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.     

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.     

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.     

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.     

Suppression of the humoral immune response by plasmacytomas: mediation by adherent mononuclear cells.

Mice bearing plasmacytomas have a severely impaired ability to mount a primary immune response; T cells from these mice, however, appear by both in vivo and in vitro criteria to function normally. This unusual pattern of immunodeficiency appears to be mediated by a regulatory cell found in the spleens and peritoneal cavities but not in the lymph nodes or thymuses of mice bearing plasmacytomas. The number of cells with suppressor activity in the spleens of plasmacytoma-bearing mice is directly proportional to the size of the subcutaneous tumor borne by the host. These cells are capable of suppressing antibody production in in vitro cultures of normal spleen cells but have no demonstrable effect on the ability of normal spleen cells to proliferate in vitro in response to phytohemagglutinin or 8-Br-guanosine-3', 5'-monophosphate (T and B cell mitogens, respectively). Characterization of the suppressor cell population on the basis of its cell surface properties, its radioresistance, its morphology, and its ability to adhere to various solid matrices suggest that these cells are adherent mononuclear cells. These data support the concept that plasma cell tumors indirectly induce an impairment in the humoral immune response of their hosts by stimulating the expression of regulatory functions in a population of splenic and peritoneal macrophages.     

Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

Characteristics of the splenic suppressor cell--target cell interaction in experimental African trypanosomiasis

We have examined further the relationship between immunosuppression and suppressor cell activity in experimental African trypanosomiasis. In the present study we describe the nature of the interaction between splenic suppressor macrophages from Trypanosoma rhodesiense-infected C57BL/6 mice and target effector cells in the primary in vitro PFC response to SRBC. Suppressor cell potential was expressed only when cell-cell contact of a noncytolytic nature was established between infected spleen cells and normal splenic responder cells. Isolation of suppressor cells from responder cells by a cell-impermeable membrane completely abrogated suppression. Similarly, supernatant fluids from infected spleen cell cultures could not passively transfer suppression. Suppressor cells did not act via prostaglandin synthesis in that indomethacin failed to restore responsiveness to infected spleen cells or to passively suppressed normal cultures. Inhibition of DNA synthesis by irradiation of mitomycin C treatment did not block suppressor cell function, but suppressor cell effects were inhibited by exposure of infected spleen cells to silica particles or to heat treatment. We conclude that suppressor cell effects in experimental African trypanosomiasis are consistent with a suppressor macrophage acting via a noncytolytic cell-cell interaction with responder target cells.     

Mouse fetal liver cells manifest antigen-specific suppressor activity

Liver cells obtained from C57B1/6J mice at different stages of development suppress the primary in vitro induced immune response. Fetal liver cells showed the strongest suppression of the PFC response, an effect which was gradually lost after birth. Thymic or splenic cells were ineffective in suppressing the PFC response under conditions where fetal liver cells from the same donors were highly active. Liver cells from newborn C57B1/6J athymic nude mice were equally suppressive as cells from their normal thymus-bearing littermates. Preculture of liver cells from 18-day-old fetuses with antigen homologous to that used in the indicator system increased suppressor activity severalfold compared with other experimental groups in which cells have been precultured in medium alone or with the addition of a heterologous antigen. The data suggest that antigen-specific suppressor activity is present in fetal liver cells. The possible relevance of these findings in relation to acquisition of self-tolerance is discussed.     

Regulation of the primary in vitro response to TNP-polymerized ovalbumin by T suppressor cells induced by ovalbumin feeding

Spleen cells from DBA/2 mice that received a single feeding of 20 mg of ovalbumin (OVA) 7 days previously were specifically hyporesponsive to primary in vitro challenge with the thymic-dependent antigen TNP-polymerized ovalbumin (TNP-POL-OVA). The tolerance observed in spleen cells from OVA-fed animals was dependent upon OVA-specific T suppressor cells, because splenic T cells from OVA-fed mice suppressed the primary response to TNP-POL-OVA of cultures containing normal T and B cells. The tolerance and suppression was OVA specific, because spleen cells from OVA-fed animals responded well to other antigens (including TNP on another carrier), and splenic T cells from OVA-fed mice did not affect the response of normal T and B cells to sheep erythrocytes. These data confirm the existence of T suppressor cells after OVA feeding and provide a direct means of assaying their activity in a primary in vitro response.     

