Proteomic characterization of human CMV-driven adaptive NK cells

Human Natural killer (NK) cells are a lymphocyte population of innate immunity, usually short-living and capable of providing rapid responses against viral infections and tumors without prior sensitization. However, upon cytomegalovirus (CMV) infection, human NK cells reveal unexpected adaptive traits, sharing features typical of T cells, such as enhanced effector function and long-term survival in response to a viral challenge (1). These peculiar CMV-driven NK cells, indicated as adaptive or memory NK cells, can be found to varying degrees in both healthy CMV-seropositive individuals and transplanted patients undergoing CMV infection/reactivation (2). CMV-driven adaptive NK cells are characterized by the expression of the specific HLA-E activating receptor NKG2C, a mature phenotype (CD57+KIR+NKG2A-), epigenetic modifications and could display anti-tumor properties (3). In view of their longevity and specialized functional features, adaptive NK cells represent suitable candidates to be exploited in cellular immunotherapy against cancer and viral infections. Adaptive NK cells have been investigated so far mainly through cytofluorimetric approaches and transcriptomic studies. Thus, to better characterize and possibly identify novel functional pathways associated to this CMV-driven NK cell population, we have set up a mass spectrometry proteome analysis on FACS-sorted adaptive NK cell subsets isolated from six CMV-seropositive healthy donors. In particular we compared the proteomic signature of adaptive NKG2C+CD57+ NK cells with conventional NKG2C-CD57- NK cells, i.e. more immature NK cells lacking CMV-driven imprinting. We identified 4618 proteins from NK cell subsets lysates, representing the largest protein catalogue reported so far for NK cells, and observed a sharply different proteome profile in adaptive compared to conventional NK cells. Indeed, we found 165 proteins that were significantly more expressed in adaptive NKG2C+ CD57+ NK cells as compared to conventional NKG2C-CD57- NK cells, with 75 proteins showing a remarkably high fold change. Interestingly, 263 proteins were significantly down-regulated on NKG2C+ CD57+ NK cells as compared to NKG2C-CD57- NK cells. The different proteome profile shown by adaptive NK cells confirmed previous data obtained by cytofluorimetric studies, such as higher expression of the inhibitory receptor LILRB1 and lower expression of CD161 characterizing adaptive NK cells. On the other hand, our proteome analysis confirmed the expression of molecules at the protein level that had been described only by transcriptomic studies, such as higher expression levels of the antiapoptotic protein Bcl-2 that is possibly responsible for long-term persistence of adaptive NK cells. Interestingly, several differentially expressed proteins have not been described in NK cell biology so far. Indeed, further analyses through the “Human Base tissue-specific network-based functional interpretation of genes and gene sets” tool revealed a significant enrichment in adaptive NK cells, compared to conventional NK cells, of proteins belonging to pathways related to regulating cell activation and proliferation (e.g. CCL5, LGALS3, LILRB1, CD3E, MNDA), involved in endosomal trafficking and cytoskeletal organization (e.g. CHMP2B, ARFGEF1, ATL1), regulating RNA transport from the nucleus to cytoplasm (e.g. FYTTD1, ATXN1), regulating viral processes/responses (e.g. TRIM14, OASL2, RELA), involved in autophagy (e.g. ATG13, GOLGA2) and also in ribosome biogenesis (e.g. ZNHIT6,EMG1). Thus, mass spectrometry proteome profiling of adaptive NK cells, combined to cytofluorimetric analyses and compared to transcriptomic data, has provided further insights in their biology and revealed novel features that could be relevant also to better harness this NK cell subset for immunotherapeutic purposes.