con A were depressed up to 88%, 78% and 83% at 4 weeks and 63%, 63% and 67% respectively, at 8 weeks. In addition, alloantigen-induced proliferation of splenocytes in a one-way mixed lymphocyte culture was suppressed up to 90% at 8 weeks. The ability to generate cytotoxic T-lymphocytes (CTL) in vitro against P815 tumor cells was depressed at both time periods (88% and 60%, respectively) as was natural killer (NK) cell cytolysis of YAC-1 tumor targets (84% and 55%, respectively). The immunosuppression noted in these parameters was similar to that observed within 3-5 days following DMBA dosing. The persistent immunosuppression induced by the PAH carcinogen DMBA, including CTL and NK cell tumoricidal functions, may represent an important epigenetic mechanism contributing to tumor outgrowth or metastasis by this class of agents.     

The immunosuppressive compound 2-acetyl-4-tetrahydroxybutyl imidazole inhibits the allogeneic mixed lymphocyte reaction by sequestration of a recirculating subpopulation of T cells.

2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)imidazole (THI) is an immunosuppressive component of caramel food colouring that causes lymphopenia in mice and rats by an unknown mechanism. In this study we investigated some of the affects of THI on the murine immune system. Initially we showed that splenic T lymphocytes from mice treated with 50 mg/l THI in their drinking water were unable to launch a mixed lymphocyte reaction (MLR) against allogeneic stimulator cells, and had decreased and delayed interleukin-2 (IL-2) production. However, these T cells exhibited a normal proliferative response to concanavalin A (Con A), immobilized anti-CD3 monoclonal antibody (mAb) and anti-CD3 plus anti-CD28 mAb. Furthermore, the MLR response could be restored by the addition of IL-2 to the MLR culture. Homing studies using intravenous injection of fluorescence-labelled splenocytes showed that THI treatment decreased absolute numbers of labelled T and B lymphocytes in the blood and the spleen. Furthermore, these labelled cells reappeared in the blood and the spleen when mice were taken off THI, indicating that lymphocyte recirculation and splenic homing were modified reversibly by THI treatment. Cessation of THI treatment also resulted in a rapid reappearance of MLR responsiveness in the spleen, indicating that THI treatment does not functionally impair recirculating T cells. Collectively these data are compatible with the concept that a rapidly recirculating population of T cells, which produce IL-2 in an allogeneic MLR, are lost from the blood and spleen following THI treatment, and are sequestered in other, yet to be identified, tissues.     

Interleukin 5 is a differentiation factor for cytotoxic T lymphocytes

The role of IL-5 on the generation of cytotoxic T lymphocytes (CTL) was analysed using a culture system in which production of T helper cell factors was abrogated by exposure of the stimulator cells to ultraviolet irradiation. Supernatants from a T helper cell line (2.19 sup), recombinant (r) IL-2, rIL-4 and rIL-5 were then tested for the capacity to replace T cell help, on the generation of CTL. The results showed that the specific CTL response, in unseparated spleen cells, could be reconstituted by either 2.19 sup, IL-2 or IL-4. However, if the responder cells were purified in nylon wool, only 2.19 sup or rIL-4 plus rIL-5, but not each lymphokine per se, reconstituted the CTL response. Because IL-5 does not support T cell proliferation, it is suggested that IL-5 induces differentiation of immature precursors into CTL. Based on these findings and in an attempt to conciliate the conflicting views that have emerged from different reports, as to whether IL-2 by itself could support generation of CTL in purified T cells, a hypothesis is formulated, suggesting that T cell at different stages of differentiation require distinct lymphokines to acquire CTL effector function.     