Granulocyte-macrophage colony-stimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells

Purified, recombinant-derived murine granulocyte-monocyte colony-stimulating factor was found to enhance the primary in vitro immune response to SRBC by murine spleen cells. In determining the mechanism of this augmentation, it was found that only splenic adherent cells and neither resting nor activated T cells nor B cells expressed specific receptors for GM-CSF. When splenic adherent cells were pulsed briefly with GM-CSF before addition to macrophage-depleted cultures, they reconstituted the PFC response to a significantly greater degree than did control macrophages. Splenic adherent cells incubated overnight with SRBC plus GM-CSF were also more efficient antigen-presenting cells than splenic adherent cells incubated with antigen alone. The mechanism of this enhanced antigen presentation was found to be due to a GM-CSF-dependent increase in the level of IL 1 secretion and Ia antigen expression. Consistent with these data was the finding that GM-CSF augmented IL 2 production by splenic T cells in response to suboptimal concentrations of Con A. Finally, the day 5 in vivo antibody response (as measured by serum titers) of mice immunized with a low dose of SRBC was enhanced by two daily inoculations of GM-CSF. Thus, the role that GM-CSF plays in augmenting immune responses may not be solely accounted for by its ability to cause the proliferation or differentiation of macrophages, but more than likely includes its ability to enhance the function of antigen-presenting macrophages.     

The evolution of immunosuppressive cell populations in experimental mycobacterial infection.

Immunosuppressor activity of considerable potency and complexity was generated during the course of chronic, progressive infection of C3H/Anf mice by Mycobacterium lepraemurium. From the 5th through 10th week after inoculation, spleen cells from infected mice mildly but reproducibly suppressed the direct plaque-forming cell response of normal spleen cell cultures to sheep erythrocytes. Suppression at this stage of infection was mediated by cells with macrophage-like characteristics. A marked increase in splenic suppressor activity at 10 to 11 weeks was associated with the appearance of a second suppressor cell subpopulation composed of T lymphocytes. The appearance of these cells was closely related in time to the onset of rapid splenic enlargement and a loss of cutaneous delayed type hypersensitivity to antigens of M. lepraemurium in mice at 10 to 11 weeks of infection. Suppressor cells were not present in peripheral lymph nodes until terminal infection at 22 to 25 weeks. Suppressor spleen cells depressed the T-dependent antibody response most severely, but there was also a direct effect upon B cells as shown by moderate suppression of responses to TNP-LPS and DNP-Ficoll. Spleen cells from 14-week-infected mice generated a soluble suppressor factor(s) that induces depression of moderate severity, however, the immunosuppression by intact cells was far greater.     

Characterization of the suppressor cell(s) responsible for anterior chamber-associated immune deviation (ACAID) induced in BALB/c mice by P815 cells

Anterior chamber-associated immune deviation (ACAID) is a complex set of immune responses induced by the inoculation of antigens into the anterior chamber of the eye. Histocompatibility antigens, tumor-specific antigens, reactive haptens, and viral antigens have been shown to induce this phenomenon, which comprises the following specific host responses: high titer humoral antibodies, primed cytotoxic T cells, but specifically, impaired skin graft rejection and delayed-type hypersensitivity (DTH). Using the model system of ACAID induced by inoculation of P815 mastocytoma cells into the anterior chambers of H-2-compatible, but minor H-incompatible, BALB/c mice, we demonstrate that the impaired capacity of these animals to develop and express DTH is due to the activation of suppressor T cells. Generation of these cells requires an intact spleen, is not inhibited by cyclophosphamide pretreatment, and is abrogated by systemic treatment of the host with anti-I-J monoclonal antibodies. This splenic suppressor cell(s) can transfer suppression of DTH adoptively to naive syngeneic mice. One suppressor cell is Thy-1.2, Lyt-2.2, and I-Jd positive. A minority of these cells (or a second population of suppressor cells) also expresses the L3T4 surface marker. Suppression is exerted on the efferent limb of DTH expression, although afferent suppression is not excluded. P815-induced ACAID suppressor cells resemble similar cells induced by haptenated spleen cells inoculated into the anterior chamber of the eye. We propose that induction of these suppressor cells, whose target of action is selective for T DTH cells, but not for other types of T cells, is responsible for the phenomenon of immune privilege in the anterior chamber of the eye.     

