Natural killer cells from patients with malignant or inflammatory pleural effusions display decreased cytotoxicity and decidual NK-like phenotype

INTRODUCTION: Natural killer (NK) cells are lymphoid cells involved in tumor recognition and eradication. NK activity is impaired in cancer patients and in non-small cell lung cancer it has been shown that they acquire a pro-angiogenic phenotype and function, similar to that of NK cells in the decidua. In this work, we characterized NK cells infiltrating malignant pleural effusions derived from subjects with primary or metastatic tumors of different origins and inflammatory pleural effusions. MATERIAL AND METHOD: We collected peripheral blood samples from healthy donors (hPB) and from patients with malignant (mPB) or inflammatory (iPB) pleural effusions in addition to malignant and inflammatory pleural effusions fluids (mPE and iPE). We performed ex vivo FACS analysis of phenotype and cytokine production of PB and PE-derived NK cells (PB-NKs and PE-NKs) and a CD107a assay to evaluate their cytotoxicity. In addition, we performed a 3-day culture with IL-2, with IL-2 and TGFβ and with IL-2 in a conditioned media (CM) containing mPE/iPE (mPE/iPE-CM) to evaluate cytotoxicity of NK cells. We’ve also isolated NKs from buffy coats of healthy donors and cultured them for 7 days with IL-15 and with IL-15 in mPE/iPE-CM to evaluate pro-angiogenic polarization. RESULTS AND DISCUSSION: We found significant levels of the CD56bright NK cell in mPE (average 60%) and iPE (average 40%), predominantly lacking CD16 Fcγ-receptor. These NKs are enriched in cells expressing CD49a decidual NK surface markers, are poorly mature (low expression of CD57) but highly activated (high expression of NKp44 and CD69) and display a deregulated expression of activating and inhibitory receptors. Furthermore, PE-NKs display a higher amount of intracellular VEGF and a lower amount of Perforin if compared with healthy and autologous PB-NKs. Moreover, PE-NK cells show greater cytotoxicity ex vivo than their counterparts found in autologous peripheral blood. However, both show lower killing capacity if compared to NK cells from peripheral blood of healthy controls. After 3-day culture with IL-2 cytotoxicity increased, with IL-2 and TGFβ increased but in lower proportion because TGFβ counteract the action of IL-2 and with IL-2 and mPE/iPE the cytotoxicity of NK cells was not affect. We’ve isolated NKs from buffy coats of healthy donors and we found that NK cells acquire pro-angiogenic characteristics after 7-day culture with IL-15 in mPE-CM or iPE-CM. Moreover, mPE/iPE-CM are able to induce in conditioned NK cells a polarization into a CD56bright CD16- phenotype and a decreased production of IFNγ and mPE-CM induced an increase in the production of VEGF. CONCLUSION: NK cells from mPE display pro-angiogenic features and mPE-CM was able to polarize healthy PB NK cells in vitro towards the pro-angiogenic CD56brightCD16- NK subset, suggesting a role of tumor microenvironment in the shaping towards pro-angiogenic and pro-tumor polarized innate immune cells.