Research ArticleHIV

Elite control of HIV is associated with distinct functional and transcriptional signatures in lymphoid tissue CD8+ T cells

See allHide authors and affiliations

Science Translational Medicine  18 Dec 2019:
Vol. 11, Issue 523, eaax4077
DOI: 10.1126/scitranslmed.aax4077
  • Fig. 1 CD8+ T cells from the LNs of ECs display superior antiviral efficacy.

    (A) Representative RNAscope images of paraffin-embedded LN biopsies from one EC and one CP (left) and data quantification (right). TCZ, T cell zone; BCF, B cell follicle. EC, n = 7; ART, n = 10; CP, n = 11. (B) Quantification of cell-associated HIV DNA and RNA from negatively selected LN CD4+ T cells by qPCR. One data point on the DNA plot was adjusted from 0 to 1 to display on the log scale. Error bars represent median and interquartile ranges. EC, n = 5; CP, n = 8. (C) Representative flow plots from a viral suppression assay using LN-derived CD8+ T cells from an EC cocultured with autologous LN-derived CD4+ T cells infected with HIV BAL virus in vitro (left) and data quantification (right). Dotted lines represent median and interquartile ranges. EC, n = 4; ART, n = 8; CP, n = 6. Flow cytometry data were pregated on single live CD8 T cells. Significance was determined using the Kruskal-Wallis test with Dunn’s correction (A), an unpaired t test with Welch’s correction (B), or Welch’s ANOVA with Benjamini-Hochberg correction (C). *P < 0.05.

  • Fig. 2 Cytolytic CD8+ T cells are rare in the LNs of ECs.

    (A) Representative flow plots showing perforin and granzyme B expression in LN memory CD8+ T cells (left) and data quantification (right). EC, n = 13; ART, n = 11; CP, n = 13; acutely infected individuals (acute), n = 7. (B) Correlation between the frequency of perforin+ granzyme B+ LN memory CD8+ T cells and pVL. Values for pVL below the limit of detection were plotted as 40 copies/ml. (C and D) Representative immunohistochemistry images of LN biopsies (left) stained for perforin (C) or granzyme B (D) and data quantification across donor groups (right). EC, n = 5; ART, n = 9; CP, n = 14. (E) Representative flow plots showing HIV-specific tetramer staining of LN memory CD8+ T cells (left) and data quantification (right). Each data point on the quantification plot represents a distinct tetramer+ population. (F) Representative flow plots showing HIV-specific tetramer+ cells (colored contours) overlaid on total LN memory CD8+ T cells (gray background) gated to display perforin and granzyme B expression (left) and data quantification (right). Each data point on the quantification plot represents a distinct tetramer+ population. EC, n = 25; ART, n = 6; CP, n = 8. (G) Representative flow plots of redirected killing assays (left) and data quantification (right). Data were pregated on single live TFL4+ cells. (H) Representative flow plots showing CXCR5 expression on LN-derived memory CD8+ T cells (left) and data quantification across donor groups (right). EC, n = 12; ART, n = 9; CP, n = 8. (I) Quantification of CXCR5 expression on LN tetramer+ memory CD8+ T cells. Each data point on the quantification plot represents a distinct tetramer+ population. EC, n = 25; ART, n = 6; CP, n = 8. (J) Representative flow plots showing perforin and granzyme B expression in LN-derived CXCR5+ and CXCR5 memory CD8+ T cells (left) and data quantification (right). Error bars represent median and interquartile ranges. EC, n = 12; ART, n = 9; CP, n = 8. Flow cytometry data, except in (G), were pregated using scheme shown in fig. S2A. Significance was determined using the Kruskal-Wallis test with Dunn’s correction (A, C, D, E, F, H, I, and J) or the Spearman’s rank correlation (B) or a two-way ANOVA (G). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Distinct transcriptomic signatures characterize HIV-specific CD8+ T cells from the LNs of ECs and CPs.

