FOXP3+Helios+ Regulatory T Cells, Immune Activation, and Advancing Disease in HIV-Infected Children

Abstract

Regulatory T cells (Tregs) are functionally suppressive CD4 T cells, critical for establishing peripheral tolerance and controlling inflammatory responses. Previous reports of Tregs during chronic HIV disease have conflicting results with higher or lower levels compared with controls. Identifying true Tregs with suppressive activity proves challenging during HIV infection, as traditional Treg markers, CD25 and FOXP3, may transiently upregulate expression as a result of immune activation (IA). Helios is an Ikaros family transcription factor that marks natural Tregs with suppressive activity and does not upregulate expression after activation. Coexpression of FOXP3 and Helios has been suggested as a highly specific marker of "bona fide" Tregs. We evaluated Treg subsets by FOXP3 coexpressed with either CD25 or Helios and their association with HIV disease progression in perinatally infected HIV-positive children. Identifying Tregs by FOXP3 coexpression with Helios rather than CD25 revealed markedly higher Treg frequencies, particularly in HIV+ children. Regardless of antiretroviral therapy, HIV-infected children had a selective expansion of memory FOXP3+Helios+ Tregs. The rise in memory Tregs correlated with declining HIV clinical status, indicated by falling CD4 percentages and CD4:CD8 ratios and increasing HIV plasma viremia and IA. In addition, untreated HIV+ children exhibited an imbalance between the levels of Tregs and activated T cells. Finally, memory Tregs expressed IA markers CD38 and Ki67 and exhaustion marker, PD-1, that tightly correlated with a similar phenotype in memory CD4 T cells. Overall, HIV-infected children had significant disruptions of memory Tregs that associated with advancing HIV disease.

Figure 1. Conventional Treg marker CD25 underestimates memory Tregs in HIV uninfected and infected children

(A) Singlet live CD4 T cells were first gated on CD45RO+ (memory) cells, then FOXP3+CD25+ or FOXP3+Helios+ cells to identify memory Tregs. Right graph shows paired comparison of %FOXP3+CD25+ with %FOXP3+Helios+ memory CD4 T cells in HIV negative children, with median with interquartile range. P-value reported for Wilcoxon matched-pairs signed rank test. (B) Paired comparison of %FOXP3+CD25+ (grey) and %FOXP3+Helios+ (black) memory Treg populations in HIV-, ART-, and ART+ children. P-values for paired comparisons between %FOXP3+CD25+ and %FOXP3+Helios+ memory Tregs were calculated with Wilcoxon matched-pairs signed rank test. (C) Cells were first gated on the FOXP3+Helios+ population in CD45RO+ CD4 T cells. Within FOXP3+Helios+ memory Tregs, CD25 negative cells were identified. Aggregate %CD25 negative cells within FOXP3+Helios+ memory CD4 T cells in HIV-, ART-, and ART+ children are shown on the right. Graph shows median with interquartile range. P-value reported for two-sided Mann-Whitney test. **** indicates p<0.0001.

Figure 2. HIV infected children have selective expansion of memory FOXP3+Helios+ Tregs that correlates with advancing HIV disease status

(A) Frequencies of FOXP3+Helios+ and FOXP3+Helios- in CD45RO+ (memory) CD4 T cells and FOXP3+Helios+ in CD45RO- (naïve) CD4 T cells in HIV-, ART-, and ART+ children. Graphs show medians with interquartile ranges. P-value reported for two-sided Mann-Whitney test with threshold significance of 0.05. (B) Percent of FOXP3+Helios+ in memory CD4 T cells vs. CD4 percent in total lymphocytes (top) and CD4:CD8 ratio (middle) in HIV-, ART-, and ART+ children. (B, bottom) Percent of FOXP3+Helios+ in memory CD4 T cells vs. plasma HIV RNA log copies/ml in ART- and ART+ children with HIV viral load above the detection limit 110 copies/ml. HIV- shown in open circles (○); HIV+ in filled circles (●). P- and r- values were calculated with Spearman's correlation test.

Figure 3. Disrupted balance between Tregs and inflammatory markers in untreated HIV infected children

Comparison of the following immune activation markers in HIV-, ART-, and ART+ children: (A) %CD38+HLA-DR+ in CD4 T cells, (B) %CD38+HLA-DR+ in CD8 T cells, and (C) sCD14 plasma levels. Graphs on the right show the ratio of %FOXP3+Helios+ in CD45RO+ CD4 T cells to each marker. Comparison graphs depict medians with interquartile ranges; p-values reported for two-sided Mann-Whitney test.

Figure 4. Elevated CD38, Ki67, and PD-1 expression in memory FOXP3-Helios- CD4 T cells and FOXP3+Helios+ Tregs of HIV+ children

(A) Comparison of percent CD38 (top), Ki67 (middle), and PD-1 (bottom) expression between HIV-, ART-, and ART+ children in (A) FOXP3-Helios- and (B) FOXP3+Helios+ in CD45RO+ CD4 T cells. Graphs depict median with interquartile range and p-value for two-sided Mann-Whitney test.

Figure 5. CD38+, Ki67+, and PD-1+ memory CD4 T cells correlate with frequency and phenotype of memory FOXP+Helios+ Tregs in HIV infected children

(A) Correlation between CD38+, Ki67+, and PD-1+ memory FOXP3-Helios- CD4 T cells and memory FOXP3+Helios+ Tregs in HIV infected children. (B) Correlation between FOXP3-Helios- and FOXP3+Helios+ memory CD4 T cells expressing CD38, Ki67, and PD-1 in HIV infected children. All data shown was gated on CD45RO+ memory CD4 T cells. P- and r- values were calculated with Spearman's correlation test.