Modulation of help and suppression in a hapten-carrier system.

Provision of beta-galactosidase (GZ) under defined conditions of dose and time can either help or suppress a subsequent response to trinitrophenyl (TNP)-GZ in CBA/J mice. The optimal helper effect occurs when 10(7) spleen cells from mice primed 9 or more days previously with 10 mug GZ are adoptively transferred to irradiated recipients which are than challenged with 10 mug TNP50GZ. Optimum suppression results from the transfer of spleen cells from mice primed 3 days previously with 100 mug GZ and challenge of recipients with TMP150GZ. Both help and suppression are carrier-specific and mediated by T cells. In experiments where helper or suppressor cells were mixed with normal cells, the anti-TNP response was proportional to the number of primed cells transferred. The results point to a wave of suppression as the initial event after immunization, which is succeeded by period in which the helper effect dominates.     

IgM-mediated, T cell-independent suppression of humoral immunity.

The immunological unreactive state occurring in (T,G)-A-L nonresponder mice after secondary antigen challenge was investigated. Syngeneic IgM anti-(T,G)-A-L antibody-containing plasma, transferred at the time of the time of primary challenge, induced persistent suppression of autologous specific antibody production. Removal of plasma IgM with goat anti-mu antisera removed the ability of the plasma to supress. The induction and maintenance of the suppressed state were not different in thymectomized or sham-thymectomized animals. Primed animals subjected to graft-vs-host reaction (GVHR) at the time of secondary challenge switched over to IgG production. Animals suppressed by passive antibody transfer reacted to GVHR, at the time of secondary challenge, with specific IgM but not IgG antibody production. Transfused normal spleen cells partially abrogated suppression only when (suppressed) hosts had been lethally irradiated. Spleen cells from antigen-plus-antibody suppressed donors, upon transfer to previously normal, syngeneic hosts, were less immunocompetent than spleen cells from untreated donors. These data are consistent with a model of IgM mediated, T cell-independent persistent suppression of humoral immunity.     

Immunologic suppression after oral administration of antigen. I. Specific suppressor cells formed in rat Peyer's patches after oral administration of sheep erythrocytes and their systemic migration.

Rats given 10(10) sheep erythrocytes (SRBC) orally were found to contain specific suppressor cells to SRBC in their Peyer's patches (PP) and mesenteric lymph nodes (MLN) after 2 days of feeding. After 4 days of feeding, similar suppressor cells were found in the thymus and spleen, but they were missing in the PP or MLN. These suppressor cells effectively blocked IgM and IgG plaque-forming cell responses to SRBC in Mishell-Dutton cultures and delayed-type-hypersensitivity responses to SRBC when transferred to syngeneic recipients, but they did not affect responses to horse erythrocytes. The orally induced specific suppressor cells appeared to be T2 cells since their activity was eliminated by in vivo treatment of SRBC-fed rats with anti-rat lymphocyte serum but not by adult thymectomy. Because carrageenan partially relieved the suppression observed in culture, the actual suppressive mechanism may also involve a macrophage.     

Specific, transient suppression of the immune response by HGG tolerant spleen cells. II. Effector cells and target cells.

