Suppressive substance produced by T cells from mice chronically infected with Trypanosoma cruzi. I. Preferential inhibition of the induction of delayed-type hypersensitivity

Culture supernatants of spleen cells from susceptible CBA mice chronically infected with Trypanosoma cruzi were able to inhibit the induction of delayed-type hypersensitivity (DTH) to a wide range of antigens as measured by 24-hr footpad swelling, bone marrow homing, and radioactivity accumulation assays. The suppressive activity, which was also present in the serum of these chronically infected mice, appears to be specific for the induction of DTH and had no effect on the 3-hr immediate-type hypersensitivity. It also failed to modify the expression of DTH in presensitized mice. Furthermore, it did not affect the synthesis in normal recipients of specific antibody or the induction of helper T cells or cytotoxic T cells. It also failed to induce DTH tolerance as recipient mice with markedly reduced DTH were able to develop a normal DTH response after secondary immunization. The suppressive activity was produced by an Ig- macrophage-depleted splenic T cell population, whose capacity to secrete the suppressive substance was completely abrogated by treatment in vitro with anti-L3T4 antibody and complement, but not with anti-Lyt-2 antibody and complement. These results therefore demonstrate that L3T4+ T cells from mice chronically infected with T. cruzi can produce substances which interfere with the induction of DTH. This finding may help to identify the differential antigenic stimulatory requirement for the activation of the various subsets of T cells.     

Delayed-type hypersensitivity to allogeneic mouse epidermal cell antigens

Footpad swelling response was used to measure the alloantigenicity of epidermal cells (ECs) in delayed-type hypersensitivity (DTH). Strong footpad swelling was oberserved 3 h after the challenge, and it continued for 48 h after the challenge. Genetical incompatibility between the recipients and the ECs was required to induce significant footpad swelling. H-2 or non-H-2 incompatibility between mice and ECs in the sensitization phase sufficed to develop significant footpad swelling. Incompatibility caused by point mutation in the A region induced strong responses when B6. C-H-2 ^bm12 mice were immunized with B6/J ECs, but the disparity in immuno-globulin h (Igh) allotype genes was insufficient. H -Y antigen on ECs could also elicit the DTH response. Semiallogeneic F₁-derived ECs sensitized the parental recipients. The responses were successfully transferred by immune lymph node cells, but not by immune sera. Treatment of these immune lymph node cells with monoclonal antibodies plus complement revealed that the cells responsible for DTH transfer were Lyt-1⁺2^–, Ia^– T cells.Abbreviations used in this paper DNFB 2,4-dinitro-1-fluorobenzene - DTH delayed-type hypersensitivity - ECs epidermal cells - HBSS Hanks' balanced salt solution - MHC major histocompatibility complex - PBS phosphate-buffered saline     

Mycoplasma pulmonis depresses humoral and cell-mediated responses in mice

Humoral and cell-mediated immune responses to sheep red blood cells (SRBC) were studied in mice infected experimentally with Mycoplasma pulmonis. The hemagglutinating (HA) antibody against SRBC was evaluated at 0, 3, 5, 7, 14, 21 and 28 days postinfection (PI). Antibody tiers during all days PI were depressed significantly (p less than 0.05) in infected mice as compared to noninfected controls. The HA antibody, which is of the IgM class, peaks at day 5 PI. There is no shift in the kinetics of the humoral response in M. pulmonis infected mice. Cellular immune responses were evaluated by a delayed-type hypersensitivity (DTH) reaction and the lymphocyte transformation technique. Mice were sensitized at 0,3,5,7,14, 21 and 28 days PI with SRBC and challenged by footpad injection of SRBC 7 days later. The DTH reaction measured at 24 hours after challenge was depressed significantly (p less than 0.05) in all infected animals. After a transient enhancement on day 3 PI, the DTH responses remained depressed through day 28 PI. The lymphocyte transformation test showed a significantly (p less than 0.05) depressed response except on days 5 and 7 PI. These results indicate that M. pulmonis infection in mice suppresses the humoral antibody and cell-mediated immune responses.     

