Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Mechanisms of antibody formation: I. Early in vivo proliferation of mouse spleen T cells as an initial step in antibody formation

Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10⁶?2 × 10⁸ sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement.Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr.Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function.In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10⁸ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.     

Immunoregulation in experimental schistosomiasis: III. Role of macrophages in soluble egg antigen-induced chronic spleen cell augmentation of baseline lymphocyte reactivity

Spleen cells from mice infected for 20 weeks with Schistosoma mansoni, exposed in vitro to soluble schistosomal egg antigens (SEA), treated with mitomycin C (Mc), and cocultured with syngeneic responder spleen cells increased the baseline proliferation of the otherwise unstimulated responder cells in cocultures. The role of macrophages in this “spontaneous” thymidine incorporation was studied directly by removal of macrophages on Sephadex G-10 columns. Removal of esterase-positive, Sephadex G-10-adherent cells (macrophages) greatly reduced the amount of SEA-induced, chronically infected spleen cell-mediated stimulation observed in cocultures. It also reduced an elevated background of spontaneous DNA synthesis seen with control cultures of spleen cells from infected animals. Depletion of T lymphocytes from chronic spleen cell populations by treatment with anti-Thy 1.2 serum and complement prior to exposure to SEA partially abrogated the augmentation effect. Comparison of these results with mitogen (concanavalin A)induced spleen cell-mediated stimulation (which is elevated, rather than reduced, by macrophage removal) and with known alterations in splenic T- and B-lymphocyte ratios in chronic murine Schistosomiasis suggests that antigen-stimulated, chronically infected splenic macrophage-de-pendent baseline augmentation may depend on specific T-lymphocyte-derived lymphokine induction. These results may reflect a general mechanism whereby animals harboring a persistent, chronic infection can respond quickly to a second or challenge infection or a flareup of the primary infection.     

BCG-induced chronic pulmonary inflammation and splenomegaly in mice: suppression of PHA-induced proliferation, delayed hypersensitivity to sheep erythrocytes, and chronic pulmonary inflammation by soluble factors from adherent spleen cells

C57BL/6 (B6), but not CBA, mice develop intense chronic granulomatous inflammation (CGI) in the lungs and spleen in response to an iv injection with killed BCG in an oil-in-saline emulsion (BCG-E). Concomitant with the development of CGI, these mice show diminished responsiveness to PHA and LPS, as well as suppression of antibody synthesis and production of delayed hypersensitivity (DH) to sheep erythrocytes (SRBC). Suppression results from the development of adherent, Thy-1^?, Ig^? spleen cells. The present study shows that cells from inflamed spleens of BCG-E-treated B6 mice elaborate factors in vitro which (a) inhibit PHA-induced proliferation of both normal syngeneic and allogencic cells, (b) suppress DH to SRBC in B6 mice, and (c) diminish the intensity of BCG-E-induced CGI in the lungs and spleens of B6 mice. These factors are produced by adherent Thy-1^? cells in BCG-injected mice but not in similarly treated CBA mice. These factors may be important in understanding the control of immunologically mediated chronic inflammation.     

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.     

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.     

Immunoregulation in experimental schistosomiasis. II. Soluble egg antigen-induced chronic spleen cell augmentation of baseline lymphocyte reactivity

Spleen cells from mice harboring infections of Schistosoma mansoni for 20 weeks exposed to Con A or to soluble schistosome egg antigenic preparation (SEA), treated with Mitomycin C (Mc), and cocultured with spleen cells from either normal or infected mice caused an augmented baseline [3H]TdR incorporation by the otherwise unstimulated responder cells. This regulation required an in vitro induction phase. SEA-exposed, Mc-treated normal spleen cells had no effect on responder cell cultures. SEA-stimulated, Mc-treated chronic spleen cell augmentation was effective on responder cell populations from either normal mice or mice infected with S. mansoni for 8 weeks. Augmentation was most pronounced when assayed on cells from infected mice assayed over a 5-day incubation. In addition, it is demonstrated that these Con A- and SEA-elicited activities are mediated by soluble mediators which lack H-2 restriction.     

