Expression of the Inhibitory Receptor TIGIT Is Up-Regulated Specifically on NK Cells With CD226 Activating Receptor From HIV-Infected Individuals

Abstract

Natural killer (NK) cells are important for maintenance of innate immune system stability and serve as a first line of defense against tumors and virus infections; they can act either directly or indirectly and are regulated via co-operation between inhibitory and stimulatory surface receptors. The recently reported inhibitory receptor, TIGIT, can be expressed on the NK cell surface; however, the expression level and function of TIGIT on NK cells during HIV infection is unknown. In this study, for the first time, we investigated the expression and function of TIGIT in NK cells from HIV-infected individuals. Our data demonstrate that the level of TIGIT is higher on NK cells from patients infected with human immunodeficiency virus (HIV) compared with HIV-negative healthy controls. TIGIT expression is inversely correlated with CD4⁺ T cell counts and positively correlated with plasma viral loads. Additionally, levels of the TIGIT ligand, CD155, were higher on CD4⁺ T cells from HIV-infected individuals compared with those from healthy controls; however, there was no difference in the level of the activating receptor, CD226, which recognizes the same ligands as TIGIT. Furthermore, TIGIT was found to specifically up-regulated on CD226⁺ NK cells in HIV-infected individuals, and either rIL-10, or rIL-12 + rIL-15, could induce TIGIT expression on these cells. In addition, high TIGIT expression inhibited the production of interferon-gamma (IFN-γ) by NK cells, while TIGIT inhibition restored IFN-γ production. Overall, these results highlight the important role of TIGIT in NK cell function and suggest a potential new avenue for the development of therapeutic strategies toward a functional cure for HIV.

Keywords: CD155; CD226; HIV; NK cell; TIGIT.

Figure 1

The expression of TIGIT on NK cells is higher in HIV-infected individuals and correlated with HIV disease progression. (A) Gating strategy used to identify total natural killer (NK) cells and NK cell subsets. Single cells were gated using the forward scatter area (FSA) and forward scatter height (FSH), then live cells were gated by Live/Dead (BV510) staining. Lymphocytes were gated according to forward scatter/side scatter properties (FSC/SSC). Total NK cells were identified from CD3-negative lymphocytes by their expression of CD16 and/or CD56. The four NK cell subsets identified were CD3⁻CD56^brightCD16^−/+, CD3⁻CD56^dimCD16⁺, CD3⁻CD56^dimCD16⁻, and CD3⁻CD56⁻CD16⁺. All the plots were based on an HIV+ individual. (B) A representative flow cytometry plot showing the different percentages of TIGIT on NK cells in the HC and HIV groups. The expression of TIGIT was gated according to an isotype control. (C) Comparison of the percentages of TIGIT on NK cells from the HIV (n = 26) and HC (n = 38) groups. (D) Comparison of the MFI of TIGIT on NK cells from the HIV (n = 26) and HC (n = 38) groups. (E) Analysis of the correlation between TIGIT expression on NK cells and absolute CD4⁺ T cell counts (cells/mm³) at the same sampling time (n = 53). (F) Analysis of the correlation between the proportion of TIGIT on NK cells and plasma levels of HIV RNA (Log–₁₀ HIV RNA copies/mL) at the same sampling time (n = 26). A Mann-Whitney U test was used for comparisons between two groups. Error bars indicate the median and interquartile range. Spearman's rank analysis was employed for evaluation of correlation. p < 0.05 was considered significant.

Figure 2

Expression of TIGIT limits the production of IFN-γ by NK cells. (A) A representative flow cytometry plot showing the different percentages of IFN-γ producing NK cells after stimulation with rIL-12 + rIL-15 + rIL-18 for 24 h in HC and HIV-infected individuals. The IFN-γ expression was gated according to an isotype control. (B) Comparison of the function of NK cells from HIV-infected (n = 8) and HC (n = 8) groups, based on their IFN-γ production on stimulation with rIL-12 + rIL-15 + rIL-18. (C) A representative flow cytometry plot showing the different percentages of IFN-γ producing TIGIT⁺ and TIGIT⁻ NK cells from HIV-infected individuals after stimulation with rIL-12 + rIL-15 + rIL-18 for 24 h. (D) Paired comparisons of IFN-γ producing TIGIT⁺ and TIGIT⁻ NK cells from HIV-infected individuals after stimulation with rIL-12 + rIL-15 + rIL-18 for 24 h (HIV: n = 11; HC: n = 12). (E) Analysis of the correlation between the percentage of TIGIT⁺ NK cells and the percentages of IFN-γ producing NK cells from HIV-infected individuals (n = 16). Mann-Whitney U tests were used for comparisons between two groups. The Wilcoxon matched-pairs signed-rank test was used for paired-group comparisons. The Spearman's rank test was employed for correlation analyses. p < 0.05 was considered significant.

