Suppressive effects of cyclosporin A on the induction of alloreactivity in vitro and in vivo

The purpose of the present study was the investigation of the effect of cyclosporin A (CsA) on the induction of alloreactivity in vitro and in vivo. Addition of CsA to mouse mixed lymphocyte cultures (MLC) not only inhibited lymphocyte proliferation but also prevented the generation of alloreactive cytolytic lymphocytes (CL). It was necessary to add CsA within the first 3 days of a 5-day MLC in order to achieve a significant suppressive effect. Lymphocytes, after being cultured in MLC with CsA for 4 days or longer, were incapable of being activated upon re-exposure to the same alloantigens although their responses to unrelated antigens remained intact, indicating antigen specificity of the suppression induced by CsA and its long-lasting effect. Furthermore, lymphocytes from mice treated with CsA after allosensitization failed to manifest primary cytotoxicity and could not be reactivated in a secondary MLC. Finally, CsA had no effect on those CL already generated, suggesting that CsA acts upon the induction of CL rather than the effector phase.     

Cyclosporine-induced tolerance in experimental organ transplantation. Evidence of diminished donor-specific cytotoxicity relative to donor-specific proliferative response

Alloreactivity of intragraft and peripheral blood lymphocytes from tolerant canine lung allograft recipients was examined. Tolerance was induced by variable periods of treatment with cyclosporine. Analysis of effector cells from lung allografts (obtained by bronchoalveolar lavage) revealed the absence of specific cytolytic T lymphocyte (CTL) activity and the presence of a low level of cytolytic activity detected in a lectin-dependent cell-mediated cytotoxicity assay. In contrast, high levels of specific CTL activity and lectin-dependent activity were detected in cell preparations from lung allografts undergoing rejection. Tolerant recipients retained normal ability to generate specific CTL activity to third party alloantigens in mixed lymphocyte cultures (MLC) but had diminished ability to generate CTL to donor alloantigens in recipient X donor MLC. Addition of exogenous interleukin 2 to these MLC was unable to restore donor-specific CTL activity. Lymphocytes from tolerant recipients were, however, capable of generating proliferative responses and lectin-dependent cytotoxicity on exposure to donor alloantigens in MLC. Evidence presented in this report suggests that the lectin-dependent cytolytic activity generated in these MLC is mediated by lymphokine-activated killer cells. Such cells are likely to be activated by interleukin 2 released in the proliferative response. The results support the proposal that the cyclosporine-induced tolerant state is characterized by the relative inability to respond against major histocompatibility complex class I antigens in contrast to class II antigens and/or minor histocompatibility antigens since MLC-induced CTL are directed, for the most part, against class I molecules.     

Human T cell subpopulations defined by a monoclonal antibody. II. Evidence for cell cooperation in the response to alloantigens and generation of cytolytic cells

It has been shown previously that the 5/9 monoclonal antibody defines a small T cell subpopulation in human peripheral blood that includes all the cells responsible for proliferation to tetanus toxoid and to alloantigens as well as the helper cells for B cell differentiation. In the present study, human peripheral blood T cells were fractionated according to their reactivity with the 5/9 monoclonal antibody and stimulated in mixed lymphocyte culture (MLC). In spite of a strong proliferative response in MLC, 5/9+ cells generated no cytolytic activity against PHA-activated lymphocytes bearing the stimulating alloantigens (CTL activity) or against the K562 human cell line (NK activity). The precursors of these cytolytic effector cells were present in the 5/9- fraction. However, 5/9+ cells or soluble factors derived from 5/9+ cells were needed to induce 5/9- cells to respond in MLC and develop cytolytic activity. Both 5/9+ and 5/9- cell populations, upon MLC stimulation, were able to lyse L1210 mouse lymphoma cells in the presence of specific antibodies (ADCC).     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Inhibition of murine suppressor cell function by progesterone

