High soluble CD163 levels correlate with disease progression and inflammation in Kenyan children with perinatal HIV-infection

Objectives: CD163 is a hemoglobin scavenger receptor on monocytes and macrophages, cleaved to soluble CD163 (sCD163) in the plasma following activation. In HIV+ adults, sCD163 is linked to non-AIDS morbidity and predicts mortality, but there is limited data in children. We investigated sCD163 levels in HIV+ children and their correlations with disease progression, immune activation and gut mucosal damage. Design and methods: We quantified sCD163 levels in Kenyan children aged 0–20 years with perinatal HIV infection, including 74 antiretroviral treatment (ART)-naïve (ART−) and 64 virally suppressed on ART (ART+), and 79 HIV unexposed-uninfected controls (HIV−). The cohort was divided into age groups 0–5 (younger) and 5–20 (older) years. Correlations between sCD163 and HIV viral load, %CD8+, CD4+ : CD8+ ratio, markers of T-cell activation and proliferation, and gut mucosal damage were also assessed. Results: ART− children have higher sCD163 levels compared with HIV− and ART+ children (P ≤ 0.01); ART+ have equivalent sCD163 levels to HIV− children. In a prospective analysis, sCD163 levels decreased in older ART− children after 12 months of treatment (P < 0.0001). Regardless of age, sCD163 levels correlate with clinical disease progression measured by %CD4+ T cells, CD4+ : CD8+ T-cell ratios and HIV viral load. sCD163 levels directly correlate with T-cell activation markers CD38, human leukocyte antigen-DR isotype, and Ki67 (P ≤ 0.01). Conclusion: High plasma sCD163 levels in HIV+ children correlate with advancing disease and T-cell activation. ART initiation normalizes sCD163 levels and may alleviate HIV-related morbidities and improve long-term pediatric outcomes

Low Peripheral T Follicular Helper Cells in Perinatally HIV-Infected Children Correlate With Advancing HIV Disease.

Background: T follicular helper (Tfh) cells are crucial for B cell differentiation and antigen-specific antibody production. Dysregulation of Tfh-mediated B cell help weakens B cell responses in HIV infection. Moreover, Tfh cells in the lymph node and peripheral blood comprise a significant portion of the latent HIV reservoir. There is limited data on the effects of perinatal HIV infection on Tfh cells in children. We examined peripheral Tfh (pTfh) cell frequencies and phenotype in HIV-infected children and their associations with disease progression, immune activation, and B cell differentiation. Methods: In a Kenyan cohort of 76 perinatally HIV-infected children, comprised of 43 treatment-naïve (ART-) and 33 on antiretroviral therapy (ART+), and 42 healthy controls (HIV-), we identified memory pTfh cells, T cell activation markers, and B cell differentiation states using multi-parameter flow cytometry. Soluble CD163 and intestinal fatty acid-binding protein plasma levels were quantified by ELISA. Results: ART- children had reduced levels of pTfh cells compared with HIV- children that increased with antiretroviral therapy. HIV+ children had higher programmed cell death protein 1 (PD-1) expression on pTfh cells, regardless of treatment status. Low memory pTfh cells with elevated PD-1 levels correlated with advancing HIV disease status, indicated by increasing HIV viral loads and T cell and monocyte activation, and decreasing %CD4 and CD4:CD8 ratios. Antiretroviral treatment, particularly when started at younger ages, restored pTfh cell frequency and eliminated correlations with disease progression, but failed to lower PD-1 levels on pTfh cells and their associations with CD4 T cell percentages and activation. Altered B cell subsets, with decreased naïve and resting memory B cells and increased activated and tissue-like memory B cells in HIV+ children, correlated with low memory pTfh cell frequencies. Last, HIV+ children had decreased proportions of CXCR5+ CD8 T cells that associated with low %CD4 and CD4:CD8 ratios. Conclusion: Low memory pTfh cell frequencies with high PD-1 expression in HIV+ children correlate with worsening disease status and an activated and differentiated B cell profile. This perturbed memory pTfh cell population may contribute to weak vaccine and HIV-specific antibody responses in HIV+ children. Restoring Tfh cell capacity may be important for novel pediatric HIV cure and vaccine strategies

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

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. J Acquir Immune Defic Syndr 2016 Aug 15; 72(5):474-84

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-Infected Children Have Elevated Levels of PD-1+ Memory CD4 T Cells With Low Proliferative Capacity and High Inflammatory Cytokine Effector Functions.

