The effect of indomethacin on the activation and effector function of suppressor cells from tumor-bearing mice

Summary The spleens of mice with large M-1 fibrosarcomas contain two populations of suppressor cells with the properties of macrophages and T cells. In this study, we tested the effect of indomethacin on suppressor cell activation and effector function. Neither the activation nor the effector function of the suppressor macrophages was inhibited by indomethacin, and the activity of suppressor macrophages correlated with the tumor size. In contrast, the treatment of tumor-bearing mice with indomethacin from the day of injection of tumor cells completely blocked the in vivo activation of suppressor T cells. Indomethacin did not, however, depress suppressor T cell activity if mice were treated only during the third week of tumor growth. The effector function of the suppressor T cells, as assessed in mixing assays, was partially blocked by indomethacin, while selective suppression by low-molecular-weight factors was completely blocked if indomethacin was present in the cultures. Furthermore, the in vitro activation of suppressor cells by soluble factors secreted by tumor-bearer spleen cells was completely blocked by indomethacin, and this inhibition was reversed by prostaglandin E₁. These data are consistent with the hypothesis that prostaglandins are involved in the activation, but not the effector function, of tumor-activated suppressor T cells.     

Modulation of the immunosuppressive effects of splenic macrophages in Fischer rats bearing adenocarcinoma 13762

Summary The nature of spleen cells in Fischer rats bearing a large size (>1 cm diameter) mammary adenocarcinoma 13762A (MAC) which block the immunostimulating capacities of MTP² (a synthetic immunomodulator) and suppress proliferation in vitro of splenic T and B lymphocytes by their respective mitogens was investigated. Splenic macrophages were recognized as the suppressor cells by (a) restoration of mitogenic responses by depletion of macrophages from spleen cell suspensions and (b) continued suppressor activity in spleen cell suspensions of tumor bearers devoid of viable T lymphocytes. Macrophage contact with T lymphocytes was required for the inhibition of T lymphocyte proliferation by concanavalin A as shown by (a) the absence of suppressor activity in supernatants derived from cultured suppressor macrophages, (b) lowering of the suppressor activity of intact macrophages after treatment with neuraminidase, (c) lowering of the suppressor activity of macrophages by addition of red cells to spleen cultures of tumor bearers indicating red cell interference with macrophage-T cell interaction and (d) lack of inhibiting action of suppressor macrophages on allogenic T lymphocyte proliferation showing macrophage T cell recognition for suppression.Animals bearing a large size tumor exhibited spleen hypertrophy and an increase in macrophage:lymphocyte ratio and a decrease in red cell:lymphocyte ratio. Splenic macrophages did not appear to be implicated in blocking antitumor immunity induction since (a) suppressor macrophages were absent in spleens during the inductive phase of the immune response and (b) MAC implanted in allogenic Wistar rats grew to about 2 cm diameter, induced splenic suppressor macrophages but the tumor was later rejected by the animals. Collectively the results suggest that suppressor macrophages are the result of increasing tumor volume rather than its cause.This study was supported by a grant from the National Cancer Institute of Canada Abbreviations used: Con A, Concanavalin A; LPS, lipopolysaccharide; PHA, phytohemagglutinin; MTP, maltose tetrapalmitate; MAC, mammary adenocarcinoma 13762; RPMI, Roswell Park Memorial Institute; TBR, tumor bearing rat; RBC, red blood cell     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

Summary We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.This paper was presented in part at the annual meeting of the American Association of Immunologists, Chicago, Illinois, 10–15 April 1983     

Immunologic significance of nonspecific suppressor cells in spleens of tumor-bearing mice.

Spleen cells from mice bearing a progressively growing syngeneic tumor failed to respond to stimulation with mitogens in vitro. This lack of reactivity was due to the presence of nylon wool-adherent cells in the population that could inhibit the mitogen response of normal lymphocytes. Paradoxically, at times when strong suppressor cell activity could be detected in tumor-bearing mice, the animals responded normally to in vivo immunization with sheep erythrocytes and allogeneic tumors, and to in vitro sensitization with allogeneic tumor cells. Regression of a highly antigenic syngeneic tumor also was unaffected by the presence of these suppressor cells. Thus, the occurrence of nonspecific suppressor cells in the spleens of tumor-bearing mice did not influence the overall immunologic competence of these animals.     

