Extended Data Fig. 7: MicrofoldTECs require SpiB for their development and regulate mTEC cellularity.

a, Sequencing tracks of chromatin accessibility near Spib by TEC subset. The range of normalized tag densities is indicated by the numbers in parentheses at the bottom left corner. UMAP highlighting Spib motif enrichment analysis of scATAC-seq data from Fig. 1c. The microfoldTEC subset is indicated by a black arrow. Each dot represents one nucleus. The Spib motif DNA sequence and P value are located to the left of the UMAP. b, Representative flow cytometry plots from four-week-old Spib^+/– (top) and Spib^–/– mice (bottom) showing the gating strategy for isolating Hetero-post TEC, mTEC-II, mTEC-I, mTEC-IV, endoTEC and corneoTEC. Numbers within or beside gates indicate the frequency of the gate from the parent population. c, Absolute counts of mTEC-II cells in Spib^+/– mice or Spib^-/– littermates (n = 4). d, Frequencies of corneoTECs from all TECs (left), endoTECs from all TEC (right) and mTEC-IV cells from all TECs (bottom) in Spib^+/– mice or Spib^–/– littermates (n = 4). e,f, Volcano plot of bulk RNA-seq of sorted EpCAM⁺CD45^–Ly51^–IA-IE^lo/negL1CAM^–ITGB4^–CD177⁺GP2⁺Ly6A⁺ TECs from Spib^+/– mice and Spib^–/– littermates. The top 50 corneoTEC (e) and top 50 endoTEC (f) gene signature as assayed in Fig. 2a is highlighted in red, all genes are in grey. g, Relative mRNA expression by qPCR of the indicated genes after ex vivo stimulation of sorted mTEC-II cells with recombinant RANKL (n = 3). h, Normalized UMI count of Tnfrsf11b (encoding OPG) in the indicated cell populations sorted in Fig. 2a (n = 3). i, Frequencies of microfoldTECs from total TECs at the specified ages (n = 4). j, Representative flow cytometry plots showing the gating strategy for identifying PGRP-S-dsRed⁺ thymic APC populations. Numbers within or beside gates indicate the frequency of the gate from the parent population. k, Frequencies of PGRP-S-dsRed⁺ thymic APC populations (n = 3). l,m, Volcano plot of bulk RNA-seq of sorted EpCAM⁺CD45^–Ly51^–IA-IE^lo/negL1CAM^–ITGB4^–CD177⁺GP2⁺Ly6A⁺ TECs from either IgM^+/+ mice and their IgM^–/– littermates (l), or Ccr6^+/+ mice and Ccr6^–/– littermates (m). The top 50 microfoldTEC gene signature as assayed in Fig. 2a is highlighted in red, all genes are in grey. n, Confocal microscopy of frozen sections of Ccr6^+/+ mice (left) and their Ccr6^–/– littermates (right) highlighting GP2⁺ microfoldTECs (green) and DCLK1⁺ mTEC-IV cells (purple) in relation to B220⁺ B cells (red). Scale bars, 200 µm. o, Confocal microscopy of frozen sections of PGRP-S-dsRed⁺ mice highlighting microfoldTECs (red), CD31⁺ endothelium (purple) and CX3CR1⁺ APCs (green). Scale bar, 200 µm. p, Representative three-dimensional reconstruction of confocal microscopy of frozen sections of PGRP-S-dsRed⁺ mice highlighting microfoldTECs (magenta), CD31⁺ endothelium (cyan) and CX3CR1⁺ APCs (green). Scale bar, 4 µm. c,d,g–i,k, n refers to the number of biologically independent mice. Data shown as mean ± s.e.m. Data were analysed using unpaired two-tailed t-tests (c,d), unpaired one-tailed t-tests (g) or one-way ANOVA (i) followed by Tukey’s multiple comparisons.

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