Figure 1. Chromatin accessibility at many T effector loci is established prior to TCR expression.

a, Heatmap of Z scores of TSS-normalized ATAC-seq fragments across the k-means clustered (k=9) T effector loci (hereafter T_EFF loci) in lung CD8⁺IV⁻CD44⁺influenza-d35-specific tissue-resident memory CD8⁺ T cells²⁰ (CD8⁺ T_RM cells), CD8⁺CD19⁻γδTCR⁻TCRβ^hiCD44^loCD62L^hi naïve T cells (T_N cells) in vitro activated with CD3ε/CD28 Abs (CD8⁺ T_{EFF in vitro} cells), CD8⁺KLRG1⁺CD127⁻LCMV-d7-specific terminal effector T cells¹⁷ (CD8⁺ T_EFF cells), CD8⁺CD44⁺CD62L⁺LCMV-d180-specific central memory T cells¹⁷ (CD8⁺ T_CM cells), IL-12/IL-4-Ab-treated CD4⁺ T_N cells¹⁸ (T_H1 cells), IL-4/IFNγ-Ab-treated CD4⁺ T_N cells¹⁸ (T_H2 cells), IL-17A-GFP⁺CD4⁺TCRβ⁺ T cells²¹ from ileum (T_H17_gut cells) or inflamed brain/spinal cord (T_H17_CNS cells), CD4⁺CXCR5⁺SLAM^lo/int T cells¹⁹ (T_FH cells) and IL-4/LPS-treated B cells (B_EFF cells)²² (n=2 each). b, Bar plots showing the developmental stages at which T_EFF loci specific to indicated T_EFF cell subtypes in a first became accessible. Serum+LIF-maintained embryonic stem cells²⁴ (ESCs), hematopoietic progenitor cells (HPCs) as aggregate of CD127⁻cKit^hiSca-1⁺Flt3⁻ hematopoietic stem cells⁸ (HSCs), CD127⁻cKit^hiSca-1⁺Flt3⁺ multipotent progenitors⁸ (MPPs) and CD127⁺cKit^hiSca-1⁺Flt3⁺ common lymphoid progenitors⁸ (CLPs); ETP-DN3 thymocytes as aggregate of γδTCR⁻CD11b⁻CD11c⁻CD19⁻B220⁻NK-1.1⁻Gr-1⁻TER-119⁻ (Lin⁻) cKit^hiCD25⁻CD4⁻CD8⁻ (ETPs), and Lin⁻cKit^hiCD25^hiCD4⁻CD8⁻ (DN2a) and Lin⁻c-Kit⁻CD25^hiCD4⁻CD8⁻ (DN3); DN4-SP thymocytes as aggregate of Lin⁻c-Kit⁻CD25⁻CD44⁻CD4⁻CD8⁻ (DN4), Lin⁻CD4⁺CD8⁺TCRβ^loCD69⁻ (DP), Lin⁻CD4⁺CD8⁻TCRβ^hi (CD4SP) and Lin⁻CD4⁻CD8⁺TCRβ^hi (CD8SP) and indicated T_EFF cells. c, Representative genome browser of ATAC-seq (1 of n=2 each) in ESC, HSC, MPP, CLP, ETP, DN2a, DN3, DN4, DP, CD8 SP, T_N, T_EFF, B_EFF at Prf1 (T_EFF=CD8⁺ T_{EFF in vitro}), Ctla4 and Icos (T_EFF=T_FH). Adjoined bars indicate transcripts-per-million (TPM) from RNA-seq in each cell type of indicated gene. d, Representative aggregate histograms (top) and heatmaps (bottom) (1 of n=2) of ATAC-seq fragments of ESC, HSC, MPP, CLP, ETP, DN2a, DN3 and CD8⁺ T_{EFF in vitro} that intersect open chromatin regions specific to CD8⁺ T_{EFF in vitro}. e, Violin plots (boxes represent 1^st, median and 3^rd quartiles; whiskers represent median +/− 1.5 interquartile range) representing the number of overlap between each ATAC-seq peak (columns in a) and all T effector ATAC-seq profiles (rows in a), grouped by the developmental windows (ESC, HPC, ETP-DN3 and respective T_EFF cells) during which the ATAC-seq peak first became accessible.