Dean S. Cunningham

TRYPANOSOMA CRUZI-INDUCED SUPPRESSION OF THE PRIMARY IMMUNE RESPONSE IN MURINE CELL CULTURES TO T-CELL-DEPENDENT AND -INDEPENDENT ANTIGENS

In vitro antisheep erythrocyte (SRBC) and antitrinitrophenyl (TNP) antibody responses of spleen cells obtained from C57BL/6 mice infected with Trypanosoma cruzi were reduced as early as 6 days postinfection and not detectable after 18 days of infection. Lymph node cells had normal antibody responses to SRBC and TNP in vitro until the 11th day of infection, after which responses were diminished. By day 31 of infection, lymph node cells were unresponsive to both SRBC and TNP in vitro. Not only were the antibody responses of spleen and lympho node cells to T-cell-dependent and -independent antigens progressively reduced as the period of infection increased, but in addition, the effect of lymphoid cell density and antigen dose on antibody production underwent several sequential changes. As the infection advanced, low densities of cultured lymphoid cells and low doses of antigen were ineffective in eliciting a detectable immune response, whereas high densities of lymphoid cells and high doses of antigen resulted in responses approximately equivalent to that observed with normal cells under the same conditions. Results of cell mixing studies have shown that a plastic-adherent, macrophage-like cell plays a major role in the suppressed humoral responses observed in this host-parasite system.

Lymphoblast transformation as a measure of immune competence during experimental Chagas' disease.

Spleen cells from normal mice and mice infected with Trypanosoma cruzi undergo mitogen-induced lymphoblast transformation in different ways. Some of these differences may be interpreted as suppression of the blastogenic response, whereas others cannot. Close examination of the culture conditions has enabled us to discriminate between in vivo-induced suppression and in vitro-determined (artifactual) suppression. Regardless of the type of suppression observed, it was shown to be dependent on duration of culture, length of labeling period, dose of mitogen, density of spleen cells, and method of expressing lymphoblast transformation data. The titration of mitomycin C-treated spleen cells from infected mice into cultures of normal spleen cells revealed two, separate, regulatory activities of spleen cells from infected mice as follow: (1) an enhancing effect which appeared late in infection and was obtained with low input of cells, and (2) a suppressive effect which appeared early in infection and was obtained with high input of cells. The enhancing effect was mediated by cells with properties characteristic of T-lymphocytes, whereas macrophages were found to be responsible for the suppressive effects.

Trypanosoma cruzi: effect on B-cell-responsive and -responding clones.

Abstract During the course of Trypanosoma cruzi infection in C57BL/6 mice, which are relatively resistant to the parasite, the hosts developed antibody activity against previously unencountered antigens. The anti-sheep erythrocyte and antitrinitrophenyl antibody levels increased rapidly from Day 7 of infection, reached a peak by the 21st day, and were maintained at this level through 120 days postinfection in these mice. In contrast, highly susceptible C3H(He) mice did not have demonstrable antibody responses to SRBC or TNP during the 24-day infection period. Autoantibody activity against the selfantigens presented on isologous erythrocytes or thymocytes, however, were reduced in infected C57BL/6 mice. No significant reduction in autoreactivity to the self-antigens on erythrocytes or thymocytes was observed in C3H(He) mice infected with T. cruzi although a trend of reduced autoresponsiveness toward erythrocytes appeared to be developing by the time of death. C57BL/6 mice immunized with sheep erythrocytes as neonates and infected with T. cruzi as adults, or adult mice primed with low doses of sheep erythrocytes prior to infection, had elevated antibody responses to sheep erythrocytes unless the mice were immunized with sheep erythrocytes during the course of infection, in which case suppression of the response against sheep erythrocytes resulted. The nonspecific synthesis of immunoglobulins in infected C57BL/6 mice was, in part, a result of the lymphocyteactivating properties of T. cruzi-associated antigens. The T. cruzi-associated antigens induced proliferative and differentiative responses in spleen cells in vitro. It is proposed that the T. cruzi-associated antigens differentially affect lymphocytes capable of responding to antigen and those lymphocytes previously stimulated by antigen.

Trypanosoma cruzi-induced suppressor substance. II. Regulatory activity.

It is reported that mice infected withTrypanosoma cruzi accumulate a suppressor substance (SS) in their sera which, when passively transferred to normal syngeneic recipients, can inhibit primary and secondary antibody responses of spleen and lymph-node cells to T-cell-dependent and -independent antigens; spleen cells were found to be suppressed earlier and to a greater degree than were lymph-node cells. Studies on the effects of the SS on normal cells in vitro also revealed that spleen cells were affected earlier than lymph-node cells, although there were no demonstrable differences in the maximum suppression effected by the SS between the two sources of lymphoid cells. Whereas normal mice could be passively immunosuppressed in vivo with the SS, it was found that serum from passively suppressed recipients did not retain measurable quantities of the SS in their sera, indicating that the substance was removed from circulation at an early stage or was diluted beyond effective concentrations. In in vivo transfers of the SS toH-2-similar and -dissimilar recipients it was found that the effectiveness of the SS related to theH-2 haplotype of the recipients.

Trypanosoma cruzi: responses by cells from infected mice to alloantigens.

Abstract 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.

