Supplementary Figure 8: TCR diversity and functional validation of ATO-derived CD4+ T cells. | Nature Methods

Supplementary Figure 8: TCR diversity and functional validation of ATO-derived CD4+ T cells.

From: Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids

Supplementary Figure 8

(a) Similar to human thymocytes, RAG1 and RAG2 are expressed in ATO-derived CD3+CD4+CD8+ (DP) but not mature CD3+CD8SP T cells. Quantitative RT-PCR for RAG1 and RAG2 are shown relative to expression of B2M in FACS sorted ATO-derived versus postnatal thymus T cell populations. Mean and SD of triplicate reactions is shown. (b) Generation of TCR diversity in CD3+TCRαβ+CD4SP T cells isolated from week 7 ATOs (n=5) or human thymi (n=4), as shown by flow cytometric analysis of the frequency of TCR Vβ family expression. (c) Cytokine production by week 12 ATO-derived CD4SP T cells treated with PMA/ionomycin for 6h. Data are representative of two independent experiments. (d) Proliferation (CTV dilution) and activation (upregulation of CD25) of cord blood (CB) and ATO-derived (week 12) CD4SP T cells after 5 days in response to anti-CD3/CD28 and IL-2. Data are representative of two individual experiments. (e) Post-ATO expansion of ATO-derived CD4SP T cells relative to starting cell number in response to anti-CD3/CD28 and IL-2 after 7 and 14 days. Mean and SD of technical triplicates are shown.

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