The effects of progesterone on murine suppressor cell function generated in allogeneic MLCs were investigated. BALB/c splenic lymphocytes stimulated in vitro with C3H/He cells significantly suppressed the proliferative response of BALB/c lymphocytes in a secondary MLC. This suppression was highly specific for the sensitizing alloantigens since the suppressor cells had no effect on the proliferative response of BALB/c lymphocytes to third-party alloantigens. In addition, BALB/c lymphocytes stimulated with syngeneic cells were observed to nonspecifically suppress the MLC response to a lesser extent. One to 10 micrograms/ml progesterone added at initiation to suppressor cell generating cultures diminished the ability of both alloantigen specific and nonspecific suppressor cell populations to suppress the proliferative response of homologous lymphocytes to alloantigens. Experiments with pyrilamine, an antihistamine, which blocks cytotoxic T lymphocyte (CTL) generation, suggests that progesterone has a direct inhibitory effect on suppressor cell function independent of its ability to block CTL induction. The effects of progesterone on suppressor cells were not due to shifts in peak response time in MLC or induction of radiosensitive cells in progesterone-treated cultures. Estradiol at doses between 5 and 10 micrograms/ml, and cortisol at dose of 1 microgram/ml, also significantly inhibited suppressor cell function. These results suggest that the steroid hormone milieu within the placenta may effect the activity of allogeneic or nonspecific suppressor cell activity.     

Proliferative and cytotoxic immune functions in aging mice. IV. Effects of suppressor cell populations from aged and young mice

The changes with age in three splenic suppressor cell populations were studied in C57BL/6 mice. Allospecific Ts cells and nonspecific non-T suppressor cells were both generated in vitro in allogeneic MLC. The presence of "pre-existing" suppressor cells in fresh spleen cells from normal mice was examined. Suppressor cell activities were assayed for their ability to suppress proliferation in a fresh allogeneic MLC after treatment to prevent their own proliferation. The ability to generate both specific and nonspecific suppressor cells decreased with age, whereas pre-existing suppressor cells were detected in spleens from the majority of the aged animals but not in spleens from young animals. The decrease in suppressor cell activity was not due to any requirement for age matching between donors of suppressor and target cells. The specific and nonspecific MLC-generated suppressor cells inhibited both the proliferative response in the assay MLC and the generation of cytotoxic cells. The pre-existing suppressor cells only suppressed the proliferative response and not the generation of cytotoxic cells. The changes seen with age in these suppressor cell populations suggest that the ability to generate suppression (both allospecific and nonspecific) to newly encountered Ag declines with age, whereas a resident splenic suppressor cell population accumulates over the lifetime of the animals.     

Dissociation of differentiation and proliferation in the primary induction of cytolytic T lymphocytes by alloantigens

The requirement for DNA synthesis in the induction of cytolytic T lymphocytes (CTL) by alloantigens has been investigated. C57BL/6 splenic T cells purified by passage on nylon wool columns were stimulated in vitro in mixed leukocyte culture (MLC) and assayed for cytotoxicity against 51Cr-labeled target cells. With this system, CTL activity was detectable after 24 hr of MLC and reached high levels after 48 hr. Addition of cytosine arabinoside (ARA-C) or hydroxyurea to such cultures at concentrations that were sufficient to inhibit DNA synthesis by greater than 98% did not reduce CTL activity measured after 24 hr; however, the increase in activity that occurred between 24 and 48 hr in control cultures was strongly reduced (or abolished) by these drugs. Velocity sedimentation analysis of MLC cells activated for 48 hr in the presence of ARA-C further revealed that CTL precursor lymphocytes had enlarged into medium- to large-sized CTL under these conditions. These studies provide direct evidence that the primary induction of CTL by alloantigens can be dissociated into a differentiation step, which occurs within 24 hr in the absence of DNA synthesis and is accompanied by blast transformation, and a subsequent proliferation.     

Nonspecific suppressor cells in patients with chronic graft-vs-host disease after marrow grafting.