Immunosuppression transfer by spleen cells from young to adult mice previous to Histoplasma capsulatum infection

The passive transfer of spleen cells from 1 month old mice into adult syngeneic mice, abrogates their resistance to histoplasmal infection. This suppressive state was detected in two cell populations, one non-adherent and another adherent with radioresistant characteristics.The transferred spleen cells were treated by different anti-sera: anti-theta, anti-adherent cells (produced in rabbits) and monoclonal anti-Thy 1.2 respectively.The irradiated and non-irradiated adult recipient mice were infected with Histoplasma yeasts utilizing the Lethal Dose₅₀ for 1 month old mice. The infection course was determined by death percentage, the histoplasmosis murine signs and the number of the fungal colony forming units (CFU) from the infected spleens. The results of the anti-sera treatment suggest that non-adherent as well as adherent cells participate in the suppressive phenomena. A lower number of CFU was identified in infected animals which received cells treated with anti-Thy 1.2 anti-sera.     

Lymphocyte function in experimental African trypanosomiasis. IV. Immunosuppression and suppressor cells in the athymic nu/nu mouse

The role of T cells in the development and expression of antigen-nonspecific immunosuppression in experimental African trypanosomiasis was addressed. Nude ($\text{nu}\text{nu}$) C57BL/ 6 NIH mice and their thymus-bearing ($\text{nu}\text{+}$) littermates were infected with Trypanosoma rhodesiense and examined for suppression of splenic B-cell responses in vitro to the mitogen LPS. All animals developed splenic unresponsiveness to LPS. Further, both nu/nu and nu/ + infected mice displayed suppressor cell activity in their spleen cell populations upon transfer to normal uninfected mouse spleen cell cultures. On the basis of these findings we suggest that both the generalized immunosuppression and the development of suppressor cell activity in the spleens of mice infected with T. rhodesiense are T-independent processes.     

Suppression of parasite antigen-specific lymphoid blastogenesis in African trypanosomiasis

Inbred C57BL/6J mice were infected with either Trypanosoma rhodesiense organisms of Walter Reed Army Trypanozoon antigenic type 3 or 5 (WRATat 3 or WRATat 5) or were immunized with soluble trypanosomal antigens. Spleen cells obtained from immunized hosts undergo blastogenesis, measured by thymidine incorporation, when exposed to trypanosomal antigens in vitro. Spleens obtained from mice infected with T. rhodesiense organisms do not respond or respond only minimally to trypanosomal antigens in vitro. Spleen cells of infected mice suppress the trypanosomal antigen-induced proliferative response of spleen cells from immunized mice in co-culture experiments. The suppressive activity was found in both the plastic adherent and plastic nonadherent spleen cell populations. The in vitro responses of normal spleen cells to LPS and Con A were also suppressed by spleen cells obtained from infected mice.     

Recombinant granulocyte-macrophage colony-stimulating factor restores deficient immune responses in mice with chronic Trypanosoma cruzi infections

Spleen cells from mice with chronic Trypanosoma cruzi infection generate a minimal plaque-forming response to SRBC in vitro. Addition of granulocyte-macrophage (GM)-CSF to cultures of spleen cells from chronically infected mice restored the plaque-forming cells (PFC) response to normal levels. Splenic adherent cells from chronically infected mice were deficient in their ability to reconstitute the PFC response of accessory cell-depleted normal spleen cells. Preincubation of splenic adherent cells from infected mice with GM-CSF restored their ability to reconstitute the PFC response of adherent cell depleted cultures. Ia Ag expression by splenic adherent cells from chronically infected mice was significantly lower compared to Ia Ag expression of cells from normal mice. Incubation of splenic adherent cells from chronically infected mice for 48 h with GM-CSF increased levels of Ia Ag expression to approximately those of uninfected mice. Peritoneal macrophages from infected mice produced IL-1 after incubation with GM-CSF at levels equivalent to those produced by similarly treated control macrophages. Spleen cells from chronically infected mice showed significant induction of IL-2 mRNA after GM-CSF treatment, and the addition of the anti-IL-2 mAb to GM-CSF supplemented cultures of spleen cells from infected mice blocked the restoration of the anti-SRBC PFC response. Thus, the ability of GM-CSF to restore the anti-PFC response to SRBC appears to involve the up-regulation of accessory cell function that includes increased Ia Ag expression and the induction of IL-1 production. These events also involve increased IL-2 production with resultant up-regulation of the response to SRBC by spleen cells from infected mice. Finally, it was shown that treatment of infected mice with rGM-CSF completely restored their depressed PFC production in vivo.     

