Research ArticleHIV

The receptor repertoire and functional profile of follicular T cells in HIV-infected lymph nodes

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Science Immunology  06 Apr 2018:
Vol. 3, Issue 22, eaan8884
DOI: 10.1126/sciimmunol.aan8884
  • Fig. 1 GC TFH cells become clonally expanded.

    (A) Representative plots showing sorting strategy to identify naïve, memory, and GC TFH cells. (B) Breakdown of the proportion of the TCR repertoire represented by clones of different sizes for sorted naïve, memory, and GC TFH cells from HIV+ LNs. TCR clone size was normalized by the total number of TCR transcripts on nucleotide sequences. (C) NSE of the TCR repertoire of sorted naïve, memory, and GC TFH cells. Gray lines link the same patient. Bars indicate means. *P < 0.05 by two-tailed Wilcoxon signed-rank test (n = 8 HIV-infected LNs).

  • Fig. 2 Antigen-driven clonal selection signature in GC TFH cells of HIV-infected LNs.

    (A) Representative degeneracy plot from sample H2. Coding degeneracy level [number of unique TCR nucleotide (nt) sequences encoding a common CDR3 amino acid sequence] of each CDR3 amino acid sequence is plotted against their frequency (measured as percentage of total TCR transcripts) in naïve, memory, and GC TFH cells. Each dot is a unique CDR3 amino acid sequence. Red dashed lines indicate cutoffs for degenerate (two or more nucleotide sequences coding for the same amino acid sequence; horizontal) and expanded (0.1% or more of TCR transcripts; vertical) clones. Red arrow points to example degenerate clone in (B). (B) Example of CDR3 amino acid degeneracy. Amino acid (top row) and nucleotide (bottom row) sequences for three distinct nucleotide sequences (0.41% of total TCR transcripts) that code for the same amino acid sequence as indicated by arrow in (A): Y = 3 and X = 0.41%. Red boxes and highlights indicate redundant codons. (C) Comparison of Q1 degenerate-abundant clone percentage in naïve, memory, and GC TFH cells. Gray lines link the same patient. Bars indicate means. *P < 0.05 by two-tailed Wilcoxon signed-rank test (n = 8 HIV-infected LNs).

  • Fig. 3 GC TFH cells exhibit HIV antigen–driven clonal expansion and selection.

    (A) Gag-specific TCR clones overlap with HIV+ LN CD4+ T cell populations. Each thin slice of the arc represents a unique TCR sequence, ordered by the clone size (darker green for larger clones, inner circle). Gray curves indicate Gag-specific TCR nucleotide sequences found in naïve (gray, outer circle), memory (blue, outer circle), and GC TFH (orange, outer circle) populations. No Gag-overlapping clones were detected for one individual, H8 (not shown). (B) Number of Gag-specific TCR clones observed in naïve, memory, and GC TFH populations. Gray lines link the same patient. Bars indicate means (P values by two-tailed paired t test). (C) Mean clone size of Gag-specific T cells, HA-specific T cells, and bulk clones of unknown specificity from the GC TFH population. (D) Number of distinct nucleotide (nt) sequences per CDR3 amino acid (aa) sequence for Gag-specific T cells, HA-specific T cells, or bulk GC TFH cells. Data from all four individuals were aggregated for (C) and (D). Error bars indicate SEM. N.S., not significant. ***P < 0.001 by two-tailed t test.

  • Fig. 4 High-dimensional analysis of lymphoid CD4+ T cells identified distinct TFH cell subsets in HIV+ patients and HCs.

    (A) Representative gates used to identify GC TFH cells for t-SNE analysis. (B) Contour plots were generated using FACS output files from Cytobank (GC TFH cells) or “cytofkit” package in R (CXCR5+CD45RO+CD4+ T cells). Data include cells from 25 HIV+ patients and seven HCs. (C) Phenotypic clusters identified from CXCR5+ memory CD4+ T cells using DensVM. (D) Heatmap shows the average staining signal of indicated markers within each of the clusters identified in (C). Memory and naïve cells are shown at the bottom of the heatmap for comparison. Red-blue scale indicates staining intensity. Green-brown scale represents the relative frequency of HIV+ cells to total numbers of cells in each cluster. (E) Frequency of CD38 expression in CXCR5+ TFH cells or GC TFH cells from HC (n = 7) or HIV+ (n = 25) patients. Data are mean ± SEM. **P < 0.005 and ***P < 0.0005 by two-tailed t test. (F) Frequency of CD57+PD1+ GC subset within CD38 or CD38+CXCR5+ TFH cells from HIV+ patients (n = 25). ***P < 0.0005 by paired two-tailed t test. (G) Relationship between IL-21 expression and CD38 frequency in CXCR5+ TFH cells. (H) Relationship between peripheral CD4+ T cell count and IL-21+ GC TFH cells frequency. For (G) and (H), data are from 25 HIV+ patients; association was determined by Pearson correlation.