In a previous report, it was shown that spleen cells from mice made tolerant to human gamma-globulin (HGG)5 could specifically inhibit the immune response of normal spleen cells after adoptive transfer to lethally irradiated recipients. However, that report also showed that the suppressive activity was only transiently associated with tolerant spleen cell populations. It was concluded from those experiments that while suppressive activity could be demonstrated in tolerant spleen cells under certain conditions, such activity was not obligatory for the maintainance of the tolerant state. The experiments presented here were performed to determine the nature of the effector cell(s) and the target cell(s) involved in this system of suppression of the immune response. Treatment of cells from tolerant animals with anti-thymocyte serum and complement to remove thymus-derived (T) cells completely abrogated suppresive activity. Removal of adherent cells from tolerant spleen cells by passage over glass wool columns resulted in partial loss of the suppression. The inhibitory activity of the suppressor cells was resistant to 900 R irradiation regardless of whether the tolerant spleen cells were irradiated before or after adoptive transfer. The cellular target(s) for the supprssor cells was examined by using lipopolysaccharide (LPS) as an alternative source of helper activity for the response to HGG. LPS, injected at the time of the initial antigenic challenge of mice that had been reconstituted with tolerant and normal spleen cells, prevented the expression of suppression against bone marrow-derived (B) cells. However, when LPS was presented only at the time of secondary antigenic challenge, it was unable to overcome suppression of the immune response of reconstituted recipients. Thus, LPS could produce a state where the B cells were resistant to suppression, but LPS could not rescue the responsiveness of B cells once the cells in the reconstituted recipient had been suppressed. In addition, the immune response to both the hapten dinitrophenol (DNP) and the carrier (HGG) were suppressed when recipients of tolerant and normal spleen cells were challenged with DNP6HGG. This indicates that T helper cells are also a target for suppression. The results presented in this paper are discussed in relation to a possible mechanism of suppression which proposes that suppressive activity represents the induction of tolerance in immunologically competent cells by HCG which is closely associated with the tolerant spleen cells.     

Long-term effects of in vitro sensitization on immune reactivity of lymphocytes: generation of suppressor and helper T cells.

Mouse spleen cells were cultured for 5 days with or without HRBC. Cultured cells were 'parked' in irradiated syngeneic recipients for 3 weeks and then tested for their immunologic reactivity in vitro. We found that spleen cells from recipients of HRBC-sensitized cells (S) as well as spleen cells from recipients of control unsensitized cells (U) possessed radiosensitive suppressor and radioresistant helper activities. Suppressor activity was observed by the capacity of unirradiated S and U spleen cells to inhibit the in vitro generation of IgM and IgG PFC by spleen cells primed in vivo to HRBC or to LacKLH. Helper activity was shown by the capacity of the irradiated S and U cells to restore IgM and IgG PFC responses of in vivo primed, T-depleted spleen cells to HRBC, LacHRBC, and LacCRBC. Both suppressor and helper activities were mediated by T cells. The possibilities that immunologically specific or nonspecific mechanisms account for these phenomena are discussed.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

Suppressor cells as an agent of immune facilitation. II. Adoptive transfer of passively induced enhancement of allografted tumors

CBA mice injected intravenously with CBA anti-A/J spleen cell antiserum, and challenged subcutaneously 24 hr later with A/J-derived sarcoma 1 (Sa 1) develop progressive tumors. “Normal” CBA mice (i.e., injected with normal CBA serum or noninjected) reject the allograft within 20 days. Spleen cells taken from mice 20–35 days after the injection of antiserum and Sa 1 challenge can specifically transfer the ability to enhance tumor growth when injected into 200 rad irradiated recipients. Spleen cells taken 8 days after antiserum and Sa 1 challenge cannot transfer suppression. The induction of suppressor cells requires both antiserum treatment and Sa 1 challenge. Serum from suppressed mice, and from control mice that are rejecting their tumors, can also transfer suppression but only when taken 20–35 days after treatment. The suppressor cells function most effectively when transferred at the time of tumor challenge, however, they also inhibit the rejection of Sa 1 when mixed with nylon-wool purified sensitized T cells. Suppressor cells are both nylon-wool nonadherent and adherent. Further purification of the column-enriched cells using antiimmunoglobulin or anti-Thy 1.2, plus complement, suggests that both T and B cells can suppress. The mixed lymphocyte response (MLR) of spleen cells from mice challenged only with Sa 1 is inhibited 8 days after challenge. A secondary response is obtained at 20–27 days. In contrast, the MLR from antiserum and Sa 1-treated mice 8 days after challenge resembles a secondary response. By 20–27 days mice with progressive tumors have only a primary-like response. In the present experimental situation, mitomycin-treated spleen cells from antiserum and Sa 1-treated mice cannot significantly inhibit the MLR of normal cells.     

