Fig. 5. TLR/MyD88 signaling increases cooperative antigen transfer between TECs and the CD14⁺moDC subpopulation.

a Two-dimensional tSNE plot from ddSEQ single-cell RNA-sequencing from FACS sorted Gr-1^–CD11c⁺TdTOM⁺ DCs from the thymus of Foxn1^CreROSA26^TdTOMATO mice. The color code represents different cell clusters based on the mRNA expression profile of each cell. b Heat-map analysis of the expression of signature genes determining each subset defined in a. c Heat-map analysis of the expression of chemokine receptors by DC subsets defined in a. d Quantification of TdTOM⁺CD11c⁺ DC subsets (defined as in Supplementary Fig. 4a) in CpG ODN or PBS intrathymically stimulated Foxn1^CreROSA26^TdTOMATO mice (representative flow cytometry plots are shown in Supplementary Fig. 6d, f) (mean ± SEM, n = 9 mice). Statistical analysis was performed using unpaired, two-tailed Student’s t-test, p ≤ 0.01 = **, p ≤ 0.001***, p < 0.0001****. e Representative flow cytometry tSNE analysis of TdTOM⁺CD11c⁺cell population in PBS or CpG ODN intrathymically stimulated Foxn1^CreROSA26^TdTOMATO mice. tSNE analysis was performed using FlowJO software, based on the FSC-A, SSC-A, CD11c, MHCII, Sirpα, Xcr1, B220, Mgl2 and CD14 markers (n = 2 independent experiments). f Quantification of frequencies of TdTOM⁺CD14⁺moDC or TdTOM⁺Mgl2⁺cDC2 from CpG ODN or PBS intrathymically stimulated Foxn1^CreROSA26^TdTOMATO mice (representative flow cytometry plots are shown in Supplementary Fig 6g) (mean ± SEM, n = 4 mice). g Flow cytometry analysis comparing the frequency of cDC2 (Sirpα⁺Mgl2⁺) and moDC (Sirpα⁺CD14⁺) between MyD88^fl/fl and MyD88^ΔTECs mice (mean ± SEM, n = 6 mice). Total Sirpα⁺ DC population was gated as shown in Supplementary Fig 4a. Statistical analysis in f and g was performed by unpaired, two-tailed Student’s t-test, p ≤ 0.01 = **, p < 0.0001****, ns not significant.