Human immunodeficiency virus type 1 infection is associated with increased NK cell polyfunctionality and higher levels of KIR3DL1+ NK cells in ugandans carrying the HLA-B Bw4 motif

Abstract

Natural killer (NK) cells are important innate effector cells controlled by an array of activating and inhibitory receptors. Some alleles of the inhibitory killer-cell immunoglobulin-like receptor KIR3DL1 in combination with its HLA class I ligand Bw4 have been genetically associated with slower HIV-1 disease progression. Here, we observed that the presence of HLA-B Bw4 was associated with elevated frequencies of KIR3DL1(+) CD56(dim) NK cells in chronically HIV-1-infected individuals from the rural district of Kayunga, Uganda. In contrast, levels of KIR2DL1(+) CD56(dim) NK cells were decreased, and levels of KIR2DL3(+) CD56(dim) NK cells were unchanged in infected subjects carrying their respective HLA-C ligands. Furthermore, the size of the KIR3DL1(+) NK cell subset correlated directly with viral load, and this effect occurred only in HLA-B Bw4(+) patients, suggesting that these cells expand in response to viral replication but may have relatively poor antiviral capacity. In contrast, no association with viral load was present for KIR2DL1(+) and KIR2DL3(+) NK cells. Interestingly, chronic HIV-1 infection was associated with an increased polyfunctional response in the NK cell compartment, and, upon further investigation, KIR3DL1(+) CD56(dim) NK cells exhibited a significantly increased functional response in the patients carrying HLA-B Bw4. These results indicate that chronic HIV-1 infection is associated with increased NK cell polyfunctionality and elevated levels of KIR3DL1(+) NK cells in Ugandans carrying the HLA-B Bw4 motif.

Fig. 1.

HLA-B Bw4 is associated with increased KIR3DL1⁺ NK cell frequency, while HLA-C group C2 is associated with decreased KIR2DL1⁺ NK cell frequency. Frozen PBMCs were thawed and stained using several multicolor flow panels to assess KIR phenotype. Plots are pregated on CD56^dim CD3⁻ CD14⁻ CD19⁻ live lymphocytes. (A) Vertical scatter plot comparing frequency of CD56^dim NK cells expressing KIR2DL1 (clone 143211) in HLA-C group C2-positive subjects with (n = 37) or without (n = 28) HIV-1 infection. (B) Vertical scatter plot comparing frequency of CD56^dim NK cells expressing KIR2DL1 in HLA-C group C2-negative subjects with (n = 11) or without (n = 1) HIV-1 infection. (C) Vertical scatter plot comparing frequency of CD56^dim NK cells expressing KIR2DL3 (clone 180071 and DX27) in HLA-C C1-positive subjects with (n = 32) or without (n = 22) HIV-1 infection. (D) Vertical scatter plot comparing frequency of CD56^dim NK cells expressing KIR2DL3 in HLA-C C1-negative subjects with (n = 16) or without (n = 4) HIV-1 infection. (E) Vertical scatter plot comparing frequency of CD56^dim NK cells expressing KIR3DL1 in HLA-B Bw4-positive subjects with (n = 26) or without (n = 21) HIV-1 infection. (F) Vertical scatter plot comparing frequency of CD56^dim NK cells expressing KIR3DL1 in HLA-B Bw4-negative subjects with (n = 20) or without (n = 5) HIV-1 infection. NS, nonsignificant.

Fig. 2.

Association between HIV-1 load and frequency of KIR⁺ NK cells in carriers with or without their corresponding HLA-ligand. (A and D) Spearman rank correlation between HIV-1 load and percentage of CD56^dim NK cell KIR3DL1 expression in Bw4-80I carriers and noncarriers, respectively. (B and E) Spearman rank correlation between HIV-1 load and percentage of CD56^dim NK cell KIR2DL3 expression in HLA-C (C1) carriers and noncarriers, respectively. (C and F) Spearman rank correlation between HIV-1 load and percentage of CD56^dim NK cell KIR2DL1 expression in HLA-C (C2) carriers and noncarriers, respectively.

Fig. 3.

Chronic HIV-1 infection is associated with increased polyfunctionality in NK cells. Frozen PBMCs were thawed and stimulated in the presence or absence of K562 cells at an effector/target ratio of 5:1, and functional responses were detected using multiparameter flow cytometry. (A) Representative pseudo-color histogram on CD56^dim NK cell discrimination. Combinations of IFN-γ, MIP-1β, and CD107a coexpression in CD56^dim NK cells after an 18-h assay with or without K562 cell stimulation are shown. Fluorescence-activated cell sorting graphs represent unstimulated (blue) density plots overlaid with K562-stimulated (red) dot plots. Boolean analysis of the three functions was performed, and unstimulated function was subtracted from the K562-stimulated condition. (B) Vertical scatter plot comparing frequency of CD56^dim NK cells coexpressing IFN-γ, MIP-1β, and CD107a in people with (n = 52) or without (n = 21) HIV-1 infection. (C) SPICE software was used to graphically present the overnight responses against K562 (with unstimulated responses subtracted out) for HIV-1-infected (n = 52) and uninfected (n = 21) individuals. Pie chart shows the distribution of a single function (blue), two functions (in green, light blue, and red), and triple functions (yellow) corresponding to the Boolean subsets in the bar graph below. Pie arcs show the relative amount of each individual function: IFN-γ (yellow), MIP-1β (green), and CD107a (red). Bar chart shows the possible combinations of three functions on the x axis and the percentage of distinct functional populations within the CD56^dim NK cells on the y axis.

Fig. 4.

KIR3DL1⁺ CD56^dim NK cells have a higher functionality in HLA-B Bw4⁺ individuals than participants homozygous for HLA-B Bw6. Frozen PBMCs were thawed and stimulated in the presence or absence of K562 cells at an effector/target ratio of 5:1, and functional responses were detected using multiparameter flow cytometry. (A) Representative pseudo-color histogram of CD56^dim NK cell discrimination. IFN-γ and MIP-1β on KIR3DL1⁺ CD56^dim NK cells after an 18-h assay with or without K562 cell stimulation are shown. Fluorescence-activated cell sorting graphs represent unstimulated (blue) density plots overlaid with K562-stimulated (red) dot plots. (B) Vertical scatter plot comparing coexpression of IFN-γ and MIP-1β both in KIR3DL1-expressing (red) and -nonexpressing (blue) CD56^dim NK cells. Study participants with the HLA-B Bw4 motif (squares) are compared to individuals homozygous for HLA-B Bw6 (triangles). (C) Vertical scatter plot comparing MIP-1β only production in KIR3DL1-expressing (red) and -nonexpressing (blue) CD56^dim NK cells. Study participants with the HLA-B Bw4 motif (squares) are compared to individuals homozygous for HLA-B Bw6 (triangles). NS, nonsignificant.