Background: During human immunodeficiency virus (HIV) disease, chronic immune activation leads to T-cell exhaustion. PD-1 identifies exhausted CD8 T cells with impaired HIV-specific effector functions, but its role on CD4 T cells and in HIV-infected children is poorly understood. Methods: In a Kenyan cohort of vertically HIV-infected children, we measured PD-1+ CD4 T-cell frequencies and phenotype by flow cytometry and their correlation with HIV disease progression and immune activation. Second, in vitro CD4 T-cell proliferative and cytokine responses to HIV-specific and -nonspecific stimuli were assessed with and without PD-1 blockade. Results: HIV-infected children have increased frequencies of PD-1+ memory CD4 T cells that fail to normalize with antiretroviral treatment. These cells are comprised of central and effector memory subsets and correlate with HIV disease progression, measured by viral load, CD4 percentage, CD4:CD8 T-cell ratio, and immune activation. Last, PD-1+ CD4 T cells predict impaired proliferative potential yet preferentially secrete the Th1 and Th17 cytokines interferon-γ and interleukin 17A, and are unresponsive to in vitro PD-1 blockade. Conclusions: This study highlights differences in PD-1+ CD4 T-cell memory phenotype and response to blockade between HIV-infected children and adults, with implications for potential immune checkpoint therapies. J Infect Dis 2017 Sep 15; 216(6):641-50

Low Peripheral T Follicular Helper Cells in Perinatally HIV-Infected Children Correlate With Advancing HIV Disease

BackgroundT follicular helper (Tfh) cells are crucial for B cell differentiation and antigen-specific antibody production. Dysregulation of Tfh-mediated B cell help weakens B cell responses in HIV infection. Moreover, Tfh cells in the lymph node and peripheral blood comprise a significant portion of the latent HIV reservoir. There is limited data on the effects of perinatal HIV infection on Tfh cells in children. We examined peripheral Tfh (pTfh) cell frequencies and phenotype in HIV-infected children and their associations with disease progression, immune activation, and B cell differentiation.MethodsIn a Kenyan cohort of 76 perinatally HIV-infected children, comprised of 43 treatment-naïve (ART−) and 33 on antiretroviral therapy (ART+), and 42 healthy controls (HIV−), we identified memory pTfh cells, T cell activation markers, and B cell differentiation states using multi-parameter flow cytometry. Soluble CD163 and intestinal fatty acid-binding protein plasma levels were quantified by ELISA.ResultsART− children had reduced levels of pTfh cells compared with HIV− children that increased with antiretroviral therapy. HIV+ children had higher programmed cell death protein 1 (PD-1) expression on pTfh cells, regardless of treatment status. Low memory pTfh cells with elevated PD-1 levels correlated with advancing HIV disease status, indicated by increasing HIV viral loads and T cell and monocyte activation, and decreasing %CD4 and CD4:CD8 ratios. Antiretroviral treatment, particularly when started at younger ages, restored pTfh cell frequency and eliminated correlations with disease progression, but failed to lower PD-1 levels on pTfh cells and their associations with CD4 T cell percentages and activation. Altered B cell subsets, with decreased naïve and resting memory B cells and increased activated and tissue-like memory B cells in HIV+ children, correlated with low memory pTfh cell frequencies. Last, HIV+ children had decreased proportions of CXCR5+ CD8 T cells that associated with low %CD4 and CD4:CD8 ratios.ConclusionLow memory pTfh cell frequencies with high PD-1 expression in HIV+ children correlate with worsening disease status and an activated and differentiated B cell profile. This perturbed memory pTfh cell population may contribute to weak vaccine and HIV-specific antibody responses in HIV+ children. Restoring Tfh cell capacity may be important for novel pediatric HIV cure and vaccine strategies

Disease Progression in Children with Perinatal HIV Correlates with Increased PD-1+ CD8 T Cells that Coexpress Multiple Immune Checkpoints.

BACKGROUND: PD-1 marks exhausted T cells, with weak effector functions. Adults living with HIV have increased levels of PD-1+ CD8 T cells that correlate with HIV disease progression, yet little is known about the role of PD-1+ CD8 T cells in children with perinatal HIV. METHODS: We enrolled 76 Kenyan children with perinatal HIV and 43 children who were HIV unexposed and quantified PD-1 levels on CD8 T cells, their coexpression with immune checkpoints (IC) 2B4, CD160 and TIM3, correlates with immune activation and HIV disease progression and HIV-specific and non-specific proliferative responses. RESULTS: PD-1+ CD8 T cell frequencies are elevated in children with perinatal HIV and associated with disease progression. The majority of PD-1+ CD8 T cells coexpress additional ICs. ART initiation lowers total PD-1 levels and coexpression of multiple ICs. The frequency of PD-1 + 2B4+CD160+TIM3- in PD-1+ CD8 T cells, predicts weaker HIV-specific proliferative responses, suggesting this subset is functionally exhausted. CONCLUSION: Children with perinatal HIV have high PD-1+ CD8 T cells that are a heterogeneous population differentially coexpressing multiple ICs. Understanding the complex interplay of ICs is essential to guide the development of PD-1 directed immunotherapies for pediatric HIV remission and cure

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