Alterations in the Immunogenic Properties of Sheep Erythrocytes by Sonic Disruption

A solubilized sheep red blood cell (SRBC) antigen (supernatant fraction obtained by centrifuging 10^7-2 × 10⁸ sonicated SRBC at 6 × 10⁴ g for 30 min [Sup-SRBC]), whose ability to inhibit anti-SRBC plaque formation was 70% of that of the original sonicated SRBC, was unable to elicit a detectable antibody response in either unprimed or SRBC-primed mice. However, Sup-SRBC as well as intact SRBC antigens generated memory for the secondary response, which was transferable to irradiated syngeneic recipients by injection of immune spleen cells. The memory generated by Sup-SRBC involved helper memory for anti-trinitrophenyl group (TNP) response to challenge with TNP-conjugated SRBC. Increase in the helper T cell memory in the spleens of Sup-SRBC-primed mice was also demonstrated by an in vitro culture experiment and by an adoptive cell transfer experiment. In contrast, no detectable B cell memory was generated by Sup-SRBC. Repeated stimulation with Sup-SRBC never induced significant antibody response but reduced the level of memory. A single injection of a low dose (10⁶) of SRBC also failed to induce a definite primary antibody response generating memory for the secondary response. However, repeated stimulation with this dose of SRBC induced a high antibody response and generated good memory. From these results it is suggested that the intact structure of SRBC is required for the activation of B cells, but is not necessary for the stimulation of T cells.     

Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.     

Antigen-induced cyclophosphamide-resistant suppressor T cells inhibit the in vitro generation of cytotoxic cells from one-way mixed leukocyte reactions.

Mice sensitized against a tumor allograft and given cyclophosmamide 6 days later failed to generate an immune response to the allograft. Spleen cells derived from these mice suppressed the generation of cytotoxic T cell response by normal spleen cells in mixed leukocyte cultures. The suppressor cells were not specific, thymus dependent, and resistant to 2000 R.     

Suppression of immune response by lung cells in experimental tuberculosis

In vitro proliferative response of lung cells from mice infected with Mycobacterium tuberculosis H37Rv against PPD and Con A was studied. It was shown that the infected lung contained immune T cells, but their response in vitro was totally inhibited by plastic and nylon wool adherent suppressor cells. The whole population of lung cells from infected, but not intact mice, efficiently suppressed the proliferative response of immune lymph node cells against various antigens (non-specific suppression). The inhibition of response again depended on the presence of plastic adherent lung cells. Our data suggest that at least two suppressor pathways are induced in the course of tuberculosis infection: one being specific for mycobacterial antigens and other non-specific. Both types of suppressor pathways depend on the plastic adherent lung cells from tuberculosis lesion.     

Contribution of Lyb 5+ and Lyb 5- B cells to the primary and secondary phosphocholine-specific antibody response

Due to a mutation on their X-chromosome, CBA/N mice lack the Lyb-5+ subset of B cells. The loss of this B cell subset results in a profound alteration in the immune response of these mice to the hapten phosphocholine (PC). Thus, when these mice are immunized with high doses of PC-KLH (200 micrograms) in CFA, they: 1) fail to produce IgM anti-PC antibodies; 2) produce little or no anti-PC antibody bearing the normally predominant T15-idiotype; and 3) produce IgG anti-PC antibodies only late in the primary response. In order to more fully delineate this defect in responsiveness to PC, the splenic focus assay was used to analyze Lyb-5- B cell precursors from both normal and immune defective mice. Lyb-5- cells were obtained from normal (CBA/N x DBA/2)F1 (CD) female spleens by treatment with anti-Lyb-5 serum and complement. These normal Lyb-5- cells and Lyb-5- cells from immune defective CD male mice were stimulated in vitro with either PC-Hy or TNP-Hy in the presence of Hy-primed T helper cells. The results demonstrate that primary Lyb-5- PC-specific B cells fail to respond in the splenic focus assay, while secondary Lyb-5- PC-specific precursors respond normally, and that both primary and secondary Lyb-5- TNP-specific precursors respond in the splenic focus assay. These data suggest that Lyb-5- PC-specific precursors must differentiate into memory cells before they can be activated to secrete antibody, and they also indicate that the Lyb-5- B cell subset may be composed of two subsets with different activation requirements.     

