A Higher Frequency of NKG2A+ than of NKG2A- NK Cells Responds to Autologous HIV-Infected CD4 Cells irrespective of Whether or Not They Coexpress KIR3DL1

Abstract

Epidemiological and functional studies implicate NK cells in HIV control. However, there is little information available on which NK cell populations, as defined by the inhibitory NK cell receptors (iNKRs) they express, respond to autologous HIV-infected CD4(+) (iCD4) T cells. NK cells acquire antiviral functions through education, which requires signals received from iNKRs, such as NKG2A and KIR3DL1 (here, 3DL1), engaging their ligands. NKG2A interacts with HLA-E, and 3DL1 interacts with HLA-A/B antigens expressing the Bw4 epitope. HIV-infected cells downregulate HLA-A/B, which should interrupt negative signaling through 3DL1, leading to NK cell activation, provided there is sufficient engagement of activating NKRs. We examined the functionality of NK cells expressing or not NKG2A and 3DL1 stimulated by HLA-null and autologous iCD4 cells. Flow cytometry was used to gate on each NKG2A(+)/NKG2A(-) 3DL1(+)/3DL1(-) (NKG2A(+/-) 3DL1(+/-)) population and to measure the frequency of all possible combinations of CD107a expression and gamma interferon (IFN-γ) and CCL4 secretion. The highest frequency of functional NK cells responding to HLA-null cell stimulation was the NKG2A(+) 3DL1(+) NK cell population. The highest frequencies of functional NK cells responding to autologous iCD4 cells were those expressing NKG2A; coexpression of 3DL1 did not further modulate responsiveness. This was the case for the functional subsets characterized by the sum of all functions tested (total responsiveness), as well as by the trifunctional CD107a(+) IFN-γ(+) CCL4(+), CD107a(+) IFN-γ(+), total CD107a(+), and total IFN-γ(+) functional subsets. These results indicate that the NKG2A receptor has a role in NK cell-mediated anti-HIV responses.

Importance: HIV-infected CD4 (iCD4) cells activate NK cells, which then control HIV replication. However, little is known regarding which NK cell populations iCD4 cells stimulate to develop antiviral activity. Here, we examine the frequency of NK cell populations, defined by the presence/absence of the NK cell receptors (NKRs) NKG2A and 3DL1, that respond to iCD4 cells. NKG2A and 3DL1 are involved in priming NK cells for antiviral functions upon encountering virus-infected cells. A higher frequency of NKG2A(+) than NKG2A(-) NK cells responded to iCD4 cells by developing antiviral functions such as CD107a expression, which correlates with NK cell killing, and secretion of gamma interferon and CCL4. Coexpression of 3DL1 on the NKG2A(+) and NKG2A(-) NK cells did not modulate responses to iCD4 cells. Understanding the mechanisms underlying the interaction of NK cells with iCD4 cells that lead to HIV control may contribute to developing strategies that harness NK cells for preventing or controlling HIV infection.

FIG 1

Gating strategy for identification of NK cell subsets and functional responses. (A) The live lymphocytic singlet population was used to gate on NK cells, which were defined as CD3⁻ CD56⁺. The NKG2A^+/− 3DL1^+/− populations were derived from the NK cell gate. Functional gates for CD107, IFN-γ, and CCL4 were set on gated NK cells in unstimulated PBMCs. (B) The frequency of NKG2A^+/− 3DL1^+/− populations, as defined by surface expression of these two inhibitory NK receptors, following stimulation with HLA-null cells (K562 or 721) compared to background stimulation with PBMCs alone (PBMC). Data from 13 individuals tested in duplicate were included in this analysis. Bar height represents the median, and error bars represent the IQR for the group. A Friedman test was used determine significance between group comparisons (P_Friedman > 0.05; data not shown). FSC-A, forward scatter area; FSC-H, forward scatter height.

FIG 2

The NKG2A⁺ 3DL1⁺ NK cell population was significantly more responsive to stimulation by HLA-null cell lines in all examined functions except degranulation. Shown on the y axes are the frequencies of the four NKG2A^+/− 3DL1^+/− NK cell populations that responded to 721 HLA-null cell stimulation with responses characterized by total responsiveness (A), trifunctional CD107a⁺ IFN-γ⁺ CCL4⁺ (B), bifunctional CD107a⁺ IFN-γ⁺ (C) and IFN-γ⁺ CCL4⁺ (D), and monofunctional IFN-γ⁺ (E) and CD107a (F and G) functional profiles. Bar height represents the median, and error bars represent the IQR for the data set. Thirteen individuals were analyzed in duplicate. Friedman (P_Friedman) and Wilcoxon (P) tests were used determine significance between group differences. P values for between-group comparisons are shown over lines linking the two groups being compared. Fxn, functional.