Forty-four human long-term survivors after marrow transplantation for aplastic anemia or hematologic malignancy were studied for the presence of circulating nonspecific suppressor cells. Twenty-two of the patients were healthy and 22 had mild to moderately severe chronic graft-vs-host disease (GVHD). Patient mononuclear cells (of donor origin) were tested for their ability to suppress the responses of lymphocytes obtained from the respective marrow donors to alloantigens in mixed leukocyte culture (MLC) and/or to concanavalin A (Con A). Tests were carried out between 199 and 2393 (median 376) days after transplantation. Cells from only 1 of 22 patients without chronic GVHD showed suppression of donor cell blastogeneis responses. In contrast, cells from 11 of 22 patients with chronic GVHD showed more than 30% suppression of donor cell responses in MLC and/or to Con A. The finding of suppressor cells was not related to the time of testing after grafting nor to immmunosuppressive therapy. Nonspecific suppressor activity was abrogated by irradiation with 1600 rads in vitro in five of six cases tested. Nonspecific suppressor cells may be one explanation for the severe combined immunodeficiency and the recurrent infectious complications characteristic of patients with chronic GVHD.     

Immunomodulating activity of p-hydroxyphenyllactic and ascorbic acids

p-Hydroxyphenyl lactic acid (PHA) in a concentration of 5 . 10(-5) M produced a significant inhibition of cell proliferation in response to alloantigens in a one-way mixed lymphocyte culture (MLC) in colonic cancer patients and in blast transformation in response to suboptimal doses of Con A. Multiple administration of ascorbic acid in an optimal concentration to the culture increased the proliferative response of lymphocytes to alloantigens and Con A. PHA and ascorbic acid did not exhibit any immunomodulating action during the use of healthy donors' lymphocytes or lymphocytes from colonic cancer patients, transformed with optimal mitogen doses. PHA did not affect the production of cytotoxic T lymphocytes in the MLC of the spleens of allogeneic mice but inhibited lymphocyte proliferation in response to alloantigens in the MLC of the spleens obtained from B6 and vitamin A deficient animals.     

Mechanism of action of cyclosporine A in vivo. II. T cell priming in vivo to alloantigen can be mediated by an IL-2-independent cyclosporine A-resistant pathway

Cyclosporine A (CsA) inhibits T lymphocyte activation in vitro by blocking at a pretranslational level the production of IL-2 and other cytokines. It is widely assumed that the effectiveness of CsA as an immunosuppressive drug is secondary to a similar mechanism of action in vivo. We have previously demonstrated that certain parameters of T cell activation in the draining popliteal lymph node in response to the injection of alloantigen in the footpad were either completely resistant or enhanced by the administration of CsA. In the present study, we have shown that the mechanism of action of CsA in vivo is identical to that seen in vitro as CsA completely suppressed the induction of IL-2 mRNA as detected in a nuclease protection assay in lymph node cells from alloantigen-primed animals. Nevertheless, T cells from CsA-treated animals appeared to have undergone both priming and differentiation. Thus, upon culture in vitro in the presence of CsA, cells from CsA-treated animals manifested a vigorous proliferative response that could not be inhibited by the addition of a large panel of anti-cytokine mAb. Furthermore, cells from CsA-treated animals demonstrated an enhanced secondary response to the priming alloantigen, which suggests that they had undergone clonal expansion in vivo. Although CTL activity was markedly suppressed in cells from CsA-treated animals, after a 36-h culture in the absence of CsA, CTL activity equivalent to that detected in cells from nontreated animals was present. Collectively, these data support the existence of an alternative IL-2-independent, CsA-resistant pathway of T cell activation/differentiation that may play a prominent role in the generation of certain T effector functions in vivo.     

