T and B lymphocyte migration into syngeneic tumors.

The migration of splenic T and B lymphocytes into syngeneic tumors undergoing immunologic rejection was investigates. Spleen cells were obtained from normal BALC/c mice or BALB/c mice bearing tumors induced by murine sarcoma virus (MSV). Either whole spleen cells or immunoabsorbent purified T and B cells were radiolabeled with sodium chromate-51 and injected i.v. into normal or MSV inducted-tumor bearing syngeneic recipients. Twenty-four hours later the recipient mice were sacrificed and radioactivity was assessed for tumor, contralateral normal muscle, the lymph nodes draining the tumor and contralateral draining lymph nodes, peripheral lymph nodes, spleen, and liver. Both T and B lymphocytes from either normal or MSV tumor-bearing animals show greatly increased migration into the tumor when compared with normal muscle. Migration of T cells from both normal and MSV tumor bearers was 30 times that of migration to normal muscle. B cells from tumor-bearing mice, on the other hand, localized in the tumor itself only 50% as frequently as did B cells from normal animals. In addition, T cells from MSV tumor bearers were found in the highest proportion in the lymph node draining the tumor site. We conclude that T and B lymphocytes from either normal or tumor-bearing mice migrate to a syngeneic tumor undergoing immunologic rejection. In contrast, the migration of both T and B cells from tumor-bearing animals was decreased to the peripheral lymph nodes at the time of maximum tumor growth.     

The B cell is the initiating antigen-presenting cell in peripheral lymph nodes

We have examined the role of B cells in antigen presentation in lymph nodes in several ways. We found that mice depleted of B lymphocytes via chronic injection of anti-mu-chain antibody do not mount peripheral lymph node T cell proliferative responses to normally immunogenic doses of antigen. Depletion of B cells by passage of immune lymph node cells over anti-immunoglobulin columns early after immunization depletes antigen-presenting function from draining lymph nodes, and this function can be restored by using B cells or splenic adherent cells to allow the remaining T cells to proliferate. Lymph node B cells present antigen very effectively to lines of antigen-specific T cells. However, unfractionated lymph node cells from anti-mu-treated mice present very poorly, if at all, whereas unfractionated spleen cells from the same mice do present antigen. This is in keeping with our previous finding that helper T cell function in the spleen is normal in B cell-deprived mice. Finally, when mice homozygous for the lymphoproliferative gene lpr are treated chronically with anti-mu-chain antibody, lymphadenopathy is greatly retarded, suggesting a role for B cells in the massive proliferation of T cells in this syndrome. From this analysis, it would appear that the initiating antigen-presenting cell in the lymph node is a B lymphocyte, and that B lymphocytes in lymph nodes may be distinct from those in the spleen. It is of interest that these results also suggest that the lymph node lacks an antigen-presenting cell that is found in the spleen, perhaps the dendritic cell.     

Mapping of SJL/J reticulum cell sarcoma tumor-associated Ia antigens by T cell hybridomas: characterization of tumor-specific and shared epitopes detected on IE+ allogeneic cells