Evidence for interaction between T cell populations of tumor-bearing and normal mice in immune suppression

Spleen cells of DBA/2 mice bearing subcutaneous implants of the syngeneic tumor L5178Y induce suppression of the in vitro antibody response of normal spleen cells to sheep erythrocytes (SRBC). Cells mediating suppression are detected in the spleens of tumor-bearing mice as early as 24 hr post-implantation but are no longer detected there 15 days post-implantation. These spleen cells are nylon wool nonadherent, sensitive to anti-Thy 1.2 + C and anti-Lyt 1.1 + C, and insensitive to anti-Lyt 2.1 + C treatment. The anti-SRBC response of the unfractionated spleen cells from the tumor-bearing mice is not itself suppressed at the cell numbers used. This along with the finding that suppression occurs in the presence of spleen cells from normal mice suggest that a cell population from the normal mouse spleen is also involved in the suppression. Spleen cells from mice inoculated with irradiated (nonproliferating) L5178Y cells are similarly capable of mediating nonspecific suppression for the same limited period of time after the inoculation. In addition, spleen cells from mice stimulated with several nontumorigenic cellular antigens interact with normal spleen cells to produce suppression. These findings suggest that suppression observed in vitro with spleen cells from these tumor-bearing mice may be the result of antigen-activated cells triggering normal immunoregulatory cells.     

Regulation of the immune response. II. Concomitant idiotope-specific enhancement and suppression can result in a phenotypically normal response

Idiotope-specific immunoenhancement or suppression was induced in C57BL/6 mice by the injection of physiological amounts (100 ng-10 micrograms) of monoclonal anti-idiotope antibody. As previously described, nanogram doses enhanced idiotope expression while a 10-micrograms dose of anti-idiotope antibody induced the activation of a population of Thy 1.2+, Lyt 1-, 2+ suppressors. Both positive and negative regulatory activities were confined to the non-mu, idiotope+ compartment of the plaque-forming cell response. Administration of intermediate doses of anti-idiotope antibody resulted in an immune state indistinguishable from that of naive mice. This apparently normal response was in fact the product of a simultaneous activation of balanced enhancing and suppressive activities. When treated with anti-Lyt 2 or Lyt 1 and complement, spleen cell populations taken from such phenotypically "naive" mice revealed latent idiotope-specific immunoenhancement or suppression, demonstrating the components of a functional regulatory equilibrium.     

Cell-mediated and humoral immune responses in mice during experimental infection with Mycoplasma pulmonis

Cell-mediated and humoral immune responses in mice after challenge exposure with Mycoplasma pulmonis were investigated. The cell-mediated immune response was determined by means of the delayed-type footpad swelling and the humoral immune response by means of the indirect haemagglutination test. Delayed-type footpad swelling and serum antibody titres were detected at one week after the challenge exposure and persisted for 7 weeks until the end of the experiment. However, there was a poor correlation between the degree of delayed-type footpad swelling and that of serum antibody titre. Delayed-type footpad swelling in mice with gross pneumonic lesions was less than that of mice with no gross lesions. A weak negative linear correlation was observed between the delayed-type footpad swelling and the number of M. pulmonis isolated from lungs.     

Requirement for a bacterial flora before mice generate cells capable of mediating the delayed hypersensitivity reaction to sheep red blood cells.

A delayed-type hypersensitivity (DTH) reaction can be elicited by an injection of 10(8) sheep red blood cells (SRBC) into a rear footpad of conventional (CV) mice previously immunized with small doses of SRBC. In contrast, immunization of germ-free (GF) mice with the same doses of SRBC produced no DTH when immunization was by the intravenous (i.v.) route, and only weak reactions when immunization was by the subcutaneous (footpad) route. Varying the immunizing dose of SRBC, or the time at which DTH was elicited, did not produce a state of DTH responsiveness in i.v. immunized GF mice. However, the transfer of lymphocytes from CV mice, immunized 4 to 5 days previously with SRBC, into GF mice, conferred on GF mice the capacity to express DTH. Although DTH was not readily demonstrable in GF mice immunized with SRBC, they nevertheless produced normal levels of hemagglutinating antibody to SRBC. Finally, it was shown that GF mice could generate a normal DTH response to SRBC if they were first monoassociated with a Gram-negative bacterial flora.     