  • Fig. 5 Gag-reactive TFH cells express IL-21 and acquire GC phenotype.

    (A) Representative plots showing IL-21 staining used to identify Gag-reactive T cells. LN cells were stimulated for 18 hours with vehicle alone using dimethyl sulfoxide (DMSO) (left) or Gag peptides (right). (B) TCR sequencing of Gag-reactive IL-21+ T cells. Each pie chart represents TCR sequences from one individual. Light gray color represents unique TCRs. Filled colors represent the fraction of cells expressing a TCR identical to that of another cell in each individual. The number of TCR sequences analyzed is indicated at the center of pie chart. (C) Phenotype of Gag-reactive IL-21+ T cells (red) overlaid onto bulk CD4+ T cells (gray). (D) Frequency of each indicated phenotypic subset within IL-21+ Gag-reactive T cells or IL-21 bulk memory T cells from HIV+ individuals (n = 11). **P < 0.005 and ***P < 0.0005 by two-tailed t test. (E) Representative plots showing identification of Gag-reactive T cells by CD25 and OX40 expression. (F) CD57+PD-1+ frequency of Gag-reactive T cells from HIV+ individuals (n = 6) identified using IL-21 capture or by CD25 and OX40 up-regulation. (G) Frequency of CD25+OX40+CXCR5+PD-1+ TFH cells that coexpressed IL-21 after Gag or HA peptide stimulation from six HIV+ individuals. (H) IL-21 expression within GC TFH subset in LN cells from 11 HIV+ patients stimulated by Gag or HA peptides. The lines connect data from the same donor. For (F) to (H), *P < 0.05 and **P < 0.005 by paired two-tailed Wilcoxon signed-rank test.

  • Fig. 6 Dominant IL-21 expression in TFH cells correlate with B cell pathology in HIV-infected LNs.

    (A) Frequency of GC TFH cells that positively stained for each indicated effector molecule as determined by CyTOF. **P < 0.005 and ***P < 0.0005 by two-tailed t test (n = 7 HCs, 25 HIV+ patients). (B) Bar graph showing the frequency of single IL-21–producing T cells as a percentage of total IL-21+ T cells within each indicated TFH cell subset. *P < 0.05, **P < 0.005, and ***P < 0.0005 by two-tailed Wilcoxon signed-rank test (PBMCs: n = 4 HCs, 16 HIV+ patients; LN: n = 6 HCs, 25 HIV+ patients). (C) Plot shows the correlation between the abundance of single IL-21+ T cells (as a percentage of total IL-21+ T cells) in paired PBMCs and LNs from four HCs and 15 HIV+ individuals. Association is determined by Pearson correlation. (D) IgD+CD27 naïve B cells were excluded (also see fig. S16). Plots show gating strategy to identify switched memory B cells (IgDCD38) and plasma cells (IgDCD38high) on non-naïve B cells. (E to H) Correlation between switched memory B cell and plasma cells with the abundance of single IL-21+ T cells in GC TFH cells (E and F) or CXCR5+CD45RO+ CD4+ T cells (G and H) (n = 7 HCs and 25 HIV+ patients). Association was measured by Pearson correlation (E and G) or Spearman’s rank correlation (F and H), depending on data normality as determined by D’Agostino-Pearson test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/22/eaan8884/DC1

    Methods

    Fig. S1. GC TFH cells are clonally expanded.

    Fig. S2. Antigen-driven clonal selection signature in GC TFH cells of HIV-infected LNs.

    Fig. S3. Identification of Gag- or HA-reactive T cells in cultured cells.