Modulation of the immune response to tumors by a novel synthetic compound, (4R)-3-benzoyl-N-[(1R)-1-phenylethyl]-4-thiazolidinecarboxamide (RS-0481)

Summary RS-0481, (4R)-3-benzoyl-N-[(1R)-phenylethyl]-4-thiazolidinecarboxamide, is a compound that can re-establish the function of certain lymphoid cell populations impaired by the presence of a growing tumor in an animal. The compound markedly augmented the tumorspecific cytotoxic T lymphocytes,Tdth (delayed-type hypersensitivity T cells), and the nonspecific lymphokine-activated-killer-cell-like cell responses. It also enhanced the tumor-inhibitory effect of macrophages in tumor-bearing mice, but not in normal mice, indicating that it enhances the antitumor immune responses. Lymphocytes from RS-0481-treated tumor-bearing mice released significantly higher amounts of macrophage-activating factor(s) (MAF) and interleukin-2(IL-2)-like factors in culture compared with lymphocytes from untreated animals. Also, sera from treated tumor bearers showed elevated colony-stimulating factor (CSF) activity. Although the compound did not influence the factor-producing activity in mice without tumor, it enhanced the responsiveness of their bone marrow cells, T cells, and macrophages to CSF, IL-2, and MAF. It seems therefore possible that the compound enhances the responsiveness of immunocompetent cells to cytokines, resulting in a marked augmentation of antitumor T cell responses in tumor-bearing mice. Consistently it inhibited the development of lymph node metastasis of transplanted X5563 plasmacytoma, and we showed that T cells play a decisive role in this inhibition. The compound also counteracted the development of suppressor T cell activity in the spleen of tumor-bearing mice.     

The role of lipocortin I in macrophage-mediated immunosuppression in tumor-bearing mice

The soluble mediators and/or mechanisms involved in immunosuppression in tumor-bearing hosts are not well characterized, although macrophages have long been recognized as major participants. We have investigated the role of lipocortin I, a phospholipid-binding protein, in macrophage-mediated immunosuppression in tumor-bearing mice. Proliferation of splenic lymphocytes in response to the mitogens (PHA, Con A, LPS, and PWM) was severely suppressed in tumor (Sqc-NH-1 carcinoma)-bearing mice. This immunosuppression was associated with a decrease in T and B lymphocytes and an increase in macrophages in these spleens. Mac-2+ macrophages were found only in spleens from tumor-bearing mice. Splenic macrophages from tumor-bearing, but not normal, mice were responsible for this immunosuppression, as revealed by negative and positive selection experiments. The levels of lipocortin I mRNA expression were markedly increased in peripheral blood cells from tumor-bearing mice as compared with those from normal mice. Lipocortin I mRNA was strongly induced in splenic mononuclear cells from tumor-bearing mice. Furthermore, these cells displayed increased expression of lipocortin I protein, as judged by Western blot analysis with polyclonal anti-lipocortin I serum. Some nonimmune organs such as the heart, submaxillary gland, muscle, and bladder also displayed increased levels of lipocortin I mRNA expression in tumor-bearing mice. Mac-2+ macrophages among the splenic mononuclear cells in tumor-bearing mice expressed lipocortin I mRNA, as judged by negative and positive selection experiments. Most of these Mac-2+ macrophages also had Mac-1 and Mac-3 Ag. Lipocortin I protein was increased in the serum of tumor-bearing mice as compared with normal mice. The culture supernatants of splenic cells from tumor-bearing mice suppressed the mitogenic responses of splenic cells from normal mice, and addition of anti-lipocortin I antiserum inhibited this suppression. Furthermore, recombinant mouse lipocortin I suppressed mitogenic responses of splenic cells from normal mice. In summary, Mac-2+ macrophage-derived lipocortin I was largely involved in immunosuppression in tumor-bearing mice.     

Altered interleukin production during Friend leukemia virus infection

Spleen cells from BALB/c mice, infected 14 to 28 days earlier with Friend leukemia virus (FLV), were shown to be inhibited in their ability to produce interleukin 2 (IL-2) when stimulated with mitogen. Likewise, these spleen cell populations failed to respond following mitogenic stimulation or exogenous addition of recombinant IL-2. By contrast, the FLV-infected spleen cell populations produced normal levels of interleukin 1 (IL-1) and thymocytes from FLV-infected mice responded normally to addition of exogenous IL-1. This suggests that FLV infection selectively affects the ability of spleen cells to produce cytokines. Spleen cell populations enriched for T lymphocytes and depleted of tumor cells by density gradient centrifugation in Ficoll were unable to produce IL-2. This indicates that the failure to detect IL-2 in cells from FLV-infected mice was not due to a dilution of T lymphocytes by tumor cells but was a functional inability to produce IL-2. Furthermore, enriched T lymphocytes from FLV-infected mice failed to respond blastogenically to exogenous IL-2. Additional studies indicate that tumor cells, but not macrophages or T lymphocytes from FLV-infected spleens, suppressed the blastogenic response to mitogens and IL-2 production by normal splenic T lymphocytes.     

