Regulatory T cells modulate monocyte functions in immunocompetent antiretroviral therapy naive HIV-1 infected people

Abstract

We previously demonstrated that the overall number of regulatory T (Treg) cells decrease proportionately with helper CD4⁺ T cells and their frequencies increase in antiretroviral therapy (ART)-naive human immunodeficiency virus type-1 (HIV-1) infected individuals. The question now is whether the discrepancies in Treg cell numbers and frequencies are synonymous to an impairment of their functions. To address this, we purified Treg cells and assessed their ability to modulate autologous monocytes functions. We observed that Treg cells were able to down modulate autologous monocytes activation as well as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) production during stimulation with polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (poly-ICLC). This activity of Treg cells has been shown to be influenced by immunocompetence including but not limited to helper CD4⁺ T cell counts, in individuals with HIV-1 infection. Compared to immunosuppressed participants (CD4 < 500 cells/µL), immunocompetent participants (CD4 ≥ 500 cells/µL) showed significantly higher levels of transforming growth factor beta (TGF-β) and IL-10 (p < 0.001 and p < 0.05, respectively), key cytokines used by Treg cells to exert their immunosuppressive functions. Our findings suggest the contribution of both TGF-β and IL-10 in the suppressive activity of Treg cells.

Keywords: Antiretroviral therapy-naive HIV-1 infection; Monocyte function; Regulatory T cells; Sustained activation/Inflammation.

Fig. 1

Purity of Treg cells in relation to the disease categorization. Dot plots of the gating strategy for magnetically purified Treg cells are shown in a. Purified CD4⁺CD25⁺ CD127^dim/− Treg cells were gated based on forward and side scatter. CD3⁺ CD4⁺ T cells were selected from live cells and then analyzed for co-expression of CD127^Low CD25⁺. CD3⁺ CD4⁺ CD127^Low CD25⁺ T cells were more than 95% pure and highly expressed FoxP3 marker (> 70%). The number of FoxP3+ Treg cells was stratified by the participants’ immunological (b) and virological (c) statuses. No significant differences (p > 0.05) were found in the number of FoxP3⁺ Treg cells across all the subgroups after purification. Horizontal bars represent the median and statistical difference was calculated using Kruskal-Wallis test

Fig. 2

Cytokine profile of Treg cells following stimulation with poly-ICLC. Figure a shows representative dot plots of the gating strategy of cytokines secreting Treg cells. Purified Treg cells obtained from PBMCs of HIV⁺ and HIV⁻ participants were stained intracellularly using mAbs specific for IL-4, IL-10, IL-17 A and TGF-β and then analyzed by multiparametric flow cytometry. Dot plots showing expression of each cytokine on CD4⁺CD25⁺CD127^Low FoxP3⁺Tregs are shown (b, c, d, e). A minimum of 100 events of each intracellular cytokine were acquired. The differences between groups were calculated using Kruskal-Wallis test followed by Dunn’s multiple comparison post-test. Treg cells from ART naive HIV-1 infected participants with helper CD4⁺ T cell counts ≥ 500 cells/mm³ showed a significant increase in TGF-β and IL-10 MFI compared to those with helper CD4⁺ T cell counts < 200 cells/ µL (p < 0.001 and p < 0.05 for TGF-β and IL-10, respectively) and those with CD4 between 200–349 cells/ µL (p < 0.05 for TGF-β). Relative to uninfected participants, there was a significant reduction of IL-4 expression in HIV-1 infected participants with helper CD4⁺ T cell counts < 200 cells/ µL (p < 0.001), between 200–349 cells/ µL (p < 0.001) and between 350–499 cells/µL (p < 0.05). Horizontal bars represent the median values. The letters on horizontal bars identify which groups are significantly different from each other. The same letter implies there is no significant difference between the compared groups, while different letters indicate a significant difference between them

Fig. 3

CD38 surface marker is more reliable than HLA-DR for monocyte activation. (a) Gating strategy for HLA-DR and CD38 expression on monocytes: following forward and side scatter selection, magnetically sorted monocytes were selected from live cells and then analyzed for CD14 expression. CD14⁺ monocytes were more than 80% pure. Next, HLA-DR and CD38 expression was monitored on the purified Monocytes. Whereas all CD38 expressing monocytes were also HLA-DR⁺, only 80% of HLA-DR expressing monocytes were CD38⁺. Relative to seronegative controls, HIV infected individuals display increased levels of immune activation as evidenced by elevated expression of HLA-DR and CD38, using the Kruskal-Wallis test followed by Dunn’s multiple comparison test (b, f). Moreover, in individuals living with HIV, higher monocyte activation levels are associated with elevated viral loads (>4 log copies/ml). Interestingly, this link is notably significant when using the CD38 marker (d), whereas the association is not statistically significant when assessed through the HLA-DR marker (h). Notably, whereas HLA-DR expression did not correlate with viro-immunological parameters (p>0.05; i, g). On the other hand, CD38 expression correlated negatively with helper CD4⁺ T cells (r= - 0.80, P < 0.0001; c) and positively with plasmatic HIV-1 viral loads (r= 0.41, p=0.01; e). Thus, making CD38 a more reliable marker for tracking monocyte activation during this study. Horizontal bars represent the median values. The letters on horizontal bars identify which groups are significantly different from each other. The same letter implies there is no significant difference between the compared groups, while different letters indicate a significant difference between them