Modulation of the IgE immune response to BSA by fragments of the antigen. I. Suppression by free fragments and by fragments conjugated to homologous gamma-globulin

Nonimmunogenic peptic fragments of bovine serum albumin (BSA), Fraction Ia, suppressed immune response to BSA in mice. Splenic T lymphocytes from mice treated with these fragments suppressed the anti-DNP response in irradiated mice reconstituted with DNP-BSA-primed cells, indicating carrier-specific suppression. The conjugate of Fraction Ia with mouse γ-globulin (MGG) was found to be an effective suppressive substance but it did not induce suppressor T cells. B cells from mice given Ia-MGG were unresponsive to BSA when transferred to irradiated recipients along with either normal or BSA-primed T cells. Thus, unresponsiveness to BSA was mediated by either T or B lymphocytes, depending whether the inducing substance was a free fragment of the antigen or fragments conjugated to homologous γ-globulin.     

Effect of murine tumors upon delayed hypersensitivity to dinitrochlorobenzene. III. In vivo activity of the nonspecific suppressor cell

Tumor-induced immunosuppression was investigated in an in vivo model of delayed hypersensitivity (DH) to the chemical sensitizer, dinitrochlorobenzene (DNCB). DH to DNCB as measured in a footpad assay was decreased in C3H/HeJ mice bearing MCA-F, a 3-methylcholanthrene-induced syngeneic fibrosarcoma. Suppressor cells from the spleens of tumor-bearing mice inhibited the induction of DH to DNCB in otherwise normal syngeneic C3H/HeJ recipients. Ten million spleen cells (SpC) harvested from mice bearing MCA-F for 10 days and adoptively transferred to tumor-free mice at the time of sensitization with DNCB suppressed the response to the sensitizer. The suppressor cells were macrophages, since they were adherent to plastic, removed by treatment with a magnet after phagocytosis of carbonyl iron, resistant to exposure to gamma radiation and to treatment with anti-Thy 1.2 serum and complement. Further, the nonspecific suppressor cells were activated by progressive tumor growth rather than by induction of tumor-specific immunity using irradiated tumor cells. Titration studies revealed that suppression of DH occurred with the transfer of as few as 10(6) SpC. Thus, nonspecific suppressor cells are effective at inhibiting in vivo DH to DNCB and suggest that nonspecific suppression in the intact host occurs through mechanisms different from those involved in suppression in vitro.     

Neonatal splenic suppressor cells in the chicken. I. In vivo suppression of the immune response to bovine serum albumin by normal and tolerized neonatal spleen cells

The presence of active splenic suppressor cells in neonatal chickens, either normal or tolerant to bovine serum albumin (BSA), was examined by assessment of their effect on both primary and adoptively transferred secondary responses to BSA or sheep red blood cells (SRC). Both normal and BSA tolerized spleen cells were shown to be highly suppressive of secondary anti-BSA responses generated by specifically primed adult spleen cells in inert recipients. Suppression of the secondary anti-BSA response by normal spleen cells was slightly less effective than that seen with BSA tolerant spleen cells. Transfer of BSA tolerant spleen cells into normal recipients, followed by BSA challenge, prevented any significant primary anti-BSA response. In contrast, transfer of normal spleen cells into normal recipients, followed by BSA challenge, failed to show any suppression of the resulting primary response. Neither normal nor BSA tolerant neonatal spleen cells were capable of suppressing either primary or secondary responses to SRC. Thus, chickens tolerized to BSA have suppressor cells specific for the tolerizing antigen. We present evidence that both the tolerance associated suppressors and the suppressors detected in normal neonatal chickens are T cells.     