FIG 3

A higher frequency of NKG2A⁺ 3DL1⁺ NK cells from carriers of a Bw4 allele respond to 721 cells than those from Bw6⁺ individuals. The frequency of all functional 721-stimulated NKG2A^+/− 3DL1⁺ NK cells from individuals belonging to the Bw4 and Bw6 groups was plotted on the y axis (A). A Mann-Whitney test was used to determine significance of between-group differences. The frequency of all functional 721-stimulated NKG2A^+/− 3DL1^+/− populations from Bw4 (B) and Bw6 (C) individuals was plotted on the y axis. Friedman (P_Friedman) and Wilcoxon (P) tests were used determine significance of between-group differences. Bar height represents the median, and error bars represent the IQR for 13 individuals analyzed in duplicate.

FIG 4

Total responsiveness of NK populations to autologous HIV-iCD4 cells. (A) Frequency of iNKR-bearing CD56⁺ NKG2A^+/− 3DL1^+/− populations following stimulation with autologous iCD4 cells and uninfected controls. (B) Frequency of total responsiveness (y axis) of NKG2A^+/− 3DL1^+/− populations following stimulation with iCD4 cells. Bar height represents the mean ± standard deviation for the group, and Friedman (P_Friedman) and Wilcoxon tests (P) were used to determine significance of between-group differences. Data from 24 individuals analyzed in duplicate were used to generate results. (C) Correlation between percentage of p24⁺ iCD4 cells used for stimulation (x axis) and total responsiveness (y axis) of each NKG2A^+/− 3DL1^+/− population. Data from 12 individuals analyzed in duplicate were used to generate these plots. Spearman's correlation test (r and P) were used to assess the significance of the association between these two measures.

FIG 5

iCD4 cell stimulation of functional subsets contributing to the total responsiveness of 3DL1^+/− NKG2A^+/− NK cell populations. (A) The frequency of CD56^Bright and CD56^Dim iNKR-bearing NKG2A^+/− 3DL1^+/− populations following stimulation with autologous iCD4 cells and uninfected CD4 controls. The inset at the top right is an expanded view of the frequency of the CD56^Bright NKG2A⁺ population in iCD4 cells versus uninfected CD4 conditions. The frequencies of iCD4 cell-stimulated NK cell populations characterized by total responsiveness (B), trifunctional (C), bifunctional CD107a⁺ IFN-γ⁺ (D), and total CD107a (E) and total IFN-γ (F) response profiles are shown on the y axis for each CD56^Bright NKG2A^+/− and CD56^Dim NKG2A^+/− 3DL1^+/− population. Bar height represents the mean ± standard deviation for each group. Data from 24 individuals analyzed in duplicate were used to generate these results. Friedman (P_Friedman) and Wilcoxon (P) tests were used to determine significance between data sets. P values for between-group comparisons are shown over lines linking the two groups being compared.

FIG 6

Blocking NKG2A and HLA-E enhances antiviral functional responses of CD56^Dim NKG2A⁺ cells. Purified NK cells were cocultured with autologous iCD4 cells or uninfected CD4 controls for 24 h. Blocking antibodies to NKG2A, HLA-E, or both were added to the corresponding cocultures overnight. These cocultures were processed as per the section describing NK cell stimulation and staining in Materials and Methods. We focused on CD56^Dim NKG2A⁺ cells and measured total responsiveness (y axis). Data for four individuals are shown, and bars represent the mean ± standard deviation.

FIG 7

iCD4 cell-stimulated CD56^Dim NKG2A⁺ 3DL1⁺ cells from Bw4⁺ individuals have greater trifunctional and total CCL4 responses than those from Bw6 homozygotes. The frequencies of iCD4 cell-stimulated CD56^Dim NKG2A^+/− 3DL1⁺ NK cells from carriers of a Bw4 allele versus Bw6 homozygotes characterized by total responsiveness (A), trifunctional response (B), and total CCL4 responses profiles are shown on the y axis. Bar height represents the mean ± standard deviation for each group. Data from 24 individuals analyzed in duplicate are plotted. A Mann-Whitney test was used to determine significance of between-group differences.