Previous studies have suggested that reticulum cell sarcoma (RCS) tumor cells of SJL/J (IA + IE-) mice express neospecificities that are related to antigenic specificities characteristic of IE+ allogeneic cells. These neospecificities have also been suggested to play a role in the strong syngeneic antitumor proliferative response as well as in regulating RCS growth in vivo. The present studies characterize four RCS tumor-specific T cell hybridoma clones prepared from the fusion of BW5147 thymoma with T cells derived from lymph nodes of tumor-bearing mice. Upon stimulation, these hybridomas secrete IL 2 in the supernatant. Two hybridomas responded to RCS to IE+k and to IE+d allogeneic cells, respectively, and the other two hybridomas were tumor specific. The specificity of these hybridomas was assessed by response to both spontaneous and transplantable RCS lines and failure to stimulate a response by either normal or LPS-induced B cell blasts from the host SJL/J cells. The epitopes recognized by the T cell hybridomas were examined by the ability of several monoclonal antibodies to inhibit the IL 2-induced response by the T cell hybridomas. Antibodies directed against the IABs polypeptide of the IA hybrid molecule blocked the antitumor response by all four hybridomas. However, the response to allogeneic IE+ cells was not blocked by anti-IAs antibody but was blocked by antibodies directed against either the IAk,d or IEk,d hybrid molecules or the corresponding alpha- or beta-chains. The response to both RCS and allogeneic cells was blocked by monoclonal antibodies directed against L3T4 antigens on the T cells. Based on the exquisite specificity of the T cell receptors, the results here demonstrate that RCS tumor cells express on their surface both tumor-specific I-A-associated epitopes and Ia-associated antigenic specificities that are shared with IE+ allogeneic cells. The present studies of adapting T cell hybridomas and blocking antibodies proved useful to characterize and map distinct tumor-associated epitopes on the surface of tumor cells. These findings, when combined with structural studies, should help unravel the molecular complexity of tumor-associated antigens.     

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.     

Tumor vaccination after allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned tumor-bearing murine host

Allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned host is supposed to provide an optimized platform for tumor vaccination. We recently showed that an allogeneic T cell-depleted graft was well accepted if the tumor-bearing host was NK depleted. Based on this finding, a vaccination protocol in tumor-bearing, nonmyeloablatively conditioned, allogeneically reconstituted mice was elaborated. Allogeneically reconstituted mice, bearing a renal cell carcinoma, received tumor-primed donor lymph node cells (LNC), which had or had not matured in the allogeneic host. Primed LNC were supported by tumor lysate-pulsed dendritic cells, which were donor or host derived. Optimal responses against the tumor were observed with host-tolerant, tumor-primed LNC in combination with host-derived dendritic cells. High frequencies of tumor-specific proliferating and CTLs were recorded; the survival time of tumor-bearing mice was significantly prolonged, and in >50% of mice the tumor was completely rejected. Notably, severe graft-vs-host disease was observed in reconstituted mice that received tumor-primed LNC, which had not matured in the allogeneic host. However, graft-vs-host was not aggravated after vaccination with tumor-primed, host-tolerant LNC. Thus, the LNC were tolerant toward the host, but not toward the tumor. The finding convincingly demonstrates the feasibility and efficacy of tumor vaccination after allogeneic reconstitution of the nonmyeloablatively conditioned host.     

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.     

Activation of T cells in tumor-bearing mice

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly+2- cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating non-specific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.     

Lymph node cell responsiveness in BALB/c mice infected with Leishmania mexicana

In the present study we measured the blastogenic response of lymph node cells from BALB/c mice infected with Leishmania mexicana throughout the course of infection. Results showed that infected mice displayed normal blastogenic responses in the lymph nodes until twenty weeks of infection. Thereafter, there was a gradual suppression. Comparison of the immunoresponsiveness in the spleen and lymph nodes, revealed normal responses in the lymph nodes several weeks after suppression in the spleen had occurred. Suppression of blastogenic responses in the lymph nodes was related to an adherent macrophage-like cell which actively suppressed normal proliferative responses to mitogens.     

CD4<Superscript>+</Superscript> T cell response against a non-tumor antigen is unaffected in melanoma-bearing mice