Complement reversal of immunosuppression induced with plasma of rats infected with Trypanosoma brucei rhodesiense

Suppression of antibody production by splenic lymphocytes from rats immunized with sheep red blood cells (SRBC) after incubation with plasma from rats infected with Trypanosoma brucei rhodesiense was confirmed. Suppressive activity became evident in plasma after the sixth day of infection and was manifested by reduction in the number of hemolytic Jerne plaques produced by the treated cells. The activity was temporally associated with increased amounts of soluble immune complex (SIC) reduced titers of lytic complement, elevated titers of immunoconglutinin (IK) and anemia. Treatment of suppressive plasma with hemolysin sensitized SRBC alexinated with horse complement to reduce IK did not reduce suppressive activity, and the activity appeared to have been enhanced when the plasma was heated to inactivate the remaining complement (C'). When fresh rat C' was added to the treated cells, the suppression was largely, though not completely, reversed. Treatment of spleen cells with SIC prepared in vitro from bovine serum albumin (BSA) and rabbit antiBSA also suppressed the plaque forming capacity of the cells. Complexes of BSA-antiBSA-C' and complexes of BSA-antiBSA-C'-IK were equally suppressive. Again, addition of fresh C' to cells treated with these complexes largely, though not completely, reversed the suppressive effect on the cells. From the results it is suggested that immunosuppression associated with experimental T. b. rhodesiense infection may be in part a suppression of the capacity of induced lymphocytes to produce antibody. It is possible that the suppression was mediated by SIC present in the plasma of the infected rats and this effect was probably enhanced by reduced levels of complement in the suppressive plasma.     

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice. I. Inhibition of proliferation of lymphocytes in primary antibody responses

The suppressor cells induced by Toxoplasma infection were shown to be macrophages, since they adhered to plastic, and their suppressive activity in anti-sheep erythrocytes (SRBC) antibody responses was abrogated by treatment with silica or carrageenan, which are selectively cytotoxic for macrophages. The suppressor macrophages strongly inhibited the uptake of tritiated thymidine ( [3H]TdR) by normal mouse spleen cells in the responses to SRBC and Toxoplasma antigens. Supernatant fluids from the suppressor macrophages could not passively transfer the suppressive effect on anti-SRBC antibody responses. Furthermore, when the suppressor macrophages were isolated by a cell-impermeable membrane from normal mouse spleen cells, the antibody responses of normal spleen cells were not suppressed. These results indicate that suppression of antibody responses in Toxoplasma-infected mice is caused by an inhibitory effect of the suppressor macrophages upon proliferation of lymphocytes via direct contact with responder target cells. The suppressive effect of the macrophages was not counteracted by indomethacin, a potent inhibitor of prostaglandin synthesis, or catalase, a catabolic enzyme for hydrogen peroxide (H2O2).     

Effect of rubomycin and carminomycin on suppressor cells participating in the regulation of the immune response

The effect of rubomycin and carminomycin on induction of suppressor cells in immunization of mice with high doses (2 . 10(9) cells) of sheep red blood cells (SRBC) or with the allogeneic spleen cells was studied. When the spleen cells of syngeneic animals hyperimmunized with SRBC were administered to intact cells, a marked specific suppression of both the antibody formation and the reaction of the delayed type hypersensitivity were observed. A decrease in the suppressor activity or its complete elimination as a result of exposure of the spleen cell donors to the antibiotics was indicative of a relatively selective effect of these drugs on the suppressor cells. This effect of the antibiotics increased after treatment of the spleen immune cells with the anti-T-serum and complement. However, the blocking effect of the antibiotics on the suppressor cells was not strictly specific, since they were detected in the syngeneic and allogenic systems with the cell transfer.     

Amelioration of delayed-type hypersensitivity responses by IL-27 administration

Interleukin (IL-) 27 is a member of the IL-12 cytokine family. Although recent analyses of WSX-1 (IL-27 receptor α chain)-deficient mice as well as in vitro studies using recombinant IL-27 revealed the immunosuppressive function of IL-27, in vivo role and therapeutic potential of IL-27 remain poorly elucidated. Here we investigated the effect of IL-27 administration on delayed-type hypersensitivity (DTH). While WSX-1-deficient mice showed higher DTH responses as shown by the degree of footpad swelling, administration of IL-27 significantly ameliorated the footpad swelling. Since the activation status of the draining lymph node cells were not affected by IL-27 deficiency, it was suggested that IL-27 affected the effector phase of the response. These results collectively indicate that IL-27 has a suppressive effect on activated T cells in the experimental model and also has a therapeutic potential for some diseases caused by immune disorder.     

