Single-cell TCR sequencing of gut intraepithelial γδ T cells reveals a vast and diverse repertoire in celiac disease

Abstract

A hallmark of celiac disease (CeD), a chronic condition driven by cereal gluten exposure, is increase of gut intraepithelial γδ T cells. This may indicate pathogenic involvement of γδ T cells and existence of disease-specific γδ T-cell receptors (TCRs) recognizing defined antigen(s). We performed high-throughput and paired γδ TCR sequencing of single intraepithelial γδ T cells of untreated CeD patients (n=8; 1821 cells), CeD patients treated with a gluten-free diet (n=5; 436 cells) and controls (n=7; 1068 cells). We found that CeD patients, both untreated and treated, had larger and more diverse γδ TCR repertoires, more frequent usage of TRDV1 and TRDV3 and different patterns of TCRγ/TCRδ-pairing compared to controls. While we observed no public CDR3δ sequences, there were several public CDR3γ sequencesmany of which were shared by the CeD patients, but also by the controls. These public CDR3s were characterized by few N/P nucleotide insertions with germline and near-germline configuration, hence being easy to generate. Previous findings of CeD-specific CDR3 motifs were not replicated. Thus, being unable to raise evidence for CeD-specific γδ TCRs in this first large, paired γδ TCR single-cell sequencing study, we project challenges for identification of CeD-relevant γδ TCR ligands.

Coeliac Disease Pathogenesis: The Uncertainties of a Well-Known Immune Mediated Disorder

“…This raises an intriguing possibility that maintenance of γ/δ T cell regulatory function could explain the phenotype of potential CD, a scenario where γ/δ IELs keep α/β IEL cytotoxicity in check. Indeed, recent studies detailing long-term genomic and functional changes in the composition of the γ/δ IEL compartment in CD has shown that a subset of Vγ4 + /Vδ1 + type γ/δ IELs, which have a role in tissue healing and homeostasis, is lost and replaced by a persistent IFNγ producing Vδ1 + T cell population, thereby supporting this hypothesis ( 21 , 22 ). The role of other lymphocyte subsets such as MAIT cells, iNKT cells and NK cells in CD is less well-understood ( 23 ).…”

Human γδ TCR Repertoires in Health and Disease

“…Importantly, celiac disease leads to a loss of the BTNL-induced Vγ4 + IEL compartment, that cannot be restored after gluten-free diet [38]. Notably, an innovative approach of single-cell TCR repertoire analysis of γδ IELs of celiac disease patients noticed a higher TCR repertoire diversity, due to loss of innate-like Vγ4 + IELs, but did not identify public γδ T cell clones that may recognize defined disease-associated ligands [39]. Moreover, in colorectal cancer patients with higher numbers of innate-like cytotoxic Nkp46 + Vγ4Vδ1 + T cells were correlated with a better clinical outcome [110].…”

“…Human Vγ4 + IELs are shaped and selected by the BTNL-like molecules, BTNL3 and BTNL8, that are exclusively expressed in the human gut epithelial [14]. TCR repertoire analysis of total or Nkp46 + γδ IELs isolated from healthy tissues gave evidence for a relatively clonal TRG and TRD repertoire, enriched for public Vγ4 + and private Vδ1 + T cell clones [38,39,110]. Importantly, celiac disease leads to a loss of the BTNL-induced Vγ4 + IEL compartment, that cannot be restored after gluten-free diet [38].…”

Our evolving understanding of the role of the γδ T cell receptor in γδ T cell mediated immunity

“…Further investigation showed remodelling of the IEL γδ T cell compartment within celiac patients with altered ability to respond to BTNL3/8 correlated with disease progression. The underlying mechanism and the importance of the H-J1 motif are still to be determined with a large scale single-cell study of γδ TCR sequences from celiac patients unable to identify the H-J1 motif [ 74 ].…”