Background
Variants in the extracellular Ig-like domain of the CD101 gene are associated with increased HIV acquisition risk, but the biological mechanism is unknown. CD101 inhibits T cell activation and is mostly expressed on antigen-presenting cells (APCs) and T cells. Here, we tested the hypothesis that CD101 regulates inflammatory homeostasis in immune cells. We also explored the impact of CD101 genetic variation on this regulation and the risk of HIV-1 infection.
Methods
We evaluated the effect of Toll-like receptor agonists (TLR1 to TLR9) on CD101 mRNA levels in peripheral blood mononuclear cells (PBMC) and purified monocytes. We used flow cytometry to measure CD101 expression on PBMC subsets. We compared individuals with (n=14) and without (n=5) candidate CD101 variants to assess the effect of CD101 variation on PBMC responses to TLR8 stimulation. We also used CRISPR to knock out CD101 in primary CD4+ T cells to test its impact on HIV infectivity.
Results
TLR2, 4, 5, 7 and 8 stimulation reduced CD101 mRNA expression in PBMC and monocytes. The strongest effect was from HIV ssRNA (a TLR8 agonist) (-77.0% in PBMC, -90.9% in monocytes; n=4). Challenge with infectious HIV also reduced CD101 mRNA (-49.1%; n=4). CD25+ CD127low Tregs and HLA-DR+ APCs were most sensitive to CD101 downregulation upon TLR8 stimulation (-41.4% and -63.0%, respectively; n=4). However, CD101 downregulation was much weaker if Tregs were purified before stimulation (-8.1%; n=5). For PBMC genotyped for CD101, following TLR8 stimulation, we observed significant mRNA increases for IFNβ1 (mean 11.1-fold), IL-6 (644.4-fold), and IFIT1 (83.8-fold), and a significant decrease for CD101 (74.1% +/- 22.5%; n=17), in 17 out of 19 donors. Cytokine increases correlated strongly with CD101 reduction (e.g. for IL-6/CD101 Spearman’s r=0.67, p=0.0017; n=19). In an exploratory analysis, CD101 mRNA reductions upon TLR8 stimulation were greater in cells from people with a CD101 variant than reference (β=-32 vs. β=-19, interaction p=0.006). Lastly, HIV replication in CD101 CRISPR-ablated CD4+ T cells was comparable to mock controls.
Conclusions
CD101 downregulation by HIV occurs primarily in monocytes and Tregs, is influenced by cell-to-cell interactions and CD101 genotype, and inversely correlates with the cells’ proinflammatory response. Our results suggest that CD101 may affect HIV infection risk indirectly, via effects on inflammation, rather than directly on HIV replication. Candidate CD101 alleles may accentuate this effect.
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