Reversal of Con A-induced suppression by a platelet-derived factor which binds to activated suppressor T cells

A platelet-derived factor found in serum as well as in platelet releasate prepared either with calcium ionophore or with thrombin was shown to reverse Con A-induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) in vivo in (CB6)F1 mice. In addition, as shown previously, lymphoma cell-induced suppression in SJL mice was similarly reversed. The factor could be injected prior to Con A on the day before SRBC injection, or on the same day as antigen with comparable results. It also enhanced PFC responses in the absence of Con A. Suppressor cell induction by Con A in vivo, as demonstrated by assay on PFC responses of normal spleen cells in vitro, was abrogated by simultaneous injection of the platelet factor. Cells from mouse spleen and lymph node, but not from thymus could absorb the factor from human serum at 4 degrees C. The phenotype of the relevant spleen cells was L3T4-, Ly1-, Ly2+, Thy1+, Ly22+, Qa1+, Qa4+, Qa5+, and Ly6.IE+. These results suggest that this factor binds to activated peripheral T cells of the suppressor cell phenotype.     

Activation of human B lymphocytes. V. Kinetics and mechanisms of suppression of plaque-forming cell responses by concanavalin A-generated suppressor cells.

The kinetics and mechanisms of suppression of the PWM-induced PFC response of human PB lymphocytes by Con A-activated suppressor cells were investigated. It was necessary that Con A suppressor cells be present early in the process of activation of human B cells toward antibody syntheses, but maximal suppression of the PFC response occurred later in the culture period. In addition, Con A-activated cells, although suppressing the PFC response to PWM greater that 90% of control, did not significantly suppress the blastogenic response to PWM after 3 or 5 days in culture. On the contrary, after 3 days in culture, background tritiated thymidine incorporation as well as tritiated thymidine incorporation to PWM stimulation was increased when Con A suppressor cells are added to fresh autologous peripheral blood lymphocytes. This increased blastogenic response after three days most likely represented an autologous mixed lymphocyte reaction (MLR) or Con A suppressor cells against fresh autologous non-T cells. The induction of autoreactive cells may be one of several modes of suppression of PFC responses by Con A activated suppressor cells.     

Effect of concanavalin A treatment on the allogeneic response of mice to challenge with P 815 mastocytoma: interleukin 2 treatment reverses concanavalin A suppression in vivo

Mice injected repeatedly with concanavalin A (Con A) prior to and following challenge with P 815 mastocytoma are suppressed in their cell-mediated cytotoxicity responses. Earlier studies showed that pretreatment of the animals with silica to affect macrophage (M phi) functions reversed the Con A suppression. In the present paper we have shown that peritoneal exudate cells (PEC) induced/activated by ip injection of Con A were able to transfer suppression to normal mice. Separation of the PEC populations into adherent and nonadherent cells abrogated their capacity to transfer suppression. It was further shown that Con A is not functioning in this in vivo system to block effector activity of cytotoxic cells on target cells, and PEC induced with Con A were not directly cytotoxic to target P 815 cells. Finally, we were able to show that the cytotoxicity response of Con A-suppressed mice could be enhanced by treatment with concentrated culture supernatants of normal mouse spleen cells, rich in interleukin 2 (IL 2) activity. Attempts to detect a recently described mouse serum inhibitor of IL 2 in normal or Con A-treated mice were unsuccessful and spleen cells from Con A-treated mice lost their capacity to generate IL 2 in vitro when cultured under appropriate conditions. Taken together, these results suggest that suppression of cell-mediated immune responses in Con A-treated mice results from interruption of the normal generation of IL 2 helper effects necessary for the activation of cytotoxic effector T cells in vivo.     