Inhibition of rat mixed lymphocyte cultures by suppressor macrophages.

Normal rat spleens contain suppressor cells which can inhibit proliferative and cytotoxic responses of lymphocytes to alloantigens in vitro. The suppressor cells are adherent, phagocytic, resistant to treatment with ATS and C, radioresistant, resistant to treatment with mitomycin C, apparently absent from the thymus, and found in very high concentrations in peritoneal exudates. These characteristics indicate that the suppressor cell is a macrophages and not a T cell. When suppressor cells were removed from spleen cell suspensions, strong in vitro proliferative and cytotoxic responses to alloantigens could consistently be observed.     

Fractionation of lymphocyte subpopulations which regulate mixed lymphocyte reactions.

Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.     

Cellular and cytokine-dependent immunosuppressive mechanisms of grm1-transgenic murine melanoma

Grm1-transgenic mice spontaneously develop cutaneous melanoma. This model allowed us to scrutinize the generic immune responses over the course of melanoma development. To this end, lymphocytes obtained from spleens, unrelated lymph nodes and tumor-draining lymph nodes of mice with no evidence of disease, and low or high tumor burden were analyzed ex vivo and in vitro. Thereby, we could demonstrate an increase in the number of activated CD4⁺ and CD8⁺ lymphocytes in the respective organs with increasing tumor burden. However, mainly CD4⁺ T cells, which could constitute both T helper as well as immunosuppressive regulatory T cells, but not CD8⁺ T cells, expressed activation markers upon in vitro stimulation when obtained from tumor-bearing mice. Interestingly, these cells from tumor-burdened animals were also functionally hampered in their proliferative response even when subjected to strong in vitro stimulation. Further analyses revealed that the increased frequency of regulatory T cells in tumor-bearing mice is an early event present in all lymphoid organs. Additionally, expression of the immunosuppressive cytokines TGF-??1 and IL-10 became more evident with increased tumor burden. Notably, TGF-??1 is strongly expressed in both the tumor and the tumor-draining lymph node, whereas IL-10 expression is more pronounced in the lymph node, suggesting a more complex regulation of IL-10. Thus, similar to the situation in melanoma patients, both cytokines as well as cellular immune escape mechanisms seem to contribute to the observed immunosuppressed state of tumor-bearing grm1-transgenic mice, suggesting that this model is suitable for preclinical testing of immunomodulatory therapeutics.     

Single administration of low dose cyclophosphamide augments the antitumor effect of dendritic cell vaccine

Single administration of low dose cyclophosphamide (CTX) was previously reported to enhance the antitumor efficacy of immunotherapies. To investigate the possible mechanisms for this effect, we examined whether a single administration of low dose CTX could augment the immunogenicity of dendritic cell (DC) vaccines. Fifty milligrams per kilogram body weight dose of CTX was administrated intraperitoneally to mice after B16 melanoma or C26 colon carcinoma tumor models were established, DC vaccine generated from mouse bone marrow and pulsed with B16 or C26 tumor cells lysates were vaccinated 4 days later. CTX treatment potentiated the antitumor effects of the DC vaccine, and increased the proportion of IFN-γ secreting lymphocytes in spleens. Furthermore, a significantly reduced proportion of CD4+CD25+FoxP3+ regulatory T (T_reg) cells was detected by flow cytometry in spleen lymphocytes from tumor-bearing mice treated with CTX. Thus, a single administration of low dose CTX could augment antitumor immune responses of DC vaccine by reducing the proportion of CD4+CD25+FoxP3+ T_reg cells in tumor-bearing mice. Our results suggested a possible mechanism of CTX-induced immunopotentiation and provided a strategy of immunotherapy combining a low dose CTX with DC vaccine. J.-Y. Liu and Y. Wu contributed equally to this work.     

