Fig. 3
CD64 and PD-L1 enrich in highly functional c1-like mDCs. a Selection of c1-specific genes encoding surface proteins for validation as c1 markers. 74 genes (listed in box) were: (1) differentially expressed between c1 and c3–5; (2) reproducibly correlated with other c1 genes across all three ECs profiled; and (3) predicted membrane proteins (see “Methods”). Candidate markers shown in green were selected for validation by FACS (Fig. 2a, Additional file 1: Figure S7). b Flow cytometry analysis of either CD64 (y-axis, left panel) or PD-L1 (y-axis, right panel) vs CD86 (x-axis) expression in mDCs from EC patient 1 (p1). Numbers above represent the percentage of CD64Hi/PD-L1Hi cells (top right gate; light blue) at 24 h in media (gray) and VSV-G pseudotyped HIV-1 virus exposure (red) conditions. c Flow cytometry plots showing analysis of CD64 vs PD-L1 expression on mDCs exposed to VSV-G pseudotyped HIV-1 for 24 h, defining two populations: CD64Hi,PD-L1Hi (Hi; blue) and CD64Lo,PD-L1Lo (Lo; green). Percentage in each gate is listed above. d Radar plots (see “Methods”) representing relative similarities of each subset (c1–5) to population-level RNA-Seq data from cells in the Hi and Lo PD-L1,CD64 gates 48 h after viral (solid line) or media exposure (dashed line). e Proportions of CD64Hi,PD-L1Hi mDCs induced from multiple ECs (n = 8), untreated CPs (n = 8), and HDs (n = 7) after 24 h of culture in media or VSV-G pseudotyped HIV-1 (*, p < 0.05; **, p < 0.01; two-tailed Wilcoxon signed-rank test). f Correlation between the proportions of CD64Hi,PD-L1Hi mDCs induced in ECs (n = 8) and untreated CPs (n = 8) or just CPs and clinical CD4 T cell count (p value = 8 × 10–3 [two-sided] and 2 × 10–2 [one-sided], respectively, Spearman correlation permutation p value) or between the proportions of CD64Hi,PD-L1Hi mDCs induced in ECs (n = 8) and untreated CPs (n = 8) or just CPs and HIV-1 viral load (p = 3 × 10–2 [two-sided] and 6 × 10–2 [one-sided], respectively, Spearman permutation p value). Diamond and square points represent indeterminate viral loads of < 20 and < 50 copies/mL, respectively. g Proportion of proliferating CD4+ (left) and CD8+ (right) T cells co-cultured with the Hi and Lo sorted virus-exposed mDCs populations (n = 6 patients). h Proportion of total IFNγ+ CD8+ T cells cultured with the Hi and Lo sorted virus-exposed mDCs populations (n = 7 patients). Statistical significance for (g, h) were evaluated using a two-tailed Wilcoxon matched pairs signed-rank test (*, p < 0.05). i Pie chart generated with data from n = 7 patients showing CD107a and TNFα expression on CD8+ T cells cultured with Hi (left) or Lo (right) mDCs. j Scatter plots of proportions of CD107a+, TNFα+ (left) and CD107a+, TNFα- (right) CD8+ T cells cultured with Hi and Lo mDCs. Statistical significance was evaluated using a two-tailed Wilcoxon matched pairs signed-rank test, n = 7 patients (*, p < 0.05)