Immune activation despite preserved CD4 T cells in perinatally HIV-infected children and adolescents

<div><p>Background</p><p>HIV disease progresses more rapidly in children than adults with mortality rates exceeding 50% by 2 years of age without antiretroviral therapy (ART) in sub-Saharan Africa. Recent World Health Organization (WHO) guidelines recommend universal treatment for all living persons with HIV, yet there is limited supporting evidence in pediatric populations. The objective of this study was to determine whether CD4 cell counts reflect immunological markers associated with disease progression in ART naïve perinatally-infected HIV+ children and adolescents and their response to ART.</p><p>Methods</p><p>PBMC and plasma samples were collected from 71 HIV negative and 132 HIV+ children (65 ART naïve and 67 on ART) between ages 1–19 years from Mombasa, Kenya. Untreated HIV+ subjects were sub-categorized by high or low CD4 T cell counts. Immune activation markers CD38, HLA-DR and Ki67 were analyzed by flow cytometry. Plasma soluble CD14 (sCD14) was quantified by ELISA.</p><p>Results</p><p>HIV-infected children and adolescents with preserved CD4 cell counts had depleted CD4 percentages and CD4:CD8 ratios, and high immune activation levels. ART initiation rapidly and persistently reversed T cell activation, but failed to normalize CD4:CD8 ratios and plasma sCD14 levels.</p><p>Conclusions</p><p>Diminished CD4 percentages and CD4:CD8 ratios along with profound immune activation occur independent of CD4 cell count thresholds in ART naïve HIV+ children and adolescents. Immediate ART initiation, as recommended in the most recent WHO guidelines may protect them from pathologic sequelae associated with persistent inflammation.</p></div

HIV disease progression in ART-CD4_hi children and adolescents.

<p>Comparison of the <b>(A)</b> CD4 percent and <b>(B)</b> HIV viral load in HIV-, ART-CD4_hi, ART- and ART+ children and adolescents. (C) Comparison of the Í38+DR+ CD8 T cells in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ children and adolescents. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test. **** p<0.0001; *** p<0.001; ** p<0.01; * p<0.05. (D) The percent of CD38+DR+ CD8 T cells vs. CD4 cell count in total lymphocytes in children and adolescents. P and <i>R</i>^<i>2</i> values are shown for a linear regression model. Shaded bar represents interquartile range of CD38+HLA-DR+ CD8 T cell frequencies in HIV-uninfected children.</p

Significant CD4 T cell activation in ART-CD4_hi children and adolescents.

<p>(A) Comparison of frequencies of the CD38+HLA-DR+ CD4 T cells in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ children and adolescents. Percentages of (B) CD38+ and (C) Ki67+ cells within in memory CD4 T cells in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ children and adolescents. To identify memory populations, CD4 T cell were first gated on CD45RO+ CD4 T cells. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test. **** p<0.0001; *** p<0.001; ** p<0.01; * p<0.05.</p

Antiretroviral therapy lowers immune activation rapidly and persistently.

<p>Comparisons of <b>(A)</b> the CD4 percentages and <b>(B-E)</b> frequencies of the following IA markers in paired longitudinal samples pre-ART (T0), 5–7 months post-ART (T1), and 10–16 months post-ART (T2): CD38+HLA-DR+ <b>(B)</b> CD8 and <b>(C)</b> CD4 T cells and <b>(D)</b> CD38+ <b>(E)</b> and Ki67+ memory CD4 T cells. Right graphs show comparison between IA markers in HIV- and the prospective cohort pre- and post-ART. Bars represent median values with IQRs. P values were calculated using the paired Wilcoxon matched-pairs signed rank test (left graphs) and the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test (right graphs). **** p<0.0001; *** p<0.001; ** p<0.01; * p<0.05.</p

Subject characteristics.

<p>Subject characteristics.</p

ART-CD4_hi children and adolescents exhibit markers of HIV progression associated with mortality.

<p>Comparison of the CD4:CD8 ratio in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ <b>(A)</b> children and <b>(B)</b> adolescents. <b>(C)</b> CD4:CD8 ratios are shown in subjects before antiretroviral treatment (T0), 5–7 months post-ART (T1), and 10–16 months post-ART (T2) and in comparison to HIV- children on the right. Plasma sCD14 levels in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ <b>(D)</b> children and <b>(E)</b> adolescents and <b>(F)</b> pre-and post-ART. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test and the Wilcoxon matched-pairs signed rank test (C and F). **** p<0.0001; *** p<0.001; ** p<0.01; * p<0.05.</p

CD8 MAIT cells and HIV disease progression.

<p>(A) Correlation between CD8+ MAIT cells and %CD4+ T cells in HIV+ (filled circles) and HIV- (open circles) children. (B) Percent CD8+ MAIT cells vs. HIV log copies/mL in viremic HIV+ children. (C) Comparison between CD8+ MAIT cells in HIV- and HIV+ children divided into groups with CD4:CD8 ratios greater than or equal to one and ratios less than one. (D) CD8+ MAIT cells vs. plasma sCD14 levels in HIV+ (closed circles) and HIV- (open circles) children.</p

CD8 MAIT cells gradually recover with ART in HIV+ children.

<p>(A) CD8+ MAIT cells in ART- children before (pre-ART) and 10–21 months after ART initiation (post-ART). (B) CD8+ MAIT cells in ART+ children at baseline (T0) and at 10–21 month follow-up visit (T1). (C) Age in years at the time of ART initiation vs. fold change in %CD8 MAIT cells in ART- (post- / pre-ART CD8 MAIT; left graph) and ART+ children (T1/T0 CD8 MAIT; right graph). (D) Log CD8+ MAIT cells in ART+ children vs. total duration of ART in years.</p

MAIT cells in HIV infected and uninfected children.

<p>(A) FACS plot showing representative gating to identify CD8 MAIT cells in an HIV- and HIV+ subject. Plot shown is gated on CD8+ T cells. MAIT cells are identified as the Vα7.2⁺CD161⁺ population. (B) CD8+ MAIT cells in HIV-, ART-, and ART+ children. (C) CD8+ MAIT cells vs. age in years in HIV- (open circles) and HIV+ (filled circles) children. CD8+ Vα7.2+CD161- non-MAIT cells (D) and CD4-CD8- Vα7.2⁺CD161⁺ MAIT cells (E) in HIV-, ART-, and ART+ children.</p

Subject Characteristics.

<p>Subject Characteristics.</p