Beyond their ability to eliminate infected cells without the need for prior sensitization, murine, non-human primate and human natural killer (NK) cells have been shown to mediate virus-specific recall responses. However, whether NK cells can mediate HIV antigen-specific immunological memory in humans remains to be demonstrated.
Using calcein acetoxymethyl ester- or flow cytometry-based cytotoxicity assays, we tested the ability of bulk and clonally expanded individual NK cells to lyse MHC-devoid K562 cell lines or autologous B-LCLs left unpulsed or pulsed with pools of self-antigen-, CMV-, CMV/EBV/influenza (CEF)-, HIV Gag- or HIV Env-derived overlapping peptides. Study participants included HIV-negative healthy donors (HD; n=6) and people living with HIV (PLWH) virally suppressed on cART (ART; n=14), untreated viremic (UT; n=14) or elite controllers (EC; n=4). Phenotypic analysis was performed using up to 28-color flow cytometry on a BD FACSymphony instrument.
Significant anti-HIV Gag activity (range: 8%-50% killing) by bulk NK cells was exclusively detected in half of all PLWH, while killing of BCLs pulsed with the CEF peptide pool, or killing of MHC-devoid K562 cell lines, was comparable between PLWH and HD. NK cells from half of EC had detectable HIV Gag-specific cytotoxic activity and displayed the most robust responses. Strikingly, 35% of all tested NKCL (n=165) generated from 22 PLWH (59% NKCL from 8 ART, 18% NKCL from 14 UT) showed positive responses to HIV (at least twice above killing of unstimulated and above killing of self-peptide-pulsed B-LCLs). Reactive NKCL displayed anti-HIV Gag cytotoxic activity up to 43% specific lysis and anti-HIV Env cytotoxic activity up to 87% specific lysis, within the range of robust cytotoxicity normally found against tumor cells. Phenotypic analysis indicated antigen-specific memory was associated with increased NKG2C and CD57 expression. Accordingly, NKG2C receptor blockade and pulsing with single HIV-derived peptides that bind HLA-E indicated memory NK cell responses likely depend on an HLA-E-dependent recognition mechanism.
Collectively, our work presents the first mechanistic evidence for HIV-specific memory NK cells induced by HIV infection in humans. These data suggest that HIV-specific responses mediated by NK cells may have the potential to be harnessed for curative or other therapeutic interventions.