Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Effect of trypsin on suppressor cell formation and function in localized staphylococcal infection

The present study has been made on (CBA X C57BL)F1 mice immunized with sheep red blood cells (SRBC) and inoculated with staphylococci (M-SRBC-S). The injection of splenic lymphocytes from syngeneic M-SRBC-S into intact mice has been found to suppress immune response to SRBC in these mice. The injection of trypsin into M-SRBC-S decreases the suppressive action of their lymphocytes on SRBC-induced immune response in syngeneic recipients. The injection of trypsin into the recipients has been found to produce no effect on the immunosuppressive action of transplanted lymphocytes obtained from M-SRBC-S. The injection of trypsin into M-SRBC-S induces the release of the factor, inhibiting the formation and function of suppressor cells, by their splenocytes. Previously formed suppressor cells block the release of the immunostimulating factor by the splenocytes of the animals receiving the injections of trypsin.     

Study of the phenomenon of specific suppression of the immune response in an adoptive transfer system]

It was revealed that the administration of the spleen cells (SC) of syngeneic animals immunized with a high dose of sheep red blood cells (SRBC) to intact mice led to a marked specific suppression of the recipients' immune response. The donors' SC obtained on the 14th day after the intraperitoneal injection of SRBC had the greatest suppressive activity. The SC of intact animals and mice given rat erythrocytes preliminarily failed to influence the immune response of the intact recipients in their SRBC immunization. Treatment of immune SC with the anti-T-serum (ATS) or the anti-B-globulin (ABG) and the complement considerably decreased or completely eliminated the suppressive activity. Administration of a mixture of two immune SC suspensions one of which was ATS- and another ABC-treated did not produce any suppression of the immune response in the intact recipients. It is supposed that the suppressor cells in the given model were T-lymphocytes expressing the antigens, common of cross-reacting with the B-cells.     

Modulation of mouse anti-SRBC antibody response by placental extracts

Mouse placental extracts (PE) and corresponding Sephadex G-200 fractions were administered to isogeneic CBA mice along with an optimal immunizing dose of SRBC. Spleen cells were harvested 8 days later and transferred to CBA recipients, subsequently immunized with SRBC. The immunoregulatory activity of spleen cells from PE-treated donors was compared to cells from liver extract (LE)-treated controls or from mice immunized with SRBC only, using Cunningham's PFC direct and indirect tests. Within the dose range used, selective modulatory activities were obtained with cells from PE, but not from LE, treated mice, the latter being comparable to cell transfer effects from donors immunized with SRBC only. Spleen cells from animals injected with low doses of PE (0.25 to 4 mg per mouse) added to immunizing SRBC had a suppressive effect on the primary IgM response of recipients immunized against SRBC. In contrast, when SRBC were given to donor animals with higher doses of PE (8 to 13 mg), transferred spleen cells potentiated the IgM response of the recipients. These opposite suppressive and potentiating activities were found in distinct Sephadex G-200 fractions of 40 and 60 kDa, respectively. When the effect of PE treatment was tested within the same animal, the indirect secondary PFC response following a challenge with SRBC was significantly modified. We observed an overall suppression of the different isotypes after treatment with lower doses of PE or with its 40-kDa fraction. PE doses of 0.5 to 2 mg resulted in a stronger inhibition of IgM than IgG₁ production. This phenomenon was also obtained with the 40 KDa fraction. IgG₂ responses were significantly reduced by all doses of this fraction. In contrast, all doses of the 60-kDa fraction gave a strong stimulation of IgG₂ and IgM responses and a constant suppression of the IgG₁ response. This shows a clear dissociation between IgG₁ and C'-fixing (IgM, IgG₂) antibody classes as far as the influence of placental substances is concerned in their regulation. These data emphasize the relevance of isogeneic placental products as a useful physiological material capable of modulating xenogeneic immune responses (as well as allogeneic systems).     

The inhibitory effect of T-2 toxin on tolerance induction of delayed-type hypersensitivity in mice.

