The tumor recall response of antitumor immunity primed by a live, recombinant Listeria monocytogenes vaccine comprises multiple effector mechanisms.

Listeria monocytogenes, a facultative intracellular bacterium, can induce a potent antitumor immune response if engineered to express a model tumor antigen also expressed by the tumor cells. The effectiveness of this approach is dependent on L. monocytogenes-induced tumor-specific CD4(+) and CD8(+) T-cells. CD8(+) T-cells may mediate tumor eradication largely through direct CTL activity, but the role of CD4(+) T-cells and other cells of the immune system is less clear. Here we investigate their role and the role of the cytokines they produce in the ability of L. monocytogenes-induced antitumor immunity to protect against tumor challenge. Our results suggest that a complex cytokine response, involving type 2 as well as type 1 cytokines, is responsible for the ability of Lm-NP-immunized mice to resist tumor challenge, potentially mediating tumor cell killing through multiple effector pathways.

Regression of established B16F10 melanoma with a recombinant Listeria monocytogenes vaccine.

We have previously shown that Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is a potent vector for targeting tumor-specific antigens to the immune system. In the present study, we extend these studies to the highly tumorigenic mouse melanoma B16F10, transduced with a model tumor antigen. We are able to induce the regression of primary tumors and established lung metastases by parenteral immunization with a L. monocytogenes recombinant that expresses the same antigen. Adjunctive therapy with granulocyte macrophage colony-stimulating factor or a vaccinia-based vaccine does not result in an improved cure rate over the L. monocytogenes vaccine alone. Tumor regression is accompanied by the expression of inflammatory cytokines in the tumor.

Listeria monocytogenes: a potent vaccine vector for neoplastic and infectious disease.

Listeria monocytogenes (L. monocytogenes) is a promising candidate vaccine vector that naturally infects antigen-presenting cells, and targets antigen delivery to both the class I MHC pathway of endogenous antigen presentation and the class II pathway of exogenous antigen presentation. At the same time, L. monocytogenes stimulates the innate immune response to produce cytokines that enhance antigen-presenting function and induce a Th1-type cytokine profile associated with cell-mediated immune responses. Immune responses with these features are considered to be particularly important for clearance of viruses, tumors, and intracellular infections. In this review, we describe the development of methods to transform L. monocytogenes to express and secrete foreign antigens and the studies that have demonstrated that genetically engineered L. monocytogenes mutants are highly effective vectors for the induction of potent immune responses against viral antigens and tumor cells. In addition, we discuss the strengths and weaknesses of L. monocytogenes as a vaccine vector.

Transforming growth factor-beta-mediated down-regulation of antitumor cytotoxicity of spleen cells from MOPC-315 tumor-bearing mice engaged in tumor eradication following low-dose melphalan therapy.

We have previously shown that treatment of mice bearing a large MOPC-315 plasmacytoma with a low dose of the anticancer drug melphalan (L-phenylalanine mustard; L-PAM) results in the acquisition of a potent CD8+ T-cell-mediated anti-MOPC-315 cytotoxic T lymphocyte (CTL) activity by the hitherto immunosuppressed tumor bearers, and this immunity contributes to complete tumor eradication. In the studies presented here, we sought to determine how the acquisition of this antitumor immunity following low-dose chemotherapy is possible, in light of the report that MOPC-315 tumor cells produce transforming growth factor-beta (TGF-beta), an immunosuppressive cytokine that can down-regulate the generation of CTL responses. We found that the acquisition of CTL activity following low-dose L-PAM therapy is not due to a chemotherapy-induced decrease in the sensitivity of MOPC-315 tumor bearer spleen cells to TGF-beta-mediated inhibition of CTL generation. Moreover, even spleen cells from MOPC-315 tumor-bearing mice, which had received L-PAM therapy 7 days earlier and had acquired CTL activity in vivo, were sensitive to the inhibitory activity of TGF-beta upon culture for as little as 1 day, with or without stimulator tumor cells. However, the production of TGF-beta by MOPC-315 tumors decreased drastically as a consequence of the low-dose chemotherapy. Thus, the curative effectiveness of low-dose L-PAM therapy for MOPC-315 tumor-bearing mice may be due, at least in part, to a reduction in TGF-beta production that enables the development of tumor-eradicating immunity.

Some approaches to improve the therapeutic effectiveness of adoptive chemoimmunotherapy with spleen cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor.

Spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells) were previously shown to be capable of bringing about the complete regression of a large (15 to 20 mm) s.c. MOPC-315 tumor in a substantial percentage of T-cell-deficient (athymic nude) mice that had been treated with low-dose L-PAM (adoptive chemo-immunotherapy; ACIT). Here we show that aggressive depletion of CD4+T-cells by treatment both of spleen-cell donors and of recipients with anti-L3T4 monoclonal antibody (MAb) greatly improved the therapeutic effectiveness of L-PAM TuB spleen cells in ACIT. In the light of the apparent importance of cytotoxic T-lymphocytes (CTLs) for tumor eradication in low-dose L-PAM-treated MOPC-315-tumor bearers, it is interesting that treatment of L-PAM TuB spleen-cell donors with anti-L3T4 MAb was found to result in the generation of enhanced splenic anti-MOPC-315 cytotoxicity. Although most athymic nude mice in which the tumor had apparently completely regressed following ACIT remained tumor-free, approximately 1/3 of the mice relapsed. However, a substantial percentage of the relapsing mice were rescued by a low dose of L-PAM, which was not effective in causing tumor regression in athymic nude mice bearing a comparably large tumor if the mice had not been subjected previously to ACIT. Almost all athymic nude mice that had been "cured" of a large MOPC-315 tumor by ACIT but did not resist a subsequent MOPC-315 tumor challenge were rescued by low dose L-PAM. Thus, the therapeutic effectiveness of L-PAM TuB spleen cells in ACIT may be improved by aggressive depletion of CD4+ T-cells, suggesting that a low dose of L-PAM, which leads to the acquisition of potent splenic-tumor-eradicating immunity in BALB/c mice bearing a large MOPC-315 tumor, does not eliminate completely (or possibly not at all) the inhibitory activity of CD4+ T-cells. In addition, athymic nude mice that are not endowed with fully protective tumor-eradicating immunity following ACIT still have a substantial residual anti-tumor immune potential that can be exploited to bring about eradication of a large tumor burden following low-dose L-PAM therapy.

Phorbol ester-induced enhancement in lytic activity of CD8+ splenic T cells from low-dose melphalan-treated MOPC-315-tumor bearers.

We have previously shown that while spleen cells from untreated mice bearing a large MOPC-315 tumor are not cytotoxic in vitro for MOPC-315 tumor cells, spleen cells obtained from such mice on day 7 after low-dose melphalan (L-phenylalanine mustard); L-PAM therapy exert a substantial anti-MOPC-315 cytotoxicity [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that this anti-MOPC-315 lytic activity is evident by day 5, and peaks on day 7 after the low-dose chemotherapy, at a time when the mice are actively engaged in tumor eradication. Short-term exposure of spleen cells from mice bearing a MOPC-315 tumor and treated with low-dose L-PAM (L-PAM TuB mice) to phorbol 12-myristate 13-acetate (PMA) was found to enhance greatly the ability of these spleen cells to lyse MOPC-315 tumor cells. The highest level of anti-MOPC-315 cytotoxicity was obtained when spleen cells from tumor-bearing mice that had received chemotherapy 7 days earlier were exposed to PMA at a concentration of 1-10 ng/ml. The exertion of the enhanced anti-MOPC-315 lytic activity by L-PAM TuB spleen cells exposed to PMA was found to require CD8+, but not CD4+, T cells. The apparent specificity of the lytic activity exerted by the PMA-stimulated L-PAM TuB spleen cells was illustrated not only by the inability of the spleen cells to lyse an allogeneic, antigenically unrelated thymoma (EL4), but also by their relatively weak lytic activity for two antigenically related syngeneic plasmacytomas. In addition, when EL4 target cells were admixed with MOPC-315 tumor cells, the lytic activity triggered in the L-PAM TuB spleen cells by the MOPC-315 tumor cells plus PMA was not effective in lysing the antigenically unrelated target cells. Moreover, even in the presence of the calcium-specific ionophore, ionomycin, L-PAM TuB spleen cells exposed to PMA were unable to lyse the EL4 target cells. Thus, fresh CD8+ splenic T cells from L-PAM TuB mice that are in the process of eradicating a large MOPC-315 tumor as a consequence of low-dose L-PAM therapy can be triggered with PMA to exert enhanced lytic activity against MOPC-315 tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Eradication of a large MOPC-315 tumor in athymic nude mice by chemoimmunotherapy with Lyt2+ splenic T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor.

