Receptor-ligand requirements for increased NK cell polyfunctional potential in slow progressors infected with HIV-1 coexpressing KIR3DL1*h/*y and HLA-B*57

Abstract

Carriage of the natural killer (NK) receptor genotype KIR3DL1*h/*y with its HLA-B*57 ligand (*h/*y+B*57) is associated with slow time to AIDS and low viral load (VL). To provide a functional basis for these epidemiological observations, we assessed whether HIV-1-infected slow progressors (SP) carrying the *h/*y+B*57 compound genotype would have increased NK cell polyfunctional potential in comparison to SP with other killer immunoglobulin-like receptor (KIR)/HLA compound genotypes and whether this enhanced polyfunctionality was dependent upon the coexpression of both KIR3DL1*h/*y and HLA-B*57. The functional potential of NK cells was investigated by stimulating peripheral blood mononuclear cells with HLA-devoid targets or single HLA transfectants. Multiparametric flow cytometry was used to detect NK cells with seven functional profiles representing all permutations of CD107a expression and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion. NK cells from individuals carrying KIR3DL1 receptor-HLA-Bw4 ligand pairs had greater trifunctional responses than those from KIR3DL1 homozygotes (hmz), who were Bw6 homozygotes. NK cells from subjects carrying the *h/*y+B*57 genotypes exhibited the highest trifunctional potential, and this was dependent on cocarriage of the NK receptor and its ligand. Trifunctional cells secreted more of each function tested on a per-cell basis than each corresponding monofunctional NK subset. Although VL influenced NK functionality, individuals with defined KIR/HLA genotypes exhibited differences in NK cell polyfunctionality that could not be accounted for by VL alone. The protective effect of HLA-B*57 on slow progression to AIDS and low VL may be mediated through its interaction with KIR3DL1 alleles to educate NK cells for potent activity upon stimulation.

Fig. 1.

Stimulated NK cells from KIR3DL1 (3DL1) homozygous HIV-infected slow progressors (SP) have higher trifunctional activity when from carriers of an HLA-Bw4 allele than from Bw6 homozygotes (hmz). (A) Comparisons of percent contributions of seven functional profiles to the total NK response by NK cells from 3DL1 hmz SP stimulated with K562. Results for 40 SP carrying at least one Bw4 allele were compared to those for 7 Bw6 hmz. Dots below the x axis refer to the presence of a measured functional marker (CD107a, IFN-γ, and TNF-α). Bar heights refer to the median percent contribution of that functional subset to the total NK response for the group. Error bars refer to the upper range for the group. Significant between-group differences are shown with an asterisk over the line linking the two bars representing the groups being compared. (B) Correlation of results reported as the percent contribution of the trifunctional (CD107a⁺ IFN-γ⁺ TNF-α⁺) NK subset to the total NK response with results reported as the frequency of this functional NK subset for the 47 SP subjects in panel A. A Spearman's correlation test was used to test the significance of these two ways of reporting results. (C) Comparisons of the mean fluorescence intensities (MFI) of IFN-γ secretion are plotted on the y axis for trifunctional (CD107a⁺ IFN-γ⁺ TNF-α⁺), bifunctional (CD107a⁺ IFN-γ⁺ and IFN-γ⁺ TNF-α⁺), and monofunctional IFN-γ⁺ NK cells. Panel D shows the MFI of TNF-α secretion plotted on the y axis for trifunctional (CD107a⁺ IFN-γ⁺ TNF-α⁺), bifunctional (CD107a⁺ TNF-α⁺ and IFN-γ⁺ TNF-α⁺), and monofunctional TNF-α⁺ NK cells. Panel E shows the MFI of CD107a expression plotted on the y axis for trifunctional (CD107a⁺ IFN-γ⁺ TNF-α⁺), bifunctional (CD107a⁺ TNF-α⁺ and CD107a⁺ IFN-γ⁺), and monofunctional CD107a⁺ NK cells. For panels C, D, and E, the line through the scatter plots represents the median. Mann-Whitney U tests were used to test the significance of between-group comparisons. P values for these are shown above the lines linking two bars representing functional subsets.

Fig. 2.

