Regulation of the immune response to tumor antigen. IV. Tumor antigen-specific suppressor factor(s) bear I-J determinants and induce suppressor T cells in vivo.

The nature and function of suppressor factor(s) elaborated by suppressor T cells in response to certain chemically induced tumors have been further defined. Thus, suppressor factor(s) specific for the S1509a methylchol-anthrene-induced fibrosarcoma have been shown to bear determinants encoded by the I-J subregion of the murine MHC since suppressive activity is removed by passage of the factor through an immunoadsorbent composed of anti-I-Jk coupled to Sepharose. No loss of activity was observed after passage of factor through control columns composed of normal mouse globulin. Furthermore, activity could be recovered from the relevant immunoadsorbent by elution with high salt. The administration of crude suppressor factor(s) to normal animals for 4 days resulted in the development of a population of suppressor cells that act in a manner analogous to the suppressor cell population used for production of factor. These factor-induced suppressor cells are T cells and exhibit an antigen specificity similar to that displayed by the tumor-induced suppressor cells. Thus, tumor-specific suppressor factor(s) bear I-J determinants and are capable of inducing the appearance of suppressor T cells in the nontumor-bearing host, which may then act in a specific manner to limit host responsiveness to tumor antigen.     

Regulation of the immune response to tumor antigens. X. Activation of third-order suppressor T cells that abrogate anti-tumor immune responses

The experiments described further define the suppressor T cell pathway in the S1509a tumor system. We demonstrated previously that S1509a-induced Ts1, TsF1, and Ts2 specifically suppress in vivo Ly1+2- T cell-dependent responses to S1509a and that Ts1 suppress in vivo Ly-1+2- T cell-mediated proliferative responses to S1509a. We have now shown that in vivo administration of either S1509a-induced TsF1 or TsF2 suppresses both in vivo and in vitro Ly-1+2- T cell-mediated responses to S1509a. Furthermore, we revealed the existence of Ts3, which are activated by S1509a tumor antigen and TsF2, in this murine tumor system. Finally, we demonstrated that cyclophosphamide abrogates the suppressive effect of TsF2 but not that of Ts3. These results are discussed with respect to T cell-mediated suppression in other murine tumor systems and the possible pivotal role for a tumor antigen-presenting cell in activating Ts3 in the S1509a tumor system.     

Involvement of antigen and I-J determinants in the induction of effector T suppressor cells by immune B cells

Immune B cells induce effector T suppressor cells in vitro. The B cells act as antigen-presenting cells, and express both I-A and I-J determinants. Antigen and I-J determinants are required for the induction of suppressor T cells by immune B cells, but I-A determinants are not. These findings indicate that precursors of suppressor T cells appear to recognize antigen in the context of I-J determinants on the surface of immune B cells.     

Persistent activity of suppressor cells in T cell-mediated immune response.

Tolerance to dinitrochlorobenzene contact sensitivity induced i.v. injection of dinitrobenzenesulfonic acid in guinea pigs is a long-lasting phenomenon (up to 1 year). The tolerogen, however, was traceable in the circulation only up to 3 months after its application. In spite of that, tolerance was adoptively transferred by parabiosis 6 months after being induced. Moreover, active suppressor cells eliminated by cyclophosphamide treatment are able to regenerate in those adoptively tolerized animals. These results indicate that the tolerogenic injection stimulates precursors of suppressor cells to generate active suppressor cells and memory cells of suppression. The further formation of active suppressor cells from memory cells seems to be tolerogen independent, but the existence of specific stimulator cells for suppression may be considered. These cells may bind undetectable small amounts of tolerogen. The recovery of suppression might, however, be also due to recovery of suppressor cells which were temporarily inactivated but not destroyed by cyclophosphamide treatment.     

Regulation of the immune response against UV-induced skin cancers: specificity of helper cells and their susceptibility to UV-induced suppressor cells

The purpose of this study was to determine the role of T helper (Th) cells in the immune response to UV-induced tumors. Repeated exposure of mice to UV radiation results in the production of suppressor T lymphocytes that facilitate tumor growth by inhibiting host immunity. To investigate whether the suppressor T cells inhibit the response to UV tumors by blocking the generation of Th, we employed an indirect method for measuring helper cell activity. We found that Th were produced in normal mice after immunization with UV-induced tumors. These Th appeared to be specific for the immunizing tumors, in contrast to the UV-induced suppressor cells, which recognize UV-induced tumors as a group. The suppressor T cells responsible for inhibiting tumor rejection have no effect on tumor-specific helper cell activity in vitro. However, UV-induced suppressor T cells transferred into unirradiated mice seem to block the generation of helper cell activity after immunization with UV-produced tumors. These results suggest the UV-induced suppressor cells may prevent tumor rejection by blocking the generation of Th.     