Inhibition of the human allogeneic mixed lymphocyte response by cyclosporin A: relationship with the IL-12 pathway

Interleukin-12 (IL-12) is an important cytokine in the control of cell-mediated immunity. We have previously shown that endogenous IL-12 plays a role in the development of human allogeneic response. In the present study, we investigated the relationship between Cyclosporin A (CsA)-inhibitory effect and IL-12 pathway during human alloreaction in vitro. CsA addition at the sensitizing phase of primary mixed lymphocyte reaction (MLR) resulted in the inhibition of both p40 and p70 IL-12 production in a dose-dependent manner. In contrast, CsA had no effect on IL-12-receptor β1 chain (IL-12 Rβ) expression in T cells induced upon allogeneic activation. Addition of exogenous IL-12 significantly restored CsA-inhibited alloreactive cytotoxic T lymphocyte (CTL) generation and had a marginal effect on T cell proliferative response. The IL-12-induced restoration of CTL generation was IFNγ-mediated, as it was significantly altered when anti-IFNywas added. The restoration of CTL activity by exogenous IL-12 correlated with the capacity of this cytokine to partially restore granzyme B mRNA expression in alloreactive CTL. This study indicates that inhibition of IL-12 production is a novel additional mechanism for the inhibitory effect of CsA on the development of human allogeneic cytotoxic response.     

Characteristics of lymphoblasts appearing in efferent lymph in response to immunization with vaccinia virus.

Efferent lymphocytes collected from a cannulated lymphatic draining a single lymph node were studied for their cytotoxic activity following the injection of live vaccinia virus s.c. into the drainage site of the lymph node. Three days after the injection of virus, there was a 40-fold increase in the output of lymphoblasts from the regional lymph node. However, antigen-reactive cells, presumably T-helper cells, cytotoxic T lymphocytes (CTLs) and CTL precursors, were first detectable in efferent lymph during the fifth day after injection of virus. After a secondary challenge with virus, both lymphoblasts and antigen-reactive lymphocytes appeared earlier in efferent lymph, but lymphoblasts were still found well before the antigen-reactive cells. Efferent lymph cells were fractionated into a blast-enriched and a blast-depleted population of cells. Antigen-proliferating cells, CTLs and CTL precursors were each found to coenrich with the lymphoblast population. These findings indicate that much of the initial lymphoblast migration from the regional lymph node into efferent lymph after immunization consists of cells that do not specifically react to the injected antigen in vitro. Previous studies using allogeneic lymphocytes as the antigen have attributed both antigen-proliferating cell and CTL activity to the small lymphocyte population. In contrast, our studies on antigen-proliferating cells, CTLs and CTL precursors, after immunization with virus, suggest that, during the first 10-12 days following immunization, these cells are large lymphoblasts rather than small lymphocytes.     

Effective anti-tumor adoptive immunotherapy: utilization of exogenous IL-2-independent cytotoxic T lymphocyte clones

To attain one of the final goals for cancer immunotherapy, cytotoxic T lymphocyte (CTL) clones were selected on the basis of exogenous IL-2 independence after limiting dilution culture from mixed lymphocyte tumor cell culture cells of FBL-3 tumor-immune spleens. About 10% of the clones could be propagated up to >5 times by weekly passages in the presence of splenic feeder and stimulating tumor cells. Two of the representative FBL-3-specific CTL clones that were able to undergo the fifth passage were expanded in large numbers for adoptive transfer by two rounds of a weekly passage with medium containing IL-2. FBL-3-specific CTL clones thus obtained showed a strong ability to eliminate the established tumors when transferred into tumor-bearing nude mice. In addition, the cells were recovered from the mouse spleen even 8 months after the transfer. The most striking differences between the CTL clones used in this experiment and those maintained conventionally in the presence of IL-2 were the abilities to produce IL-2 by themselves and the high expression level of the integrin molecule, VLA-4, that disappeared when cultured completely in the continuous presence of IL-2 in vitro during 12 weeks. In addition, concomitant with the disappearance of exogenous IL-2 independence and VLA-4 expression, the CTL clones lost their capacity to eradicate the tumor in vivo. Thus, the higher capacity of CTL clones to produce IL-2 on their own seemed to be correlated with the in vivo efficacy for tumor eradication and the long-term maintenance of their physiological profiles typical of memory T cells.     