FCS-activated blast T cells inhibit the in vitro response of memory CTL precursors to alloantigens by removal of factor(s) from MLC supernatant

The inhibitory effect of spleen cells, precultured in the presence of FCS, was assayed on the memory cytolytic T lymphocytes (CTL) response to alloantigens. For this, we have used in vitro conditions in which both particulate alloantigen and MLC SN are required to allow the generation of CTL. It was shown that the CTL response was totally inhibited in the presence of 5 to 7 days precultured spleen cells. This inhibitory effect was partly due to removal, by those precultured cells, of relevant factor(s) contained in the MLC SN. After velocity sedimentation at unit gravity, it was shown that the T cells able to inhibit the cytolytic response and to remove MLC SN factor(s) are found in the fractions containing the large proliferating cells. It was further demonstrated that in the presence of inhibiting cells, a significant CTL response may be obtained after addition of concentrated MLC SN. However, in this way, this inhibitory effect was not totally circumvented, which suggests that the memory CTL response is also impaired by other mechanisms.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. III. Effect of priming on precursor frequencies.

The effect of specific priming with alloantigens on the frequency of cytolytic T lymphocyte precursors (CTL-P) has been investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2b) mice primed with DBA/2 (H-2d) tumor cells or from 14-day unidirectional mixed leukocyte cultures (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2d alloantigens were estimated by limiting dilution analysis in a sensitive micro MLC system. Under these conditions, an apparent increase of 3 to 4-fold in CTL-P frequency was observed in alloimmune (as compared with normal) C57BL/6 spleen cells. Evidence was obtained suggesting that this increase was specific for the priming alloantigens. A much greater increase in CTL-P frequency (25 to 100-fold) was observed after alloimmunization of C57BL/6 spleen cells in unidirectional MLC. Under the latter conditions, 5 to 20% of the surviving splenic MLC cells could be identified operationally as CTL-P. A similar enrichment in CTL-P frequency was obtained when lymph node, peripheral blood, or thymus cells were cultured for 14 days in MLC. These studies provide direct evidence that the pool of specific CTL-P can be expanded after alloimmunization. Furthermore, the very high frequencies observed after in vitro priming indicate that this system should be particularly useful for future studies of the progeny of individual CTL-P.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. IV. High frequency of cytolytic T lymphocyte precursor cells in MLC blasts separated by velocity sedimentation

A sensitive limiting dilution microculture system was used to obtain minimal estimates of the frequency of cytolytic T lymphocyte precursor cells (CTL-P) directed against DBA/2 alloantigens, after priming of spleen cells in unidirectional mixed leukocyte cultures (MLC, C57BL/6 anti-DBA/2). The mean CTL-P frequency in day 4 to 5 MLC populations was found to be approximately 50- to 100-fold greater than the frequency in normal spleen, and up to 25% of the cells present in such MLC could be identified operationally as CTL-P. Even higher frequencies (up to 50%) of CTL-P were obtained in a population of large-sized cells separated from day 4 MLC by velocity sedimentation. Furthermore, since a strikingly quantitative correlation was observed between CTL activity and CTL-P frequency in such separated MLC populations, it is likely that mature CTL in MLC are not end cells, but can further proliferate and thus behave operationally as CTL-P.     

Analysis of cloned T cell function. I. Dissection of cloned T cell proliferative responses using cyclosporin A

CsA interferes in a specific manner with the expansion of T cell clones in that it inhibits the antigen-driven component of the proliferative responses made by cloned helper T cells, cloned conventional cytolytic T cells, and cloned helper-independent cytolytic T cells. Cloned helper T cells and helper-independent cytolytic T cells, which share the ability to proliferate when cultured with specific alloantigen, fail to proliferate when cultured with specific alloantigen, fail to proliferate in response to this stimulus in the presence of CsA (10 to 100 ng/ml). In contrast, the proliferation observed when these cells are cultured with exogenous growth factors (but not alloantigen) is little influenced by as much as 1000 ng/ml CsA. When cloned helper T cells or helper-independent cytotoxic T cells are cultured with alloantigen plus exogenous growth factor, additive or synergistic proliferation occurs. However, CsA (10 to 1000 ng/ml) blocks only the component of proliferation induced by alloantigen, and leaves the lymphokine-driven component intact. CsA has similar effects on the proliferation of cloned conventional cytolytic T cells. Thus, CsA separates cloned T cell proliferation into two components: one driven by contact with alloantigens, the other driven by contact with mitogenic lymphokines.     