Intact B-cell activity is essential for complete expression of experimental allergic encephalomyelitis in Lewis rats

The mechanisms of the inhibitory effect of the serum thymic factor (FTS) on allograft immunity have been studied on both cellular and humoral immune responses of skin allografted mice. FTS-induced suppression of allogeneic skin graft rejection was correlated with a transient diminution of in vivo alloreactive CTL production in the spleen, whereas the generation of allo-anti-H-2 antibodies was not affected. The involvement of suppressor cells in the effect of FTS was supported by the observation that irradiated spleen cells from FTS-treated recipients (bearing a 9-day allograft) suppressed the in vitro CTL generation.     

The immunological role of the thymus in the pathogenesis of virus-induced lymphomas. Suppression of cytolytic T-cell function

Thymectomy of young adult mice has been found to prevent virus-induced lymphomas which develop as the animals age. Thymectomy protects mice by removing a source of suppressor T cells which inhibit the generation of cytolytic T cells against autochthonous tumors. Furthermore, suppression is specific since T cells are regulated in their capacity to respond to syngeneic but not allogeneic tumor cells. To determine if suppression could be adoptively transferred, lethally irradiated, bone-marrow-reconstituted mice were inoculated with T cells from either normal or thymectomized mice. Only T cells from thymectomized animals transferred enhanced T-cell reactivity to syngeneic tumor cells. More importantly, T cells from thymectomized mice injected with virus protected recipients challenged with lethal doses of syngeneic tumor cells. We conclude that thymectomy protects mice from developing virus-induced T-cell lymphomas by removing a source of suppressor T cells which regulates the activity of specific cytolytic T cells directed against autochthonous tumor cells.     

Trypanosoma cruzi-induced suppressor substance

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.     

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Studies on the mechanism of suppression of experimental allergic encephalomyelitis induced by myelin basic protein-cell conjugates

The mechanism of suppression of experimental allergic encephalomyelitis (EAE) induced in Lewis rats by pretreatment with myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) was examined. Studies on the kinetics of the tolerance induction showed that pretreatment with MBP-SL suppressed EAE if given 7 but not 3 days before the disease-inducing injection of MBP in Freund's complete adjuvant. Treatment with cyclophosphamide 48 hr before administration of MBP-SL completely abolished the suppression of EAE. Transfer of lymph node and spleen cells from MBP-syngeneic erythrocyte conjugate (MBP-RBC) but not MBP-SL-pretreated rats resulted in suppression of disease in recipients subsequently given a disease-inducing injection of MBP. Administration of MBP coupled to SL from the histocompatible rat strain F344 resulted in suppression of the MBP-induced proliferative response of spleen cells from Lewis rats which had been given a disease-inducing injection of MBP. Taken together these results are consistent with the suppression of EAE induced by MBP-SL being mediated by suppressor T cells.     

Suppressor T cells induced by soluble immune complexes can adoptively transfer inhibition of cytophilic antibody receptors on macrophages.

Immune complexes (soluble antigens of L1210 and antibody to L1210) when given to allogeneic C3H mice generated suppressor cells that inhibited receptors for cytophilic antibody on macrophages. Thymocytes or nylon-nonadherent splenic T cells (4 × 10⁷) from immune-complex-treated mice transferred this suppressive activity when injected into normal syngeneic mice. Maximal suppression of macrophages occurred 4 to 6 days after transfer. In contrast, even 5 × 10⁷ nylon-adherent, non-T spleen cells from immune-complex-treated (“suppressed”) mice failed to induce macrophage suppression in the syngeneic recipients. When T-cell-depleted “B” mice were used as recipients, neither thymocytes nor splenic T cells from suppressed mice were able to transfer suppressive activity. However, the admixture of 2 × 10⁷ normal syngeneic thymocytes with 4 × 10⁷ thymocytes from suppressed mice restored the latter's ability to elicit suppression of macrophages in T-cell-deprived recipients. Peritoneal monocytes from recipients of suppressor thymocytes (to L1210) could not attach cytophilic antibody to L1210 but could attach cytophilic antibody to EL-4 and sheep erythrocytes. Thus, suppressor T cells induced by immune complexes can transfer immunologically specific macrophage suppression (inhibition of cytophilic antibody receptors) to syngeneic recipients. The suppressor cells required the cooperation of normal T cells, suggesting either recruitment of suppressor cells from, or a helper effect by, the normal T cells, in order to produce their effect.     