Fig. 4

In coculture with autologous monocytes, Treg cells suppress their activation by poly- ICLC. As shown in a, a significant increase in CD38 expression was observed in poly-ICLC treated monocytes compared to monocytes cultured alone. However, the addition of autologous Treg cells resulted into a significant suppression of CD38 expression. The stimulation index (SI) was calculated by dividing the MFI of CD38 expression of poly ICLC treated monocytes by the MFI of CD38 expression in unstimulated monocytes. The suppressive activity of Treg cells defined as index of suppression (IS) was assessed based on the following formula: 1-(MFI of CD38 expression of poly ICLC treated monocytes in the presence of Treg cells / MFI of CD38 expression of poly ICLC treated monocytes in the absence of Treg cells). The SI and IS correlated positively with the helper CD4⁺ T cell count (r = 0.75, P = 0.0001 and r = 0.68, p = 0.0001, respectively for the SI and IS; b, c, d, e) and negatively with HIV-1 plasmatic viral loads (r = − 0.43, P = 0.01 and r = − 0.45, p = 0.01, respectively for the SI and IS; f, g, h, i), demonstrating a strong correlation between SI and IS (r = 0.83, P < 0.000; j). Horizontal bars represent the median values that were first compared using the Kruskal Wallis test, then followed by Dunn’s multiple comparison post-test. Correlation coefficients and their significance were calculated by Spearman test. The letters on horizontal bars identify which groups are significantly different from each other. The same letter implies there is no significant difference between the compared groups, while different letters indicate a significant difference between them

Fig. 5

In coculture with monocytes, Treg cells suppress IL-6 production following stimulation with poly-ICLC. In a, representative plots of IL-6 forming monocytes are shown for both HIV⁺ and HIV^- participants. Monocytes stimulation with poly-ICLC resulted into increased IL-6 production relative to unstimulated monocytes. Upon co-culture with autologous Treg cells, there was a significant reduction in IL-6 production indicating the inhibitory effect of Treg cells on poly-ICLC stimulated monocytes. The SI and IS correlated positively with helper CD4⁺ T cell counts (r = 0.40, P = 0.008 and r = 0.61, p = 0.0001, respectively for the SI and IS; b, c, g, f) and negatively with HIV-1 plasmatic viral loads (r = - 0.52, P = 0.031 and r = - 0.46, p = 0.0089, respectively for the SI and IS; d, e, h, i) demonstrating a strong correlation between SI and IS (r = 0.40, p = 0.0093; j). Horizontal bars represent the median values that were compared using the Kruskal Wallis test followed by Dunn’s multiple comparisons post-test. Correlation coefficients and their significance were calculated using the Spearman test. The letters on horizontal bars identify which groups are significantly different from each other. The same letter implies there is no significant difference between the compared groups, while different letters indicate a significant difference between them

Fig. 6

In coculture with monocytes, Treg cells suppress TNF-α production following stimulation with poly-ICLC. In a, representative plots of TNF-α forming monocytes are shown for both ART naive HIV-1 infected and uninfected participants. The addition of poly-ICLC in the culture medium enhanced the production of TNF-α by monocytes compared to monocytes cultured alone, without significant difference (P > 0.05; b, c, d, e). Upon co-culture with autologous Treg cells, a significant reduction of TNF-α production was observed. This suppressive activity of Treg cells correlated positively with helper CD4⁺ T cell counts (r = 0.67, P < 0.0001; f, g) and negatively with HIV-1 plasmatic viral loads (r = − 0.5, p = 0.0042; h, i). In contrast to IL-6, no correlation was observed between the stimulation and the suppression index (P > 0.05; j). Horizontal bars represent the Median values that were compared using the Kruskal Wallis test followed by Dunn’s multiple comparisons post-test. Correlation coefficients and their significance were calculated using the Spearman test. The letters on horizontal bars identify which groups are significantly different from each other. The same letter implies there is no significant difference between the compared groups, while different letters indicate a significant difference between them