Tumor-Experienced Nk Cells Inhibit T Cell Proliferation And Activation Through Pd-L1

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. Nunez, 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.