Research ArticleHIV

Nonprogressing HIV-infected children share fundamental immunological features of nonpathogenic SIV infection

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Science Translational Medicine  28 Sep 2016:
Vol. 8, Issue 358, pp. 358ra125
DOI: 10.1126/scitranslmed.aag1048
  • Fig. 1. Normal CD4 T cell counts for age and low immune activation despite high viral loads in PNPs, in association with evidence of low levels of microbial translocation.

    (A to C) Absolute CD4 T cell count, CD4% (percentage of peripheral blood lymphocytes expressing CD4), and viral load in ART-naïve pediatric subject 517-C over the first 10 years of life. The 10th, 50th, and 90th centiles of absolute CD4 T cell counts and CD4% are shown in (A) and (B) for uninfected children over the first 10 years of life 18, 19. (D) Longitudinal viral load data from 170 ART-naïve PNPs. Viral load declines with age over the first 5 years (r = −0.34, P < 0.0001) but then plateaus thereafter. (E) Current absolute CD4 T cell counts and viral loads in 170 PNPs. (F) Lack of correlation between CD4 T cell count and viral load in 170 PNPs. (G and H) Immune activation (CD38/HLA-DR expression) on CD4+ T cells (G) and CD8+ T cells (H) is inversely correlated with absolute CD4 T cell count in ART-naïve children aged >5 years (n = 163 HIV-infected children and n = 21 HIV-uninfected children). (I) Levels of sCD14 are significantly lower in PNPs (absolute CD4 T cell count >750 cells/mm3; n = 14) than in progressors (absolute CD4 T cell count <500 cells/mm3; n = 16) and similar to HIV-uninfected children (n = 21). (J) Levels of iFABP are lower in PNPs (n = 14) and HIV-uninfected children (n = 21) compared to progressors (n = 19). Comparisons between groups were calculated by Mann-Whitney tests (*P < 0.05; **P < 0.01). P and r values for bivariate associations were calculated by Spearman’s rank correlation tests.

  • Fig. 2. CD4 T cell differentiation and function in nonprogressing pediatric infection.

    (A to C) IFN-γ, IL-2, and TNF-α intracellular cytokine staining (ICS) response to SEB and Gag in PNPs. (A) Representative fluorescence-activated cell sorting (FACS) staining in a progressor and a nonprogressor. (B) ICS responses to SEB in nonprogressors (n = 14), progressors (n = 32), and uninfected pediatric controls (n = 19), gating on bulk CD4+ T cells. (C) ICS responses to a Gag peptide pool in nonprogressors (n = 26) and progressors (n = 11), gating on bulk CD4+ T cells. (D and E) CD4 T cell differentiation in pediatric progressors and nonprogressors (n = 161). (D) Representative FACS staining in a progressor and a nonprogressor. (E) Percentage of CD4 T cells by absolute CD4 T cell count defined as naïve, central memory, effector memory, or terminally differentiated effector memory, by CCR7 and CD45RA expression as shown in (D). Correlation between absolute CD4 T cell count and percent CD4 T cells in each subset shown as Spearman’s rank correlation coefficient and P value. Temra, effector memory RA. (F and G) Functional differences between distinct CD4 T cell subsets. (F) Representative FACS staining in response to SEB showing that IFN-γ–producing cells are predominantly effector memory, and IL-2–producing cells are predominantly naïve and central memory in phenotype. (G) Contribution of IL-2, IFN-γ, and TNF-α to the total CD4 T cell SEB response. The proportion of cells producing each cytokine to the total CD4 T cell SEB response was determined (data from 11 nonprogressors). (H to J) PD-1 expression on CD4 T cell subsets in pediatric progressors, nonprogressors, and uninfected controls (H). Increased PD-1 expression on CD4+ T cells in pediatric progressors (n = 20) compared to nonprogressors (n = 32) and uninfected pediatric controls (n = 22). (I) Increased PD-1 expression on CD4+ T cell subsets in pediatric progressors (n = 20) compared to nonprogressors (n = 30). (J) Representative FACS plots showing differential cytokine staining in relation to PD-1 expression. (K and L) T cell activation by memory subsets. Immune activation (CD38/HLA-DR expression) on CD4+ central memory T cells (Tcm) (K) and other CD4+ and CD8+ T cell memory subsets (l) is inversely correlated with absolute CD4 T cell count in ART-naïve children aged >5 years (n = 97). Comparisons between groups were calculated by Mann-Whitney tests (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). P and r values for bivariate associations were determined by Spearman’s rank correlation tests.

  • Fig. 3. Associations between immunological and clinical variables.

    (A) Selected bivariate associations between immunological and clinical measurements in HIV-infected, ART-naïve children aged >5 years (n = 163, Spearman’s rank correlation tests). (B) Correlation matrix in a subset of n = 45 with available data for all regarded parameters from the same study visit. Positive correlations are indicated in blue and inverse correlations are indicated in red. Darker color shades indicate higher r values on the basis of Spearman’s rank correlation tests. Clustering of variables is based on principal components analysis using the R package corrgram and reveals two well-differentiated groups of parameters, one associated with disease nonprogression (upper left quadrant) and the other associated with disease progression (lower right quadrant).

  • Fig. 4. Neutralization of a panel of 16 tier 2 and tier 3 subtype C, B, and A viruses by pediatric and adult plasma samples.