We have previously shown that spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose (2.5 mg/kg) melphalan (L-phenylalanine mustard) therapy are effective in preventing tumor progression upon adoptive transfer into BALB/c mice bearing a barely palpable tumor that had been treated with a subcurative dose of melphalan [Mokyr et al. (1989) Cancer Res 49:4597]. Here we show that such spleen cells in conjunction with a subcurative dose of drug (adoptive chemoimmunotherapy, ACIT) can cause the complete regression of a large (15-20 mm) s.c. MOPC-315 tumor in a large percentage of T-cell-deficient (athymic nude) tumor-bearing mice. Spleen cells that were effective in ACIT of athymic nude mice displayed in vitro a substantial direct lytic activity against MOPC-315 tumor cells, and the lytic activity was greatly enhanced when the spleen cells were cultured for 5 days with or without mitomycin-C-treated MOPC-315 stimulator tumor cells. The cells responsible for the therapeutic effectiveness of the spleen cells in ACIT of athymic nude mice, as well as the cells responsible for the direct in vitro anti-MOPC-315 lytic activity of the spleen cells, were of the Lyt 2 and not the L3T4 phenotype. Most of the athymic nude mice that completely eradicated a large MOPC-315 tumor as a consequence of ACIT were capable of rejecting a challenge with 30-100 times the minimal lethal tumor dose for 100% of normal BALB/c mice administered more than 1 month after the ACIT. The ability of these athymic nude mice to resist the tumor challenge was associated with the presence of a greatly elevated percentage of cells expressing T cell surface markers in their spleens. Thus, it is conceivable that splenic Lyt 2+T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor mediate their therapeutic effectiveness in ACIT of athymic nude mice bearing a large MOPC-315 tumor, at least in part, through direct cytotoxicity for MOPC-315 tumor cells. In addition, eradication of a large MOPC-315 tumor through cooperation between antitumor immunity and melphalan toxicity endues the athymic nude mice with an elevated percentage of T cells in their secondary lymphoid organs, and these T cells are probably responsible for the long-lasting protective antitumor immunity exhibited by these mice.

Importance of Lyt 2+ T-cells in the curative effectiveness of a low dose of melphalan for mice bearing a large MOPC-315 tumor.

We have previously demonstrated that the curative effectiveness of a low dose (2.5 mg/kg) of melphalan (L-phenylalanine mustard; L-PAM) for mice bearing a large s.c. (approximately 20 mm in diameter) MOPC-315 tumor and extensive metastases requires the participation of T-cell-dependent antitumor immunity in tumor eradication (S. Ben-Efraim et al., Cancer Immunol. Immunother., 15: 101-107, 1983). Here we show that the Lyt 2+ T-cells, and not the L3T4+ T-cells, participate in the cure of such tumor-bearing mice by a low dose of L-PAM. Specifically, depletion of Lyt 2+ T-cells from mice bearing a large MOPC-315 tumor by treatment with monoclonal anti-Lyt 2.2 antibody abolished the curative effectiveness of the low dose of drug. In contrast, depletion of L3T4+ T-cells from mice bearing a large MOPC-315 tumor by treatment with monoclonal anti-L3T4 antibody did not reduce significantly the curative effectiveness of the low dose of drug. Histological examination of tumor nodules on various days following low-dose L-PAM therapy revealed widespread lymphocytic infiltration by Day 5 following the chemotherapy, and this infiltration was drastically reduced when the L-PAM-treated tumor bearers were treated with either anti-Thy 1.2 or anti-Lyt 2.2 antibody but not with anti-L3T4 antibody. The antitumor immunity exhibited by Lyt 2+ T-cells derived from mice which were in the process of eradicating a large MOPC-315 tumor following low-dose L-PAM therapy was exploited successfully to confer systemic antitumor immunity to mice bearing a barely palpable tumor. Specifically, the adoptively transferred Lyt 2+ splenic T-cells, in conjunction with a subcurative dose of L-PAM, brought about the cure of most mice. The Lyt 2+ splenic T-cells from L-PAM-treated MOPC-315 tumor bearers were also found to be capable of exerting a direct potent lytic effect against MOPC-315 tumor cells in an antigen-specific manner. Thus, it is conceivable that the direct cytotoxic activity of Lyt 2+ T-cells for MOPC-315 tumor cells is responsible, at least in part, for the ability of the Lyt 2+ T-cells from L-PAM-treated MOPC-315 tumor bearers to bring about the eradication of the tumor burden not eradicated through the direct antitumor effects of the low dose of drug.