Immunoregulation in the rat: requirements for in vitro B cell responses to classical TI-1 and TI-2 antigens

The presence of suppressor cells and their mediators has made it difficult to induce B cell mitogenic or immune responses in rat spleen cell cultures. In the present study, we have defined culture conditions required for induction of in vitro thymic independent (TI) immune responses in the rat. Rat spleen cell cultures support low responses to various trinitrophenyl (TNP) haptenated antigens including TNP-Brucella abortus (TNP-BA), TNP-lipopolysaccharide [LPS; either phenol (Ph)- or butanol (Bu)-water extracted], TNP-Ficoll, and TNP-dextran. However, all of these antigens induced good splenic anti-TNP PFC responses when given at appropriate doses in vivo. When spleen cells were depleted of adherent cells and cultured with TI antigens in vitro, good anti-TNP PFC responses were seen with TNP-BA, whereas, lower responses were induced by TNP-LPS (Ph or Bu). No responses were observed in cultures incubated with either TNP-Ficoll or TNP-dextran. Purified splenic B cell cultures [prepared by panning on plates coated with anti-rat F (ab')2] supported good responses to TNP-LPS (Ph or Bu) and TNP-BA. The addition of irradiated splenic adherent cells (macrophages, M phi) to either M phi-depleted or purified B cell cultures completely abrogated in vitro responses to TNP-BA or TNP-LPS (Ph or Bu). Purified splenic B cell cultures generally responded poorly to TNP-Ficoll or TNP-dextran. Addition of indomethacin (IM) to spleen cell cultures abrogated suppression and allowed anti-TNP PFC responses to TNP-BA, TNP-LPS (Ph or Bu), TNP-Ficoll, and TNP-dextran. Furthermore, nude spleen cell cultures treated with IM, also allowed significant TNP-Ficoll and TNP-dextran immune responses; however, untreated cultures did not respond to these antigens. Our studies indicate that rat splenic B cell cultures are responsive to TI antigens, and highest responses occur with the murine TI-1 class, e.g., TNP-BA and TNP-LPS. Inhibition of suppression with IM restored splenic B cell responses to the murine TI-2 class, i.e., TNP-Ficoll and TNP-dextran.     

Suppression of heterologous immunity by Nematospiroides dubius antigens in vitro

The direct effect of the soluble antigens in the homogenate of adult Nematospiroides dubius (AH) on spleen cells from uninfected NIH mice was investigated using a Mishell-Dutton culture system. Parasite antigens were shown to reduce the plaque-forming cell (PFC) response to sheep red blood cells (SRBC) in a dose-dependent manner in vitro. A population of suppressor cells was demonstrated in the spleens of infected mice. Furthermore naive spleen cells cultured in the presence of AH gave rise to cells which depressed the PFC response of naive cells when subsequently cultured together in vitro. Treatment of these cell populations with anti-thy 1.2 plus complement did not impair suppressor activity, and it was concluded that cells expressing the T-cell phenotype were not involved.     

Biological expressions of lymphocyte activation. IV. Concanavalin A-activated suppressor cells in mouse mixed lymphocyte reactions.

Thymus-derived lymphocytes (T cells) from mouse spleen, activated in vitro or in vivo with concanavalin A (Con A), suppress proliferative responses of syngenic lymphocytes in mixed lymphocyte reactions (MLR). Replication in vitro was not required for expression of suppressor activity by Con A-activated cells and was blocked in MLR by treating suppressor cells with mitomycin C or irradiation. Kinetics of MLR responses and viability of cultures were not altered by addition of activated suppressor cells. The data are consistent with a direct inhibitory effect of suppressor T cells on antigen-induced DNA replication. These observations extend a model previously described for regulation of antibody synthesis by Con A-activated T cells to control of cell-mediated immune responses. This model should be particularly useful in further definition of regulatory T cell subpopulations, and in investigation of interactions and relationships between such populations.     

Natural suppressor (NS) activity from murine neonatal spleen is responsive to IFN-gamma

