Immune exhaustion occurs concomitantly with immune activation and decrease in regulatory T cells in viremic chronically HIV-1-infected patients

Abstract

Background: Chronic HIV-1 infection is associated with excessive immune activation and immune exhaustion. We investigated the relationship of these 2 phenotypes and frequency of regulatory T cells (Tregs) in controlled and uncontrolled chronic HIV-1 infection.

Methods: Immune exhaustion marker PD-1, its ligand PD-L1, CD4CD25 FoxP3 Tregs, HLA-DR, and CD38 coexpression as activation markers were investigated in peripheral blood lymphocytes of 44 HIV-1-infected patients and 11 HIV-1-uninfected controls by multicolor flow cytometry.

Results: Activated and PD-1 expressing T cells were increased, and Tregs were decreased in HIV-1-infected patients as compared with controls, and alterations were greatest in viremic patients. The proportion of activated CD8 T cells exceeded activated CD4 T cells. Tregs had an inverse correlation with activated T cells and PD-1 expressing T cells. PD-L1 was highly expressed on monocytes and to a lesser extent on T lymphocytes of patients. These abnormalities partially reversed with virologic control after potent antiretroviral therapy.

Conclusions: Immune exhaustion is a component of aberrant immune activation in chronic HIV-1 infection and is associated with loss of Tregs and ongoing virus replication. These defects are corrected partially with effective virologic control by potent antiretroviral therapy.

FIGURE 1. Expression of activation markers, HLA-DR and CD38 and their relationship with viral load and CD4+ T cell counts

Percentage of CD8+HLA-DR+CD38+ cells show a significant positive correlation with viral load (r = 0.76, P<0.05, dotted line) and a negative correlation with CD4+ T cell count (r = − 0.27, P = ns, bold line) (A); Longitudinal analysis of eight patients show a significant decrease in the expression of activation markers on CD8+ T cells at week 48 in comparison to week 0 (B); ns, not significant

FIGURE 2. Expression of PD-1 on CD8+ T cells in HIV-1 infected patients and healthy controls (HC) in relation to viral load, CD4+ T cells and markers of immune activation

A. Representative flow cytogram from a healthy control showing the gating scheme for PD-1, coexpression of PD-1 and PD-L1 on CD4+ T cells and PD-L1 expression on monocytes; B. Expression of PD-1 (MFI) on CD8+ T cells in viremic and aviremic patients and healthy controls (HC); C. PD-1 expressing CD8+ T cells had a positive correlation with viral load (r = 0.366, P<0.05, dotted line) and negative correlation with CD4+ T cell counts (r = − 0.11, ns, bold line); D. PD-1 expressing CD8+ T cells had a positive correlation with HLA-DR and CD38 on CD8+ T cells (r = 0.5, P<0.05); E. PD-1 expression on CD8+ T cells decreased after 48 weeks on ART; ns, not significant; *, P<0.05.

FIGURE 2. Expression of PD-1 on CD8+ T cells in HIV-1 infected patients and healthy controls (HC) in relation to viral load, CD4+ T cells and markers of immune activation

A. Representative flow cytogram from a healthy control showing the gating scheme for PD-1, coexpression of PD-1 and PD-L1 on CD4+ T cells and PD-L1 expression on monocytes; B. Expression of PD-1 (MFI) on CD8+ T cells in viremic and aviremic patients and healthy controls (HC); C. PD-1 expressing CD8+ T cells had a positive correlation with viral load (r = 0.366, P<0.05, dotted line) and negative correlation with CD4+ T cell counts (r = − 0.11, ns, bold line); D. PD-1 expressing CD8+ T cells had a positive correlation with HLA-DR and CD38 on CD8+ T cells (r = 0.5, P<0.05); E. PD-1 expression on CD8+ T cells decreased after 48 weeks on ART; ns, not significant; *, P<0.05.

FIGURE 3. Expression of PD-L1 and of both PD-L1 and PD-1 on monocytes, CD8+ T cells and CD4+ T cells

A. Expression of PD-L1 on monocytes of viremic and aviremic patients and healthy controls; B. Decrease in PD-L1 expression on monocytes at week 48 compared to week 0 in 8 patients followed longitudinally. Expression of PD-L1 on CD4+ T cells (C) and on CD8+ T cells (D) in viremic and aviremic patients and healthy controls. Coexpression of PD-1 and PD-L1 on CD4+ T cells (E) and CD8+ T cells (F); *, P<0.05,**, P<0.01.

FIGURE 4. Regulatory CD4+ T cells and their relationship to activated T cells and to CD8+ PD-1 T cells

A. Identification of Tregs defined as CD4+CD25^brightFoxP3+ cells in a viremic patient. First, upper bright portion of CD25+CD3+CD4+ cells were gated to identify CD4+CD25^bright cells. Next the FoxP3+CD4+CD25^bright cell subset was identified. CD4 negative cell subset does not express FoxP3, this subset was used to set gates for FoxP3+ cells in CD4+CD25^bright population as shown. B. Pie charts depicting the proportion of Tregs (CD3+ CD4+CD25^bright FoxP3+ and activated non Treg cells (CD3+ CD4+CD25^bright FoxP3 negative) in viremic and aviremic patients and healthy controls. Treg cells show a negative correlation with CD8+PD-1+ T cells (r = − 0.23, P<0.05) (C), and with CD8+HLA-DR+CD38+ cells (r = − 0.41, P<0.05) (D); E. Increase in Tregs at week 48 as compared to week 0 in patients followed longitudinally; *, P<0.05.

FIGURE 4. Regulatory CD4+ T cells and their relationship to activated T cells and to CD8+ PD-1 T cells

A. Identification of Tregs defined as CD4+CD25^brightFoxP3+ cells in a viremic patient. First, upper bright portion of CD25+CD3+CD4+ cells were gated to identify CD4+CD25^bright cells. Next the FoxP3+CD4+CD25^bright cell subset was identified. CD4 negative cell subset does not express FoxP3, this subset was used to set gates for FoxP3+ cells in CD4+CD25^bright population as shown. B. Pie charts depicting the proportion of Tregs (CD3+ CD4+CD25^bright FoxP3+ and activated non Treg cells (CD3+ CD4+CD25^bright FoxP3 negative) in viremic and aviremic patients and healthy controls. Treg cells show a negative correlation with CD8+PD-1+ T cells (r = − 0.23, P<0.05) (C), and with CD8+HLA-DR+CD38+ cells (r = − 0.41, P<0.05) (D); E. Increase in Tregs at week 48 as compared to week 0 in patients followed longitudinally; *, P<0.05.

FIGURE 4. Regulatory CD4+ T cells and their relationship to activated T cells and to CD8+ PD-1 T cells

A. Identification of Tregs defined as CD4+CD25^brightFoxP3+ cells in a viremic patient. First, upper bright portion of CD25+CD3+CD4+ cells were gated to identify CD4+CD25^bright cells. Next the FoxP3+CD4+CD25^bright cell subset was identified. CD4 negative cell subset does not express FoxP3, this subset was used to set gates for FoxP3+ cells in CD4+CD25^bright population as shown. B. Pie charts depicting the proportion of Tregs (CD3+ CD4+CD25^bright FoxP3+ and activated non Treg cells (CD3+ CD4+CD25^bright FoxP3 negative) in viremic and aviremic patients and healthy controls. Treg cells show a negative correlation with CD8+PD-1+ T cells (r = − 0.23, P<0.05) (C), and with CD8+HLA-DR+CD38+ cells (r = − 0.41, P<0.05) (D); E. Increase in Tregs at week 48 as compared to week 0 in patients followed longitudinally; *, P<0.05.