Elevated Foxp3+ double-negative T cells are associated with disease progression during HIV infection

Abstract

Persistent immune activation, which occurs during the whole course of HIV infection, plays a pivotal role in CD4⁺ T cells depletion and AIDS progression. Furthermore, immune activation is a key factor that leads to impaired immune reconstitution after long-term effective antiretroviral therapy (ART), and is even responsible for the increased risk of developing non-AIDS co-morbidities. Therefore, it's imperative to identify an effective intervention targeting HIV-associated immune activation to improve disease management. Double negative T cells (DNT) were reported to provide immunosuppression during HIV infection, but the related mechanisms remained puzzled. Foxp3 endows Tregs with potent suppressive function to maintain immune homeostasis. However, whether DNT cells expressed Foxp3 and the accurate function of these cells urgently needed to be investigated. Here, we found that Foxp3⁺ DNT cells accumulated in untreated people living with HIV (PLWH) with CD4⁺ T cell count less than 200 cells/µl. Moreover, the frequency of Foxp3⁺ DNT cells was negatively correlated with CD4⁺ T cell count and CD4/CD8 ratio, and positively correlated with immune activation and systemic inflammation in PLWH. Of note, Foxp3⁺ DNT cells might exert suppressive regulation by increased expression of CD39, CD25, or vigorous proliferation (high levels of GITR and ki67) in ART-naive PLWH. Our study underlined the importance of Foxp3⁺ DNT cells in the HIV disease progression, and suggest that Foxp3⁺ DNT may be a potential target for clinical intervention for the control of immune activation during HIV infection.

Keywords: Foxp3; HIV; double-negative T cell; immune activation; immune regulation.

Figure 1

Increased proportions of Foxp3⁺ DNT cells associated with progressive HIV disease. Flow cytometry analysis of Foxp3 expression was performed on PBMCs collected from HCs and different TNs groups. (A) Representative flow data showed the expression of Foxp3 on DNT cells from TNs.  (B) Violin plots of the percentage of Foxp3⁺ DNT cells from HCs and different TNs groups (n=20-66 each group). P values were obtained by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (C–E) Correlation analysis of the percentages of Foxp3⁺ DNT cells with CD4⁺ T cell count (C), CD4/CD8 ratio (D), HIV viral load (E) in PHIV infected patients without treatment. The percentage of Foxp3⁺ DNT cells was represented on a log2 scale. The correlation was calculated using Spearman’s non-parametric test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

Foxp³⁺ DNT cells correlated with the phenotypic profile of activation and systematic inflammation. (A, B) Correlation analysis between the percentage of Foxp3⁺ DNT cells and (HLA-DR⁺CD38^hiCD4⁺ T cells (A) and HLA-DR⁺CD38^hiCD8⁺ T cells (B) from TNs. Spearman’s non-parametric test was used to test for correlations. (C) Heatmap depicting the relative concentrations of 12 differentially expressed serum proteins in TNs. Each column of the heatmap indicated a sample, while the rows represented different serum proteins. The color scale in the heatmap represented scores standardized across rows.(blue represented low levels; red, high levels) (D) Violin plots of the concentration of twelve differentially expressed serum proteins in the Foxp3-low group (n = 40) and Foxp3-high group (n = 40). The concentration of different serum was shown on a log10 scale. P values were obtained by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (E) Radar map of 12 differentially expressed serum proteins in Foxp3-low group (n = 40) and Foxp3-high group (n = 40). The radius is the percentage of expression. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Foxp3⁺ DNT cells showed a unique phenotypic characteristic compared with their circulating Foxp3^- counterpart in TNs. Flow cytometry analysis of expression of CD73 (A), CD39 (B), GITR (C), Ki-67 (D), CD25 (E), FasL (F), LAG-3 (G) and CTLA-4 (H) on Foxp3^- vs. Foxp3⁺ DNT cells from TNs with baseline CD4⁺ T cell count ≤ 200 cells/µl. Representative histograms (left) and plots (right) displayed the expression of the above markers on Foxp3^- vs. Foxp3⁺ DNT cells. P values were obtained by Wilcoxon matched-pairs signed rank test.

Figure 4

Foxp3⁺ DNT cells secreted different level of cytokine and intracellular proteins compared with their circulating Foxp3^- counterpart in TNs. Flow cytometry analysis of expression of GRA (A), GRB (B), Perforin (C), IDO (D) and IL-10 (E) on Foxp3^- vs. Foxp3⁺ DNT cells from TNs with baseline CD4⁺ T cell count ≤ 200 cells/µl. Representative histograms (left) and plots (right) displayed the expression of the above markers on Foxp3^- vs. Foxp3⁺ DNT cells. Statistical tests were performed using the Wilcoxon matched-pairs signed rank test.

Figure 5

The frequency of Foxp3⁺ DNT cells was partly restored after ART. (A)Violin plots of the frequencies (left) and absolute numbers (right) of DNT cells from healthy donors, TNs with baseline CD4⁺ T cell count < 200 cells/µl, ARTs who have been treated more than 4 years with matched nadir CD4⁺ T cell count. (B) Comparison of Foxp3⁺ DNT cells frequencies (left) and absolute numbers (right) in healthy donors, TNs, ARTs. P values were obtained by the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.