Suppression of Mitogen-Induced Blastogenesis by the Trypanosoma cruzi-Induced Suppressor Substance

Serum from mice infected with Trypanosoma cruzi-suppressed (=SSS) lipopolysaccharide (LPS)-, phytohemagglutinin (PHA)-, and concanavalin A (Con A)-induced lymphoblast transformation when added to cultures of spleen cells or lymph node cells. This serum maximally suppressed blastogenic responses in spleen cell and lymph node cell cultures that contained supportive fetal bovine serum concentrations of 2% and 4%, respectively. Preincubation of lymphoid cells with SSS for 18 to 48 hr prior to initiation of the blastogenesis assay led to suppression of LPS-, PHA-, and Con A-induced proliferation at the optimal concentration of supportive fetal bovine serum (5%), whereas adsorption of lymphoid cells with SSS at 4 C for 30 min before stimulation with mitogen led to suppression of LPS-induced proliferation in spleen cells only. There was a close temporal correspondence between the induction and manifestation of suppression to the T-cell mitogens (PHA and Con A), but the manifestation of suppression preceded the induction of suppression to the B-cell mitogen (LPS) by approximately 12 hr. The SSS-induced suppression of proliferative responses, except in spleen cell cultures stimulated with LPS, was shown to be dependent on the presence of macrophages during the preincubation and stimulation phases of the assay system. The combined results of experiments in which macrophages were preincubated with SSS, or in which macrophages from the spleen were cultured with lymphocytes from the lymph nodes and vice versa (before and after preincubation with SSS), clearly demonstrated the presence of SSS-activated suppressor cells in the spleen, but not in the lymph nodes. Furthermore, the activation of these suppressor macrophages was reliant upon interactions with splenic lymphocytes.

INCREASED RESISTANCE TO AEROMONAS HYDROPHILA IN MICE EXPERIMENTALLY INFECTED WITH TRYPANOSOMA CRUZI

Mice that differ in susceptibility to Trypanosoma cruzi were shown to differ in susceptibility to Aeromonas hydrophila as well when infection with A. hydrophila succeeded an established infection with T. cruzi. Challenge with A. hydrophila had no effect on longevity in T. cruzi-infected mice. Mice which are highly susceptible to T. cruzi (C3H(He)) exhibited increased resistance to an intravenous challenge with A. hydrophila on the 7th day of infection; then resistance waned, and by the 21st day of infection, C3H(He) mice were more susceptible than uninfected mice to challenge with A. hydrophila. Mice that are relatively resistant to T. cruzi (C57BL/6) expressed increased resistance to an intravenous challenge with A. hydrophila from the 7th through the 28th day of infection, although no increase in resistance occurred from the 7th day forward. Neither C3H(He) nor C57BL/6 mice infected with T. cruzi developed altered susceptibility to an intraperitoneal challenge with A. hydrophila. While the growth of A. hydrophila in the blood, liver, and spleen increased during the first 36 hr in normal mice, marked retardation of growth occurred at the times of increased resistance in both strains of mice. In C3H(He) mice, bacterial growth returned to normal as resistance was lost. The degree of resistance to A. hydrophila in mice increased as the T. cruzi infection inoculum increased from 102 to 104; however, at the highest dose tested (5 x 104), both strains of mice showed lowered resistance, yet were still more resistant than uninfected mice. Even though resistance to A. hydrophila increased and decreased in C3H(He) mice, and increased and plateaued in C57BL/6 mice, titers of agglutinating antibody to A. hydrophila continued to increase throughout the course of infection with T. cruzi in both strains of mice. Passive suppression of mice with the T. cruzi-induced suppressor substance prior to challenge with A. hydrophila rendered C57BL/6 mice more resistant than normal mice, had no effect on the susceptibility of C3H(He) mice, but lowered the anti-A. hydrophila agglutinating antibody titers in both strains of mice.

Trypanosoma cruzi -induced suppressor substance. III. Activation of suppressor cells.

Serum from mice infected with Trypanosoma cruzi (SSS) is known to interact with normal spleen cells to induce an immunosuppressed condition and activate splenic suppressor cells. The induction of immunosuppression by SSS was shown to be independent of, and precede the activation of, suppressor cells. Suppressor-cell activation, however, was demonstrable only after the induction of immunosuppression. Furthermore, mice that were given two aliquots of SSS at different intervals of time, exhibited suppression of humoral responses of similar duration and magnitude, regardless of the SSS transfer regimen, whereas both the length and degree of suppressor-cell activity was critically dependent on the interval of time between SSS transfers. SSS interacted with spleen cells via a trypsin-sensitive membrane site which was regenerable within a 4- to 5-hr period, yet the suppressive effects of SSS on spleen cells following interaction was resistant to treatment with trypsin. The interaction between SSS and spleen cells during brief adsorption protocols leads to immunosuppression only because extensive washing of SSS-treated spleen cells did not reverse the immunosuppression process, but did prevent the development of detectable suppressor cells. The phenomenon of suppressor-cell activation was further distinguished from immunosuppression in that supernates from culture of spleen cells derived from SSS-treated mice or T. cruzi-infected contained a factor that activated suppressor cells, but did not directly induce a state of suppression in the responding cell population.