Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.
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Microarray data generated during the current study were deposited in the Gene Expression Omnibus public depository with the accession number GSE128297 for the SuperSeries, and the following accession numbers for the Subseries: GSE129872 (HIV-specific CD4+ T cells samples from CPs, VCs and ECs), GSE128280 (CXCR5mem and CXCR5neg HIV-specific CD4+ T cells from CPs and ECs), GSE128296 (HIV-specific CD4+ T cells samples from CPs before/after ART and ECs). mRNA expression data by high-throughput RT–qPCR are available in the Supplementary Material. All the datasets that support the findings of this study are available from the corresponding author upon reasonable request.
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We thank J. Girouard, the clinical staff at the McGill University Health Centre in Montreal, the Ragon/MGH clinical and technical staff and all study participants for their invaluable role in this project; B. Walker for providing clinical samples; D. Gauchat and the CRCHUM Flow Cytometry Platform for technical assistance; N. Hamel and McGill University and Génome Québec Innovation Centre for microarray analysis; and J. Boss and S. Crotty for their input on this manuscript. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 IIIB p24 Recombinant Protein from ImmunoDX, LLC. This study was supported by the National Institutes of Health grants (no. HL092565 to D.E.K.; no. AI100663 CHAVI-ID to D.E.K. and R.T.W. (Consortium PI: D. Burton); nos. OD011103 and OD011132 to R.P.J.); the Canadian Institutes for Health Research grants (nos. 137694 and 152977 to D.E.K.; no. MOP-93770 to C.T.; and foundation no. 352417 to A.F.); the Canada Foundation for Innovation Program Leader grant (no. 31756 to D.E.K.); and the FRQS AIDS and Infectious Diseases Network. This work was funded in part by the Intramural Program of the National Institutes of Health (D.C.D., S.D.). D.E.K. and C.T. are supported by Senior Research Scholar Awards of the Quebec Health Research Fund (FRQS). J.-P.R. is the holder of the Louis Lowenstein Chair in Hematology & Oncology, McGill University. A.F. is a Canada Research Chair on Retroviral Entry.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Analysis of HIV-specific CD4+ T cells identified by the CD69/CD40L (CD154) Activation-Induced Marker (AIM) assay.
(a) Gating strategy for sorting HIV-specific CD4+ T cells based on co-upregulation on the cell surface of the activation markers CD69 and CD154 9h after stimulation with an HIV Gag peptide pool. (b) Flow cytometry plots comparing the use of CD69 alone or in combination with CD154 as AIM markers for detection of antigen-specific CD4+ T cells. Numbers in plots represent the percentage of CD69+CD154+ Gag-specific CD4+ T cells over unstimulated cells. (c) Statistical comparison of frequencies of CD69+ CD154+ CD4+ T cells between unstimulated and Gag-stimulated PBMCs in the three groups of HIV-1+ infected people (two-tailed Wilcoxon matched paired test). Bars represent median with interquartile range. For (a,b,c) n= (11 CPs, 11 VCs, 13 ECs). (d) Statistical comparison of frequencies of CD69+ IL-2+ or CD69+ IFNγ+ CD4+ T cells detected by intracellular staining 6h after Gag stimulation with the frequencies of CD69+ CD154+ CD4+ T cells detected by surface staining 9h after Gag stimulation (two-tailed Wilcoxon matched paired test) (n=17 subjects). (e,f) Two-tailed Spearman correlation of net frequencies of Gag-specific CD4+ T cells (background in No Antigen condition subtracted) with (e) CD4+ T cell count (cells/μl) and (f) Viral Load (vRNA copies/ml) (n=38 subjects).
Supplementary Figure 2 Transcriptional expression and correlation of genes associated with Thelper differentiation and function.
(a) Heatmap of two-sided CAMERA enrichment analysis for CD4+ T cell lineage or exhaustion signatures in CP (n=11), VC (n=9) and EC (n=12) subjects. Color and intensity indicate the directionality of the enrichment and -log10FDR by the Benjamin Hochberg procedure. White boxes represent results with FDR>0.05. (b) Statistical comparison of transcriptional levels of master regulators of CD4+ T cell subsets in sorted HIV-specific CD4+ T cells of CPs (n=11) compared to ECs (n=12) as assessed by RT-qPCR (Fluidigm) by Mann-Whitney test. Bars represent median with interquartile range. (c,d) Correlograms illustrating gene correlations between (c) functional factors and surface molecules, and (d) functional factors and transcriptional factors in sorted HIV-specific CD4+ T cells. Transcriptional expression values (−ΔCt values) assessed by RT-qPCR (Fluidigm) were used for generation of the correlograms. Blue and red circles denote positive and negative correlation (two-sided Spearman), respectively. Color intensity and the size of the circle are proportional to the correlation coefficients. Only gene correlations with p<0.05 are displayed. (n= 11 CPs, 9 VCs, 12 ECs).
Supplementary Figure 3 Isolation and characterization of CXCR5mem and CXCR5neg HIV-specific CD4+ T cells.
(a) Gating strategy for the identification and live-cell sorting of CXCR5mem vs CXCR5neg HIV-specific specific CD4+ T cells 9 h after stimulation with a Gag peptide pool (n= 6 CPs, 6 ECs). (b) Statistical comparison of the frequencies of CXCR5mem in total CD4+ T cells compared to HIV-specific CD4+ T cells in CPs (n=10 CPs, upper panel) and ECs (n=9 ECs, lower panel) by two-tailed Wilcoxon matched paired test. Bars represent median with interquartile range. (c) Transcriptional expression of TFH-associated genes in sorted CXCR5mem or CXCR5neg HIV-specific CD4+ T cells assessed by real-time quantitative reverse transcription PCR on a Fluidigm platform. (n = 6 CPs, 6 ECs) Bars represent median with interquartile range. Statistical comparison by two-tailed Mann Whitney or two-tailed Wilcoxon matched paired test were used to verify significance. Only p ˂ 0.05 are displayed for clarity.