Alloantigens placed into the anterior chamber of the eye induce specific suppression of delayed-type hypersensitivity but normal cytotoxic T lymphocyte and helper T lymphocyte responses

Intracameral inoculation of allogeneic B16F10 melanoma cells (C57BL/6) into LP/J mice resulted in progressively growing intraocular tumors and impaired delayed-type hypersensitivity (DTH) reactivity. Additional experiments showed that DTH responses were specifically down-regulated by splenic T suppressor cells. By contrast, subcutaneous inoculation of B16F10 melanoma cells induced significant DTH responses to the alloantigens expressed on the tumor cells and stimulated brisk rejection of the subcutaneously injected tumor cells. In spite of the T suppressor cell inhibition of DTH reactivity, significant cytotoxic T lymphocyte activity could be demonstrated in lymphoid cell suspensions from hosts harboring allogeneic intraocular tumors. The demonstrated cytotoxic T lymphocyte activity is particularly noteworthy because it occurs in the face of severely suppressed DTH responsiveness and thus implies that the intracameral presentation of alloantigens evokes a precise immunoregulatory process that selectively and concomitantly modulates specific cellular immune components; one immune process (cytotoxic T lymphocyte function) is stimulated whereas the other (DTH responsiveness) is down-regulated.     

Role of colony-stimulating factor-1 in macrophage activation in tumor-bearing mice.

We previously reported a dramatically increased number of macrophages in tumor-bearing mice. In this study, we investigated the involvement of CSF in that phenomenon. CSF-1 responding cells as macrophages precursors increased significantly in number in the spleens of tumor-bearing mice as compared with those in normal mice. Splenic cells and sera from the tumor-bearing mice respectively expressed CSF-1 in mRNA and serum protein levels, but failed to express the other CSF (granulocyte-macrophage-CSF or IL-3). Nonadherent splenic mononuclear cells (< 0.5% macrophages) from normal mice proliferated and differentiated into mature macrophages in culture within 7 days with recombinant mouse CSF-1 (rCSF-1). Both macrophages harvested from tumor-bearing mice and those activated in vitro with rCSF-1 expressed mostly Mac-1, -2 (and -3) Ag, showed yeast phagocytosis, produced IL-1 but not IL-2 or IL-3, and displayed potent cytotoxicity against NK cell resistant Meth-A tumor cells. These macrophages also expressed lipocortin I mRNA and secreted lipocortin I protein, and suppressed mitogenic responses of splenic lymphocytes. rCSF-1-activated macrophages derived from nonadherent splenic cells expressed both CSF-1 and CSF-1 receptor (c-fms) mRNA. Administration of rCSF-1 into normal mice induced hemopoietic and immunologic alternations similar to those observed in tumor-bearing mice. These results suggest that CSF-1 is involved in the dramatic increase of macrophages in tumor-bearing mice, possibly through an autocrine or paracrine loop.     

The antimetastatic function of concomitant antitumor immunity. II. Evidence that the generation of Ly-1+2+ effector T cells temporarily causes the destruction of already disseminated tumor cells

Progressive growth of the P815 mastocytoma in an immunocompetent host evokes the generation of an antitumor immune response that can be measured in terms of the production of cytolytic Ly-1+2+ T cells in the draining lymph node and spleen. This immunity, designated concomitant immunity, is present on day 6 of tumor growth, peaks on day 9, and decays progressively thereafter. It fails to develop in mice made T cell deficient by thymectomy and lethal whole-body gamma-radiation, and reconstituted with syngeneic bone marrow cells (TXB mice). Employment of a mouse survival assay, capable of enumerating metastatic P815 cells in cell suspensions, showed that the P815 tumor metastasizes to the draining lymph node and spleen at the same rate in normal and TXB mice for the first 6 days of growth of an intradermal P815 tumor. By day 6 of tumor growth there were approximately 10(3) P815 cells in the draining lymph node in both types of mice. However, during the generation of concomitant immunity between days 6 and 9, the number of metastatic P815 cells in the draining lymph nodes and spleens of normal tumor-bearing mice declined by nearly 90%. After day 12, however, the number of tumor cells in the nodes and spleens increased concordantly with the decay of concomitant immunity. These findings, together with the demonstration that T cell-deficient mice failed to restrain the number of metastatic P815 cells in the draining lymph node and spleen, suggest that concomitant immunity is an important defense mechanism against the development of systemic disease. Additional evidence consistent with this interpretation was provided by studies which showed that adoptive immunization with spleen cells from concomitant immune donors significantly prolonged the median survival time of TXB tumor-bearing mice by destroying a substantial proportion of P815 tumor cells already seeded in the draining lymph node. Adoptive immunization also delayed the appearance of metastatic tumor cells in the spleen.     

The T-cell receptor: just another hypothesis.

