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

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

Reduction of lung HIV-1 RNA levels and HIV-1 envelope mutations associated with aerosol IFN-γ treatment.

<p>(<b>A</b>) HIV-1 RNA levels in bronchoalveolar lavage (BAL) fluids of 5 HIV-1/<i>M. tb</i> co-infected patients before (pre IFN-γ) and after (post IFN-γ) treatment with aerosolized IFN-γ. HIV-1 RNA viral load at post-treatment was significantly reduced in BAL fluids (p<0.05; mean±SE). (<b>B</b>) Nucleotide mutations around the V3 region of HIV-1 envelope post IFN-γ treatment. The percentages of mutations found in the V3 region were calculated by comparing virus sequences before and after IFN-γ therapy. A to G and G to A mutation occurred significantly as compared with other mutations (p<0.01).</p

ADAR1 overexpression and knock down in a chronically HIV-1 infected macrophage cell model system.

<p>(<b>A</b>). Chronic HIV-1 infection induced ADAR1 <i>in</i><i>vitro</i>. Expression of 150 kD and 110 kD ADAR1 was measured in OM10.1 cells and YS+OA cells treated with and without indinavir (IDV). OM10.1 is a latently HIV-1 infected macrophage cell line (HL-60 cell is the parental uninfected cell line). YS+OA cells are OM10.1 cells that stably over-express ADAR1. YS+OA cells have increased expression of the ADAR1 150 kDa isoform. Densitometry data are shown below the lanes. (<b>B</b>) HIV-1 RNA copies in the culture supernatant from ADAR1-overexpressing YS+OA were significantly lower than those of ADAR1-knocked down YS-OA. (p<0.05; mean±SE, n = 6). YS-OA is OM10.1 cells that are stably transfected with shRNA to knock down of ADAR1. (<b>C</b>) Intra-cellular p24 gag and its precursor Pr55 protein in ADAR1-overexpressing YS+OA as compared to ADAR1-knocked down YS-OA and untreated OM10.1. (<b>D</b>) Nuclear run-on transcription assays of YS+OA cells and YS-OA cells. Synthesis of HIV-1 nascent RNA was calculated as the ratio of HIV-1 RNA to GAPDH RNA.</p

A diagram showing the distinct targets of adenosine and cytidine deaminase in the HIV-1 life cycle.

<p>IFN-γ induces both adenosine deaminase (ADAR1L) and cytidine deaminase (APOBEC3G). Adenosine deaminase may inhibit the step after viral transcription whereas cytidine deaminase acts on reverse transcription immediately after virus entry.</p