Background
The HIV-1 Nef has been shown to alter MHC-1 presentation on infected T cells, and infection of cell lines suggest upregulation of non-canonical HLA-E may be advantageous for Natural Killer (NK) cell evasion. Conflicting clinical reports leave the role of NK cells in infection control unclear. True mechanistic understanding of HIV replication and control in primary T cells has been challenging due to technical limitations efficiently genetically modifying and infecting true primary cells ex vivo.
Methods
We recently developed a strategy for efficient knock-out (~99%) and knock-in (~80% of alleles) in purified human peripheral blood CD4+ T cells, and supports in vitro infection challenge with both CCR5-tropic and CXCR4-tropic HIV-1. We therefore generated HLA-E knock-out primary T cells, challenged them with NL4-3, and quantified spreading infection with or without donor-matched NK cells from 5 healthy human blood donors.
Results
HLA-E KO caused loss of cell surface HLA-E on T cells from all donors, without any alteration of CD4, CXCR4, or CCR5 protein levels. HLA-E knock-out cells from all 5 donors showed increased control of virus replication during co-culture, suggesting that NL4-3 uses HLA-E to inhibit NK-mediated killing of infected cells. Control knock-out cells demonstrated a slight drop (average ~3-fold) in infection during T:NK co-culture, while HLA-E knock-out cells demonstrated a 28-fold average drop in supernatant p24 concentration, with one donor showing as much as an 82-fold reduction in p24 concentration. The level of virus control directly correlated with the ratio of HLA-E cognate inhibitory/activating receptors (NKG2A/NKG2C) present on the NK cell surface in all donors except one donor containing a naturally occurring homozygous deletion in NKG2C. To identify additional pathways that facilitate HLA-mediated evasion in the absence of NKG2C, we did single-cell RNA seq to identify NK cell genes upregulated during infection co-culture after NKG2C knock-out in NK cells.
Conclusions
These data highlight that the ratio of NKG2A/NKG2C on individual patient cells may influence the level of HIV HLA-E mediated control, and redundant pathways exist for recognition of HIV infection in the absence of NKG2C. These data may explain discrepancies in previous studies on the role of NKG2C on viral load. Importantly, these data highlight a role for HLA-E in HIV-1 immune evasion, and highlight the potential of novel gene editing and infection models in true primary immune cells.