Natural suppressor (NS) cell activity is the ability of apparently unprimed "null" cells to nonspecifically suppress immune responses. Previously we have shown that NS cell activity from the spleens of mice undergoing chronic graft-vs-host disease (GVHD) is enhanced in vitro by activated T cell signals (e.g., Con A supernatant [CAS]). Here we asked if the naturally occurring suppressor activity found in the neonatal mouse spleen is caused by NS cells, and if so whether this NS activity is also responsive to T cell signals. Finally, we wanted to identify the material in the CAS to which the NS cells respond. Spleen cells from (BALB/c X B10.D2)F1 neonates contain potent, genetically unrestricted suppressor activity toward normal mitogen responses. The cells responsible for this suppression are nonadherent, Thy-, Ig- and are thus by definition NS cells. Neonatal spleen NS cells suppress the indicator Con A response of all mouse strains tested, but their behavior with regard to LPS responses is different. They significantly inhibit the indicator LPS response of allogeneic strains, but are less inhibitory of LPS-stimulated syngeneic (BALB/c X B10.D2)F1 and parental strains. However, the addition of CAS to these latter cultures enhances the NS inhibition of the LPS response to the level of suppression seen with a Con A response. Two lymphokines were able to replace the CAS. Recombinant interferon-gamma (rIFN-gamma) closely mimics the activity found with whole CAS, with low concentrations (1 U/well) being capable of enhancing the neonatal NS activity to near-maximal levels. Recombinant interleukin 2 (rIL 2) is also capable of stimulating the neonatal NS activity to near maximum. However, the rIL 2 must be added at much higher concentrations, taking greater than 50 U/well to get maximum activation of NS suppression. The addition of anti-IFN-gamma antiserum to these LPS suppression assays removes the ability of CAS to activate the neonatal NS cells. Anti-IFN-gamma antiserum also removes the ability of rIL 2 as well as rIFN-gamma to activate the NS cells. It thus appears that the rIL 2 is working by its ability to stimulate IFN-gamma production. Anti-IFN-gamma also removes the ability of the neonatal NS cells to suppress a Con A response. Therefore, it appears that neonatal splenic NS cells respond to, and are activated by, IFN-gamma to carry out their suppressive activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective impairment of B cell function by Neisseria meningitidis

Spleen cells from CBA/J mice infected with Neisseria meningitidis displayed depressed in vitro plaque-forming cell (PFC) responses to T-dependent (sheep red blood cell; SRBC) and T-independent (TNP-LPS, TNP-Ficoll) antigens. The inhibition was observed over a wide range of antigen concentrations. The decreased responsiveness of splenocytes from infected mice was due to a selective impairment of B-cell function since helper-T-cell activity was intact in infected mice as shown by the ability of T-enriched lymphocytes to cooperate with normal B-enriched lymphocytes in the generation of an anti-SRBC response, accessory macrophage function was preserved since adherent spleen cells from bacteria-injected mice were shown to produce normal or increased levels of IL-1 and were able to cooperate with normal non-adherent spleen cells in the generation of PFC against SRBC. Addition of peritoneal cells from normal animals or extraneous IL-1 both failed to restore normal PFC responses in cultures of splenocytes from infected mice. Finally, B-enriched lymphocytes from infected mice produced poor anti-SRBC responses when cultured with either Con A supernatant or T-enriched lymphocytes from normal or infected mice. Cell-mixing experiments failed to detect the presence of suppressor cells in cultures of unfractionated spleen cells or B-enriched lymphocytes from infected mice. Therefore, the immunological unresponsiveness associated with a Neisseria meningitidis infection was attributed to a meningococcus-induced defect(s) in B-cell function. In vivo polyclonal B-cell activation leading to clonal exhaustion did not play a major role in the depression of humoral responses since meningococcal infection induced little or no polyclonal Ig secretion.     

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions

Responder cells from primary syngeneic and allogeneic one-way mixed-lymphocyte cultures (MLC) specifically inhibit the development of cytotoxic T lymphocytes (CTL) directed against the major histocompatibility complex (MHC) antigens of the MLC responder cells. This special kind of suppressor activity is known as veto suppression. Ia+ cells with veto activity obtained from H-2 recombinant mouse strains were shown to downregulate alloantigen (class II)-specific helper activity for class I-specific CTL development in a primary MLC provided that the veto cells expressed the same I-E alpha subregion as the MLC stimulator cells. The veto-induced suppression of allo-help was prevented by the addition of supernatant from concanavalin A-stimulated spleen cells (Con A-SN) and was inhibited considerably by very high amounts of recombinant interleukin-2 (IL-2). In the presence of Con A-SN, CTL precursors recognizing either the K end or the D end of the veto cell MHC were found to be inactivated. Thus, our results indicate that MLC responder cells include active veto cells expressing Ia region-encoded restriction elements for allospecific T helper cells, as well as K- or D-encoded restriction elements for allospecific T cytotoxic cells.     

Effect of concanavalin A on lymphocyte interactions involved in the antibody response to type III pneumococcal polysaccharide. II. Ability of suppressor T cells to act on both B cells and amplified T cells to limit the magnitude of the antibody response.

