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Fig. 1 | Retrovirology

Fig. 1

From: Identification of novel HIV-1 dependency factors in primary CCR4+CCR6+Th17 cells via a genome-wide transcriptional approach

Fig. 1

Identification of a molecular signature associated with HIV permissiveness in CCR4+CCR6+ Th17 cells. Total CD4+ T-cells were isolated from PBMCs of HIV-uninfected subjects by negative selection using magnetic beads. Cells were labeled with a cocktail of CD45RA, CD8, CD19, CD56, CCR4, CCR6, and CXCR3 Abs. a Shown is the gating strategy used for the FACS sorting of the following four memory (CD45RA) CD4+ subsets lacking CD8, CD19, and CD56 expression: CXCR3+CCR4CCR6 (Th1), CXCR3CCR4+CCR6 (Th2), CXCR3CCR4+CCR6+ (Th17), and CXCR3+CCR4CCR6+ (Th1Th17). Shown are results from one donor representative of results generated with cells from >10 HIV-uninfected donors. b Highly pure matched Th1, Th2, Th17, and Th1Th17 subsets were sorted by FACS (n = 5) and stimulated via CD3/CD28 for 3 days. Total RNA was reverse transcribed into cDNA and hybridized onto the Human HT-12 v4 Expression BeadChip (Illumina) for genome-wide transcriptional profiling. One-way ANOVA analysis was performed to identify differentially expressed genes based on p value < 0.05 and fold change (FC, cut-off 1.3). Shown are volcano plots for all probes in each linear model with the FC on the x axis and the negative logarithm of the adjusted p values adjusted for false discovery rate (FDR) on the y axis. Red/green color code is based on the 5 % FDR threshold. c, d Cell subsets were stimulated via CD3/CD28 for 3 days and exposed to replication competent NL4.3BAL-GFP (c) and single round VSVG-HIV-GFP strains (d). Shown is real-time quantification of HIV-DNA integration at day three post-infection (mean ± SD of triplicate wells) in matched subsets isolated from n = 3 different donors. e Shown are the top 50 pathways up regulated in Th17 versus Th1 cells; heat map of the individual enrichment statistics (ES) from a gene set variation analysis (GSVA) for pathways differentially expressed between Th1 and Th17 cells with a FDR inferior to 1 %. Transcriptional profiles in a, b, and e were generated with cells stimulated via CD3/CD28 but unexposed to HIV

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