In depth characterization of human CMV-driven adaptive NK cells through mass spectrometry and flow cytometry analyses

Human Natural killer (NK) cells are a population of innate lymphocytes, usually short-lived and rapidly responding against viral infections and tumors. However, upon cytomegalovirus (CMV) infection, NK cells reveal unexpected adaptive traits typical of T cells, such as enhanced effector function and longevity. CMV-driven adaptive NKs show high NKG2C (i.e. a HLA-E-specific activating receptor) levels, a mature phenotype (CD57+KIR+NKG2A-), epigenetic modifications and could display anti-tumor properties. In view of their longevity and specialized functional features, adaptive NK cells are suitable candidates in cellular immunotherapy. 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 NK cell subset, 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 and 263 proteins that 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 LILRB1 and lower expression of CD161. On the other hand, our proteome analysis confirmed at the protein level data from transcriptomic studies, such as higher expression of the antiapoptotic protein Bcl-2, possibly responsible for adaptive NK cell longevity, and of the signaling protein CD3e. Based on the hints from mass spectrometry data, we further investigated the most significant differentially expressed proteins through intracellular flow cytometry to validate our results in a larger donors cohort and to explore in detail NK cell subsets not included in the proteomic analysis. We could thus reveal that high Bcl-2 expression mainly characterizes NKG2C+ NK cells including not only adaptive NKG2C+CD57+ but also the less differentiated NKG2C+CD57- NK cells. In addition, we confirmed that cytoplasmic CD3e expression marks adaptive NKG2C+CD57+ NK cells highlighting the phenotypic proximity that this NK cell subset displays with T lymphocytes. Thus, mass spectrometry proteome profiling of adaptive NK cells, combined to cytofluorimetric analyses, 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.