When administered 2 days after immunization with 0.5 microgram Type III pneumococcal polysaccharide (SSS-III), the T lymphocyte mitogen concanavalin A (Con A) stimulates a 2.6-to 7-fold enhancement of the plaque-forming cells (PFC) response to SSS-III in vivo. This enhancement requires the presence of amplified T cells, which act by driving PFC or their precursors to extra rounds of proliferation. The extra proliferation that can be stimulated by Con A is not seen in the normal primary response to SSS-III; but treatment with anti-lymphocyte serum (ALS) to remove suppressor T cells will permit the additional proliferation to occur. This indicates that in the primary response to SSS-III, suppressor T cells act on amplifier T cells to limit the magnitude of the antibody response. Only suppression of B cells can account for the further suppression induced by Con A given at the time of immunization or by low-dose paralysis of the SSS-III response. The relatively late development of amplified activity compared to suppressor activity appears to account for the absence of amplifier activity after primary immunization with SSS-III. It is apparent that one can explain the regulatory effects observed during the development of an immune response to SSS-III only by considering both T cell- B cell and T cell- T cell interactions, together with the temporal relationships involved in those interactions.     

Suppression of Con A mitogen-induced proliferation of normal spleen cells by macrophages from chickens with hereditary muscular dystrophy

Spleen cells from chickens with hereditary muscular dystrophy (MD) give low blastogenic responses to the T cell mitogen concanavalin A (Con A) while exhibiting normal mitogen stimulated blastogenic responses to the T cell mitogen phytohemagglutinin (PHA). The addition of MD spleen cells to normal spleen cells caused a marked suppression of the Con A response of the normal cells while not affecting the PHA response of the normal cells. The suppressive activity by the MD spleen cells requires viable cells and is contact mediated. The suppressive activity is attributed to the presence in MD spleens of a population of suppressor cells with characteristics typical of macrophages. The suppressor cell activity was not removable by complement-mediated lysis using anti-T or anti-B sera, but it was reversible by treatment with carrageenan or carbonyl iron magnet, by passage through a Sephadex G-10 column, and by adherence to plastic petri dishes or glass beads. MD spleen cells depleted of the suppressor cell population remained unable to respond to Con A.     

Induction of suppressor T cells of antibody formation under conditions that preferentially stimulate DTH

These studies describe the conditions under which antibody-forming cells and TDTH cells are selectively induced in vitro. TDTH cells are preferentially stimulated when high doses of antigen are included in the culture. Antibody-forming cells, on the other hand, are optimally stimulated with a 100 to 1000-fold less concentration of antigen. The conditions that optimally stimulate TDTH cells also induce a population of suppressor T cells that inhibit the antibody response. However, although their inductive requirements are similar, the suppressor T cells of antibody formation are a distinct subpopulation of cells from the TDTH cells. Whereas the suppressor T cells are LY-1-, 2+, 4-, 6+, and Ia+; the TDTH cells are Ly-1+, 2+/-, 4-, 6+, and Ia-. Furthermore, the DTH cells are sensitive to high doses of irradiation, whereas the suppressor cells are resistant. Based on the Ly phenotype and the kinetics of suppression, the suppressor T cells are not the "feedback suppressors" that have been identified in other systems. The system described in this paper provides a means whereby the cells that regulate humoral and CMI can be studied in vitro.     

Human suppressor T cells induced by concanavalin A: suppressor T cells belong to distinctive T cell subclasses.

Human peripheral blood lymphocytes were stimulated by concanavalin A (Con A) and then evaluated by their suppressive activity for thymus-derived (T) cell- and bone marrow-derived (B) cell-proliferative responses to mitogen and allogeneic cells. Con A-activated T cells markedly suppressed these responses, but Con A-activated B cells failed to demonstrate suppressor activity. Discontinuous bovine serum albumin (BSA) density gradient separation of T cells which had been activated by Con A demonstrated that a fraction containing blast cells as well as fractions containing unproliferated cells manifest the same degree of suppressor capabilities. However, when density gradient separation of T cells followed by subsequent incubation with Con A was performed, fractions of proliferating cells of low density exhibited no suppression; a fraction containing high density T cells produced marked suppression, but this fraction incorporated only little thymidine in response to Con A. Thus, these studies indicate that Con A-induced suppressor T cells belong to a distinctive subpopulation which has already been programmed to express this function before exposure to Con A and that cell proliferation may not be a prerequisite for the development of such suppressor T cells.     