Regulation of the immune response to alloantigens: suppressor and helper T cells generated in the primary MLR of the rat

The primary MLR of the rat was used to generate suppressor, cytotoxic, and helper T cells from lymph node cells of the WF (RT1 mu) inbred strain. They were assayed in 51Cr-release cytotoxic assays and by their effect on proliferation of fresh unprimed responder cells. Suppression by MLR cellular products was antigen-specific and generation and functional expression were directed to class II (RT1.B,D) antigens of stimulator cells in the strains tested. In contrast, help was not antigen-specific. The monoclonal antibodies OX8 and W3/25 were used to separate the primed products of the MLR into the constitutive subsets, suppressor/cytotoxic (OX8+) and helper/inducer (W3/25+). Gamma irradiation of OX8+ MLR-primed cells caused modest reductions in suppressive activity, but had no effect on the helper activity of W3/25+ cells. MLR-derived suppressor cells are effective only when added in the early stages of the test primary MLR, whereas helper cells can augment proliferation even when added late. Feedback suppression is not mediated by classical cytotoxic T cells, because of differences in kinetics of development, cell numbers required, susceptibility to freezing, and expression of the RT6 differentiation antigen.     

Regulation of the immune response to tumor antigens. IX. In vitro Lyt-1+2- cell proliferative responses to cellbound or subcellular tumor antigen

We investigated the cellular basis of immune reactivity to the S1509a fibrosarcoma in tumor-immune A/J mice. In a Winn assay, immune Lyt-1+2- T cells are capable of retarding S1509a tumor growth in naive A/J mice. In vitro proliferation to S1509a is also mediated by tumor-immune Lyt-1+2- T cells. This response is specific to the immunizing tumor and appears 5 to 7 days after reexposure to the tumor in vivo. Proliferation also requires the presence of a population of adherent cells. In fact, adherent peritoneal exudate cells pulsed with tumor membrane fragments derived from S1509a cells can stimulate proliferation. Proliferation is blocked by the addition of anti-I-Ak monoclonal antibody to the culture medium without complement or by treatment of the responder population with anti-I-Ak and complement. In vitro responsiveness is also inhibited by the presence of tumor-specific suppressor T cells in vivo. These observations suggest in vitro proliferation may provide a potential means of defining tumor antigens and cell-surface structures involved in tumor immunity.     

Regulatory T cells dynamically control the primary immune response to foreign antigen

The population dynamics that enable a small number of regulatory T (T(R)) cells to control the immune responses to foreign Ags by the much larger conventional T cell subset were investigated. During the primary immune response, the expansion and contraction of conventional and T(R) cells occurred in synchrony. Importantly, the relative accumulation of T(R) cells at peak response significantly exceeded that of conventional T cells, reflecting extensive cell division within the T(R) cell pool. Transfer of a polyclonal T(R) cell population before immunization antagonized both polyclonal and TCR transgenic responses, whereas blocking T(R) cell function enhanced those responses. These results define an inverse quantitative relationship between T(R) and conventional T cells that controls the magnitude of the primary immune response. The high frequency of dividing T(R) cells suggests degenerate TCR specificity enabling activation by a broad spectrum of Ags.     

Regulation of the immune response to tumor antigen. VIII. The effects of host specific anti-I-J antibodies on the immune response to tumors of different origin

The efficiency of in vivo therapy using alloantisera produced to interact specifically with I-J subregion encoded determinants has been investigated in two etiologically distinct syngeneic tumor systems, both of which have been shown to evoke suppressor T-cell host responses. Administration of 2 μl/day of anti-I-J^k alloantisera caused a significant reduction in the growth of the P815 methylcholanthrene (MCA)-induced mastocytoma or the 1316 ultraviolet (uv) radiation-induced fibrosarcoma in the syngeneic host. Inhibition of tumor growth with anti-I-J antibody treatment occurred in normal as well as in subcarcinogenically uv-treated hosts given the uv-induced 1316 fibrosarcoma, even though the normal host is capable of spontaneously rejecting the tumor graft in the absence of external manipulation. Evidence is also provided that the effects of anti-I-J antibody treatment are not due to direct interactions with the tumor cells, or to contaminating antiviral antibody activity within the antiserum. We have previously demonstrated the reduction of tumor growth in two antigenically distinct MCA-induced tumor systems (S1509a, SAI) using similar treatments. The data presented herein thus reinforce the possibility that such means of therapy may be beneficial to the treatment of a wide variety to tumor types where suppression represents a detrimental component of the host response, and may also provide some insight into the mechanisms underlying the effects of uv radiation on the immune response to tumor antigen.     