Mice injected intravenously with 1 X 10(9) sheep red blood cells (SRBC) showed no delayed-type hypersensitivity (DTH) response to SRBC and were unresponsive to DTH induction by sc injection of an optimal dose of SRBC. However, when treated with T-2 toxin, a mycotoxin, 2 days after the iv injection, mice became to show significant DTH response and to be responsive to the DTH induction by the sc injection. When the spleen cells of the mice receiving the iv injection were transferred to unsensitized syngeneic recipients, the DTH response of the recipients to SRBC was suppressed. However, the suppressor activity of the spleen cells was decreased by T-2 toxin treatment. By the iv injection, cell population of the spleen was increased and that of the thymus decreased. In contrast, by T-2 toxin treatment 2 days after the iv injection, cell population of the spleen was not increased and that of the thymus was markedly decreased. The ratio of theta-bearing cells was increased in the spleen by the iv injection. However, such increase was not observed after the T-2 toxin treatment. The ratio of Ig-bearing cells in the spleen was not changed by the iv injection and the T-2 toxin treatment after the iv injection. T-2 toxin seems to interfere with generation of suppressor cells for the DTH response.     

Cyclophosphamide-induced specific suppression of the protective immune response to rodent malaria.

Nonspecific and specific chemosuppression of the immune response to Plasmodium berghei protective antigens were investigated. Specific immunosuppression was defined operationally as the selective suppression of the protective response to the parasite in mice injected with a combination of gamma-irradiated infected mouse erythrocytes (gammaPb) and cyclophosphamide (CY) with continued responsiveness to sheep erythrocytes (SRBC). After initial treatment (gammaPb + CY), mice were injected with gammaPb in potentially immunogenic doses. These and appropriate control animals were later challenged with nonirradiated infected mouse erythrocytes. The influence of the initial treatment regimens on the protective response was evaluated by parasitemia, and mortality was observed after challenge. Specificity of suppression was measured by evaluating the ability of mice to produce antibody to SRBC. Both specific and nonspecific suppression of the protective response to malaria were noted. Initial treatment with drug alone resulted in increased parasitemia and mortality and suppression of the SRBC antibody synthesis in drug-pretreated immunized mice as compared with immunized mice not pretreated with the drug. On the other hand, suppression of the response to the parasite, but not to SRBC, in animals pretreated with gammaPb + CY was clearly greater than that induced by drug alone. Thus, animals treated with malarial antigen and cyclophosphamide develop a measurable specific immunosuppression. These studies indicate that immunity to malaria is influenced by both cyclophosphamide alone (general immunosuppression) and cyclophosphamide in combination with antigen (specific immunosuppression) in a manner analogous to other immune responses.     

Induction of immunological tolerance against sheep red blood cells in A/J mice by maternal immunization

Female A/J mice were immunized with sheep red blood cells (SRBC) before mating and boosted a few days before delivery. The progeny of these mothers was immunologically tolerant against SRBC at the level of plaque forming cells (PFC). The state of unresponsiveness was antigen specific. Exchange of the newborn mice between control mothers and immunized ones shows clearly that the tolerance is induced by factors present in the milk or the colostrum, respectively. Some others findings suggest that antibodies of the mothers and not small amounts of the injected antigen are responsible for the nearly complete suppression of the immune response of the offspring.     

Antigen-specific helper activity in serum of mice primed with sheep red cells. I. Definition of the test system and comparison with other systems

An adoptive transfer system is described to measure serum helper activity in the primary antibody response to sheep red blood cells (SRBC). Mice injected with a high dose of cyclophosphamide and reconstituted with rabbit anti-thymocyte serum-treated spleen cells were used as recipients. Serum obtained 9 hr after ip injection of normal mice with 2 × 10⁸ SRBC (S(SRBC)) injected i.v. in the recipients caused a significant enhancement of the antibody response to 2 × 10⁷ SRBC. The serum helper activity was not generated in thymectomized animals and could be absorbed from S(SRBC) by normal and formalinized SRBC. The SRBC-specific serum helper activity (SSHA) is heat labile (30 min 56 °C) and shows allogeneic restriction. Another test system described in literature for measuring T-cell help in vivo was less suited to measure SSHA in the response to 2 × 10⁷ SRBC. A system using normal mice injected with 10⁵ SRBC for determining specific immune response-enhancing factor (SIREF), demonstrated SIREF activity in S(SRBC). It did, however, not measure SSHA, as absorption of S(SRBC) with formalinized SRBC did not abolish the activity in that system.     

