Susceptibility of primary cDCs to <i>ex-vivo</i> infection with HIV-1.

<p>(A): Flow cytometry gating strategy for defining cDCs in bulk PBMC cultures. (B): Representative flow cytometry dot plots indicating GFP expression in gated CD11c⁺ HLA-DR⁺ cDCs from Neg, CP, EC and HAART individuals after 24, 48 and 96 hours of <i>ex-vivo</i> infection with GFP-encoding HIV-1. Numbers in dot plots reflect the proportion of GFP-positive cells within gated cDCs. (C–D): Proportion (C) and GFP MFI (D) of GFP⁺ cDCs from Neg, CP, EC and HAART subjects at 96 hours after infection with HIV-1 (n = 20 tested subjects for each cohort). Horizontal lines represent the median for each specific cohort and experimental condition. Differences among cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test (* p<0.05; *** p<0.001) or using Mann Whitney U test (# p<0.05; ## p<0.01).</p

Type I IFN secretion and activation in cDCs after <i>ex-vivo</i> exposure to HIV-1.

<p>(A): IFNα and IFNβ mRNA levels in isolated BDCA1⁺ cDCs from HIV-negative persons (Neg), individuals with chronic progressive HIV-1 infection (CP), Elite controllers (EC) and HAART-treated HIV-1 patients (HAART) at 24 and 48 hours after exposure to HIV-1 (HIV) or to media only (Med) as negative control. Horizontal lines represent the median for each specific cohort and experimental condition. (B): Mean Fluorescence Intensity (MFI) reflecting surface expression of CD86, CD83 and CD40 in cDCs from the different study cohorts at 24 hours after infection with HIV-1 (HIV) or after exposure to poly(I:C) (PIC). MFI values are expressed as fold-changes in comparison to baseline levels. Intra-individual differences were tested for statistical significance using Wilcoxon matched-pairs signed rank tests (above each cohort), differences between cohorts were tested using a Kruskal-Wallis test with post-hoc Dunn’s test; * p<0.05; ** p<0.01; *** p < 0.001; **** p< 0.0001. Horizontal lines represent the median for each specific cohort and experimental condition. (C): Heatmaps reflecting gene expression patterns of 28 interferon-stimulated genes (ISG) in cDCs from Neg (n = 6), CP (n = 6) and EC (n = 6) at 48 hours after infection with HIV-1. (D): Heatmaps reflecting correlations among gene expression intensities of all 28 ISGs in indicated study cohorts. Color-coding reflects Pearson’s correlation coefficient indicating strengths of statistical association between expression intensities of given gene pairs.</p

Antigen-presenting properties of cDCs after exposure to HIV-1.

<p>(A): Representative flow cytometry plots reflecting proliferation of CD4⁺ T cells after stimulation with allogeneic uninfected (Med) or HIV-1 infected (HIV) cDCs from Neg, CP, EC and HAART patients. Numbers in flow cytometry plots represent the proportion of proliferating CFSE^low T cells. (B–C): Induction of allogeneic CD4 (B; n = 8) and CD8 T (C; n = 7) cell proliferation after exposure to HIV-1 infected cDC from indicated study cohorts. Horizontal lines represent the median for each specific cohort and experimental condition. (D): Representative flow cytometry dot plots reflecting IFN-γ secretion in an HLA-A2-SL9 (SLYNTVATL)-specific CTL cell line after 16 hours of co-culture with uninfected or HIV-1-infected cDCs from Neg, CP, EC and HAART individuals. (E): Proportion of IFNγ⁺ cells in the SL9 CTL cell line after exposure to HIV-1-infected cDCs from indicated study groups. Cumulative data from n = 8 experiments are shown. (B,C,E): Differences within and among study groups were tested for statistical significance using a Wilcoxon matched-pairs signed-rank test rank test or a Mann Whitney test corrected for multiple comparisons using Bonferroni method, respectively. * p<0.05; ** p<0.01. (F): Induction of allogeneic CD4 (left; n = 7) and CD8 (right; n = 7) T cell proliferation after exposure to cDC from EC cultured in the presence of media (Med) or infected with HIV-1 in the presence or absence of AZT or RAL. Statistical significance of differences was tested using a Wilcoxon matched-pairs signed rank test. Bonferroni correction was applied for multiple comparisons.* p<0.05. (E-F): Horizontal lines represent the median for each specific cohort and experimental condition.</p

Induction of cytosolic DNA sensors in cDCs from EC.

<p>(A): Fold change in cGAS, STING and IFI16 mRNA expression levels in indicated study cohorts at 24 (upper panels) and 48 (lower panels) hours after <i>ex-vivo</i> infection with HIV-1. Induction of mRNA expression in comparison to baseline levels was tested for statistical significance using Wilcoxon matched-pairs signed-rank test tests. Significant differences between distinct cohorts were calculated using a Mann Whitney test. No correction for multiple comparisons was applied (B): IFNα and IFNβ expression in primary cDCs nucleofected with scrambled (SC) or cGAS-specific siRNAs, followed by infection with HIV-1. Data from n = 4 experiments are shown. Data were normalized to results from experiments with scrambled siRNA sequences. Differences in type I IFN responses between untreated or SC- or cGAS-nucleofected cDCs were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test * p<0.05; ** p<0.01.</p

HIV-1 replication patterns in cDCs from EC.

<p>(A): Early and late HIV-1 reverse transcripts (RT) and 2-LTR circles in cDCs from indicated study subjects at 48 hours after <i>ex vivo</i> infection with HIV-1. (B): Analysis of integrated HIV-1 DNA in primary cDCs from the different study cohorts. Basal levels of integrated HIV-1 DNA are shown in the left panel, right panel shows <i>de novo</i> HIV-1 integration in cDCs after subtraction of baseline levels of integrated HIV-1 DNA. (C): Analysis of late HIV-1 RT products and 2-LTR circles normalized to levels of <i>de novo</i> integrated HIV-1 DNA in cDCs at 48 hours after infection. (A–C): Differences between different cohorts were tested for statistical significance using a Kruskal-Wallis test with post-hoc Dunn’s test or using Mann Whitney U test (# p<0.05; ## p<0.01). (A,B,C). Horizontal lines represent the median for each specific cohort and experimental condition. (D): Inhibition of IFNα and IFNβ mRNA expression in cDCs from EC cultured in media (Med) or infected with HIV-1 (HIV) in the presence or absence of AZT, Efavirenz (EFV) or Raltegavir (Ral). Data reflect mean and standard error from qPCR values of IFNα and IFNβ mRNA levels after normalization to β-actin endogenous expression from n = 5 experiments. Numbers above bars represent the mean percentage of inhibition induced by each drug. Differences were tested for statistical significance using a one-tailed Wilcoxon matched-pairs signed rank test, * p<0.05.</p