Foxp3(+) regulatory T cells and natural killer cells distinctly infiltrate primary tumors and draining lymph nodes in pulmonary adenocarcinoma.

INTRODUCTION: Regulatory T cells (Tregs) can play a key role in suppressing T-cell-mediated immunity in patients with cancer. In this study, the immune cell composition of the lung tissue and draining lymph nodes from patients with non-small cell lung cancer was analyzed. METHODS: Samples (solid tumor, tumor border, and tumor-free lung tissue, as well as intrapulmonal N1 and mediastinal N2 lymph nodes) from 30 patients subjected to curative resection were analyzed by immunohistochemistry and flow cytometry. RESULTS: Immunohistochemistry showed the presence of Foxp3(+) Tregs in tumor-infiltrated lung tissue, scattered Tregs in tumor-free lung samples, and a large number of these cells in metastatic lymph nodes. Using flow cytometry, we observed a significant enhancement of CD4(+) T cells and Foxp3(+) Tregs in the tumor center of adenocarcinoma samples, when compared with tumor-free lung tissues and tumor periphery. This enrichment was associated with a drastic decrease in natural killer cell amounts. Metastatic lymph nodes also showed higher Treg numbers than tumor-free ones in patients with lung adenocarcinomas. In contrast, patients with squamous cell carcinomas displayed less profound accumulation of Tregs. CONCLUSION: Accumulation of Tregs in the center of lung tumors and in metastatic lymph nodes in combination with a decrease in the natural killer cell numbers suggests a critical role of Treg in the formation of immunosuppressive tumor microenvironment. Therefore, lung cancer immunotherapy may be improved by a specific Treg elimination or suppression.

Distinct metabolism of cyclic adenosine monophosphate in regulatory and helper CD4+ T cells.

Regulatory T cells (Treg) are crucial for the suppression of antigen-specific immune responses by activated conventional T cells (Tcon). It has been recently reported that this suppression is mediated by cyclic adenosine monophosphate (cAMP) transported from Treg to Tcon via gap junctions. However, the underlying biochemical mechanisms of cAMP accumulation in activated Treg are still unclear. Here we reported that although non-activated murine Treg and Tcon displayed similar intracellular cAMP amounts, both subpopulations showed distinct expression of enzymes regulating cAMP metabolism. Thus, in Treg, activities of both anabolic (adenylyl cyclase, AC) and catabolic (phosphodiesterase, PDE) enzymes were lower than in Tcon. Furthermore, we demonstrated for the first time the expression of the PDE11 protein in murine Treg and Tcon. Treg activation by IL-2 induced a strong AC7 activation and cAMP accumulation in Treg. In contrast, Tcon showed a significant decrease in the AC7 activity and cAMP amounts under these conditions. Our data suggest that the mechanism of cAMP accumulation in stimulated Treg involves the AC7 activation and provide new insight into the modulation of Treg activities via AC inhibition or stimulation in various pathological processes like tumor and autoimmune diseases.

Skin melanoma development in ret transgenic mice despite the depletion of CD25+Foxp3+ regulatory T cells in lymphoid organs.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) known to mediate self-tolerance were also shown to contribute to tumor progression. In mouse melanoma transplantation models, Treg depletion resulted in the stimulation of antitumor immune responses and tumor eradication. To study Treg in conditions close to the clinical situation, we used a ret transgenic mouse spontaneous melanoma model, which, in contrast to transplantation models, resembles human melanoma regarding clinical development. Significantly higher numbers of Treg were found in skin tumors and metastatic lymph nodes at early stages of melanoma progression compared with more advanced stages accompanied by the elevated CCR4 expression on Treg and higher production of its ligand CCL2 in tumor lesions. Numbers of tumor infiltrating Treg inversely correlated with Treg amounts in the bone marrow, suggesting their possible recruitment to melanoma lesions from this organ. The immunosuppressive function of Treg from transgenic tumor-bearing mice was similar to that from transgenic tumor-free mice or nontransgenic littermates. Although anti-CD25 mAb injections resulted in the efficient Treg depletion from lymphoid organs of transgenic mice, melanoma development was not significantly delayed. Furthermore, the treatment of mice with macroscopical tumors also failed to inhibit tumor progression, which correlated with the inability to deplete intratumoral Treg. We suggest that in the autochthonous melanoma genesis, other immunosuppressive cells could play an important role and replace immunosuppressive, tumor-promoting functions of Treg. Therefore, effective melanoma immunotherapy should include the inhibition of Treg migration into the tumor combined with neutralization of other immunosuppressive cells and factors in the tumor microenvironment.

NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo.

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.