Figure 3

Association between the expression of CD155 on CD4⁺ T cells and IFN-γ production by NK cells from HIV-infected individuals. (A) Gating strategy used to identify the CD4⁺ T cells. Single cells were gated using the forward scatter area (FSA) and forward scatter height (FSH) (P1), then live cells were gated by Live/Dead (BV510). Lymphocytes were gated according to forward scatter/side scatter properties (FSC/SSC) (P2). CD4⁺ T cells were identified from the CD3⁻CD4⁺ gate. A representative flow cytometry plot showing the expression of CD155 on CD4⁺ T cells in HIV-infected and HC individuals. (B) Comparison of the percentages of CD155 expressing CD4⁺ T cells between HIV-infected (n = 18) and HC (n = 13) groups. (C) A representative flow cytometry plot showing production of IFN-γ in NK cells after treatment with 20 μg/mL anti-human CD155 antibody or purified mouse IgG (as a negative control) from HIV-infected individuals; the production of IFN-γ was gated according to an isotype control. (D) Paired comparison of the production of IFN-γ by NK cells after treatment with 20 μg/mL anti-human CD155 antibodies or purified mouse IgG (as a negative control) from HIV-infected individuals (n = 8). The Mann-Whitney U test was used for comparisons between two groups. The Wilcoxon matched-pairs signed-rank test was used for paired-group comparisons. p < 0.05 was considered significant.

Figure 4

TIGIT is primarily expressed on CD226⁺ NK cells in HIV-infected individuals. (A) A representative flow cytometry plot showing the expression of CD226 on total NK cells in HIV-infected and HC individuals. (B) Comparison of the percentages of CD226⁺ NK cells in HIV-infected (n = 26) and HC (n = 38) groups. (C) A representative flow cytometry plot showing the proportion of IFN-γ producing NK cells after treatment with 20 μg/mL anti-human CD226 antibodies or purified mouse IgG (as a negative control) from HIV-infected individuals. The expression of IFN-γ was gated according to an isotype control. (D) Paired comparisons of the proportion of IFN-γ producing NK cells from HIV-infected individuals (n = 8) after treatment with 20 μg/mL anti-human CD155 antibodies or purified anti-mouse IgG (as a negative control). (E) Comparison of TIGIT expression on CD226⁺ NK cells in the HIV (n = 13) and HC (n = 9) groups. (F) Paired comparisons of the expression of TIGIT on CD226⁺ NK cells unstimulated or stimulated with rIL-10, rIL-12 + rIL-15, or rTGF-β. A Mann-Whitney U test was used for comparisons between two groups. A Wilcoxon matched-pairs signed-rank test was used for paired-group comparisons. p < 0.05 was considered significant.

Figure 5

Blocking TIGIT can restore the function of NK cells in HIV-infected individuals. (A) A representative flow cytometry plot showing the proportion of IFN-γ producing NK cells after treatment with 5 μg/mL anti-human TIGIT antibodies or purified mouse IgG (as a negative control) from HIV-infected individuals. The expression of IFN-γ was gated according to an isotype control. (B) Paired comparisons of the proportions of IFN-γ producing NK cells after treatment with 5 μg/mL anti-human TIGIT antibodies or purified mouse IgG (as a negative control) from HIV-infected individuals (n = 8). (C) Paired comparisons of the proportion of CD107a expressed by NK cells after treatment with 5 μg/mL anti-human TIGIT antibodies or purified mouse IgG (as a negative control) from HIV-infected individuals (n = 7). (D) A representative flow cytometry plot demonstrating the effects of different doses of rTIGIT (0 (medium only), 20, 50, and 100 ng/mL) on IFN-γ and CD107a production by NK cells. (E) Proportion of IFN-γ⁺ NK cells from HIV-infected individuals after treatment with different concentrations of rTIGIT (0, 20, 50, and 100 ng/mL; n = 3). (F) Proportion of CD107a⁺ NK cells from HIV-infected individuals after treatment with different concentrations of rTIGIT (0, 20, 50, and 100 ng/mL; n = 3). (G) Paired comparisons of the proportion of IFN-γ producing NK cells after treatment with 50 ng/mL rTIGIT or rIgG from HIV-infected individuals (n = 7). (H) Paired comparisons of the proportion of CD107a-expressing NK cells after treatment with 50 ng/mL rTIGIT or rIgG from HIV-infected individuals (n = 7). Wilcoxon matched-pairs signed-rank tests were used for comparisons between paired groups. p < 0.05 was considered significant.