Single-cell cloning of untreated HIV-specific natural killer cells in humans

Abstract

Abstract Beyond their ability to eliminate infected cells without the need for prior sensitization, natural killer (NK) cells have been shown to exhibit adaptive immune functions. In particular, our lab demonstrated that antigen-specific memory NK cells responses against SIV can be induced by infection and vaccination in non-human primates. These data suggested that a subset of NK cells in humans may also mediate immunological memory responses to HIV. To study human HIV-antigen-specific NK cells in more detail, we adapted limiting dilution assays to clonally expand individual peripheral blood NK cells (NKCL) from untreated HIV-infected individuals. Using a calcein acetoxymethyl ester cytotoxicity assay, the ability of each NKCL to lyse MHC-devoid K562 cell lines or autologous B-LCLs pulsed with a pool of MOG- (human self-peptide), HIV Gag- or HIV Env-derived overlapping peptides was assessed. As expected, all tested NKCL could potently lyse K562 cells (median=56% killing) but did not kill B-LCL pulsed with the MOG control. Strikingly, 37.5% of NKCL displayed anti-HIV Gag cytotoxic activity up to 31% specific lysis (range: 1%–31%), while 25% of NKCL displayed anti-HIV Env cytotoxic activity up to 72% specific lysis (range: 1%–72%). Furthermore, pre-treatment with an anti-NKG2C blocking antibody decreased the specific lysis, thereby confirming the importance of NKG2C receptor in the function of antigen-specific NK cells. Thus, we were able to isolate single NK cells displaying potent HIV-specific killing, within the range of robust cytotoxicity normally found against tumor cells. Together these data suggest that HIV-specific responses mediated by NK cells exist in humans and may have the potential to be harnessed for therapeutic modalities.