The tumor microenvironment is complex and creates an immunosuppressive network to tolerize tumor-specific immune responses; however, little information is available regarding the response against non-tumor antigens in tumor-bearing individuals. The goal of the present study was to evaluate if tumor burden could influence a CD4⁺ T cell response against a soluble protein, not expressed by the tumor, in the absence of in vitro stimulation. Using an experimental system in which we can compare CD4⁺ T cell responses to the Ea antigen when it is either expressed by B16F10 melanoma cells (B16EaRFP cells) or is an exogenous, non-tumor antigen (soluble EaRFP protein), in immunizations of B16F10 tumor-bearing mice, we observed that the tumor can modulate the CD4⁺ T cell-specific response to the antigen when it is expressed by the tumor cells. TEa cells proliferated poorly and produced less IFN-γ in mice bearing B16F10 melanoma expressing Ea peptide, and tumor growth was impervious to this response. However, in mice bearing 7 days B16F10 tumors, not expressing the Ea antigen, priming of TEa cells was similar to that observed in tumor-free mice, based on the total number of cells recovered and proliferation assessed by CFSE dilution after EaRFP immunization. We also investigated if tumor burden could influence recall responses of already differentiated effector cells. We immunized mice with EaRFP antigen and after a few days injected B16F10 cells. After 10 days of tumor growth, we challenged the mice with the non-tumor antigen. We found that the number of TEa cells producing IFN-γ in tumor-bearing mice was not different compared to tumor-free mice. No differences in antigen presentation, assessed by YAe antibody staining, were verified in the draining lymph node of these two groups. Collectively, our data indicate that tumor burden does not affect immune responses to non-tumor antigens. These results have important implications in the design of anti-cancer therapy.     

Kinetics of lymphoid cells in tumor-bearing mice

The distribution patterns of ⁵¹Cr-labeled lymphoid cells from normal C3H and tumor-bearing 6C3HED mice were studied by the method of Bainbridge and Gowland (1). An increased localization of normal recirculating lymphocytes was observed in draining lymph nodes of tumor bearing animals which reached a maximum of 8.4% by the 8th–10th day following transplantation. The proportion of recirculating cells from draining nodes of tumor-bearing animals decreased with the progression of the tumor although their actual numbers increased. This decrease is thought to be related to the invasion of draining nodes by tumor cells.     

Characterization of the spontaneous murine B cell leukemia (BCL1). III. Evidence for monoclonality by using an anti-idiotype antibody.

We have raised an anti-idiotypic antibody against the cell surface IgM of the murine BCL1 tumor cells. This antiserum reacts exclusively with the IgM expressed on the tumor cells and detects a unique population of cells in the spleen and blood of the tumor-bearing mice. When these cells are stimulated in vitro with LPS, they secrete an IgM bearing the same idiotype as the cell surface Ig. These results are discussed in terms of a model for the immunotherapy of a chronic lymphocytic leukemia-like syndrome in mice.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

Lymphocyte subsets involved in tumor-activated nonspecific feedback suppression

Cells from the spleen, lymph nodes, and peritoneum of DBA/2 mice bearing a subcutaneous tumor mediate nonspecific suppression of an in vitro antibody response to sheep red blood cells (SRBC) when cocultured with a normal T-cell subset(s). The spleen cells from the tumor-bearing mouse required for the suppression bear the Lyt 1 and Ala 1 surface markers characteristic of "inducer" T cells and activated cells, respectively. The activity of this cell population is also sensitive to irradiation. The normal T-cell subset which cooperates in the suppression bears the Qa-1 surface antigen which has been associated with suppressor cell precursors in several systems but lacks detectable surface Lyt 1 and 2 markers. Suppression of antibody responses in spleen cell cultures from tumor-bearing mice alone could also be elicited, but only when increased numbers of cells were cultured. These data are consistent with the theory that a tumor-activated, Lyt 1+ T-cell subset has the capacity to nonspecifically suppress immune responses by activating a Qa-1+ subset(s) of T suppressor cells, perhaps via feedback signals.     

The role of dendritic cells in the initiation of immune responses to contact sensitizers. I. In vivo exposure to antigen

Twenty-four hours after skin painting mice with picryl chloride (PIC) there was a four- to fivefold increase in the numbers of dendritic cells (DC) isolated from the lymph nodes. These DC initiated primary proliferative and cytotoxic responses when added to cultures of normal syngeneic lymph node cells. The proliferative response was enhanced when the donors of the responding lymph node cells were sensitized with the same antigen. Contact sensitivity developed in syngeneic mice injected into the footpads with 30,000-50,000 DC from lymph nodes of mice painted with picryl chloride 1 day previously. Thus, 1 day after skin painting mice, there were dendritic cells in the draining lymph nodes which were able both to initiate primary stimulation of lymphocytes in vitro and to sensitize recipient mice to give specific delayed hypersensitivity reactions.     