HLA-A匹配的人胃癌细胞系诱生胃癌特异性T淋巴细胞

目的 用ＨＬＡ－Ａ匹配的人胃癌细胞系诱导对胃癌细胞具有特异性杀伤作用的Ｔ淋巴细胞。方法 分离胃癌患者及正常人外周血淋巴细胞（ＰＢＬ）,用ＨＬＡⅠ类型别已知、经照射的异体胃癌细胞系刺激,进行筛选性的混合淋巴细胞肿瘤细胞培养（ＭＬＴＣ）,初步获得ＨＬＡ－Ａ可能匹配的淋巴细胞供体,血清法测定其ＨＬＡ－Ａ、Ｂ分子确认匹配后,进一步进行ＭＬＴＣ,＾３Ｈ掺入法评估其增殖能力,直接免疫荧光法分析其细胞表型,＾５     

Secondary cytotoxic allograft responses in vitro. II. Differentiation of memory T cells into cytotoxic T lymphocytes in the absence of cell proliferation.

Murine cytotoxic T lymphocytes (CTL) were induced in "one-way" mixed lymphocyte cultures and their physical characteristics investigated by velocity sedimentation at 1 X g. The in vitro differentiation of progenitors of CTL into primary and secondary CTL was paralleled by characteristic changes in the size of the responder cells. Fractionated cells enriched for primary blast CTL reverted into clonally restricted "nonlytic" secondary T lymphocytes. Upon antigenic reexposure, these lymphocytes differentiated into secondary CTL within 18-32 h. This took place in the absence of cell proliferation and could be triggered by UV light irradiated allogeneic stimulator cells. It is suggested the different characteristics for the induction of either a primary or secondary cytotoxic T cell response reflect qualitative differences between unprimed T cells and memory T lymphocytes.     

Cytotoxic T lymphocyte recognition of a xenogeneic major histocompatibility complex antigen expressed in transgenic mice

Introduction of a porcine major histocompatability complex (MHC) class I gene (PD1) into the genome of a C57BL/10 (B10) mouse has been shown to lead to cell surface expression of the porcine MHC antigen, SLAPD1 in a transgenic mouse. The PD1 product expressed on spleen cells from the transgenic mice stimulated B10 spleen cells in a mixed lymphocyte culture to generate PD1-specific cytotoxic T lymphocytes (CTL). The CTL were PD1 specific since they lysed transgenic splenic blast cells and PD1-transfected L cells, but not B10 blasts or control L cells. The CTL were L3T4-, Lyt-2+ and their activity was partially inhibited by either anti-Lyt-2 antibody or by anti-swine MHC alloantibodies. The repertoire of responding B10 anti-transgenic CTL was assessed by examining their cross-reactivity on a series of murine allogeneic targets. The B10 anti-transgenic CTL showed some cross-reactivity on conventional allogeneic targets, but reacted strongly on a series of mutant H-2Kbm blast cells. In addition, B10 anti-B6.cH-2bm6 CTL cross-reacted extensively on the transgenic target cells. These results demonstrated that normal B10 CTL possess a repertoire specific for the products of the xenogeneic class I gene PD1, that this repertoire is cross-reactive with the conventional alloreactive CTL repertoire, and that there exists an unanticipated relationship between PD1-specific CTL and CTL specific for Kb mutant determinants.     

The relationship of lytic effects against autologous and allogeneic PHA blasts induced in human mixed lymphocyte cultures

In addition to the cytotoxic T lymphocytes (CTL) killer cells which lyse autologous PHA lymphoblasts are also generated in mixed lymphocyte cultures (MLC). By modifying various tissue culture conditions, we demonstrate that the generation of allospecific effectors can be reduced but not the lytic activity against autologous PHA lymphoblasts. Cold target inhibition experiments showed a cross reaction between autologous and allogeneic PHA lymphoblasts. No inhibition was seen with fresh lymphocytes. It seems, therefore, that the measured lytic activity of specific CTL generated in a MLC is in fact partially mediated by cytotoxic cells which kill also autologous PHA lymphoblasts.     

