FOXP3 expression is upregulated in CD4T cells in progressive HIV-1 infection and is a marker of disease severity

Abstract

Background: Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.

Methodology: FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.

Principal findings: HIV infected individuals had significantly higher frequencies of CD4(+)FOXP3(+) T cells (median of 8.11%; range 1.33%-26.27%) than healthy controls (median 3.72%; range 1.3-7.5%; P = 0.002), despite having lower absolute counts of CD4(+)FOXP3(+) T cells. There was a significant positive correlation between the frequency of CD4(+)FOXP3(+) T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = -0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/microl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%-26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4(+) T cells following antigenic or other stimulation.

Conclusions/significance: FOXP3 expression in the CD4(+) T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression.

Figure 1. FOXP3 expression at baseline in HIV infected patients.

Panel A: Subgroup analysis of the HIV and HIV/TB coinfected groups showed no difference in FOXP3 expression as a percentage of CD3⁺CD4⁺ T lymphocytes between the HIV and HIV/TB co-infected groups. Panel B: Representative plots of baseline FOXP3 expression in CD3⁺CD4⁺ lymphocytes plotted against CD25 for a control and an HIV positive sample. Panel C: Expression of FOXP3, CD25, CTLA-4 and GITR as a percentage of the CD3⁺CD4⁺ population. Panel D: Absolute numbers of total and FOXP3 expressing CD4⁺ T cells at baseline.

Figure 2. Representative plot illustrating CD25 gating strategy.

Lymphocytes were gated according to forward and side scatter. CD3 positive lymphocytes were selected. CD25 expression on the x-axis was plotted against CD4 on the y-axis. A gate was set according to CD25 expression on the CD4⁻ population. This gave a percentage of CD3⁺CD4⁺CD25⁺ lymphocytes as a percentage of CD3⁺ (figure in top-right quadrant). This percentage was then used to calculate the percentage of CD3⁺CD4⁺CD25⁺ lymphocytes as a percentage of the CD3⁺CD4⁺ population (CD4 population being top-left and right quadrants added together).

Figure 3. Correlation of FOXP3 with CD4 count and viral load.

Panel A: CD4 count was negatively correlated with day 1 FOXP3 percentage in the HIV infected group. Panel B: The control group showed no correlation between CD4 count and FOXP3 percentage. Panel C: FOXP3 percentage stratified by CD4 count. Panel D: FOXP3 percentage was positively correlated with viral load.

Figure 4. FOXP3 expression after T cell receptor stimulation.

Panel A: FOXP3 expression as a percentage of T lymphocytes at baseline and after 4 days of cell culture, with and without T cell receptor stimulation with anti-CD3. Panel B: Proliferation of total CD4⁺ T cells and FOXP3⁺ expression in proliferated CD4⁺ T cells following T cell receptor stimulation with aCD3. Panel C: Representative plot of FOXP3⁺ expression in proliferated T cells (left-hand plot) following anti-CD3 stimulation compared with an unstimulated sample(right-hand plot). The small plots above show ancestry – lymphocytes were gated; followed by exclusion of events with high CFSE; followed by selection of CD3⁺CD4⁺ T cells. Proliferation is demonstrated by halving of CFSE fluorescence in cells that have divided (large plots below). In the anti-CD3 stimulated sample, FOXP3 expression is noted in cells which have proliferated (top-left quadrant) as well as those that have not proliferated (top-right quadrant).