Cellular and molecular mediators of thymic dendritic cell homeostasis
To determine changes in cellular composition and gene signatures of hematopoietic antigen-presenting cells (APCs) in thymi harvested from mice where T-cell development is blocked at different maturation stages. Overall design: 1) CD45+ MHC-II lo to + hematopoietic APCs were FACS-isolated from WT (C57BL6/J), Rag2-/-, B2m-/- and MHC-II-/- mice to determine the role of thymocytes in regulating DC homeostasis and activation (N=2 experimental repeats per genotype). We used scRNA-seq to determine the transcriptional heterogeneity of DCs across the four genotypes. 2) To determine if our flow-validated DC subsets correlate with transcriptional DC clusters, we FACS-isolated 7 DC subsets and labeled each DC subset with a unique TotalSeqBTM hashtag antibody (BioLegend), prior to pooling all subsets for scRNA-seq and hashtag-derived oligonucleotide (HTO) amplification.