Functional activities of T and B lymphocytes and the kinetics of hematopoietic stem cells were studied in mice with inoculated or spontaneous tumors. The development and growth of the tumor inhibited B cells and helper T cells, while the activity of killer T cells and spleen suppressor cells was markedly enhanced. The processes of stem cell migration from the bone marrow were considerably intensified and altered in tumor-bearing mice. Data were obtained suggesting that helper T cells and killer T cells represent nonidentical compartments within the population of thymus-dependent lymphocytes. Immunosuppression during tumor bearing is probably due to an impairment of T lymphocytes cooperating in immune responses, B-lymphocytes and their precursors.     

Mechanism for the suppression of gamma-interferon responsiveness in mice after thermal injury

As reported previously, gamma-interferon production was decreased after the administration of inducers to thermally injured mice as compared with noninjured controls. Similarly, spleen cells from injured mice had decreased ability to produce interferon in vitro after stimulation with inducers. The present study demonstrated that the decrease in interferon production was associated with the presence of suppressor cells in the spleen of burned mice that were capable of inhibiting interferon production by normal splenic lymphocytes in vitro. Passive transfer of spleen cells containing suppressor cell activity derived from injured mice induced suppression in normal mice, and the time of the appearance of suppressor cell activity in injured mouse spleens closely approximated the time of the appearance of the suppression of interferon production observed in mice after thermal injury. The suppressor cells were characterized as a population of macrophages by the following: they adhered to plastic surface and could be removed from spleen cells by carbonyl-iron treatment; treatment of plastic-adherent cells with anti-Thy-1.2 and anti-mouse immunoglobulin antisera followed by complement failed to abrogate the suppression produced by these cells.     

Therapeutic effects of tumor-reactive type 1 and type 2 CD8+ T cell subpopulations in established pulmonary metastases.

Cytolytic CD8+ T cells fall into two subpopulations based on cytokine-secretion. Type 1 CD8+ cells (Tc1) characteristically secrete IFN-gamma, whereas type 2 CD8+ cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 CD8+ cells in mice bearing established OVA-transfected B16 melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a reduction in lung tumor growth that subsequently prolonged survival times in mice with both early (day 7) and more advanced (day 14) levels of tumor development. CD8+ T cell populations recovered from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing activities that correlated with delays in tumor cell growth and progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells accumulated at the tumor site with nearly equal frequency. Tc1 cells persisted, whereas Tc2 cell numbers progressively diminished over time. Titration of Tc1 and Tc2 effector cells showed that protection was dose dependent with the former being 5-fold more effective. Tc2 cells achieved a comparable reduction in lung tumor cell growth at higher concentrations of cell transfer. Tc1 effectors from IFN-gamma-deficient mice were less therapeutically effective than wild-type mice, but there was no significant reduction in activity between corresponding Tc2 populations. We speculate that the effectiveness of Tc1 and Tc2 cells may depend on different mechanisms. These studies suggest a potential role for Tc1 and Tc2 CD8+ subpopulations in tumor regression and immunotherapy.     

Suppressor cell activity in tumor-bearing mice. I. Dualistic inhibition by suppressor T lymphocytes and macrophages.

Spleen cells from mice bearing methylcholanthrene-induced fibrosarcomas impaired mitogen responses of normal syngeneic lymphocytes. Nylon wool column and other depletion techniques were utilized to characterize the cellular source of suppressive activity in tumor-bearing host (TBH) spleens. Evidence is presented for two distinct suppressor cell systems operating in the spleens, but not lymph nodes, of BALB/c mice bearing transplanted tumors. Spleens from TBH were shown to have greatly increased numbers of macrophages over their normal counterparts. TBH macrophages were observed to have suppressive activity at low in vitro concentrations. Anti-Thy 1 serum treatment of TBH macrophages abrogated low dose inhibition but not suppression due to high numbers of macrophages. No functional difference was detected between anti-Thy 1 serum-treated TBH and normal splenic macrophages. In a macrophage-depleted culture system, mildly nylon wool adherent, anti-Thy 1 serum, and hydrocortisone succinate-sensitive suppressor cells could be detected. Soluble supernatant products of TBH spleen and thymus cells were also found to inhibit in vitro mitogen responses, whereas TBH macrophages and lymph node cells demonstrated no soluble suppressive activity. The major source of soluble inhibitor of DNA synthesis (IDS) seems to be an anti-Thy 1 serum, hydrocortisone-sensitive population.     

Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1+, Lyt 1+2+ and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.