Restimulation of cytolytic T-lymphocytes in long-term mixed leukocyte cultures. 1. Physical and proliferative characteristics of cytolytic T-lymphocytes restimulated at low cell density.

Some physical and proliferative characteristics of cytolytic thymus-derived lymphocytes (CTL) have been investigated in long-term mixed leukocyte cultures (MLC). Velocity sedimentation analysis of MLC cells restimulated by homologous alloantigens at low responding cell density indicated that a shift from large cycling CTL to much smaller (probably non-cycling) CTL occurred between the third and sixth day in secondary cultures. This change in physical characteristics as a function of growth phase was accompanied by a parallel change in the responsiveness of secondary MLC cells to a further alloantigenic stimulus; restimulated Day 3 secondary cells gave rise to a transient CTL response (peaking after 2–3 days) whereas the response of restimulated Day 6 secondary cells increased for 4 days and reached much higher peak levels. Repeated stimulation of MLC cells under the latter conditions led to dramatic increases in both CTL activity and viable cell number. In particular, four sequential restimulations at 7-day intervals resulted in a calculated absolute increase of approximately 500,000-fold in both parameters. Cells derived from such extensive proliferation retained their original lytic specificity and were uniquely T cells as determined by surface markers. These results raise interesting questions regarding the extent and regulation of CTL proliferation in MLC.     

Alloantigen-specific cytotoxic and suppressor T lymphocytes are derived from phenotypically distinct precursors

Although Leu-2+ (OKT8+) T cells activated in the mixed lymphocyte reaction (MLR) mediate both alloantigen-specific cytotoxicity and suppression of alloantigen-induced proliferation, it is not known whether these functions derive from a single cell type or phenotypically distinct cells. This study was undertaken to examine the alloantigen-specific cytolytic and suppressor potential of two subpopulations of Leu-2+ cells distinguishable from one another on the basis of their binding to the monoclonal antibody 9.3. Leu-2+, 9.3+ and Leu-2+, 9.3- populations were purified from peripheral blood, cultured for 7 days with autologous helper/inducer (Leu-3+) cells and allogeneic non-T cells, and reisolated before testing for cytotoxicity and suppression. All detectable alloantigen-specific cytolytic activity was confined to the Leu-2+, 9.3+ subpopulation. Killing by this subset was specific for the HLA-A and B (class I) major histocompatibility complex (MHC) antigens of the priming cell. By contrast, suppression of proliferation was mediated predominantly by the Leu-2+, 9.3- cells, and suppression by this subpopulation was specific for the HLA-DR (class II) MHC antigens of the priming cell. The development of suppression by Leu-2+, 9.3- cells was unaffected by cyclosporin A (CsA), an agent shown previously to block the development of cytolytic but not suppressor cells in MLR. Alloactivated Leu-2+, 9.3+ cells were slightly inhibitory of fresh MLR, but this effect as well as the development of cytolytic cells was completely abrogated by CsA. These results indicate that suppressor and cytolytic Leu-2+ T cells activated in MLR are derived from distinct precursors separable by antibody 9.3.     