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.     

Evidence for interaction between T cell populations of tumor-bearing and normal mice in immune suppression

Spleen cells of DBA/2 mice bearing subcutaneous implants of the syngeneic tumor L5178Y induce suppression of the in vitro antibody response of normal spleen cells to sheep erythrocytes (SRBC). Cells mediating suppression are detected in the spleens of tumor-bearing mice as early as 24 hr post-implantation but are no longer detected there 15 days post-implantation. These spleen cells are nylon wool nonadherent, sensitive to anti-Thy 1.2 + C and anti-Lyt 1.1 + C, and insensitive to anti-Lyt 2.1 + C treatment. The anti-SRBC response of the unfractionated spleen cells from the tumor-bearing mice is not itself suppressed at the cell numbers used. This along with the finding that suppression occurs in the presence of spleen cells from normal mice suggest that a cell population from the normal mouse spleen is also involved in the suppression. Spleen cells from mice inoculated with irradiated (nonproliferating) L5178Y cells are similarly capable of mediating nonspecific suppression for the same limited period of time after the inoculation. In addition, spleen cells from mice stimulated with several nontumorigenic cellular antigens interact with normal spleen cells to produce suppression. These findings suggest that suppression observed in vitro with spleen cells from these tumor-bearing mice may be the result of antigen-activated cells triggering normal immunoregulatory cells.     

Tumor-associated CD8+ T cell tolerance induced by bone marrow-derived immature myeloid cells

T cell tolerance is a critical element of tumor escape. However, the mechanism of tumor-associated T cell tolerance remains unresolved. Using an experimental system utilizing the adoptive transfer of transgenic T cells into naive recipients, we found that the population of Gr-1+ immature myeloid cells (ImC) from tumor-bearing mice was able to induce CD8+ T cell tolerance. These ImC accumulate in large numbers in spleens, lymph nodes, and tumor tissues of tumor-bearing mice and are comprised of precursors of myeloid cells. Neither ImC from control mice nor progeny of tumor-derived ImC, including tumor-derived CD11c+ dendritic cells, were able to render T cells nonresponsive. ImC are able to take up soluble protein in vivo, process it, and present antigenic epitopes on their surface and induce Ag-specific T cell anergy. Thus, this is a first demonstration that in tumor-bearing mice CD8+ T cell tolerance is induced primarily by ImC that may have direct implications for cancer immunotherapy.     

Lymphocyte function in experimental African trypanosomiasis. VIII. Loss of suppressor T cell function in lymph nodes

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.     

A vaccine based on exosomes secreted by a dendritic cell line confers protection against T. gondii infection in syngeneic and allogeneic mice

Our results show that exosomes secreted by SRDC pulsed in vitro with Toxoplasma gondii-derived antigens (Exo-TAg) induced protective responses against infection with the parasite in both syngeneic and allogeneic mice. After oral infection, syngeneic CBA/J mice exhibited significantly fewer cysts in their brains and allogeneic C57BL/6 mice survived. This protection was associated with strong humoral responses in vivo in serum from both CBA/J and C57BL/6 mice, and with high levels of anti-TAg IgA antibodies in intestinal secretions from CBA/J mice alone. Furthermore, strong cellular responses in vivo were observed in both mouse models. Cellular proliferation was associated with cytokines production by spleen and mesenteric lymph node cells. The results presented here show that exosomes are nucleic acid free vesicles that are able to induce immune responses correlated with protection against parasitic infections in both syngeneic and allogeneic mice. They could constitute an efficient tool for use in vaccination and antitumor strategies based on exosomes.     

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells

 Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D₃ eliminated natural suppressor activity. In mice that were first treated with vitamin D₃ and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D₃/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D₃/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D₃ to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor. Received: 15 December 1995 / Accepted: 22 March 1996