    Fig. S4. HA-specific CD4 T cell clones detected in HIV-infected LNs.

    Fig. S5. Batch normalization of CyTOF data using calibration beads.

    Fig. S6. Identification of GC TFH cells from LN samples.

    Fig. S7. High-dimensional analysis of CXCR5+CD45RO+CD4+ T cells.

    Fig. S8. Signal intensity of individual markers on t-SNE plots.

    Fig. S9. Correlation between IL-21 frequency and CD38 expression with viral load and antiviral treatment.

    Fig. S10. IL-21 surface capture effectively identified IL-21–producing T cells.

    Fig. S11. Correlation between the frequency of IL-21+ GC TFH cells and signal intensity of CD57 staining.

    Fig. S12. TFH cells respond to Gag or HA peptides.

    Fig. S13. Identification of effector molecule–producing CD4+ T cells on CyTOF.

    Fig. S14. Identification of effector molecule–producing CD4+ T cells by flow cytometry.

    Fig. S15. Frequency of IL-21–producing subsets in TFH cells in HIV+ and HC samples.

    Fig. S16. Identification of B cell subsets.

    Table S1. TCRβ sequencing primers.

    Table S2. TCR repertoire sequencing cell and transcript counts.

    Table S3. Gag and HA TCR sequence reference panel.

    Table S4. CyTOF antibody staining panel.

    Table S5. Clinical characteristics and demographic information of LN samples.

    Table S6. Gag-reactive TCR sequences from single-cell TCR sequencing of IL-21+ cells.

    Table S7. Tabulated raw data sets.

  • The receptor repertoire and functional profile of follicular T cells in HIV-infected lymph nodes

    Ben S. Wendel, Daniel del Alcazar, Chenfeng He, Perla M. Del Río-Estrada, Benjamas Aiamkitsumrit, Yuria Ablanedo-Terrazas, Stefany M. Hernandez, Ke-Yue Ma, Michael R. Betts, Laura Pulido, Jun Huang, Phyllis A. Gimotty, Gustavo Reyes-Terán, Ning Jiang,* Laura F. Su*

    *Corresponding author. Email: laurasu{at}upenn.edu (L.F.S.); jiang{at}austin.utexas.edu (N.J.)

    Published 6 April 2018, Sci. Immunol. 3, eaan8884 (2018)
    DOI: 10.1126/sciimmunol.aan8884

    This PDF file includes:

    • Methods
    • Fig. S1. GC TFH cells are clonally expanded.
    • Fig. S2. Antigen-driven clonal selection signature in GC TFH cells of HIVinfectedLNs.
    • Fig. S3. Identification of Gag- or HA-reactive T cells in cultured cells.
    • Fig. S4. HA-specific CD4 T cell clones detected in HIV-infected LNs.
    • Fig. S5. Batch normalization of CyTOF data using calibration beads.
    • Fig. S6. Identification of GC TFH cells from LN samples.
    • Fig. S7. High-dimensional analysis of CXCR5+CD45RO+CD4+ T cells.
    • Fig. S8. Signal intensity of individual markers on t-SNE plots.
    • Fig. S9. Correlation between IL-21 frequency and CD38 expression with viral load and antiviral treatment.
    • Fig. S10. IL-21 surface capture effectively identified IL-21–producing T cells.
    • Fig. S11. Correlation between the frequency of IL-21+ GC TFH cells and signal intensity of CD57 staining.
    • Fig. S12. TFH cells respond to Gag or HA peptides.
    • Fig. S13. Identification of effector molecule–producing CD4+ T cells on CyTOF.
    • Fig. S14. Identification of effector molecule–producing CD4+ T cells by flow cytometry.
    • Fig. S15. Frequency of IL-21–producing subsets in TFH cells in HIV+ and HC samples.
    • Fig. S16. Identification of B cell subsets.
    • Table S1. TCRβ sequencing primers.
    • Table S2. TCR repertoire sequencing cell and transcript counts.
    • Table S3. Gag and HA TCR sequence reference panel.
    • Table S4. CyTOF antibody staining panel.
    • Table S5. Clinical characteristics and demographic information of LN samples.
    • Table S6. Gag-reactive TCR sequences from single-cell TCR sequencing of IL-21+ cells.

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