Fig 1: Single-Cell Trajectory of pDC Cell State Conversion during Stimulation(A) Wanderlust trajectory of fresh (day 0), 2-, or 6-day CD40L-stimulated bona fide pDCs analyzed by CyTOF; each point represents 1 cell (1 experiment of 3–4).(B) Normalized expression of pDC and cDC markers plotted along Wanderlust trajectory axis.(C) As in (A), but colored by expression of key markers.(D) Statistical Scaffold maps of CyTOF data from fresh (day 0), 2-, or 6-day CD40L-stimulated pDCs (1 representative donor).(E) Summary graph of frequency of pDCs mapped to each landmark node (n = 2–3 donors in 3–4 experiments).(F) Protein expression in fresh (day 0), 2-, or 6-day CD40L-stimulated bona fide pDCs analyzed by flow cytometry and CyTOF (n = 3–18 donors in 3–16 experiments). Statistics determined by Kruskal-Wallis with Dunn’s multiple comparisons test.(G) Functional analysis of pDCs that mapped to each landmark node. Two-day CD40L-stimulated pDCs were re-sorted based on phenotype. Left: IFN-a in culture supernatant after 24 h CpG-A, measured by ELISA. Right: T cell proliferation in MLR (n = 2–3 donors in 2–3 experiments).Bar graphs show means ± SDs. **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figure S6 and Table S2.
Fig 2: CD40L-Stimulated pDCs Share Chromatin Accessibility Landscape with tDCs and cDCs(A) Modified GSEA to test for enrichment of DC subset chromatin signatures among CD40L- or IMIQ-stimulated pDCs (see Quantification and Statistical Analysis). The bubble size represents the Spearman’s rank correlation coefficient. The color indicates the normalized enrichment score (NES).(B) Top Gene Ontology terms enriched in CD40L-stimulated pDCs compared to freshly isolated (day 0) pDCs. The terms are colored and ranked by -log10 FDR. The bubble size represents the term fold enrichment.(C) Left: histogram of difference in TF scores between CD40L-stimulated pDCs and day 0 pDCs. The significantly different TF motifs (?TF score > |0.08| and p-adj < 0.05) are colored. Right: boxplots of TF scores.(D) Heatmap of differentially active TFs from (C). The color indicates the scaled TF score for each subset.(E) HINT-ATAC footprint plots for indicated TFs. The data are pooled from 3–4 donors per condition.(F) Bar graphs of scaled scores for select TFs from (D) (n = 4–17 samples per cell type).(G) Top: frequency of pDCs expressing high levels of TCF4 protein measured by flow cytometry (n = 13–17 in 10–14 experiments). Bottom: ZBTB18 transcript variant 1 expression measured by RT-PCR (n = 3–4 in 3–4 experiments). The statistics are determined by t test.(H) Top: representative histogram of ID2 mRNA expression in 2-day CD40L-stimulated pDCs measured by PrimeFlow. The unfilled histogram represents the control. Bottom: frequency of ID2+ pDCs (n = 2 in 2 experiments).(I) Scatterplot comparing changes between CD40L stimulation and TCF4 silencing. x axis: FC of mRNA expression between TCF4 and control small hairpin RNA (shRNA) conditions in the pDC cell line CAL-1 (microarray) (Ceribelli et al., 2016). y axis: ?TF score between freshly isolated (day 0) and CD40L-stimulated pDCs (ATAC-seq). Shown are TFs that were significantly different in both analyses.Bar graphs show means ± SDs. *p < 0.05 and ****p < 0.0001. See also Figure S5.
Fig 3: Analysis of Chromatin Landscapes Reveals Alternative pDC Cell States following Stimulation(A) Experimental workflow for analysis of freshly isolated (day 0) and stimulated pDCs. Bona fide pDCs (AXL-) were sorted and analyzed immediately (day 0) or stimulated in vitro for 2 days, followed by re-sorting live cells for ATAC-seq analysis (see Figures S3A and S4A for gating strategy).(B) Left: protein levels of HLA-DR and CD80 in freshly isolated (day 0) or 2-day stimulated bona fide pDCs measured by flow cytometry (n = 3–9 in 3–8 experiments). The statistics are determined by Kruskal-Wallis with Dunn’s multiple comparisons test. Right: IFN-a measured by ELISA in culture supernatant after 2 days (n = 5 in 5 experiments). The statistics are determined by Mann-Whitney test.(C) Genome tracks from 1 representative donor.(D) Left: PCA based on ATAC-seq signal in all cis-elements. Each point represents 1 sample (n = 3–4 per condition). Right: scatterplot comparing all cis-elements between CD40L- and IMIQ-stimulated pDCs. The colored points indicate significantly differentially accessible cis-elements (FC > 2 and p-adj < 0.05).(E) Heatmap of scaled ATAC-seq signal in cis-elements identified in (D).(F) Genome tracks from 1 representative donor.(G) Left: PCA of sorted bona fide pDCs analyzed by CyTOF, including three time points (days 0, 2, and 6) and conditions (media alone, CD40L, IMIQ) subsampled and merged. The color indicates the branch cluster determined by Wishbone (n = 1 representative of 2 experiments). Center: percentage of pDCs in each Wishbone branch at day 6. Right: PCA colored by expression of select markers.(H) Top Gene Ontology terms enriched in CD40L and IMIQ differentially accessible cis-elements. The bubble size represents the fold enrichment. The color indicates -log10 false discovery rate (FDR).(I) Cytokines in culture supernatant of 2-day stimulated pDCs (n = 5 in 5 experiments). The statistics are determined by t test.(J) Left: frequency of Ki67+ cells among fresh (day 0) or 2-day stimulated pDCs. Right: number of CellTrace Violet low (CTVlo) cells among fresh, 2-, or 6-day stimulated pDCs (n = 4–11 in 2–7 experiments). The statistics are determined by t test.(K) MLR using fresh or 2-day stimulated pDCs (DC:T cell ratio 1:20, n = 3–4 donors in 3 experiments). The statistics are determined by 1-way ANOVA against day 0 or t test.Bar graphs shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S4 and Table S2.
Supplier Page from R&D Systems, a Bio-Techne Brand for CD40 Ligand/TNFSF5 Protein
Available conjugates: Sizes Available: 25 ug