    (A) Neutralization breadth in PNPs (n = 66) and progressors (n = 19) compared with adults (n = 21). (B) The frequency of bnAbs among PNPs (median: age, 6.6 years; absolute CD4 T cell count, 1050 cells/mm3; viral load, 14,000 copies/ml), pediatric progressors (median: age, 8.2 years; absolute CD4 T cell count, 225 cells/mm3; viral load, 71,803 copies/ml), and adults (5 years after infection; median: absolute CD4 T cell count, 449 cells/mm3; viral load, 31,200 copies/ml). (C) Correlation between viral load and percent viruses neutralized among pediatric subjects. P and r values were calculated by Spearman’s rank correlation tests. (D to F) Geometric means of neutralization titers for pediatric and adult samples against subtype C (n = 6), B (n = 6), and A (n = 4) viruses. Comparisons between groups were calculated by Mann-Whitney tests.

  • Fig. 5. HIV-specific CD8+ T cell responses in pediatric infection in relation to viral load and absolute CD4 count.

    (A to C) Breadth of IFN-γ ELISPOT responses correlates with viral load in a protein-specific fashion. (A and B) Total breadth and Gag-specific breadth of IFN-γ ELISPOT response (n = 90). (C) Env-specific breadth of IFN-γ ELISPOT response (n = 90). (D and E) Total breadth and Pol-specific breadth of IFN-γ ELISPOT response in ART-naïve pediatric subjects are inversely correlated with absolute CD4 count (n = 90). (F) Total breadth of IFN-γ ELISPOT response directly correlates with CD8+ T cell activation in pediatric infection (n = 30, Spearman’s rank correlation tests).

  • Fig. 6. CCR5 expression and HIV infection are lower in central memory CD4+ T cells in PNPs than in progressors.

    (A) Representative FACS data of CCR5 expression in pediatric progressors versus nonprogressors. (B) CCR5 expression on CD4+ T cell subsets in ART-naïve children aged >5 years by absolute CD4 T cell count (n = 59). P and r values were calculated by Spearman’s rank correlation tests. (C) Absolute CD4 T cell counts and viral loads in PNPs and adult future progressors (FP). (D) HIV infection in Tn, Tscm, Tcm, and Tem in PNPs and adult future progressors, determined by quantitative PCR (qPCR) of HIV DNA in sorted CD4 T cell subsets. P values were determined by Mann-Whitney tests.

  • Table 1. Association of clinical and immunological parameters with CD4 T cell count.

    Selected regression parameter estimates with the three LASSO approaches implemented in the R packages grplasso, penalized, and glmnet are shown in the first four columns. Standardized regression parameter estimates for the selected set of covariates by an unregularized conventional GLM using the R-function glm are shown in the last three columns.

    LASSOGLM
    VariablegrplassoPenalizedglmnetVariableβ-CoefficientP
    CD4 CD38+DR+−0.1701−0.1754−0.1718CD4 CD38+DR+−0.3123<10−10
    CD4 Tem−0.0549−0.0603−0.0567CD4 Tem−0.1630<10−10
    Age at visit−0.0632−0.0674−0.0646Age at visit−0.1582<10−10
    CD8 Tnaïve0.10000.10080.1003CD8 Tnaïve0.1105<10−10
    sCD14−0.0472−0.0498−0.0481sCD14−0.1098<10−10
    CD4 Tnaïve0.13220.12740.1305CD4 Tnaïve0.04750.0147
    iFABP000
    CD8 CD38+DR+000
    CD8 Tem000
    Sex000
    Viral load000
    CD4 PD1000
    CD8 PD1000
    Gag CD4 IFN-γ000
    Gag CD4 IL-2000
    Gag CD8 IFN-γ000
    Gag CD8 IL-2000

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/358/358ra125/DC1

    Fig. S1. Decreased CD4 T cell function and increased expression of exhaustion markers in pediatric progressors.

    Fig. S2. nAb potency in pediatric and adult subjects.

    Table S1. CD8+ T cell activation is independently associated with total breadth of HIV-specific cytotoxic T lymphocyte responses and viral load.

    Table S2. List of pseudoviruses tested for neutralizing sera activity by HIV-1 subtype.

    Source data

  • Supplementary Material for:

    Nonprogressing HIV-infected children share fundamental immunological features of nonpathogenic SIV infection

    Maximilian Muenchhoff, Emily Adland, Owen Karimanzira, Carol Crowther, Matthew Pace, Anna Csala, Ellen Leitman, Angeline Moonsamy, Callum McGregor, Jacob Hurst, Andreas Groll, Masahiko Mori, Smruti Sinmyee, Christina Thobakgale, Gareth Tudor-Williams, Andrew J. Prendergast, Henrik Kloverpris, Julia Roider, Alasdair Leslie, Delane Shingadia, Thea Brits, Samantha Daniels, John Frater, Christian B. Willberg, Bruce D. Walker, Thumbi Ndung'u, Pieter Jooste, Penny L. Moore, Lynn Morris, Philip Goulder*

    *Corresponding author. Email: philip.goulder{at}paediatrics.ox.ac.uk

    Published 28 September 2016, Sci. Transl. Med. 8, 358ra125 (2016)
    DOI: 10.1126/scitranslmed.aag1048

    This PDF file includes:

    • Fig. S1. Decreased CD4 T cell function and increased expression of exhaustion markers in pediatric progressors.
    • Fig. S2. nAb potency in pediatric and adult subjects.
    • Table S1. CD8+ T cell activation is independently associated with total breadth of HIV-specific cytotoxic T lymphocyte responses and viral load.
    • Table S2. List of pseudoviruses tested for neutralizing sera activity by HIV-1 subtype.
    • Source data

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