Replacement of the helper function of T cells by an RNA-containing antigen-specific lysis factor]

The lymph node viable cells suspension of immunized mice was centrifugated. The supernatant was chromatographed in Sephadex G-200, and fractions were deproteinized. The deproteinized third fraction (Mol wt 30000) stimulated specifically the plaque-forming cells of intact mice immunized by SRBC. It restored the capacity to antibody production in the lethally irradiated intact mice protected by the syngeneic bone marrow. The activity of this fraction disappeared following treatment with RNA-ase, but not with DNA-ase or trypsin. The first and the second deproteinized fractions of the supernatant inhibited non-specifically the viable lymph node cells of the immunized animals in the intact mice immunized with SRBC.     

Antigen-dependent induction of a non-specific humoral factor blocking rosette-forming cells in experiments in vitro and in vivo

Isologous serum of CBA mice immunized with rabbit immunoglobulins (ARIS) contained a factor capable of inactivating rosette-forming splenocytes (RFC) in vitro from the same strain of mice immunized with SRBC. When mouse SRBC immunization was carried out against the background of ARIS injection the court of RFC to SRBC at the peak of immune response was 30% of that of mice injected with SRBC and normal isologous serum. A decrease of RFC count was the result of disappearance of the theta-negative RFC. Passive ARIS immunization failed to influence the antigen-induced proliferation of the antibody-forming cells and the synthesis of antibodies against SRBC.     

Suppressive action of Candida albicans on the immune response in mice.

This study was carried out to determine whether Candida albicans infection has a suppressive effect on the immune response in mice and, if so, whether the suppressive effect influences the response towards T-dependent or T-independent antigens. ICR mice were injected with SRBC with or without C. albicans, or with bacterial LPS with or without C. albicans. The immune response of the mice towards SRBC or towards the LPS was compared by the assay for PFC, hemagglutination and hemolysis tests. The results showed a decrease in the number of PFC in spleens of mice inoculated with SRBC and C. albicans as compared to mice inoculated with SRBC alone, but no decrease in animals injected with LPS and C. albicans as compared to those immunized with LPS alone. No significant differences in the titers of hemagglutinins and hemolysins in sera of mice inoculated with SRBC or with SRBC and C. albicans were observed. C. albicans infection had no effect at all on the hemagglutinins and hemolysins titers in sera of mice inoculated with LPS. These data indicate that C. albicans affects the early phase of the immune response primarily towards T dependent antigens.     

The effect of Bordetella pertussis lymphocytosis-promoting factor (LPF) on antibody response in mice: its enhancing and suppressive effects

The effect of lymphocytosis-promoting factor (LPF) on antibody response in mice was estimated under different sets of experimental conditions. Four- and 6-week-old mice were intravenously inoculated with LPF. Three days later these mice were inoculated either intraperitoneally or intravenously with sheep red blood cell (SRBC) or human serum albumin (HSA) as an antigen. The adjuvant effect of LPF was demonstrated on antibody response in 6-week old mice to intraperitoneally inoculated SRBC but not to intravenously-inoculated one. When 4-week-old mice were immunized, hemagglutinin production in response to intraperitoneally inoculated SRBC was not enhanced by LPF. In addition, a rather suppressive effect of LPF at a comparatively high dose was demonstrated on hemagglutinin production in response to intravenously inoculated SRBC. Anti-HSA production was enhanced by inoculation of LPF in any combination of the mouse age and the route of antigen administration. These findings indicate that the adjuvant effect of LPF on antibody response in mice depends upon experimental conditions: the age of mice, the quality of antigen and the route of antigen administration used for immunization.     

