Mesenteric lymph node (mLN) T cells undergo tissue adaptation upon migrating to intestinal lamina propria (LP) and intraepithelial (IE) compartments, ensuring appropriate balance between tolerance and resistance. By combining mouse genetics with single-cell and chromatin analyses, we addressed the molecular imprinting of gut epithelium on T cells. Transcriptionally, conventional and regulatory (Treg) CD4+ T cells from mLN, LP and IE segregate based on the gut layer they occupy; trajectory analysis suggests a stepwise loss of CD4-programming and acquisition of an intraepithelial profile. Treg fate–mapping coupled with RNA– and ATAC–sequencing revealed that the Treg program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of CD4 lineage–defining transcription factor ThPOK results in premature acquisition of an IEL profile by mLN Tregs, partially recapitulating epithelium imprinting. Thus, coordinated replacement of circulating lymphocyte program with site–specific transcriptional and chromatin changes is necessary for tissue imprinting.
Overall design: Atac-Seq: ATAC-Seq was performed on 5,000-40,000 FACS-purified cells from 2-9 mice. In brief, cells were lysed in lysis buffer for 1 minute and transposed with Tagment DNA Enzyme 1 (Illumina) for 30 minutes. DNA was cleaned up using a MinElute DNA purification Kit (Qiagen), followed by barcoding and library preparation by the Nextera DNA Library preparation kit (Illumina) according to manufacturer’s guidelines and sequenced on an Illumina Illumina NextSeq 500.
ChIP-Seq: Cells were fixed in 1% formaldehyde for 20min, quenched with 0.15M glycine and washed in PBS. 2x106 were then sorted and lysed for 30min at 4ºC. Cells were then sonicated for 22 minutes at 30 seconds on/off using the Bioruptor sonicator (Diagenode) and spun down. 10% was frozen for input, the remaining 90% was incubated with anti-ThPOK antibody bound to goat anti-rabbit M280 magnetic beads (Invitrogen) overnight at 4ºC prior to washing with RIPA buffer and overnight decrosslinking at 65ºC. DNA was then eluted off beads into TE buffer and, along with input, purified using the Zymogen DNA clean & concentrator kit. DNA was sequenced using NextSeq2500. To obtain Tregs, Foxp3RFP reporter mice were injected with IL-2/aIL-2 complex as previously described for 3 consecutive days for Treg expansion. CD4+ T cells were isolated from the spleen and mLNs and sorted as Thy1+TCRb+CD4+CD8a–RFP+.
RNA-Seq: Sorted cells (300-800) were lysed in TCL buffer (Qiagen, 1031576) supplemented with 1% βmercaptoethanol. RNA was isolated using RNAClean XP beads (Agentcourt, A63987), reversibly transcribed, and amplified. Uniquely barcoded libraries were prepared using Nextera XT kit (Illumina) following manufacturer’s instructions. Sequencing was performed on an Illumina NextSeq 550.
Single Cell Sequencing: Lymphocytes were sorted, counted for viability and immediately subjected to library preparation. The scRNA-Seq library was prepared using the 10x Single Cell Chromium system, according to the manufacturer’s instructions at the Genomics core of Rockefeller University and was sequenced on an Illumina NextSeq 550 to a minimum sequencing depth of 50,000 reads per cell using read lengths of 26 bp read 1, 8 bp i7 index, 98 bp read 2.
Please note that the processed data generated from both replicates is linked to the corresponding rep1 sample (with the lower GSM accn number).
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