Single-cell cloning of NK cells from rhesus macaques: a new methodology for in-depth analysis of MHC-E-dependent adaptive functions

Abstract Natural killer (NK) cells provide rapid and robust responses against a wide range of viral infections. More recently, multiple studies have shown that subsets of NK cells are also capable of antigen-specific recall to eliminate infected cells. In humans and nonhuman primates (NHP), antigen-specific memory NK cell responses can at least be partially delineated by expression of NKG2C, a ligand for MHC-E. Due to MHC-E limited polymorphism, MHC-E-presented viral antigens may induce universally antigen-specific memory NK cells. To study antigen-specific NK cells in NHP, we developed a new in vitromethod to clonally expand single rhesus macaque NK cells (rhNKCL) from blood and spleen. To define the effector functions of rhNKCL, we used a calcein acetoxymethyl ester (CAM) cytotoxicity assay and intracellular staining assay in combination with K562 cell lines or autologous BLCL as targets. We also identified, by in silicomethodology, peptides from influenza, SIV, and SARS-CoV-2 viruses predicted to display strong binding to MHC-E. Our procedure showed for the first-time clonal expansion of single rhesus macaque NK cells. We observed a conserved NHP NK cell phenotype of CD3 −NKG2A/C +CD16 +CD56 +following expansion with expression of additional NK cell surface receptors CD8, NKp80, and NKG2D. Upon stimulation with K562 cells, rhNKCL effector functions (cytotoxicity, CD107a, IFNγ, TNFα) were like primary NK cells. Altogether, this new method of single-cell cloning in rhesus macaques could provide key information about antigen-specific NK cells, including epigenetic, metabolic, transcriptional, and phenotypic profiles, which could be exploited to further enhance antiviral NK cell activity in future vaccine strategies or cell therapies. Supported by grants from NIH (R01 AI158516, R01 DK130472, R01 AI161010, NIH-funded BEAT-HIV Martin Delaney Collaboratory to cure HIV-1 infection by Combination Immunotherapy UM1 AI164570).