Suppression of delayed-type hypersensitivity to syngeneic testicular cells in mice: involvement of suppressor T cells which act at the induction stage

Suppressor cells for delayed footpad reaction (DFR) against syngeneic testicular cells (TC) were detected in the spleen cells of donor mice immunized intravenously (iv) with viable syngeneic TC. Cyclophosphamide (CY)-pretreated recipients were given spleen cells from donors iv, immunized subcutaneously (sc) with syngeneic TC, and the footpad reaction at 24 hr was elicited with syngeneic TC 6 days after immunization. DFR in the recipients was suppressed by the transfer of spleen suppressor cells. The suppressor cells induced were Thy-1+, CY-sensitive, adult thymectomy (ATx)-resistant and act only at the induction stage. They directly suppress the generation of effector T cells for delayed-type hypersensitivity (DTH). When mice pretreated with CY were actively immunized with syngeneic TC, DFR could be provoked to a measurable level only when they were immunized sc. However, peritoneal exudate cells of those tolerant mice immunized sc without CY pretreatment or immunized iv with CY pretreatment also passively transferred DFR locally, suggesting the existence of effector T cells for DTH even in tolerant mice.     

The isolation of immunogenic molecular entities from immunogenic and nonimmunogenic tumor homogenates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Summary YAC, a Moloney-virus-induced tumor of A-strain mice, is a nonimmunogenic tumor. Mice injected with the inactivated neoplastic cells and challenged with viable tumor cells did not survive longer than mice that received the challenge dose alone. The homogenate of this nonimmunogenic tumor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel slices containing isolated molecular entities were injected into various groups of mice. The mice were challenged with low doses of viable tumor cells (10–30 cells) and their survival time was recorded. Small but significant numbers of mice injected with apparent 80–90 K SDS-PAGE-isolated molecular entity rejected the tumor or survived longer than the control groups of mice. Spleen cells from mice injected with 80–90 K molecular entity inhibited the YAC tumor cotransferred with them to naive recipients (Winn assay). Spleen cells from mice injected with monoclonal antibody against nonspecific T-cell helper factor and immunized with 80–90 K SDS-PAGE-isolated molecular entity failed to inhibit the tumor growth in naive recipients, indicating that helper T cells are involved in induction of the antitumor resistance. Nylon-wool-passed splenocytes from mice injected with 80–90 K inhibited tumor growth in some of the recipient mice. Spleen cells from these mice treated with anti-Thy-1 and complement also inhibited the tumor growth in some of the recipients, suggesting that the effector cells were both T and non-T cells. C57BL/6 mice immunized with apparent 20 K SDS-PAGE-isolated molecular entity of RBL5 tumor also induced in vivo resistance to the syngeneic viable RBL5 cells, but not to the syngeneic B16 melanoma cells, indicating the specificity of the protective effect. The practical and theoretical implications of these findings are discussed.     

Supressor activity of spleen cells during medically induced immunologic tolerance]

SRBC tolerance was induced in mice (CBA X C57BL/6) F1 by single intraperitoneal injection of 6 X 10(9) SRBC and of cyclophosphamide (100-200 mg/kg) in 44-46 hours. Spleen cells of tolerant mice obtained at various periods after the tolerance induction (in 12-26 days) failed to decrease their immune response to SRBC after administration to intact syngeneic recipients. Contrary to intact mice, tolerant animals were incapable of producing suppressor cells after a single SRBC immunization. Only when 3 additional injections of high SRBC doses (6 X 10(9)) were given to tolerant mice the spleen cells in them acquired the capacity to inhibit the immune response after administration to normal mice. It is supposed that the absence of suppressor cells in induction of the immunological tolerance by means of cyclophosphane was caused by the processes of clone elimination. Suppressor cells can originate in tolerant animals under the effect of intensive antigenic stimulation, this leading to enhancement of the tolerance state as a result of additional SRBC injections.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Antibody formation in mouse bone marrow. VIII. Dependence on potentially circulating memory cells: a study with parabiotic mice.

