Immunotherapy may provide valid alternative therapy for patients with hormone-refractory metastatic prostate cancer. However, if the tumor environment exerts a suppressive action on antigen-specific tumor-infiltrating lymphocytes (TIL), immunotherapy will achieve little, if any, success. In this study, we analyzed the modulation of TIL responses by the tumor environment using collagen gel matrix–supported organ cultures of human prostate carcinomas. Our results indicate that human prostatic adenocarcinomas are infiltrated by terminally differentiated cytotoxic T lymphocytes that are, however, in an unresponsive status. We demonstrate the presence of high levels of nitrotyrosines in prostatic TIL, suggesting a local production of peroxynitrites. By inhibiting the activity of arginase and nitric oxide synthase, key enzymes of L-arginine metabolism that are highly expressed in malignant but not in normal prostates, reduced tyrosine nitration and restoration of TIL responsiveness to tumor were achieved. The metabolic control exerted by the tumor on TIL function was confirmed in a transgenic mouse prostate model, which exhibits similarities with human prostate cancer. These results identify a novel and dominant mechanism by which cancers induce immunosuppression in situ and suggest novel strategies for tumor immunotherapy.

Previous studies have suggested that T-lymphocyte dysfunction might be attributable to nitrative stress induced by reactive nitrogen species (RNS). In this manuscript, we explored this hypothesis and provided a direct demonstration of the inhibitory effects of RNS on human T-cell signaling, activation, and migration. We found that short exposure of human T cells to RNS induced tyrosine phosphorylation of several proteins, including the CD3f chain of the TCR complex, and release of Ca 21 from intracellular stores. When the exposure to RNS was prolonged, T cells became refractory to stimulation, downregulated membrane receptors such as CD4, CD8, and chemokine receptors, and lost their ability to migrate in response to chemokines. Since substantial protein nitration, a hallmark of nitrative stress, was observed in various human cancers, intratumoral generation of RNS might represent a relevant mechanism for tumor evasion from immune surveillance.

Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.

A soluble recombinant form of Neisseria meningitidis adhesin A (NadAΔ351–405), proposed as a constituent of anti-meningococcal B vaccines, is here shown to specifically interact with and immune-modulate human monocyte-derived dendritic cells (mo-DCs). After priming with IFN-γ and stimulation with NadAΔ351–405, mo-DCs strongly up-regulated maturation markers CD83, CD86, CD80, and HLA-DR, secreted moderate quantities of TNF-α, IL-6, and IL-8, and produced a slight, although significant, amount of IL-12p70. Costimulation of mo-DCs with NadAΔ351–405 and the imidoazoquinoline drug R-848, believed to mimic bacterial RNA, increased CD86 in an additive way, but strongly synergized the secretion of IL-12p70, IL-1, IL-6, TNF-α, and MIP-1α, especially after IFN-γ priming. CD86/CD80 overexpression correlated with the occupation of high-(kd ∼ 80 nM) and low-(kd ∼ 4 μM) affinity binding sites for NadAΔ351–405. Alternatively, secretion of IL-12p70 and TNF-α, IL-6, and IL-8 corresponded to the occupation of high- or low-affinity receptors, respectively. Mo-DCs matured by IFN-γ and NadAΔ351–405 supported the proliferation of naive CD4+ T lymphocytes, inducing the differentiation of both IFN-γ and IL-4 producing phenotypes. Our data show that NadA not only is a good immunogen but is as well endowed with a proimmune, self-adjuvating, activity.

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