The immune response to Schistosoma mansoni infections in inbred rats. VI. Regulation by T cell subpopulations

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. CDF rats were depleted of RT 7.1+ (anti-Pan-T), W3/25+ (anti-T helper/inducer), or OX8+ (anti-T suppressor) cells by the in vivo administration of monoclonal antibodies (mAb). The development of parasites and immunity to challenge by S. mansoni were compared with results in undepleted normal and congenitally athymic rats. Discrete subpopulations of T lymphocytes were adoptively transferred to ascertain effects upon parasite development and the protective immune response. In vitro studies, involving utilizing cocultivation of cell subpopulations +/- cyclosporin A, were utilized to dissect mechanisms. Depletion of T lymphocytes by anti-RT7.1 mAb and anti-W3/25 mAb resulted in augmented initial worm development, suboptimal resistance, and decreased antibody and delayed-type hypersensitive reactivity directed against schistosome antigens. Depletion with OX8 mAb produced opposite effects. The adoptive transfer of T cell subpopulations produced concordant results with T cell regulation expressed B cell-dependent effector mechanisms. The coadoptive transfer of cells resulted in the suppression of resistance afforded by the W3/25+ cells by OX8+ cells, which could be augmented in vitro by cyclosporin A. Thus, protective immunity to S. mansoni in rats is regulated by discrete subpopulations of T lymphocytes. The findings suggest the possibility of selective immune regulation of resistance based on the manipulation of specific T cell subpopulation.     

Regulatory function of T lymphocytes in the immune response to polyvinyl pyrrolidone. I. Two categories of suppressor T cells.

The regulation of antibody response of mice to polyvinyl pyrrolidone (PVP) was investigated using three preparations of PVP (K90, K30, and K15) differing from each other in molecular weight. The immunogenicity of PVP was higher as the molecular weight increased. The depletion of thymus-derived cells resulted in the augmentation of anti-PVP response. On the other hand, the response of intact mice to the most immunogenic PVP (K90) was suppressed more or less by the injection of any preparation of PVP 4 days prior to K90. This was most pronounced when the smallest PVP (K15) was preinjected. The suppression, however, was not observed in thymectomized-irradiated-bone marrow reconstituted mice.These results indicated that anti-PVP response was regulated by two different categories of thymus-derived cells, that is, “intrinsic” and “induced” suppressor cells. The activity of the latter was transferrable, PVP-specific, and eliminated by anti-Thy 1 serum and complement. In addition, the mean affinity of anti-PVP plaque-forming antibodies was found to be reduced by the action of “induced” suppressor cells.     

Regulation of the class of immune response induced by antigen. I. Specific T cells switch the in vivo response from a cell-mediated to humoral mode

Unprimed murine spleen cells, when administered intravenously to irradiated recipients together with antigen for 7 days, are induced to display either DTH reactivity or to mount a humoral (IgM and IgG) response. The class induced depends on the number of spleen cells given to the irradiated host. A low number of cells does not support the induction of any response, a medium number only gives rise to substantial DTH reactivity, whereas a high number only mounts a humoral (IgM and IgG) response. Observations show that the higher number of T cells in a large inoculum of spleen cells, compared to the number present in a medium one, is responsible for the absence of DTH reactivity and the mounting of a humoral response. This finding suggests that the induction of DTH precursor cells may occur when fewer antigen-specific helper-T-cell-dependent signals are generated than the number of signals required to induce B-cell precursors of the IgM and IgG classes. This possibility is favored by further observations. The administration of in situ irradiated, primed helper T cells to mice reconstituted with a medium number of normal spleen cells, results both in the specific suppression of the DTH response that occurs in the absence of these primed cells and in the mounting of a humoral response.     

Suppression of the in vitro secondary response to syngeneic tumor and of in vivo tumor therapy with immune cells by culture-induced suppressor cells.