Mouse bone marrow barely contains antibody-producing plaque-forming cells (PFC) during the primary response to sheep red blood cells (SRBC). However, during the secondary response, the number of IgM, IgG, and IgA PFC in the bone marrow can rise to a level which surpasses the number of PFC in all the other lymphoid organs together. In the present paper we investigated whether the capacity of immune mice to react upon a booster injection of SRBC with a bone marrow PFC response can be transferred from immune to nonimmune mice. Therefore, mice primed with SRBC 6 months previously and nonprimed syngeneic mice were joined for parabiosis and were separated from each other at various intervals after joining. These separated mice were subsequently immunized with SRBC. It was found that, after 3 weeks of parabiosis, the nonprimed members reacted upon an injection of SRBC with a bone marrow IgM, IgG, and IgA PFC response as high as did the previously primed members. Furthermore it could be demonstrated by means of cell transfer experiments that, after a period of parabiosis of 3 weeks, the bone marrow and spleen of the normal mice contained about as many memory cells as the bone marrow and spleen of the immune mice. These results suggest that antibody formation in mouse bone marrow is dependent on a population of potentially circulating memory cells.     

Can tolerant allogeneic cells restore nude mice?

BALB/c-nunu mice have been injected with allogeneic or tolerant allogeneic spleen or thymus cells and sheep red blood cells (SRBC). From day 3 to day 10 the mice were bled daily and the antiSRBC antibody was assayed by hemagglutination. No difference was found between recipients of normal allogeneic cells or tolerant allogeneic cells. Both showed a transient response but the response was maintained only in recipients of congenic cells.     

Primary and secondary immune response in animals following spontaneous loss of tolerance

Mice subjected to tolerogenic treatment by sheep red blood cells (SRBC) and cyclophosphamide were immunized at various intervals (from 1 to 8 weeks after treatment) either by a single injection of 5 X 10(8) SRBC or by a double-injection of 1 X 10(6) SRBC. In control mice both immunization methods proved to be equally successful. In the experimental animals the immunological memory formation and/or its realization was destroyed to a greater extent and was restored more slowly than the capacity to the primary response.     

Delayed-type hypersensitivity (DTH) in BCG-sensitized mice I. Lack of suppressor T cell activity on DTH to sheep red blood cells.

Mice pretreated with an intravenous (i.v.) injection of BCG (BCG-sensitized mice) and then immunized intravenously with a high dose (10(8)--10(9)) of sheep red blood cells (SRBC) 2 weeks later developed strong delayed-type hypersensitivity (DTH) to SRBC, as in mice pretreated with cyclophosphamide (CY) (CY-treated mice) and then immunized with SRBC 2 days later; normal mice given the same dose of SRBC did not show such DTH. The mechanism of this strong DTH to SRBC which developed in BCG-sensitized mice was studied, by comparing it with that in CY-treated mice. The transfer of either whole spleen cells or thymus cells, but not serum, obtained from mice immunized with i.v. injections of 10(9) SRBC 4 days previously (hyperimmune mice) did not suppress either the induction or the expression of DTH to SRBC in BCG-sensitized mice, but suppressed those in CY-treated mice. The suppressor cells were SRBC-specific T cells. Adoptive transfer of DTH to SRBC by spleen cells from either BCG-sensitized mice of CY-treated mice to hyperimmune recipients failed. The adoptive transfer of DTH from BCG-sensitized mice to normal recipients also failed if the spleen cells from hyperimmune mice were cotransferred. Whole body irradiation (600 rad) of mice 2 hr before or after the time of immunization with SRBC reduced significantly DTH to SRBC in both BCG-sensitized and CY-treated mice. It was noticed that the total number of spleen cells in BCG-sensitized mice was 3--4 times larger than that in CY-treated mice. From these results, we conclude that the entity of effector T cells of DTH to SRBC induced in BCG-sensitized mice and in CY-treated mice was not different in terms of susceptibility to suppressor T cells and irradiation, but that the total numbers of effector T cells generated in these mice differed remarkably, resulting in the above-described different responsiveness to suppressor T cells transferred passively.