Supplementary Figure 4 TFH, TH17 and TH22 cytokine expression by HIV-specific CD4+ T cells in CPs compared to ECs.
(a) Statistical comparison of transcriptional levels of TFH cytokines and TH17 and TH22 cytokines in sorted HIV-specific CD4+ T cells compared to CMV-specific CD4+ T cells from the same donors, 9 h after stimulation with a gag or pp65 peptide pool (two-tailed Wilcoxon matched paired test). Transcriptional expression was assessed by RT-qPCR (Fluidigm). Bars represent median +/- interquartile range transcriptional expression (n= 9 CPs, 10 ECs). (b,c) Representative flow cytometry plots of CXCL13 and IL22 mRNA+ CD69+ CD4+ T cells after 12 h stimulation with a gag peptide pool, SEB or no antigen. Numbers in plots represent the percentage of CD69+ mRNA+ CD4+ T cells (n= 8 CPs, 8 ECs). (d-i) Statistical comparison of frequencies of CD69+ mRNA+ CD4+ T cells after stimulation with gag peptide pool or SEB detected by RNA-Flow-FISH Cytometry. (two-tailed Mann Whitney test, n= 8 CPs, 8 ECs).
Supplementary Figure 5 TFH,TH17 and TH22 cytokine expression by HIV-specific CD4+ T cells compared to CMV-specific CD4+ T cells in CPs and ECs and in the absence of CD40 blockade and CD40L pre-gating.
(a) Comparison of CXCL13 protein expression in the supernatant of CD8-depleted PBMCs of CPs versus ECs by two-tailed Mann-Whitney test. The cells were stimulated with a Gag-peptide pool for 48 h and CXCL13 protein concentration was measured by ELISA. Bars represent median with +/- interquartile range (n= 8 CPs, 7 ECs). (b,c) Statistical comparison of frequency of PD-1/TIGIT subpopulations in (b) CXCL13 mRNA+ or (c) IL21 mRNA+ HIV-specific CD4+ T cells in CPs compared to ECs by two-tailed Mann-Whitney test (b,c; n = 6 CPs, 5 ECs). Bars represent median with +/- interquartile range. (d) Correlation of quantification of p24-specific antibodies in plasma by Biolayer Interferometry (BLI) binding analysis or ELISA (two-tailed Spearman correlation test, n = 12 CPs, 15 ECs). Semi-quantitive scale for anti-p24 antibody binding index: -, -/+, +, ++, +++ symbols correspond to peak values at <0.2, 0.2-0.3 0.3-0.5, 0.5-0.8, >0.8 nm, respectively (two-tailed Spearman correlation test, n=12 CPs, 15 ECs). (e,f) Correlation of weighted Mean Fluorescence Intensity for IL22 and IL17F detected by RNA-Flow-FISH cytometry to protein concentration detected by a magnetic bead-based assay (Luminex) (n= 6 CPs, 6 ECs) (two-tailed Spearman correlation). (g) Detection of IL-17A protein in the supernatant of CD8-depleted PBMCs 48 h after stimulation with a Gag-peptide pool by Luminex. Bars represent median with interquartile range and statistical comparison was performed by two-tailed Mann-Whitney test (n = 8 CPs, 8 ECs). (h) SPICE analysis of phenotyping of IL22 mRNA+ and IL17F mRNA+ antigen-specific CD4+ T cells from the CP group. Pie charts represent median percentages of CCR6/CXCR3 subpopulations (n= 6 CPs, 6 ECs). (i,j) Correlation of IL22 and IL17F mRNA levels assessed by RT-qPCR (Fluidigm) with the protein expression of the activation markers HLA-DR and CD38 on total unstimulated CD8+ T cells by two-tailed Spearman correlation (n = 11 CPs, 6 VCs, 12 ECs). (k,l) Correlation of bacterial diversity (Shannon Entropy) with the protein expression of the activation markers HLA-DR and CD38 on total unstimulated CD4+ and CD8+ T cells by two-tailed Spearman correlation (n = 8 CPs, 6 ECs). (m) Hierarchical clustering of Morisita-Horn dissimilarity indexes illustrates that samples largely segregate by disease status based on Genus TPM values (n= 12 CPs, 8 ECs).
(a) Barcode plots of enrichment of the LCMV exhaustion signature in comparisons CPpre vs. EC, CPpost vs EC and CPpost vs EC by CAMERA. Red and blue lines denote positive and negative enrichment. Barcode plots were generated using microarray data by sorted HIV-specific CD4+ T cells from 8 CPs before and after ART and 12 ECs. (Exhaustion Signature; GSE41866, LCMV Clone 13 D30 vs. Armstrong D30 - Exhausted vs Memory) (two tailed p-values by CAMERA followed by the Benjamin Hochberg method). (b) Top 50 DEGs in HIV-specific CD4+ T cells of CPs before and after treatment compared to ECs. Red and purple denote logarithmic fold change for comparisons CPpre vs EC and CPpost vs EC, respectively, by microarray analysis in 8 CPs before/after ART and 12 ECs.
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Morou, A., Brunet-Ratnasingham, E., Dubé, M. et al. Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease. Nat Immunol 20, 1059–1070 (2019) doi:10.1038/s41590-019-0418-x
Nature Immunology (2019)