The characteristics of the complete study population have been reported by us before (Teer et al. 2019). However, we summarized participant data for samples here used for Treg marker analyses in Tables 1 and 2. There were no significant differences in age between the study groups based on treatment status. CD4 count and VL were not significantly different between the HIV-positive groups based on cART treatment status.
Control(n = 12) HIV-positive Treatment naïve (n = 22) First-line cART treated (n = 15) Second-line cART treated (n = 18) Age (years) 49 (32–54) 36 (30–42) 37 (31–45) 34 (28–49) Gender (male/female) 5/7 10/12 5/10 2/16 Data presented as medians (interquartile ranges). These data are for specific samples used for Treg marker analyses in this study. Refer to Teer et al. (2019)for the complete sample set. cART: combination antiretroviral therapy.
Table 1. Study participant characteristics.
Treatment naïve First line cART treated Second line cART treated CD4 count (cells/μL) 503 (297–677) 337 (192–711) 364 (154–521) Viral load (C/mL) 586 (40–18, 855) 582 (20–85, 076) 4524 (69–109, 383) Data presented as median (IQR). These data are for specific samples used for Treg marker analyses in this study. Refer to Teer et al. (2019)for the complete sample set. cART: combination antiretroviral therapy; C/mL: copies per millilitre.
Table 2. CD4 count and viral load in HIV-positive study participants.
The median percentage of FOXP3-expressing cells increased in the CD4+CD25- T cell population in HIV-treated group with relatively low CD4 counts (< 500) [0.42 (0.09-1.24) vs. treatment naïve [0.078 (0.046-0.118)], P < 0.01, Fig. 1A) and high VL (> 1000) [0.66 (0.23-1.93) vs. treatment naïve [0.078 (0.046-0.118)], P < 0.001, Fig. 1D). GARP-expressing cells displayed an increase in the CD4+CD25- T-cells in HIV-treated patients with a relatively low CD4 count [1.21 (0.12-9.77)] vs. treatment naïve [0.034 (0.009-1.63)], P < 0.01, Fig. 1B) and high VL [2.47 (0.057-28.7)] vs. treatment naïve [0.034 (0.009-1.63)], P < 0.01, Fig. 1E). There were no significant differences between the other groups or for the SATB1-expressing CD25-negative cells (Fig. 1C, 1F). There was a significant, strong positive correlation between GARP and FOXP3 expressed on CD4+CD25- cells (r = 0.57; P < 0.0001), and a significant, moderate positive correlation between CD25-FOXP3+ and CD25++GARP+ (r = 0.38; P = 0.0005) (Table 3).
Figure 1. Expansion of FOXP3 and GARP in CD4+CD25- cells in immune compromised HIV-infected individuals with virological failure. A-C classification according to CD4 count; D-F classification according to viral load; (A and D) percentage of CD4+CD25-FOXP3+ cells; (B and E) percentage of CD4+CD25-- GARP+ cells; (C and F) percentage of CD4+CD25-SATB1+ cells. ** P < 0.01; ***P < 0.001.
CD4+CD25-FOXP3+ (%) CD4+CD25++FOXP3+ (%) CD4+CD25-GARP+ (%) r = 0.57; P < 0.0001 r = 0.37; P = 0.001 CD4+CD25++GARP+ (%) r = 0.38; P = 0.0005 r = 0.24; P = 0.02 CD4 count (cells/μL) r = - 0.53; P < 0.0001 r = - 0.38; P = 0.001 Viral load (copies/mL) r = 0.43; P = 0.0007 r = 0.23; P = 0.04 CD8+CD38+ (%) r = 0.21; P = 0.04 NS
Table 3. Correlation of CD25- and CD25++ Tregs expressing FOXP3 with immunological markers.
The CD4+CD25-FOXP3+ Treg subset showed a significant, strong negative correlation with CD4 count (r = — 0.53; P < 0.0001) and a significant, strong positive correlation with VL (r = 0.43; P = 0.0007) and CD38 expression on CD8+ T-cells (r = 0.21; P = 0.04) (Table 3).
The percentage of FOXP3-expressing cells remained unaltered in the CD4+CD25++ T cell population for all study groups (Fig. 2A and 2D), while SATB1 expression in the CD4+CD25++ subset was not significantly different between study groups (Fig. 2C and 2F). However, there was a significant increase of the GARP+ subset in the HIV-treated group with low CD4 counts (CD4 count < 500; P < 0.05) (Fig. 2B), while there was no significant increase for the GARP+ subset in the HIV-treated group with high VL (VL > 1000) (Fig. 2E).
Figure 2. Expansion of GARP in CD4+CD25++ cells in immune compromised HIV-infected individuals. A–C classification according to CD4 count; D–F classification according to viral load; (A and D) percentage of CD4+CD25++FOXP3+ cells; (B and E) percentage of CD4+CD25++GARP+ cells; (C and F) percentage of CD4+- CD25++SATB1+ cells. *P < 0.05.
There was a strong positive correlation of pro-inflammatory CD4+CD25-SATB1 + T cells with immune activation markers (CD8+CD38+ T-cells) (r = 0.50; P < 0.0001) and significant, weak positive correlations with the percentage of coagulation marker tissue factor-expressing CD8+ T cells (CD8+CD142+) (r = 0.24; P = 0.02) and D-dimer (r = 0.24; P = 0.03), respectively (Table 4).
CD25-SATB1+ (%) CD8+CD38+ (%) r = 0.50; P < 0.0001 CD8+CD142+ (%) r = 0.24; P = 0.02 Plasma D-dimer (μg/mL) r = 0.24; P = 0.03
Table 4. Correlation of CD25- immunological markers.
There was a significant increase in FOXP3+ cells in both CD4+CD25- and CD4+CD25++ T cell subsets for all study groups stratified according to CD4 count and VL (P < 0.0001) (Fig. 3A and 3D). The differences in the GARP (Fig. 3B and 3E) and SATB1 (Fig. 3C and 3F) expressing subsets in the CD4+CD25- vs. CD4+CD25++ populations were not statistically significant.
Figure 3. Comparison of CD25- vs. CD25++ Tregs expressing FOXP3, GARP and SATB1 in immune compromised HIV-infected individuals with virological failure. A-C classification according to CD4 count; D-F classification according to viral load; (A and D) percentage of FOXP3-expressing T cells; (B and E) percentage of GARP-expressing T cells; (C and F) percentage of SATB1-expressiing T cells. *** P < 0.001; **** P < 0.0001.
Circulating IL-10 levels did not differ significantly between the study groups (refer Fig. 4). IL-10 showed a significant, weak positive correlation with CD4+CD25-- FOXP3+ T-cells (r = 0.27; P = 0.03). It also showed significant, moderate positive correlations with immune activation (CD38 expression) in CD8+ T-cells (r = 0.33; P = 0.01) and with D-dimer (r = 0.30; P = 0.03; Table 5).
Figure 4. Plasma interleukin-10 levels. A classification according to CD4 count; B classification according to viral load.
IL-10 (pg/mL) CD4+CD25-FOXP3+ (%) r = 0.27; P = 0.03 CD8+CD38+ (%) r = 0.50; P < 0.0001 Plasma D-dimer (μg/mL) r = 0.24; P = 0.03
Table 5. Correlation of IL-10 with CD25- Tregs expressing FOXP3, immune activation of CD8+ T cells and plasma D-dimer.