Mice with advanced disseminated syngeneic tumor can be successfully treated with a combination of chemotherapy and adoptively transferred syngeneic immune cells. We have previously demonstrated that in vivo primed cells secondarily sensitized in vitro became more effective in tumor therapy, whereas primed cells cultured for 5 days without tumor stimulation became less effective than an equal number of uncultured fresh primed cells. Therefore, we examined stimulated and unstimulated cultures of tumor-primed cells for the presence of culture-induced suppressor cells, and determined whether in vivo tumor therapy with immune cells could be inhibited by concurrent inoculation of immune effector cells and cultured normal spleen cells, which contain culture-induced suppressor cells but are devoid of additional effector cells. The in vitro primary allogeneic response was suppressed by cultured normal spleen cells, or tumor-primed spleen cells previously cultured for 5 days with or without tumor stimulation. In vitro secondary sensitization to syngeneic tumor was suppressed by normal or tumor-primed cells that had previously been cultured for 5 days without stimulation. The majority of this suppression was mediated by T cells in the cultured populations. The efficacy of fresh tumor-primed cells, as well as primed cells secondarily sensitized in vitro, in adoptive chemoimmunotherapy of advanced tumor was diminished by concurrent inoculation of cultured normal cells. The cells mediating suppression of in vivo therapy required previous in vitro culture for induction, and were radiation sensitive.     

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). VI. T cell fine specificity

In the companion paper it was demonstrated that the T cell proliferative response to HBsAg was controlled by I region genes as was previously shown for in vivo anti-HBs production. In this paper, the structural requirements for T cell recognition of HBsAg were compared with B cell (antibody) recognition of HBsAg. Secondly, we attempted to map determinants on HBsAg required for activation of HBsAg-primed T cells, and we examined the influence of I region genotype on the observed T cell antigenic fine specificity. The results of these studies indicate clear differences between T cell and B cell recognition of HBsAg. T cell activation required significantly less native structure as compared with antibody binding to HBsAg. Reduced and alkylated HBsAg, the subunit polypeptide P25, tryptic fragments of P25, and synthetic peptide analogues of HBsAg were all capable of eliciting a T cell proliferative response, whereas these "denatured" forms of the antigen bind anti-HBs marginally or not at all. Furthermore, the results suggest that T cell recognition sites on HBsAg do not necessarily overlap with B cell recognition sites. Examination of T cell fine specificity in a series of H-2 congenic strains, with the use of HBsAg, P25, tryptic fragments of P25, and synthetic peptides, revealed multiple T cell recognition sites on HBsAg, and the particular site(s) recognized is dependent on the H-2 genotype of the responding strain. Finally, preliminary results indicate that the specificity of human, HBsAg-primed T cells appear to be variable among individuals.     

Modulation of immune responses by suppressor T cells.

The activity of suppressor T cells has been demonstrated in almost every phase of the immune response. These regulatory cells modulate both humoral and cell-mediated immunity utilizing antigen-specific and nonspecific mechanisms. For comparative purposes two murine models are described, the nonspecific suppressor T cell stimulated by the mitogen concanavalin A and the antigen-specific suppressor T cell stimulated by injection of the synthetic terpolymer acid 60-L-alanine30-L-tyrosine10 (GAT) in nonresponder mice. These two T cells are similar to expression of Ly alloantigens, ability to inhibit antibody responses, and the mediation of suppression, at least in part, by soluble products. However, differences in radio-resistance and antigenic specificity of the suppressor T cells, as well as differences in molecular characteristics of the soluble factors and their targets suggest that these T cells regulate the immune response by different mechanisms. The relationship of these two suppressor T cells to other nonspecific and antigen-specific suppressor T cells is discussed.     

Feedback suppression of the immune response in vivo. I. Immune B cells induce antigen-specific suppressor T cells

Stimulated lymphocytes are capable of synthesizing and secreting a variety of lymphokines which can affect the functions of several types of target cells. We report here the existence of a soluble factor released by activated human mononuclear leukocytes which produces a selective inhibition of human pulmonary fibroblast migration. This fibroblast migration inhibitory factor (FIF) was produced by antigen- or mitogen-stimulated human peripheral blood mononuclear leukocytes (PBML) and purified T cells. It inhibited the migration of ⁵¹Cr-labeled fibroblasts in a dose-dependent fashion with optimal effect (65–70% inhibition) obtained at 1:10 dilution and 8–20 hr of incubation. Sephadex G-100 fractionation revealed most activity to be found between 28,000 and 34,000 daltons. FIF was stable at 56 °C for 15 min, but destroyed at 80 °C or at low pH. This factor may play an important role in the modulation of fibrogenesis and healing processes by the immune system.     

