Extended Data Fig. 5: Transcriptome and selected phenotype comparison of MHC+ and MHC− OP9-DL4 cells and select gene expression profiles for the DN4 unusual and DPbl abnormal populations.
From: Pre-T cell receptor self-MHC sampling restricts thymocyte dedifferentiation
a. Comparison of MHC+ and MHC− OP9-DL4 stromal cells for transcriptome and phenotypic differences. 93.6% of transcripts detected shared by MHC+ and MHC− stroma. b. Correlation between cell transcriptomes. Square of two-tailed Pearson correlation coefficient (R2 = 0.958) ideally greater than 0.92 under optimal experimental conditions. c. Differential gene expression is <4% of all transcripts detected. d. Loss of CD1d surface expression in B2m/Tap2 KO MHC− OP9-DL4 and confirmation of lack of MHC Class II expression in MHC+ and MHC− OP9-DL4. e. Raet expression in MHC+ and MHC− OP9-DL4. f. Select transcripts significantly differentially expressed between the MHC− DN3b/4 cluster and the DN4 “unusual” cluster. Heatmap depicts log2-fold change (L2FC) of the DN4 “unusual” cluster relative to the DN3b/4 cluster. Actual L2FC values are listed within the heatmap. g. Co-expression of Cd4 transcript with Cd8a and/or Cd8b1 transcripts in an overlay of the MHC− libraries focussed on the DN4 unusual, DPbl, and DP abnormal clusters. h. Characteristic myeloid gene transcript expression maps to the MHC− DP abnormal cluster. i. Full-length clonotypic TCR β chain transcript expression in 82 of 221 Spi1+ cells (37.1%) in the MHC− DP abnormal cluster. j. Mpo-expressing cells in the DP abnormal cluster and the Mpo+Spi1+ subset co-express T lineage Lck and/or Cd3e. k. DP cell yields after 12 d for DN3a and DN4 cells seeded onto MHC+ or MHC- stromal cells. l. Relative expression by qRT-PCR (normalised to Actb = 1000) of Mpo and Spi1 in DP cells developing from DN3a cells seeded 12 d earlier onto MHC+ or MHC− stromal cells (Cells pooled from 3 separate cultures; n = 7 qRT-PCR replicates; Mpo: P < 0.00001, Spi1: P = 0.000655). m. Relative expression (normalised to Actb = 1000) of Mpo and Spi1 in DP cells developing from DN4 cells seeded 12 d earlier onto MHC+ or MHC− stromal cells (Cells pooled from 3 separate cultures; Mpo: n = 8 qRT-PCR replicates; Spi1: n = 4 qRT-PCR replicates); for l, m: mean ± s.d.; P from two-tailed t-test; representative of 2 independent experiments; Mpo: P = 0.000013, Spi1: P = 0.000233).