    (A) Setup of the scRNAseq experiment, including data exclusion criteria. ERCC, External RNA Controls Consortium. (B) tSNE visualization of the unsupervised kernel-based algorithm. (C) Violin plots of cytolysis-related genes showing z-normalized expression. (D) Average percent error of k-fold cross-validation of support vector machine (SVM) models using subsets of the L0 norm–ranked gene list. Each cross-validation was reiterated 100 times. (E) Heatmap showing z-normalized expression of the 200 feature genes. (F) GO analysis of the feature gene list using topGO. The top 25 enriched terms were reported, and P values were calculated using the classic Fisher method. (G) tSNE feature plots showing the distribution of gene expression. tSNE coordinates were calculated using the first 50 principal components and iterated 1000 times. The color gradient displays relative log-normalized gene expression. Significance was determined using scDD calculations (C and G). **P < 0.01 and ***P < 0.001. The color of asterisks indicates the group with higher average expression of the indicated gene.

  • Fig. 4 Polyfunctionality is associated with protein translation efficiency in HIV-specific CD8+ T cells from the LNs of ECs.

    (A) Representative flow plots showing the production of TNF and IL-2 by HIV-specific CD8+ T cells from the LNs of one EC and one CP in response to cognate peptide stimulation. Plots were pregated on total memory CD8+ T cells using the scheme shown in fig. S2A. (B) Quantification of cytokine production by HIV-specific CD8+ T cells from LNs. The total response for each readout was determined by summing the corresponding frequencies after background subtraction of all Boolean gates using permutations of CD107a, IFNγ, TNF, MIP-1β, and IL-2. Error bars represent mean and SEM. (C) Polyfunctionality plot of HIV-specific CD8+ T cells from LNs. Only combinations of four and five functions are shown. Error bars represent mean and SEM. EC, n = 49; ART, n = 10; CP, n = 27. (D) Pie charts summarizing all combinations of functions. (E) Heat map showing z-normalized expression of genes from the feature list identified by GO protein translation terms. (F) GSEA of the scRNAseq data with reference to the Reactome Pathway Database, reporting the top 10 enriched pathways in ECs and CPs. UTR, untranslated region; NES, normalized enrichment score. (G) Quantification of protein translation efficiency in LN-derived CD8+ T cells. LNMCs were incubated with HPG in methionine-free medium for 6 hours in the presence or absence of HIV-derived peptides or anti-CD3. Responding cells were defined using Boolean gating for IFNγ+ and/or TNF+ events. CM, central memory; EM, effector memory. EC, n = 3; ART, n = 3; CP, n = 4. Error bars represent mean and SEM. Significance was determined using a one-way ANOVA (D and G) or a two-way ANOVA (B). *P < 0.05, **P < 0.01, and ***P < 0.001. MFI, mean fluorescence intensity. SEM, standard errors of the mean.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/523/eaax4077/DC1

    Fig. S1. HIV RNA is present in B cell follicles and the T cell zone in LNs.

    Fig. S2. Relatively few CD8+ T cells from the LNs of ECs express perforin and granzyme B.

    Fig. S3. CD8+ T cells from the LNs of ECs do not up-regulate cytolytic molecules in response to cognate antigen exposure.

    Fig. S4. Active caspase-3 detection effectively captures killed target cells.

    Fig. S5. Differential expression analysis of the scRNAseq data.

    Table S1. Clinical characteristics of the donors included in this study.

    Table S2. Complete list of 2264 genes that were differentially expressed between HIV-specific CD8+ T cells from the LNs of ECs and HIV-specific CD8+ T cells from the LNs of CPs.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. HIV RNA is present in B cell follicles and the T cell zone in LNs.
    • Fig. S2. Relatively few CD8+ T cells from the LNs of ECs express perforin and granzyme B.
    • Fig. S3. CD8+ T cells from the LNs of ECs do not up-regulate cytolytic molecules in response to cognate antigen exposure.
    • Fig. S4. Active caspase-3 detection effectively captures killed target cells.
    • Fig. S5. Differential expression analysis of the scRNAseq data.
    • Table S1. Clinical characteristics of the donors included in this study.
    • Table S2. Complete list of 2264 genes that were differentially expressed between HIV-specific CD8+ T cells from the LNs of ECs and HIV-specific CD8+ T cells from the LNs of CPs.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Stay Connected to Science Translational Medicine

Navigate This Article