Regulation of immune responses by T suppressor cells and by serum in chronic paracoccidioidomycosis

Regulation of cellular responses was studied during the course of chronic murine disseminated paracoccidioidomycosis. Regulation of peripheral blood lymphocyte (PBL) proliferative responses to concanavalin A (Con A) was studied in vitro by mixing PBL from infected and noninfected mice. PBL from mice infected for 18 weeks had depressed responses to Con A and they depressed the Con A responses of PBL from noninfected mice by 95% when they were mixed in a 1:1 ratio. After treatment of PBL from infected mice with anti-Lyt-2.2 antibody plus complement, the responses to Con A were increased to normal values. The percentage of T-cell subpopulations in PBL from infected mice did not differ significantly from those of normal mice. Immunoregulation of delayed-type hypersensitivity (DTH) responses to antigen by serum from infected animals was studied in mice 1 week after intranasal (i.n.) infection, a time when DTH responses were maximal. DTH responses to antigen 7 days after i.n. infection (10(7) CFU Paracoccidioides brasiliensis) were significantly reduced when 0.5 ml of immune mouse serum (ELISA antibody titer to P. brasiliensis antigens 1:10,240) was given i.v. 1 day before infection (P less than 0.01) or 1 day before skin testing (P less than 0.001). Normal mouse serum did not have this effect. The results indicate that progression of chronic disseminated paracoccidioidomycosis was associated with the development of T-cell suppressor activity for Con A responses of PBL, and that DTH responses to antigen were depressed by the administration of serum with specific high titer antibodies.     

Regulation of the cellular p53 tumor antigen in teratocarcinoma cells and their differentiated progeny.

F9 embryonal carcinoma cells express high levels of a 53,000-molecular-weight cellular tumor antigen called p53. When F9 cell cultures are treated with retinoic acid and dibutyryl adenosine 3',5'-phosphate, they differentiate, predominantly into endoderm-like cells. This differentiation is accompanied by a marked decrease in the levels of p53. The mechanism(s) responsible for this decline in the level of p53 in differentiated cells was investigated. The results demonstrate that the high levels of p53 in F9 cells relative to their differentiated progeny were not due to alterations in the stability or turnover of this protein. Rather, the regulation during differentiation involved a marked decrease in the amount of in vitro translatable p53 mRNA detected in the differentiated cell cultures. This mechanism is unlike the one operating during the simian virus 40 infection or transformation, where the increased levels of p53 are largely due to the increased stability of the p53 protein.     

Regulation of the immune response to tumor antigens. III. Characterization of thymic suppressor factor(s) produced by tumor-bearing hosts.

Immunosuppressor T cells (IST),6 capable of inhibiting the rejection of a methylcholanthrene-induced fibrosarcoma (S1509a) in A/Jax mice immune to this tumor, produced soluble factors with similar suppressive activity. The immunosuppressive factor(s) (ISF) has been shown to be immunologically specific, as demonstrated by the complete loss of suppressive activity after absorption with S1509a cells, but not with cells of an unrelated syngeneic tumor. From its behavior on gel filtration, the size of ISF was deduced to be less than 70,000 daltons. The ISF was not removed by passage through a rabbit anti-mouse F(ab')2 reverse immunosorbent and, hence, it was concluded that ISF was not likely to be an immunoglobulin. The (immunosuppressive) activity of ISF was destroyed by treatment with Pronase, but not with RNase. The ISF was found to share the antigenic determinant(s) of the product(s) of the K end of the major histocompatibility complex of the mouse. Moreover, antibodies to ISF were induced by immunization with ISF-tumor cell complexes. Thus, IST and their factor(s) appear to play an important role in the regulation of the immune response to tumor antigens.     

Regulation of the immune response to tumor antigens. II. The nature of immunosuppressor cells in tumor-bearing hosts.

Immunosuppressor (IS) cells were found to be generated in tumor-bearing animals (TBA) within 24 hr after inoculation of tumor cells of the methylcholanthrene-induced Sarcoma 1509a and appeared to persist in the hosts as long as the tumor was progressing. However, IS cells disappeared with 5 days after extirpation of the tumor. Increasing doses of thymus cells of TBA increased the degree of suppression of tumor rejection in immune syngeneic animals. Ten million thymus cells of TBA were capable of suppressing significantly the tumor rejection. The IS cells were detected in the thymus, spleens, and draining lymph nodes, as well as in bone marrow of TBA, but could not be detected in the peripheral circulating blood. Since immunosuppressive activity of bone marrow cells from TBA was entirely abolished by the in vitro treatment of the cells with anti-theta serum and complement, the observed immunosuppression appears to be mediated by the T cell population.