Immunoprotection occurs T-cell-mediated activation and M1 polarization of TAMs, making them cytotoxic to neighboring tumor cells (2, 13)
Immunoprotection occurs T-cell-mediated activation and M1 polarization of TAMs, making them cytotoxic to neighboring tumor cells (2, 13). and presentation on tumor-infiltrating antigen-presenting cells (APCs). Activation of the APCs can induce tumor rejection, but the mechanisms underlying tumor killing by such cells have not been established. To elucidate the molecular basis of CD4+ T-cell-mediated tumor rejection, we utilized a murine model of multiple myeloma, in which the T cells recognize a secreted tumor neoantigen. Our findings demonstrate that T cell recognition triggers inducible nitric oxide synthase activity within tumor-infiltrating macrophages. Diffusion of nitric oxide into surrounding tumor cells results in intracellular accumulation of toxic secondary oxidants, notably peroxynitrite. This results in tumor cell apoptosis through activation of the mitochondrial pathway. We find that this mode of cytotoxicity has strict spatial limitations, and is restricted to the immediate surroundings of the activated macrophage, thus limiting TAK-700 (Orteronel) bystander killing. These findings provide a molecular basis for macrophage-mediated anti-tumor immune responses orchestrated by CD4+ T cells. Since macrophages are abundant in most solid tumors, evoking the secretion of nitric oxide by such cells may represent a potent therapeutic strategy. the Fas/Fas ligand (9) or perforin/granzyme pathway (3). For other tumor cell types, including the MOPC315 plasmacytoma cell line used in the present study, the tumor cells do not themselves express MHC class II, even in the presence of interferon gamma (IFN-) (2, 10, 11). The tumor cells are therefore unable to directly interact with tumor-infiltrating T cells (2), and antigen TAK-700 (Orteronel) presentation is dependent on uptake in host antigen-presenting cells (APCs) (12). Hence, CD4+ T cell recognition of tumor antigen occurs in an indirect manner (2, 10, 12, 13). We have previously exhibited that CD4+ T cells reactive against a secreted myeloma protein tumor antigen can mediate protection against tumor development upon challenge with MOPC315 myeloma cells (2, 6, 7, 12). Immunoprotection occurs T-cell-mediated activation and M1 polarization TAK-700 (Orteronel) of TAMs, rendering them cytotoxic to neighboring tumor cells (2, 13). Such indirect tumor antigen recognition results in a change in the cytokine profile of the tumor microenvironment toward a Th1-type inflammatory response (13). Despite these observations, the molecular mechanism(s) underlying macrophage-mediated killing of tumor cells is not known. We have here performed an in-depth characterization of macrophage-mediated cytotoxicity against MOPC315. Our results demonstrate that activated macrophages rapidly induce apoptosis in tumor cells the mitochondrial pathway. This occurs in a cell contact-independent, but spatially limited fashion. Cytotoxicity is dependent on short-lived factors, and is completely abrogated in the absence of TAK-700 (Orteronel) inducible nitric oxide synthase (iNOS) in TAMs. Further assays reveal a critical role of peroxynitrite formed within the tumor cells, pointing to short-lived reactive nitrogen species (RNS) as mediators of macrophage cytotoxicity. Materials and Methods Reagents, Cells, and Viral Transduction Apocynin, taurine, and superoxide dismutase (SOD) (Sigma-Aldrich, St. Louis, MO, USA). Manganese (III) meso-tetrakis(Experiments DO11.10, CByJ.129P2(B6)-Nos2tm1Lau/J and wild-type (WT) BALB/c mice were obtained from Jackson (The Jackson laboratory, Bar Harbor, ME, USA). Homozygous Id-specific T cell receptor-transgenic (TCR-Tg) BALB/c mice have been previously described (18). Heterozygous TCR-Tg SCID mice (6) and SCID littermates were kept on a BALB/c background. TCR-transgenic BALB/c SCID and BALB/c Rag?/? mice hemizygous for the TCR transgenes were bred in-house. Offspring (50% transgenic, 50% non-transgenic) were typed by staining of blood CD4+ lymphocytes using the TCR clonotype-specific mAb GB113 (18). All mice were bred and maintained under special pathogen-free conditions. All experiments were approved by the Norwegian Animal Research Authority (Mattilsynet), and performed in accordance with institutional and Federation of European Laboratory Animal Science Associations guidelines. Tumor challenge experiments were performed by subcutaneous (s.c.) injection of 1 1.6??105 MOPC315 cells dissolved in 100?L PBS. For some experiments, cells were embedded in 250?L Matrigel to form a tumor bed of defined size, as previously described (13). Tumor development was followed by palpation and digital caliper measurement, and mice were euthanized upon developing tumors with IL18R1 largest diameter >10?mm. Isolation of cells from explanted Matrigels was performed as previously described (13). For adoptive transfer, mice were sub-lethally irradiated (500?cGy) at day ?2, injected i.v. with 2??106 na?ve Id-specific.