Splenic I-J-bearing antigen-presenting cells in activation of suppression: further characterization

A set of I-J-bearing murine splenic antigen-presenting cells (APC) has been found to be responsible for first order suppressor cell (Ts1, afferent suppressor cell) activation in the azobenzenearsonate (ABA) hapten system after intravenous administration. Suppressor cells induced by this set of hapten-coupled cells do not function in the efferent phase of the delayed hypersensitivity (DTH) response. The functional activity of this novel APC to activate afferent suppressor cells was resistant to a dose of ultraviolet radiation (UVR) sufficient to largely abrogate the ability of splenic APC to immunize for a DTH response. It was also found that the previously described splenic I-J-bearing APC needed for third-order suppressor cell (Ts3, effector-suppressor cell) activation is adherent and UVR resistant. The sets of I-J-bearing APC appear to be crucial elements in the activation of suppression and thus in determining the balance between immunologic reactivity and unresponsiveness. Furthermore, the UVR resistance of this set of novel APC may be relevant to the in vivo effects of UVR exposure to mice.     

Surgical influence on murine immunity and tumor growth: relationship of body temperature and hormones with splenocytes.

Adrenalectomy predisposed the C3HeB/FeJ Mouse to tumor from a low dose of tumor cells, derived from a C3H spontaneous mammary adenocarcinoma. Sham surgery had a similar effect. In contrast, ovariectomized females, intact females, and male mice did not allow the low dose of cells to develop into a tumor. In order to better understand the role of hormones on the immune system controlling tumor growth, normal C3HeB/FeJ mice were studied for the effect of corticosterone or estradiol on splenic lymphocyte surface antigen expression. Adrenalectomy and ovariectomy caused a decrease in the percentage of all T cell subclasses and an increase in absolute numbers of immunoglobulin-bearing cells. Reconstitution of ovariectomized mice with estradiol did not significantly alter lymphocyte cell surface antigen expression. In contrast, injection of corticosterone into adrenalectomized mice brought these values to normal. Further study on normal mice placed on a 12:12-hr light:dark schedule showed that the hours after lights on (HALO) had a significant effect (analysis of variance) on body temperature, percentage of splenic B cells, T pan, T helper and T suppressor cells, and absolute numbers of T pan cells. Brain dehydroepiandrosterone sulfate correlated positively with T pan lymphocytes, but showed no significant effect on HALO. In contrast, body temperature showed a strong circadian rhythm (P less than 0.001). In addition, the presentation of estrus was circadian rhythmic (P = 0.003) with 58% of mice in estrus at 16 HALO and only 8% at 4 HALO. Multiple regression analysis revealed body temperature was strongly associated with absolute numbers of splenic T lymphocytes and their subsets, as well as percentage of B lymphocytes, Thy 1.2-, and Lyt-2-bearing cells. Similarly, HALO and estrous cycle stage were associated with percentage of T helper cells. The data showed that body temperature and hormones were associated with the cell surface antigens on lymphocytes and suggest that they affect lymphocyte function.     

Effect of a staphylococcaL infection on the development of an immune response after physical loading

The infection of rats with staphylococci enhances the suppressing effect of physical loading on the development of immune response induced by the injection of sheep red blood cells. Intensive physical loading is accompanied by the release of the substance activating the function of splenic suppressor cells into the blood. Staphylococcal infection increases the action of the serum substance of rats performing physical work on splenic suppressor cells, as well as on the action of the suppressor factor released by these cells on immunocompetent cells.