Higher percent contribution of trifunctional NK cells to the total NK cell response in *h/*y+B*57 than Bw6 homozygotes (hmz) SP requires coexpression of the *h/*y 3DL1 receptor genotype and the HLA-B*57 ligand. Scatter plots show the percent contributions of trifunctional NK cells to the total K562-stimulated NK response for HLA-B*57 (B*57)-positive SP versus those with Bw4 alleles other than HLA-B*57 (Non-B*57-Bw4) (A), for carriers of HLA-Bw4 and 3DL1*h/*y genotypes (*h/*y+Bw4) versus 3DL1*l/*x genotypes (*l/*x+Bw4) (B), and for carriers of a 3DL1*h/*y genotype with HLA-B*57 (*h/*y+B*57), a 3DL1*l/*x genotype with HLA-B*57 (*l/*x+B*57), a 3DL1*h/*y genotype with an HLA-Bw4 allele other than HLA-B*57 (*h/*y-Other Bw4), and Bw6 hmz (C). The line through each scatter plot is the median for the group. Mann-Whitney U tests were used to test the significance of between-group differences. P values for comparison are shown over the line linking two groups.

Fig. 3.

Influence of HIV viral load (VL) and KIR/HLA genotype on NK functional potential. (A) Correlation between the percent contribution of trifunctional NK cells to the total K562-stimulated NK response and log₁₀ VL. A Spearman's correlation test was used to assess the significance of the association between these two parameters. (B) Scatter plots show the distribution of the percent contribution of trifunctional NK cells to the total K562-stimulated NK response in HIV-infected individuals meeting the criteria for classification as either elite controllers (EC; <50 HIV copies/ml plasma) or viral controllers (VC; <3,000 HIV copies/ml plasma) that carry a 3DL1*h/*y genotype with HLA-B*57 (*h/*y-B*57) versus a 3DL1*l/*x genotype with HLA-B*57 (*l/*x-B*57). (C) Scatter plots show the distribution of the percent contribution of trifunctional NK cells to the total K562-stimulated NK response in HIV-1-negative individuals, HIV-1-infected EC and VC, and untreated viremic progressors matched for the *h/*y+B*57 genotype. (D) Scatter plots are the same as in panel C for untreated viremic progressors carrying the *h/*y+B*57 genotype versus the Bw6 hmz genotype. For panels B to D, the line through each scatter plot is the median for the group. Mann-Whitney U tests were used to test the significance of between-group differences. P values for comparison are shown over the line linking two groups.

Fig. 4.

Cell line 721.221 (221) transfectants expressing HLA-B*57:01 and B*27:02 but not B*35:02 suppress 3DL1⁺NK cell function. (A) Flow cytometry plots of peripheral blood mononuclear cells (PBMC) from a representative individual carrying the 3DL1*h/*y genotype with HLA-B*57 (*h/*y+B*57) following stimulation with medium alone, the parental 221 cell line, and 221 cells transfected with single HLA alleles. The 221 stimulus is indicated over the plots with the parental cell line designated 221, 221-B*57:01 as B*57, 221-B*27:02 as B*27, and 221-B*35:02 as B*35. NK cells staining positive for the Z27 monoclonal antibody (3DL1⁺) cells were gated in the upper panels, and Z27⁻ (3DL1⁻) NK cells were gated in the lower panels. The number in each plot shows the frequency of the IFN-γ⁺ 3DL1⁺ (upper) or 3DL1⁻ (lower) response in the boxed area following stimulation. Panels B to D show pooled data from 10 *h/*y+B*57 SP. Panels B and C show the results for the percent contribution of total IFN-γ secretion within the 3DL1⁺ (B) and 3DL1⁻ (C) compartments, respectively. Mann-Whitney U tests assessed the significance of differences in the percent contributions of IFN-γ to the 3DL1⁺ response and 3DL1⁻ response upon stimulation with 221-B*57:01, 221-B*27:02, and 221-B*35:02 transfectants versus the 221 parental cell line. Panel D shows results from 10 *h/*y+B*57 subjects for the trifunctional 3DL1⁺ NK response following culture with the 221 transfectant panel expressed as a percentage of the response to the 221 parental cell line. Paired t tests were used to test the significance of the frequencies of trifunctional 3DL1⁺ NK cells upon stimulation with 221-B*57:01 and 221-B*27:02 transfectants versus the 221-B*35:01 transfectant. For panels B to D, bar and error bar lengths represent the mean and standard error for the group. *, P < 0.05; **, P < 0.01.