Segregation of mechanisms for cytotoxic T lymphocyte killing between lungs and regional lymph nodes subsequent to intratracheal delivery of adenovirus 2 vector

Recombinant adenovirus (Ad) vectors, highly effective for targeting the respiratory epithelium, have been investigated for proposed application in mucosal immunization. For rendering successful use of Ad vectors, it is imperative to understand the host immune responses in affected organs. We investigated the mechanisms of cytotoxic T lymphocyte (CTL) killing following intratracheal instillation with recombinant Ad2/betagal-2 vector. From the analysis of CTL responses, it became apparent that the lung CTLs were more Fas-dependent, whereas pulmonary lymph nodes (LN) and splenic CTLs were more perforin-dependent. Although there was a segregation in the mode of CTL killing, both mechanisms of cytolysis were used in the described tissues, and the observed dominance in CTL killing was maintained irrespective of the target evaluated. Restimulation of LN and spleen cells did not change dominance in the CTL mechanism utilized. Absence or blockage of perforin or Fas did not result in reciprocal compensation by the other effector mechanism except in Fas ligand-deficient LN and spleens. In vitro restimulation of immune lymphocytes from each mouse group tested showed segregation in the types of cytokines generated. Ad2-restimulated cells showed bias toward IL-2 and IFN-gamma, while betagal-restimulated cells showed bias toward IL-4 and IL-10.     

Allospecific proliferative human T-cell clones acquire the cytotoxic effector function after three months in culture, in IL-2 conditioned medium

Allostimulated T lymphocytes were cloned by micromanipulation and expanded in IL-2 conditioned medium. Three T3+,T4+,T8-, clones called BJ1, BJ4, and BJ37, were extensively studied. The BJ1 cells were able to proliferate and kill the specific target. The BJ4 and BJ37 cells were able to proliferate with the specific restimulator but could not kill even in lectin-dependent cell-mediated cytotoxic assay; however, they acquired the specific cytolytic activity in the 6-day culture when fresh irradiated autologous peripheral blood mononuclear cells as feeder cells were added to the specific irradiated Epstein-Barr virus transformed cell line, in the presence of recombinant IL-2. This observation strongly suggested that the culture conditions could be involved in the differentiation of proliferative clones into cytotoxic T lymphocyte (CTL) clones, by the lymphokines, either present in the IL-2 conditioned medium or secreted by the mixed allogeneic irradiated feeder cells. Moreover, it was shown that the acquisition of the cytolytic function could be blocked by the monoclonal antibody LeoA1, previously described and which recognized the TLiSA1 structure involved in the CTL differentiation.     

Interleukin-1 synthesis and activity in aged mice

Recent studies have provided evidence that deficient interleukin-2 (IL-2) production by helper T cells contributes to the impaired T-cell-mediated functions observed in aged mice. Since most of these responses depend upon the presence of macrophages, a deficit in the functional capacity or in cell cooperation of macrophages may result in a decrease in immune reactivity. We found in the present study, that in vitro the cytostatic activity of macrophages from aged C57BL/6 (B6) mice is affected only slightly, but that in vivo their number increases with age. The synthesis of IL-1 is reduced when macrophages from aged mice are stimulated in vitro by lipopolysaccharide, but addition of exogenous IL-1 apparently does not restore either the mixed lymphocyte reaction or cytotoxic T lymphocyte generation. Co-cultures of young splenic macrophages with aged T lymphocytes do not restore to normal level the impaired proliferative response to T mitogens of aged B6 mice, but aged splenic macrophages provide a full accessory help for mitogenesis of young T cells. Thus, absorption of IL-1 by phytohemagglutinin-activated T cells is slightly altered in aged mice. IL-2 responsive T cells are not altered since exogenous IL-2 supply in vitro completely reconstitutes cytotoxic T lymphocyte generation after an allogeneic stimulation. Moreover, the number of Lyt 1⁺ cells is not modified in aged B6 mice. These results suggest that the impaired capacity of macrophages to release IL-1 and of blast T cells to bind IL-1 may contribute to the depression of cell-mediated immune reactivity associated with aging but also that the main defect is a functional lesion of IL-2 production by Lyt 1⁺ helper T cells.