The distribution of the Qa-2 alloantigen on functional T lymphocytes

The expression of Qa-2 on functional lymphocytes was investigated in vitro and in vivo by using a monoclonal anti-Qa-2 antibody. In vitro treatment of T cells with antibody and complement demonstrated that T cells mediating help or delayed-type hypersensitivity for anti-SRBC responses were Qa-2+. In addition, cytotoxic T cells and either their precursors or cells involved in their generation were Qa-2+, as were anti-HGG suppressor T cells. Panning techniques were also used to show that secondary suppressor T cells were Qa-2+ and that there may be heterogeneity in suppressor T cells defined by Qa-2 expression. In vivo treatment of mice with anti-Qa-2 resulted in decrease in immune responsiveness seen by i) prolongation of skin grafts with either H-2D or I-A differences, ii) suppression of delayed-type hypersensitivity, and iii) inhibition of T cell-mediated suppression. Finally, IgG, but not IgM, anti-body-forming cells were Qa-2+.     

Genetic Control of Immune Responses to HIV-1 env DNA Vaccine

We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4⁺8^? and Ia— T cells. However, CD8⁺ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.     

Effect of antiserum against isologous aggregated immunoglobulins on the delayed-type hypersensitivity in mice immunized with sheep red blood cells

The mouse antiserum against isologous aggregated immunoglobulins (MAAS) injected to mice sensitized with 10(5) sheep red blood cells (SRBC) did not influence the delayed-type hypersensitivity (DTH) tested on the peak of sensitization (the 4th day) but enhanced significantly DTH tested on the 6th day. MAAS completely abolished the DTH suppression observed after sensitization with 5 x 10(7) SRBC. In transfer experiments the number of the DTH suppressor cells decreased in the spleen of sensitized mice under the MAAS action. MAAS did not affect the proliferation of antibody-forming cells (AFC) and hemagglutinin production but reduced by 70% the number of rosette-forming cells (RFC) in the spleen on the peak of the initial immune response. The data obtained may indicate that RFC participate in DTH suppression.     

Difference in thymus dependency between effector and suppressor T cells for delayed footpad reaction to sheep erythrocytes in mice

Effects of thymectomy at various times after birth on effector and suppressor T cells for a delayed footpad reaction were determined in 6-week-old mice immunized intraperitoneally (ip) with sheep erythrocytes (SRBC). Mice thymectomized 1 day after birth (Tx-1 mice) gave delayed footpad reactions weaker than those of mice thymectomized 7 days after birth (Tx-7 mice) or sham operated (SH mice) after immunization with a low dose of SRBC. After immunization with a high dose of SRBC, on the other hand, Tx-1 mice showed reactions stronger than those of Tx-7 or SH mice. Pretreatment with cyclophosphamide (CY) augmented the delayed footpad reaction in Tx-7 or SH mice, but not in Tx-1 mice, immunized with a high dose of SRBC. The presence of T cells suppressive for the delayed footpad reaction in the spleen of Tx-7 or SH mice was confirmed by cell transfer experiments. These results suggest that effector T cells responsible for a delayed footpad reaction to SRBC are less thymus dependent and require the presence of the thymus for a shorter period in their development compared to suppressor T cells.     

B-cell-mediated regulation of delayed-type hypersensitivity

We obtained immune sera from mice which received suppressor B cells induced in vitro, injected them into immunized mice, and measured suppression of the delayed-type hypersensitivity (DTH) of these recipient mice. In the recipients, effector-phase suppressor T (Ts) cells were induced, and the action of these Ts cells was antigen-nonspecific. The suppressive material of the sera was adsorbed on a Sepharose column coated with anti-mouse immunoglobulin antibody and acid elution of the column yielded the elute fraction that showed significant suppressive activity. The suppressive activity of the sera was also adsorbed by an antigen-coated Sepharose column, and the eluate from the column had suppressive activity. Moreover, we established antigen-specific monoclonal antibodies, some of which suppressed the DTH in an H-2-nonrestricted way. The isotype or specificity of the antibodies was not related to the suppression, because suppressive and nonsuppressive antibodies belonged to the same immunoglobulin isotype and because the antibodies that recognized the same epitope had different suppressive activities. The Fc portion was not the functional site, because the F(ab')2 fragment had the activity. The suppressive antibody induced effector-phase Ts cells, which had the anti-idiotypic receptor. These findings suggested that antigen-specific antibodies in the immune sera mediated the suppression of DTH by the induction of effector-phase Ts cells in vivo and the idiotype of the antibody stimulated the anti-idiotypic receptor of these Ts cells.     