Suppression of the in vitro secondary antibody response of rabbit lymphoid cells by Concanavalin A.

The effect of various concentrations of concanavalin A (Con A) on the in vitro secondary antibody response of rabbit lymph node and spleen cells to sheep red blood cells (SRBC) was studied. Complete suppression of the IgM plaque-forming cell (PFC) response of both lymph node and spleen cultures was observed when 10 mug/ml of Con A was added at the time of initiation of the cultures whereas only partial suppression was observed when 1 mug/ml of Con A was added. Moreover, marked suppression of the immune responses of both spleen and lymph node cultures was observed when 10 mug/ml of Con A was added at 24 hr after antigenic challenge and to a lesser extent when added at 48 hr. Suppression of the IgM PFC response was also detected when spleen cultures were exposed to 10 mug/ml of Con A for as little as 2 hr after antigenic challenge. However, substantial increases in DNA synthesis were observed only in those cultures which were in contact with Con A for at least 24 hr. Finally evidence is presented that the Con A-induced suppression is mediated by a soluble substance(s).     

Long-term effects of in vitro sensitization on immune reactivity of lymphocytes: generation of suppressor and helper T cells.

Mouse spleen cells were cultured for 5 days with or without HRBC. Cultured cells were 'parked' in irradiated syngeneic recipients for 3 weeks and then tested for their immunologic reactivity in vitro. We found that spleen cells from recipients of HRBC-sensitized cells (S) as well as spleen cells from recipients of control unsensitized cells (U) possessed radiosensitive suppressor and radioresistant helper activities. Suppressor activity was observed by the capacity of unirradiated S and U spleen cells to inhibit the in vitro generation of IgM and IgG PFC by spleen cells primed in vivo to HRBC or to LacKLH. Helper activity was shown by the capacity of the irradiated S and U cells to restore IgM and IgG PFC responses of in vivo primed, T-depleted spleen cells to HRBC, LacHRBC, and LacCRBC. Both suppressor and helper activities were mediated by T cells. The possibilities that immunologically specific or nonspecific mechanisms account for these phenomena are discussed.     

Establishment of a nucleoside-specific suppressor T cell line from SLE prone (NZB/NZW)F1 mice

A long-term cultured suppressor T cell line (GTS-124) was established from an autoimmune mouse strain, (NZB X NZW)F1, by a two-part procedure: a) B/W F1 mice were made tolerant to guanosine (G) by administration of a tolerogen, the G-modified copolymer of D-glutamic acid and D-lysine (G-D-GL); and b) the spleen cells obtained from tolerant mice were repeatedly stimulated with mitomycin C-treated G-modified syngeneic spleen cells. The GTS-124 cells suppressed the secondary in vitro response to G-keyhole limpet hemocyanin (G-KLH) but did not suppress the response to unrelated antigens, sheep erythrocytes (SRBC), or trinitrophenyl-KLH (TNP-KLH). The expression of Thy-1 antigen on the cell surface of GTS-124 was demonstrated by flow cytometry. Growth of GTS-124 cells was dependent on IL 2. To determine whether GTS-124 cells could suppress the response to nucleosides other than G, KLH coupled with four nucleosides (adenosine [A], G, cytidine [C], and thymine riboside [T]) collectively (AGCT-KLH) was first used as the antigen in the assay system. The PFC response to the individual nucleosides (anti-A, -G, -C, and -T PFC) were effectively inhibited by GTS-124 cells, suggesting that the GTS-124 cells mediated cross-suppression toward all four nucleosides. A more stringent cross-suppression test was conducted by using only the T moiety bound to KLH (T-KLH) as antigen. The results showed that GTS-124 cells were capable of suppressing the T-specific response. The cross-suppression could be seen after repeated selection on a G-BSA-coated dish. These results provide direct evidence that the suppressor T cells induced by in vitro stimulation with G-modified self can indeed suppress the response to nucleosides other than G.     

