Supplementary Figure 6: Recapitulation of thymopoiesis and naive T cell phenotype in ATOs.

(a) Progressive differentiation of naïve CD3+TCRαβ+CD8SP and CD3+TCRαβ+CD4SP cells in ATOs between weeks 6-10. ATOs were cultured in parallel using CB HSPCs from a single donor and analyzed at the indicated weeks. Cells are gated on CD14-CD56-TCRαβ+CD3+ cells, and sequential sub-gates (CD8SP or CD4SP) are indicated above plots. The corresponding week 12 timepoint is shown in Fig. 2. (b,c) Additional markers characterizing the phenotype of week 12 ATO-derived CD3+TCRαβ+ (b) CD8SP and (c) CD4SP T cells compared with corresponding populations in the human thymus (see also Fig. 2). (d) Frequency of HLA-DR+ cells in CB ATOs compared with postnatal thymi (gated on total CD45+ cells). (e) Multiple HLA-DR+ antigen presenting cell (APC) populations are present in week 6 ATOs. Sequential gates are shown above each plot. HLA-DR+ populations include monocytes (CD14+), granulocytes (CD66b+), B cells (CD19+), HSPCs (CD34+), plasmacytoid DC (CD303+CD123+), CLEC9A+ DC (CD141+CLEC9A+), and CD1c+ DC (CD1c+CLEC9A-). Paired analysis from a postnatal thymus is shown for comparison. Data in (d) and (e) are representative of three independent experiments.