Tissue-specific butyrophilin-like proteins are γδTCR selecting ligands distinct from antigens

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Tissue-resident γδ T cells are a population of unconventional lymphocytes able to mount rapid immune responses following tissue damage. It has long been debated whether selecting elements for γδ T cells exist, akin to MHC for αβ T cells. In mice, γδ T lymphocytes show tissue-restricted T cell receptor (TCR) repertoires, which are dependent, at least in the epidermis and gut, on the tissue-specific expression of butyrophilin-like proteins (Btnl). Hence, we hypothesised that the Btnl family of proteins are physiological ligands modulating γδ T cell selection and function. We have previously established in vitro that BTNL3 and BTNL8, specifically expressed in the human intestinal epithelium, induce selective TCR-dependent responses in a subset of colonic γδ T cells. The aim of this project was to investigate the mechanism of interaction between BTNL3 plus BTNL8 and the TCR of responding cells.
To this end, responding, human colonic intraepithelial lymphocytes (IELs) co-cultured with BTNL3 plus BTNL8-expressing cells were single cell-sorted and sequenced. Majority of responding cells were clonally diverse Vγ4+ lymphocytes and several paired γδTCR sequences were expressed in TCR-deficient Jurkat cells. In co-culture assays, Vγ4-expressing Jurkats downregulated their TCR and upregulated CD69 when exposed to BTNL3 plus BTNL8. Site-directed mutagenesis showed unexpectedly, that the hypervariable loop 4 (HV4) of the Vγ4 chain was critical for BNTL3-specific recognition. Intriguingly, CD1c-phosphatidylcholine-reactive Vγ4Vδ1 and endothelial protein C receptor (EPCR)-reactive Vγ4Vδ5 cells were capable of mounting a response against BTNL3 plus BTNL8 deeming them dually reactive. Importantly, CD1c and EPCR recognitions were CDR3-mediated.
Thus, human Vγ4+ TCRs appear to have two spatially distinct regions involved in antigen recognition. The adaptive, non-germline encoded γδCDR3 loops are required for binding to clonally-restricted ligands whereas the innate, germline-encoded HV4γ loop is required for interaction with the tissue-specific complex BTNL3/8. The same phenomenon is true for murine intestinal Vγ7+ cells which require Btnl1/4/6 for their development and tissue retention. Although these molecules are not direct human orthologues, an evolutionarily conserved mechanism emerges. BTNL3/8 are perhaps selecting elements of intestinal Vγ4+ IELs and future studies should investigate the biological significance of these interactions in humans. Of note, early studies presented in this thesis suggest that BTNL3 and BTNL8 heteromeric complex is partially depleting the Vγ4 TCR from the lymphocyte surface, suggesting an immunoregulatory role for this interaction
Date of Award1 Jul 2020
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorAdrian Hayday (Supervisor) & James Arnold (Supervisor)

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