Adherent cells (macrophages?) in tumor-bearing mice suppress MLC responses

The spleens of mice bearing transplanted methylcholanthrene (MCA)-induced fibrosarcomas (MCA-1425 and MCA-1460) were shown to contain cells capable of suppressing the generation of cytolytic T lymphocytes (CTL) in mixed leukocyte cultures (MLC). The suppressive activity was first detected 21 days after tumor transplantation. No suppression was seen with lymph node cells taken at the same time as the spleen cells. The cells responsible for the suppressive activity were adherent to nylon wool and plastic dishes and they were not lysed by anti-T-cell serum plus complement. The suppressor cells were phagocytic and were resistant to irradiation (3000 rads) in vitro. Spleen cells from tumor-bearing nude mice were as suppressive as were spleen cells from tumor-bearing conventional mice. We conclude from these findings that T cells were not involved either as inducers or as effectors of the suppression observed, although the responsible adherent cells may have exerted their effect by interacting with a T-suppressor cell population in the MLC mixtures. While spleen cells of tumor-bearing mice were suppressive when added at any time during the first 4 days of a 5-day MLC, they showed no effect on the cytotoxicity of fully differentiated CTL. Indomethacin reversed suppression, suggesting that prostaglandins may have been involved.     

Depressed cytotoxic T-cell responses in previously treated Hodgkin's and non-Hodgkin's lymphoma patients

Summary Peripheral blood lymphocytes from 62 previously treated Hodgkin's and non-Hodgkin's lymphoma patients were tested for their ability to generate cytotoxic T lymphocytes in response to stimulation with allogeneic cells in mixed leukocyte culture. In most patients, including some in long-term unmaintained remission, extremely low cytotoxic responses were generated. To test whether these patients have circulating cells that suppress autologous lymphocytes from responding to alloantigens, patients' responding cells were passaged over columns of sepharose beads conjugated with histamine-rabbit serum albumin (Hist-RSA). This procedure has been shown to remove mouse suppressor cells and Concanavalin A(ConA)-induced human suppressor cells. Passage of patients' cells, prior to allogeneic stimulation, over columns of sepharose beads conjugated with Hist-RSA but not over control RSA columns, resulted in the isolation of lymphocytes that generated increased cytotoxic responses to alloantigens in 18 of 22 patients with initially low cytotoxic responses. These results suggest that the impaired ability of treated Hodgkin's and non-Hodgkin's lymphoma patients' lymphocytes to differentiate into cytotoxic T lymphocytes is at least in part due to the presence of circulating suppressor cells that bear histamine receptors.Scholar of the Leukemia Society of America     

Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine

L-Ornithine was shown to inhibit the development of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Lymphokines were unable to reverse the suppressive effect, and cytotoxic activity was not revealed by coupling ornithine-inhibited MLC cells to target cells with phytohemagglutinin (PHA). If addition of ornithine to MLC were delayed, sensitivity of CTL to inhibition was reduced after 24 hr and lost by 48 hr. Suppression of CTL development was not due to a toxic effect. MLC washed free of ornithine after 3 days produced detectable cytolytic activity within 24 hr of secondary culture, and to the same degree as the uninhibited MLC control within 48 hr. Cytotoxic cells generated in secondary cultures were Lyt-2+, did not kill the natural killer-sensitive YAC-1 cell line, and were shown to be antigen-specific by virtue of the findings that cytolysis and cold target inhibition were observed only with cells carrying the original, inducing H-2 haplotype. Cytolysis of target cells by normal CTL effector cells was not inhibited by L-ornithine. MLC depleted of accessory cells so that CTL activation was dependent upon addition of lymphokines remained susceptible to inhibition by ornithine. Our findings indicate that in the ornithine-inhibited MLC, CTL precursors undergo clonal expansion, but their maturation is arrested at a precytolytic stage. L-Arginine and putrescine also suppressed generation of CTL in primary MLC, and cells recovered from arginine- and putrescine-inhibited MLC developed control levels of CTL within 48 hr of secondary culture. Inhibition by putrescine was observed in tissue culture medium supplemented with human serum but not with fetal calf serum, presumably due to the presence of diamine oxidase activity in fetal calf serum. Similar to ornithine, the suppressive effects of arginine and putrescine on T lymphocytes were apparently selective for CTL because they did not inhibit mitogen activation with concanavalin A or the production of interleukin 2 and interleukin 3. These findings are consistent with a hypothesis that the inhibitory effects of ornithine, arginine, and putrescine are mediated by polyamines, and exerted on the differentiative stage of CTL development.