Influence of NKG2C Genotypes on HIV Susceptibility and Viral Load Set Point

Abstract

NKG2C is an activating NK cell receptor encoded by a gene having an unexpressed deletion variant. Cytomegalovirus (CMV) infection expands a population of NKG2C⁺ NK cells with adaptive-like properties. Previous reports found that carriage of the deleted NKG2C^- variant was more frequent in people living with HIV (PLWH) than in HIV^- controls unexposed to HIV. The frequency of NKG2C⁺ NK cells positively correlated with HIV viral load (VL) in some studies and negatively correlated with VL in others. Here, we investigated the link between NKG2C genotype and HIV susceptibility and VL set point in PLWH. NKG2C genotyping was performed on 434 PLWH and 157 HIV-exposed seronegative (HESN) subjects. Comparison of the distributions of the three possible NKG2C genotypes in these populations revealed that the frequencies of NKG2C^+/+ and NKG2C^+/- carriers did not differ significantly between PLWH and HESN subjects, while that of NKG2C^-/- carriers was higher in PLWH than in HESN subjects, in which none were found (P = 0.03, χ² test). We were unable to replicate that carriage of at least 1 NKG2C^- allele was more frequent in PLWH. Information on the pretreatment VL set point was available for 160 NKG2C^+/+, 83 NKG2C^+/-, and 6 NKG2C^-/- PLWH. HIV VL set points were similar between NKG2C genotypes. The frequency of NKG2C⁺ CD3^- CD14^- CD19^- CD56^dim NK cells and the mean fluorescence intensity (MFI) of NKG2C expression on NK cells were higher on cells from CMV⁺ PLWH who carried 2, versus 1, NKG2C⁺ alleles. We observed no correlations between VL set point and either the frequency or the MFI of NKG2C expression. IMPORTANCE We compared NKG2C allele and genotype distributions in subjects who remained HIV uninfected despite multiple HIV exposures (HESN subjects) with those in the group PLWH. This allowed us to determine whether NKG2C genotype influenced susceptibility to HIV infection. The absence of the NKG2C^-/- genotype among HESN subjects but not PLWH suggested that carriage of this genotype was associated with HIV susceptibility. We calculated the VL set point in a subset of 252 NKG2C-genotyped PLWH. We observed no between-group differences in the VL set point in carriers of the three possible NKG2C genotypes. No significant correlations were seen between the frequency or MFI of NKG2C expression on NK cells and VL set point in cytomegalovirus-coinfected PLWH. These findings suggested that adaptive NK cells played no role in establishing the in VL set point, a parameter that is a predictor of the rate of treatment-naive HIV disease progression.

Keywords: HIV exposed seronegative; HIV load set point; adaptive NK cells; human immunodeficiency virus; injection drug users; men who have sex with men; people living with HIV.

FIG 1

Evaluation of the frequency of NKG2C⁺ NK cells and mean fluorescence intensity (MFI) of NKG2C expression. (A) Shown is the gating strategy used to detect the frequency and MFI of NKG2C expression. Peripheral blood mononuclear cells were stained for viability and cell surface CD3, CD56, CD14, CD19, and NKG2C. CD3⁻ CD14⁻ CD19⁻ CD56^dim NK cells were gated on from the live singlet lymphocyte gate. From these, we determined the frequencies of NKG2C⁺ CD56^dim NK cells and MFI of NKG2C expression on NK cells. The y axes show the frequency (B), MFI (C), and fold change over background in the MFI (D) of NKG2C expression on CD56^dim NK cells from CMV⁺ people living with HIV (HIV⁺ CMV⁺) carrying the NKG2C^+/+ (n = 32), NKG2C^+/− (n = 19), and NKG2C^−/− (n = 6) genotypes and from CMV-monoinfected (HIV⁻ CMV⁺) individuals carrying the NKG2C^+/+ (n = 43) and NKG2C^+/− (n = 18) genotypes. Each point represents a single individual. Bar graph heights and error bars represent medians and interquartile ranges for the group. FSC-A, forward scatter area; SSC-A, side scatter area; LD, live/dead; FSC-H, forward scatter height; Pk-w, P value for the Kruskal-Wallis test used to analyze the significance of differences between groups: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 2

Log₁₀ viral load (VL) set points in people living with HIV (PLWH) carriers of the NKG2C^+/+, NKG2C^+/−, and NKG2C^−/− genotypes. Shown are violin plots of the median and interquartile range of the treatment-naive log₁₀ VL set point in each NKG2C genotype group. The number of subjects included in each group is shown above each data set. A Kruskal-Wallis test was used to assess the significance of between-group differences in log₁₀ VL set point.

FIG 3

Correlation between log₁₀ VL set point and frequency of NKG2C⁺ NK cells, mean fluorescence intensity (MFI) of NKG2C expression and fold change in NKG2C MFI over background in cells from HIV⁺ CMV⁺ NKG2C^+/+, NKG2C^+/−, and NKG2C^−/− carriers. Correlations between the frequency (A to D) MFI (E to H) of NKG2C expression and fold change in NKG2C MFI over background (I to K) on NK cells from CMV⁺ PLWH with log₁₀ VL for carriers of all NKG2C genotypes tested (A, E, and I) and stratified by NKG2C^+/+ (B, F, and J), NKG2C^+/− (C, G, and K), and NKG2C^−/− (D and H) genotypes. The number of subjects tested, the correlation coefficients (r), and the P values for each correlation are shown in the top left corner of the graphs.

FIG 4

Correlation between log₁₀ (VL) viral load set point and frequency of NKG2C⁺ CD57⁺ NK cells from CMV⁺ PLWH carrying the three possible NKG2C genotypes. (A) From the live singlet lymphocyte gate, CD56^dim CD3⁻ CD14⁻ CD19⁻ NK cells were gated on. From these NKG2C⁺ CD57⁺ NK cells were gated onto assess the frequency of these cells among CD56^dim NK cells. Correlations between the frequency of NKG2C⁺ CD57⁺ (B to E) CD56^dim NK cells with log₁₀ viral load set point from for CMV⁺ PLWH carrying all NKG2C (B), NKG2C^+/+ (C), NKG2C^+/− (D), and NKG2C^−/− (E) genotypes. The number of subjects tested, the correlation coefficients (r), and the P values for each correlation are shown in the top left corner of the graphs.

FIG 5

Correlation between log₁₀ VL set point and frequency of NKG2A⁺ NKG2C⁻ CD56^dim NK cells from CMV⁺ PLWH carrying the three possible NKG2C genotypes. Correlations between the frequency of NKG2C⁺ CD56^dim and NKG2A⁺ CD56^dim NK cells from CMV⁺ PLWH for carriers of all NKG2C (A), NKG2C^+/+ (B), and NKG2C^+/− (C) genotypes. The number of subjects tested, the correlation coefficients (r) and the P values for each correlation are shown in the inset at the top left corner of each graph. (D) Shown is the strategy used to gate on CD56^dim CD3⁻ CD14⁻ CD19⁻ NK cells, from which NKG2A⁺ NKG2C⁻ cells were gated onto assess their frequency among CD56^dim NK cells. Correlations between the frequency of CD56^dim NKG2A⁺ NKG2C⁻ NK cells with log₁₀ VL set point from for CMV⁺ PLWH carrying all NKG2C (E), NKG2C^+/+ (F), NKG2C^+/− (G), and NKG2C^−/− (H) genotypes. The numbers of subjects tested, the correlation coefficients (r), and the P values for each correlation are shown in the top left corner of the graphs.