Suppression of responses to cryptococcal antigen in murine cryptococcosis

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.     

Plasmodium yoelii: the thymus-dependent lymphocyte in mice immunodepressed by malaria

Euthymic mice, athymic nude mice, and mice treated with antithymocyte serum were infected with Plasmodium yoelii and immunized 10 days postinfection with pneumococcal polysaccharide (SSSIII). As a control, uninfected mice were also immunized with SSSIII. Splenic plaque-forming cells as well as serum antibody titers to SSSIII were measured 5 days after immunization. In infected euthymic mice, both plaque-forming cells (PFC) and serum antibody were severely depressed. In contrast, plaque-forming cells and serum antibody were approximately normal in infected nude mice and in infected mice treated with antithymocyte serum. Splenic adherent cells from infected euthymic mice failed to function as accessory cells in the in vitro antibody response to a second antigen, the sheep erythrocyte. Moreover, they lacked suppressor activity when cultured with spleen cells from uninfected mice. In contrast, adherent spleen cells from infected mice treated with antithymocyte serum displayed accessory cell function.     

Deficiency in the incorporation of labeled thymidine and inhibition in the biosynthesis of interleukin-2 in lymphocytes obtained from Histoplasma capsulatum infected mice

The incorporation of (³H) thymidine and the biosynthesis of interleukin-2(IL-2) were investigated in Concanavalin A (ConA) and histoplasmin stimulated lymphocytes from spleen of infected Balb/c mice with the yeast phases of Histoplasma capsulatum. The ability to incorporate (³H) thymidine of Con A stimulated lymphocytes in culture from spleen of Histoplasma capsulatum infected mice, as well as the IL-2 content present in the supernatants of that cultures, were depressed along the first three weeks of the experiments, but starting week five, normal values were restored or even discretly increased. Incorporation of (³H) thymidine in histoplasmin stimulated lymphocytes remained inhibited along the seven weeks the experiment lasted. Results showed that inoculation of H. capsulatum yeast in mice provoked a temporary immunosuppression on cell mediated immunity, that can be explained by means of the inability of T cells to produce enough IL-2 necessary for the proliferation of T cells in culture.     

Role of interleukins 1 and 2 on human thymocyte mitogen activation

The effects of d-penicillamine on proliferation and polyclonal activation of lymphocytes were studied in cultures of spleen cells from a variety of murine strains. Inclusion in serum-free or serum-containing medium of optimal concentrations of d-penicillamine resulted in the uptake of tritiated thymidine in a dose-dependent fashion, with both lower and higher doses causing less marked effects. The kinetic peak of these responses was found to occur at Day 2 of culture. Experiments examining the responsiveness of B-cell-enriched and T-cell-enriched populations demonstrated that d-penicillamine, like 2-mercaptoethanol, stimulates both types of cell. The magnitude of the response remained unchanged in populations depleted of adherent cells. In correlation with previous results seen for 2-ME, d-penicillamine was mitogenically active both in reduced and oxidized forms. The oxidized form failed to enhance the response to LPS, in contrast to the marked effect of the reduced form. Additionally, d-penicillamine failed to evoke a mitogenic response from B cells of CBA/N mice, a strain characterized by a deficit in the function of a particular set of mature B cells. Young mice from autoimmune strains responded to d-penicillamine as well as normal mice did. No relationship could be observed between responsiveness to d-penicillamine and the H-2 phenotype of the cell donor, and in A/J mice hyporesponsiveness was shown to be a function of their background rather than H-2. d-Penicillamine was found to function as a polyclonal B-cell activator, and to significantly enhance the primary humoral immune response to sheep erythrocytes in vitro. These immunomodulatory effects of d-penicillamine are discussed in relation to possible pathogenetic mechanisms of its spectrum of autoimmune side effects.     

Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Murine delayed hypersensitivity (DHS): modulation of in vitro antigen-induced responsiveness of lymph node cells from mice expressing DHS to human gamma-globulin by lymphocyte populations from normal syngeneic animals

Using a chemically defined, protein-free medium, the modulatory effect of normal (N) lymphocytes on in vitro antigen-induced proliferation by lymph node cells (LNC) from mice immunized to express delayed hypersensitivity (DHS) to human γ-glogulin (HGG) was quantitated in coculture. LNC from normal syngeneic animals exerted little if any effect on immune-LNC proliferation. Compared with immune-LNC plus N-LNC coculture response. N thymus cells (TC) were consistently suppressive while N spleen cells (SC) varied in their effect from a marginal to a marked potentiation of radiolabeled thymidine incorporation. Inactivation of N-SC suspensions by X irradiation prior to coculture with immune LNC abrogated the increased responsiveness. It therefore appeared that interaction of immune LNC and antigen resulted in recruitment of N-SC to proliferate. Separation of N-SC suspensions to provide enriched populations of thymic-independent (B) and thymic-dependent (T) lymphocytes showed that B cells augmented and T cells suppressed HGG-induced incorporation of [3H] thymidine when cocultured with immune LNC.     

Immune unresponsiveness of spleen cells from lipopolysaccharide-sensitized mice to particulate thymus-dependent antigens. II. Evidence for an abortive cooperation between T lymphocytes and antigen-presenting cells

LPS-induced immune unresponsiveness has been shown to be related to an impaired production of differentiation signal factor(s). The mechanism underlying this phenomenon was analyzed. The in vitro anti-SRBC response of spleen cells from normal mice was not suppressed by addition of LPS-treated spleen cells, ruling out a possible implication of active suppressor cells. Immune responsiveness concomitant with TRF production was restored in LPS-sensitized cells upon addition of 2-ME. The role of adherent and nonadherent cells was also investigated; both cell populations from LPS-treated mice were able to collaborate with their normal counterparts showing that the defective TRF production results from synergistic effects of LPS-induced alterations concerning both adherent and T-cell populations.     

Nude mice--a model system for studying the cellular basis of the humoral immune response

Mice homozygous for the nu gene fail to develop a thymus. In comparison to spleen cells from +/nu mice spleen cells from nu/nu mice have a deficient 19S PFC response to SRBC when tested in culture or in vivo. This deficiency is due to a lack of “helper” T cells in nu/nu spleen; A cells and B cells appear to be normal. The capacity of nu/nu spleen cells to produce a PFC response in culture can be corrected by the addition of T cells obtained from either the thymuses or the spleens of +/nu mice. In contrast to “helper” T cells obtained from the spleen, “helper” T cells obtained from the thymus appear to require the capacity for proliferation during the response to SRBC.     

Inhibition of in vitro antibody synthesis by cyclophosphamide-induced suppressor cells

A population of suppressor lymphocytes appears in the spleens of mice 5 to 14 days after treatment with a high dose of cyclophosphamide (100–200 mg/kg body wt). Removal of carbonyl iron adherent cells or Ig^? cells from cyclophosphamide (CP)-treated spleen cells does not abolish suppressive activity. These suppressors are, however, sensitive to removal by treatment with anti-Thy-1.2 and rabbit complement. CP-treated spleen cells can suppress the in vitro primary response of normal spleen cells to the soluble hapten-protein conjugate DNP-MON or the particulate antigen HRBC when added at time of culture initiation or up to the second day of culture. CP-treated spleen cells can themselves respond in vitro to DNP-MON, as well as to HRBC, but with altered kinetics from that of normal spleen cells. Collectively, the data suggest that the CP-induced suppressors act late in the in vitro antibody response, possibly by prematurely shutting off antibody synthesis by B cells.