Spontaneously Induced Suppressor Cells In Vitro: Nonspecific Suppression of In Vitro Antibody Formation

Nonspecific suppressor cells were induced during in vitro culture of normal mouse spleen cells (SPC) using the Marbrook culture system. The suppressor cells inhibited both the primary and secondary antibody-formation responses antigen nonspecifically in vitro, and both IgM- and IgG-responses were inhibited. The supernatants from suppressive precultured cells were not suppressive. The suppressor cells also inhibited the response of allogeneic SPC beyond H-2 compatibility. The induction of the suppressor cells did not require the presence of antigen but required fetal calf serum (FCS) or both FCS and 2-mercaptoethanol (2-ME). The suppressor cells were generated from the nylon-wool adherent, radiation-sensitive T cell population. On the other hand, the suppressor cells were nylon-wool nonadherent, relatively radiation-sensitive T cells. Actively antibody-producing cells were not affected by the suppressor cells. The suppressor cells inhibited the mitogenic responses of normal SPC to phytohemagglutinin-P (PHA), bacterial lipopolysaccharide (LPS) and concanavalin A (Con A). The suppressor cells themselves inhibited the growth of EL4 cells (T-cell leukemia of C57BL/6 mouse origin) and MOPCll cells (B cells, plasmacytoma of BALB/c mouse origin) even at a low effector-to-target cell ratio (E:T ratio = 1:1), but did not kill these tumor cells. These results indicate that the target cells of the suppressor cells are both T and B cells, and that the mechanism of action of the suppression is either inhibition of proliferation or inhibition of early events in the course of the immune response.     

Immunosuppression induced by attenuated Salmonella. Reversal by IL-4

We previously demonstrated that an aroA- strain of Salmonella typhimurium, which provides excellent protection against virulent Salmonella challenge, also rendered immunized mice unable to mount in vivo and in vitro antibody responses to heterologous Ag. Coculture studies using transwell plates indicated that suppression was mediated by soluble factors. The suppressive cells were identified as belonging to the monocytic linkage. Macrophage precursors as well as mature adherent macrophages mediated the observed suppression. In the present study, the mechanism of immunosuppression was investigated. Suppression was found to be genetically nonrestricted as spleen cells from immunized C3HeB/FeJ mice (H-2k) suppressed the anti-SRBC plaque-forming cell (PFC) responses of normal spleen cells from two MHC noncompatible mouse strains, BALB/c (H-2d) and C57BL/6 (H-2b). Time course experiments demonstrated that the addition of spleen cells from immunized mice to normal splenocytes as late as day 4 of a 5-day assay was still markedly suppressive. Furthermore, suppression of the PFC responses was accompanied by a profound inhibition of the capacity of immune splenocytes to produce IL-2 in response to in vitro stimulation by Con A. Coculture studies showed that immune spleen cells were able to suppress IL-2 production by normal splenocytes in a dose-dependent fashion. However, the suppressed PFC responses of immune spleen cells could not be reversed by the exogenous addition of up to 200 U/ml of IL-2, suggesting that immune splenocytes are also defective in their ability to respond to IL-2. In marked contrast, suppression of PFC responses was reduced by more than 50% by the addition of as little as 1 U/ml of IL-4 and was completely abrogated when 5 U/ml of IL-4 were added to in vitro cultures of spleen cells from immunized mice. The antisuppressive action of IL-4 appeared to be via its inhibitory effect on activated macrophages. The implications of the above findings are discussed.     

BCG-induced suppressor cells. I. Demonstration of a macrophage-like suppressor cell that inhibits cytotoxic T cell generation in vitro.

Spleen cells obtained from mice 5 to 40 days after infection with viable BCG organisms (BCG-spleens) were found to be unresponsive in vitro to both mitogenic and alloantigenic stimuli. Moreover, suppressor cells could be demonstrated in the spleens from these infected animals. When spleen cells from BCG-infected mice were added to either syngeneic or allogeneic normal spleen cells, the mixtures neither proliferated nor developed cytotoxic activity when cultured with alloantigen or with concanavalin A (Con A). The development of unresponsiveness post-infection paralleled the onset of suppressive activity. Spleen cells obtained from mice given heat-killed BCG were neither suppressive nor unresponsive. The suppressive activity of BCG-spleen cells was associated with an adherent, phagocytic cell that lacked membrane-associated Thy-1 antigen. Removal of this cell by passage through nylon wool columns resulted in a cell population that was no longer capable of suppression and that responded normally to alloantigen and to Con A. It would thus appear that BCG infection results in the development of a "suppressor" macrophage-like cell population within the spleen. The role of this cell type in regulation of the immune response in BCG-infected animals is as yet undefined.