CD8aa+ intraepithelial lymphocytes recruited from peripheral CD4+ and CD8+ T cells represent functionally distinct lineages [TCR-seq]

The TCRaß CD8aa+ intraepithelial lymphocytes (IELs) play a significant role in primary immune defence in the small intestine. The canonical model of their development distinguishes TCRaß IELs into induced and natural (TCRaß CD8aa+) types, each influenced by distinct developmental pathways and antigen encounters. It is believed that natural CD8aa+ IELs differentiate solely from triple positive (TP) CD4+CD8aß+CD8aa+ thymic progenitors that recognized self MHC-peptide complexes with high affinity but avoided deletion during selection. Our investigation reveals that, in addition to this central commitment, the CD8aa+ IELs undergo in situ enrichment through recruitment from peripheral CD4+ and CD8aß+ T cell populations. We found that the functional makeup of individual clones within the CD8aa+ IEL subset is dictated by their origin. We demonstrate that expression of Bmp7 or Ly49 (Ly49C, F, H, and I) serve as markers indicating the CD8aa+ IELs' peripheral provenance: CD8aß+ and CD4+ T lymphocytes, respectively. Overall design: To generate Next-Generation Sequencing (NGS) libraries encompassing the entire T cell receptor (TCR) repertoire, total RNA was extracted from sorted TCRß CD8aa+ Ly49+, TCRß+ CD8aa+ Bmp7+, TCRß+ CD8aa+ DN IEL and peripheral CD4+ and CD8+ T cells. The reverse transcriptase reaction employing template-switching primers with the sequence TAAGAGACAGCAACTACTACTGCrGrGrGr (where r ,indicates ribonucleotide). Two rounds of amplification for the TCR cDNAs were conducted using Q5® High-Fidelity DNA Polymerase (NEB, Massachusetts, USA) following the manufacturer's instructions. The primers used in the first PCR round: For-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACTACTACTGC Rev-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTACACAGCAGGTTCTGG