Extended Data Fig. 2: Identification of transcriptionally distinct cDC clusters and their correlation with FACS-sorted cDC subsets.

Related to Fig. 1. (a) Flow cytometric gating strategy to identify distinct DC subsets shown in Fig. 1d. (b-e) To determine if DC subsets identified by flow cytometry correlate with the expected transcriptional signatures, DC subsets were FACS-purified, hashtagged to informatically distinguish the sorted subsets, pooled, and evaluated by scRNA-seq. (b) UMAP visualization of scRNA-seq transcriptional data from pooled FACS-sorted cDC subsets, colored by their hashtag labels. (c) Same UMAP plot as (b), colored by transcriptionally distinct cDC clusters defined by the Seurat pipeline. (d) Dot plot showing normalized expression levels (z-score) of select genes associated with the hashtagged FACS-sorted cDC subsets. Expression is normalized per row (representing a gene) across different FACS-sorted cDC subsets. (e) Frequency of each transcriptionally distinct cDC cluster contained within the FACS-sorted hashtagged cDC subsets. (f-i) To determine intrathymic localization of DC subsets, immunofluorescence staining and histocytometry were carried out on N = 3 independent biological thymic replicates. (f) Representative immunofluorescence image of MertK⁺ macrophages and CD63⁺SIRPa⁺MerTK⁻ aDC2s in thymic sections. Centroids are placed over CD63⁺ SIRPα⁺ cells that also express MertK: the frequency of MertK-expressing cells among CD63⁺SIRPα⁺ myeloid cells was used to correct aDC2 densities from data as in (h). Scale bar, 250 µm. Lines demarcate the cortex (C), medulla (M), and CMJ in the thymic sections. Centroids with 40% opacity were overlaid on the quantified cells. (g) Representative immunofluorescence images of DC subsets identified by the indicated markers. White boxes indicate the regions of interest displayed in Fig. 1e. Scale bars, 1,000 µm. (h) Quantification of cell densities of each of the indicated DC subsets in distinct anatomical regions of the thymus, based on histocytometry analyses of data as in (f-g). Dots represent biological thymic replicates. Statistical analysis was performed using Tukey’s Honest Significant Difference test. (i) Quantification of cortical cell densities of each of the indicated DC subsets, based on histocytometry analyses of data as in (f-g).