Natural Killer Cell Receptors and Ligands Are Associated With Markers of HIV-1 Persistence in Chronically Infected ART Suppressed Patients

Abstract

The latent HIV-1 reservoir represents a major barrier to achieving a long-term antiretroviral therapy (ART)-free remission or cure for HIV-1. Natural Killer (NK) cells are innate immune cells that play a critical role in controlling viral infections and have been shown to be involved in preventing HIV-1 infection and, in those who are infected, delaying time to progression to AIDS. However, their role in limiting HIV-1 persistence on long term ART is still uncharacterized. To identify associations between markers of HIV-1 persistence and the NK cell receptor-ligand repertoire, we used twin mass cytometry panels to characterize the peripheral blood NK receptor-ligand repertoire in individuals with long-term antiretroviral suppression enrolled in the AIDS Clinical Trial Group A5321 study. At the time of testing, participants had been on ART for a median of 7 years, with virological suppression <50 copies/mL since at most 48 weeks on ART. We found that the NK cell receptor and ligand repertoires did not change across three longitudinal samples over one year-a median of 25 weeks and 50 weeks after the initial sampling. To determine the features of the receptor-ligand repertoire that associate with markers of HIV-1 persistence, we performed a LASSO normalized regression. This analysis revealed that the NK cell ligands CD58, HLA-B, and CRACC, as well as the killer cell immunoglobulin-like receptors (KIRs) KIR2DL1, KIR2DL3, and KIR2DS4 were robustly predictive of markers of HIV-1 persistence, as measured by total HIV-1 cell-associated DNA, HIV-1 cell-associated RNA, and single copy HIV-RNA assays. To characterize the roles of cell populations defined by multiple markers, we augmented the LASSO analysis with FlowSOM clustering. This analysis found that a less mature NK cell phenotype (CD16⁺CD56^dimCD57^-LILRB1^-NKG2C^-) was associated with lower HIV-1 cell associated DNA. Finally, we found that surface expression of HLA-Bw6 measured by CyTOF was associated with lower HIV-1 persistence. Genetic analysis revealed that this was driven by lower HIV-1 persistence in HLA-Bw4/6 heterozygotes. These findings suggest that there may be a role for NK cells in controlling HIV-1 persistence in individuals on long-term ART, which must be corroborated by future studies.

Keywords: HIV cure; HIV latency; human immunodeficiency virus (HIV); natural killer cell receptor ligands; natural killer cells.

Figure 1

Description of cohort and workflow. We obtained samples from 50 individuals chronically infected with HIV-1 and on long term ART with successful viral suppression, at three timepoints. PBMCs were purified, and cryogenically stored. Samples were then thawed and divided, with a portion of each sample stained with our NK ligand panel of antibodies, and the rest used to purify NK cells, which were then stained with our NK receptor panel of antibodies. Following staining, samples were acquired by CyTOF. Our CyTOF data were analyzed by UMAP, MDS, and FlowSOM. We also obtained virological and immunological measurements from each participant at the first timepoint. These were combined with our CyTOF measurements for analysis by LASSO. Created with Biorender.com.

Figure 2

Deep characterization of NK receptor and ligand expression in patients on long term ART reveals no longitudinal changes. (A) UMAP embeddings of gated NK cells based on expression of NK receptors, and gated monocytes, CD4⁺ T-cells, and CD8⁺ T-cells based on expression of NK ligands. Colored by week of sample. (B, C) MDS embedding, where each point represents the mean expression of NK markers on all NK cells and ligand markers on each population. After filtering for low cell count files, n = 50 participants with n = 142 total samples. In (B) points are colored by timepoint. In (C) shaded convex hulls connect the points of each participant. Each axis is scaled by the percent variance explained by that axis.

Figure 3

LASSO normalized regression reveals novel associations between clinical variables, NK receptors and ligands, and the HIV-1 latent reservoir. (A–C) Plots showing the LASSO coefficient estimates for the optimal models predicting (A) CA-DNA (n = 47), (B) CA-RNA (n = 46), or (C) SCA (n = 45). (D) Boxplot showing the association between KIR2DS4 genotype and CA-DNA.

Figure 4

FlowSOM and LASSO analysis reveals an association between immature NK cells and reduced CA-DNA. (A) Estimated LASSO coefficients for clusters with large coefficients included in the least stringent model within one standard error of the zero coefficient model (n = 47). (B) Scatterplot showing the association between CD8 cluster 2 and CA-DNA. (C) Scatterplot showing the association between NK cluster 2 and CA-DNA. (D) Histograms showing the phenotype of NK cluster 2 compared to all other NK cells. (E) Scatterplot showing the association between the frequency of gated CD16⁺ CD56^dim CD57^- LILRB1^- NKG2C^- NK cells and CA-DNA. (F) UMAP showing the location of cluster 2 on the total NK cell UMAP, with cells not in cluster 2 shown in grey and the density of cluster 2 shown in color. (G) Expression of the markers used to define cluster 2 shown on the UMAP of total NK cells.

Figure 5

UMAP embeddings and genetic analysis reveal an association between HLA-Bw4/6 phenotype/genotype and the latent HIV-1 reservoir. (A) UMAP embedding of CD4+ T-cells colored by participant CA-DNA at entry. (B) UMAP embedding of CD4+ T-cells colored by HLA-Bw6 expression (C–E) Boxplots showing (C) CA-DNA, (D) CA-RNA, and (E) SCA in participants with low vs. high HLA-Bw6 expression as measured by CyTOF. Dashed horizontal lines indicate the limit of detection for each assay. Points are colored by Bw6 hi vs. low as determined in Supplemental Figure 11 (F–H) Boxplots showing (F) CA-DNA, (G) CA-RNA, and (H) SCA in participants with Bw4/4, Bw4/6, and Bw6/6 genotypes. Dashed horizontal lines indicate the limit of detection for each assay. Points are colored by Bw6 hi vs. low as measured by CyTOF.