Regulatory action of immune B cells on delayed-type hypersensitivity in mice

Suppressor cells in delayed-type hypersensitivity (DTH) to soluble protein antigens were induced in vitro from BALB/c spleen cells. Transfer of these cells into syngeneic recipients resulted in suppression of the hosts' DTH responses in an antigen-specific manner. These suppressor cells were characterized as B cells by their adherence to nylon-wool columns, resistance to treatment of anti-Thy 1, -Ly 1, and -Ly 2 antibodies plus complement, adherence to anti-mouse immunoglobulin-coated dishes, and nonadherence to uncoated plastic dishes. In addition to being radiation sensitive, these suppressor B cells showed the capability of binding to the primed antigen. Thus, it was demonstrated that our in vitro-induced suppressor cells were antigen-specific B cells. When these suppressor B cells were transferred into the recipients, serum titers of specific antibodies were elevated and effector phase suppressor T cells were induced in the recipients. These results suggest that suppressor B cells exert their suppressor activity through the idiotype-anti-idiotype network.     

Selective suppression of primary IgM responses by induction of low dose paralysis to type III pneumococcal polysaccharide.

Pretreatment of mice with a low dose of Type III pneumococcal polysaccharide (S3)² decreases the response elicited by an optimal dose of S3 (low dose paralysis). The types of S3-specific responses which are affected by the induction of low dose paralysis were examined in the present study. The results indicate that pretreatment with low doses of S3 selectively suppresses responses of virgin IgM-producing B cells (Bμ). This was apparently due to a direct suppression at the level of the B cells, since S3-specific IgM responses were also suppressed when pretreated mice were challenged with S3 coupled to a thymus-dependent carrier, S3-HRBC. Pretreatment with low doses of S3 does not suppress primary IgG or secondary IgM or IgG responses to S3. Spleen cells from S3-pretreated mice did not suppress responses of normal cells after transfer to irradiated or intact recipients.     

Involvement of suppressor cells in active enhancement of allografted tumors. In vivo (adoptive transfer) and in vitro (MLR) evaluation. Synergy with enhancing antibodies

Specific enhancement of allografted A/J mouse tumor Sa 1 can be transferred not only passively by serum but also adoptively by lymphoid cells from syngeneic donors actively treated for enhancement. This can be done mainly by thymocytes in untreated recipients and by splenocytes in irradiated recipients, the transfer being more efficient in the latter. Results are more striking when the donors' treatment has led to a successful enhancement (AE⁺) demonstrated by an allografted tumor test. The responsible cells, studied in the donors' spleens, were found to be nonadherent to nylon wool, sensitive to specific anti-IJ serum and complement, and sensitive to anti-specific recognition structure serum with complement. Inactive doses of passively transferred serum added to subactive doses of adoptively transferred spleen cells, both from AE⁺ donors, lead to a substantial enhancement of allografted tumors, indicating a synergistic action of enhancing antibodies and suppressor cells. Similar results were obtained in vitro in a MLR system. MLC reactivity of AE⁺ donor cells is impaired. Furthermore, when mitomycin-treated, these cells have a regulatory (suppressor) effect on a MLR of syngeneic spleen cells directed against grafted tumor strain mitomycin-treated spleen cells. This suppressor effect is impaired by pretreating the regulatory cells with anti-IJ serum and complement or with anti-cell recognition structure serum (which has an opposite effect on AE^? cells).