Effects of suramin on the immune responses to sheep red blood cells in mice. II. In vitro studies

The effect of Suramin on the secondary in vitro response to sheep erythrocytes (SRBC) was studied. Spleen cells from mice which were treated with Suramin immediately prior to sensitization with SRBC failed to respond to an in vitro SRBC challenge. This Suramin-induced immunosuppression is not related to a defect in macrophage or B-cell function(s). Suramin does not interfere with the induction by SRBC of radioresistant and radiosensitive helper-T-cell subpopulations. Cell separation studies, using wheat germ agglutinin, showed radiosensitive helper-T-cell function in the nonagglutinated fraction while the radioresistant helper activities are carried out by the agglutinated subpopulation. Evidence is presented that Suramin administration results in a suppressive T-cell activity which can be demonstrated in the subpopulation agglutinated by wheat germ agglutinin. The role of such suppressive T cells in the inhibitory effect exerted by Suramin on the cell-mediated delayed-type hypersensitivity response to SRBC is discussed.     

Genetic restrictions of transfer of delayed-type hypersensitivity to sheep erythrocytes: local versus systemic transfer.

Regulation of the transfer of delayed-type hypersensitivity (DTH) reactions to SRBC was studied using two assays. In the systemic transfer, SRBC immune cells were transferred intravenously and the recipient challenged by injecting antigen into the footpad. In the local transfer assay, SRBC immune cells were mixed with antigen before transfer into the footpad of the recipient. These studies utilized B10.D2 and B10.BR mice which are congenic strains differing only at H-2 region. DTH reactions can be transferred across H-2 barriers using a local transfer assay. When the immune cells were transferred intravenously or depleted of adherent cells prior to local transfer, DTH reactions cannot be transferred to an H-2 congenic recipient. Spleen cells from naive mice syngeneic to the intravenously transferred cells supply the necessary accessory cell when mixed with the antigen prior to injection into the footpad. This accessory cell may be a macrophage.     

Effects of pertussis toxin on delayed-type hypersensitivity responses and on the activity of suppressor T cells on the responses

Experiments were performed on mice to investigate the effects of pertussis toxin (PT) on delayed-type hypersensitivity (DTH) to ovalbumin (OA) and on the activity of suppressor T cells on the DTH (DTH-Ts). Mice immunized with alum-precipitated ovalbumin showed a transient DTH, which was determined as footpad swelling which disappeared 2 weeks after immunization. Maximal footpad swelling was observed 24 hr after DTH elicitation. On the other hand, when mice received PT (2 micrograms/mouse) at the time of immunization, the transient DTH became an enhanced and persistent DTH, which persisted for at least 4 weeks. In addition, the time of maximum footpad swelling was delayed from 24 to 48 hr after DTH elicitation. The immune spleen T cells from PT-treated mice showed a persistently high ability to transfer DTH into syngenic naive mice. DTH-Ts was induced in spleens of mice injected iv with OA-coupled syngeneic spleen cells. However, when these mice received PT at the time of suppressor induction, their spleen cells revealed considerably reduced suppressor activity. The activity of DTH-Ts was also reduced when DTH-Ts were either treated in vitro with PT or transferred into PT-injected recipient mice. From these results, interference with the suppressor function of DTH-Ts from PT was considered to be, at least in part, as an enhancing mechanism of DTH.     

Suppression of Hen Egg-White Lysozyme (HEL)-Specific Delayed-Type Hypersensitivity Responses in Mice by Suppressor T Cells after Neonatal Administration of Anti-Idiotypic Antibodies

Anti-idiotypic rabbit antiserum (anti-Id) directed to the idiotypes of anti-hen egg-white lysozyme (HEL) antibody from a single C3H mouse (No. 2) was shown to be capable of recognizing only a fraction of the anti-HEL antibody populations produced by other C3H mice. Experiments were performed to examine the effect of this particular anti-Id on the delayed-type hypersensitivity (DTH) response specific for the same protein antigen. A group of 60-day-old C3H mice which had been administered anti-Id within 24 hr after birth were tested for HEL-DTH response. The results indicated that the DTH response was completely suppressed by the anti-Id treatment. The inhibition of DTH reactivity is due to active suppression and involves the generation of suppressor T cells. Thus, the suppression induced with a single injection of anti-Id was transferable with both spleen cells and thymocytes from mice that received anti-Id. These suppressor cells are T cells since their ability to suppress DTH is completely abrogated by treatment in vitro with anti-Thy 1.2 serum and complement.