Extended Data Fig. 6: Marker validation of Penk+ and Postn+ ThyMCs.
From: Mesenchymal thymic niche cells enable regeneration of the adult thymus and T cell immunity
(A) Gating strategy for FACS analysis of mouse thymus tdTomato+ (Penk + ) ThyMCs in Penk-Cre-tdT mice. Percentages refer to percent of parent gate. (B) FACS plots showing overlap between tdTomato+ cells and thymic epithelium (EpCam), endothelium (CD31), hematopoietic cells (CD45), and myeloid cells (F4/80, CD11b) in Penk-Cre-tdT mice. (C) Representative histograms from flow cytometric analysis of intracellular Cre protein staining in hematopoietic cells (CD45) and ThyMC (CD248-). (D) Bar graphs showing Postn and Ccl19 expression as determined by qPCR on sorted tdTomato+ (Penk ThyMC) and tdTomato- (Postn+ ThyMC) cells from Penk-Cre-tdT mice (n = 4, mean ± SEM). Two independent experiments. (E) Gating strategy for flow cytometric analysis of mouse thymus tdTomato+ (Postn + ) ThyMCs after in vivo administration of 4-hydroxytamoxifen in Postn-CreERT-tdT mice. Percentages refer to percent of parent gate. (F) Flow cytometric analysis of overlap between tdTomato+ cells and thymic epithelium (EpCam), endothelium (CD31), and hematopoietic cells (CD45) in Postn-CreER-tdT mice. (G) Bar graphs showing Postn and Ccl19 expression as determined by qPCR on sorted tdTomato+ (Postn+ ThyMC) and tdTomato- (Penk+ ThyMC) cells from 4-hydroxytamoxifen induced Postn-CreERT-tdT mice (n = 4, mean ± SEM). Two independent experiments. (H) Detailed ThyMC annotation presented as ThyMC specific UMAP embedding (left). UMAP showing expression of marker genes DPP4 (Dpp4), PDPN (Pdpn), LTBR (Ltbr), S100A4 (S100a4) in human (top) and mouse (bottom). (I) Representative histogram showing staining of S100A4 and Ltbr on thymic ThyMCs from Penk-Cre-tdT mice. (J) Bar graphs showing mean fluorescent intensity (MFI) on CD248+ ThyMC, Penk+ ThyMC and Postn+ ThyMC as determined by flow cytometry in Penk-Cre-tdT mice (n = 3, mean ± SEM). Two independent experiments. (K) FACS plots showing staining for DPP4 and Pdpn on CD248+ ThyMC, Penk+ ThyMC and Postn+ ThyMC from Penk-Cre-tdT mice. (L) Bar graphs showing the percentage of phenotypic cortical fibroblasts (cFib; DPP4+ Pdpn + ) and medullary fibroblasts (mFib; Dpp4- Pdpn + ) within CD248+ ThyMC, Penk+ ThyMC and Postn+ ThyMC subsets from Penk-Cre-tdT mice. (n = 3, mean ± SEM). Two independent experiments. Statistical significance is based on beta regression with Benjamini-Hochberg FDR control of these comparisons. Statistical significance is using a two-sided Beta regression-based Wald test with Benjamini-Hochberg FDR control of these comparisons. CD248+ ThyMC: cFib vs. mFib **** p = 3.8×10−136. Penk+ ThyMC: cFib vs. mFib **** p = 2.1×10−65. Postn+ ThyMC: cFib vs. mFib p = 0.21.
