Natural Killer (NK) cells play a key role in cancer immunosurveillance. However, NK cells from cancer patients display an altered phenotype and impaired effector functions. In addition, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation, and autoimmunity. Here, we analyzed clear cell renal cell carcinoma (ccRCC) datasets from The Cancer Genome Atlas (TCGA) and observed that a higher expression of NK cell signature genes is associated with reduced survival. Analysis of fresh tumor samples from ccRCC patients unraveled the presence of a high frequency of tumor-infiltrating PD-L1+ NK cells, suggesting that these NK cells might exhibit immunoregulatory functions. In vitro, PD-L1 expression was induced on NK cells from healthy donors (HD) upon direct tumor cell recognition through NKG2D and was further up-regulated by monocyte-derived IL-18. Moreover, in vitro generated PD-L1hi NK cells displayed an activated phenotype and enhanced effector functions compared to PD-L1- NK cells, but simultaneously, they directly inhibited CD8+ T cell proliferation in a PD-L1-dependent manner. Our results suggest that tumors might drive the development of PD-L1-expressing NK cells that acquire immunoregulatory functions in humans. Hence, rational manipulation of these regulatory cells emerges as a possibility that may lead to improved anti-tumor immunity in cancer patients.
BackgroundNatural killer and cytotoxic CD8+T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity.MethodsWe generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase fromBrucellaspp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect.ResultsImmunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+T cell recruitment.ConclusionsImmunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.
Natural killer (NK) cells play a key role in cancer immunosurveillance and in the elimination of infected cells, however, NK cells from cancer patients show an altered phenotype and impaired effector functions. Moreover, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation and autoimmunity. In the tumor context, we have recently described an immunoregulatory population of PD-L1-expressing NK cells arising in tumor-bearing mice that inhibits CD8+ T cell priming. Those results led us to examine PD-L1 expression on human NK cells after tumor recognition and their regulatory potential. First, we analyzed PD-L1 expression on tumor-infiltrating or peripheral blood NK cells (CD56+CD3- cells) from patients with kidney cancer by flow cytometry. We found an increased frequency of PD-L1+ tumor-infiltrating NK cells compared to autologous peripheral blood NK cells. Moreover, PD-L1 was up-regulated on peripheral blood NK cells from those cancer patients compared to healthy donors. Further experiments were performed in vitro to investigate the mechanisms involved in tumor-induced PD-L1 up-regulation on NK cells. After culture of human PBMCs from healthy donors with the susceptible K562 cell line, PD-L1 expression was induced on NK cells. Transwell and receptor blockade experiments showed a dependence on cell-to-cell contact that involved NKG2D engagement. PD-L1 expression on isolated NK cells cultured with K562 tumor cells could be further increased by soluble factors derived from tumor-experienced PBMCs; neutralization of different cytokines identified IL-18, produced by monocytes, as the main factor responsible for PD-L1 up-regulation. Analysis of NK cells after incubation with K562 cells showed that relative to PD-L1- NK cells, PD-L1+ NK cells expressed increased levels of the activation markers CD25 and CD69 and the effector molecules TRAIL, FasL, IFN-γ and CD107a, suggesting that PD-L1 is preferentially up-regulated on activated NK cells. Finally, to evaluate the immunosuppressive potential of these NK cells, CFSE-labeled autologous T cells were stimulated with anti-CD3/anti-CD28 antibodies (Ab) and cultured in the presence of sorted tumor-experienced NK cells or control NK cells, and in the absence or in the presence of anti-PD-L1 blocking Ab. After 5 days, T cell proliferation and activation (evaluated as CFSE dilution and CD25 expression by flow cytometry) were diminished in the presence of tumor-experienced NK cells compared to control NK cells, and this inhibition was reverted by PD-L1 blockade. Our results indicate that tumor recognition results in upregulated expression of PD-L1 on NK cells leading to a direct inhibition of T cell priming trough PD-L1. Thus, rational manipulation of these regulatory cells could lead to improved anti-tumor immunity in vivo. Citation Format: Jessica M. Sierra, Ximena L. Raffo Iraolagoitia, Florencia Secchiari, Andrea Ziblat, Sol Y. Nuñez, Nicolas I. Torres, Maria V. Regge, Carolina I. Domaica, Norberto W. Zwirner, Mercedes B. Fuertes. Tumor-experienced NK cells inhibit T cell proliferation and activation through PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 704.
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