Disease-driving CD4+ T cell clonotypes persist for decades in celiac disease

Abstract

Little is known about the repertoire dynamics and persistence of pathogenic T cells in HLA-associated disorders. In celiac disease, a disorder with a strong association with certain HLA-DQ allotypes, presumed pathogenic T cells can be visualized and isolated with HLA-DQ:gluten tetramers, thereby enabling further characterization. Single and bulk populations of HLA-DQ:gluten tetramer-sorted CD4+ T cells were analyzed by high-throughput DNA sequencing of rearranged TCR-α and -β genes. Blood and gut biopsy samples from 21 celiac disease patients, taken at various stages of disease and in intervals of weeks to decades apart, were examined. Persistence of the same clonotypes was seen in both compartments over decades, with up to 53% overlap between samples obtained 16 to 28 years apart. Further, we observed that the recall response following oral gluten challenge was dominated by preexisting CD4+ T cell clonotypes. Public features were frequent among gluten-specific T cells, as 10% of TCR-α, TCR-β, or paired TCR-αβ amino acid sequences of total 1813 TCRs generated from 17 patients were observed in 2 or more patients. In established celiac disease, the T cell clonotypes that recognize gluten are persistent for decades, making up fixed repertoires that prevalently exhibit public features. These T cells represent an attractive therapeutic target.

Resident memory CD8 T cells persist for years in human small intestine

“…In the last PCR reaction, TRAC and TRBC barcoding primers were added together with Illumina PairedEnd primers. Cycling conditions, concentrations and primer sequences for all three PCR reactions can be found in (Risnes et al, 2018), and original protocol in (Han et al, 2014). Products in each well were combined, purified and sequenced using the Illumina paired-end 250 bp MiSeq platform at the Norwegian Sequencing Centre (NSC), at Oslo University Hospital.…”

“…Products in each well were combined, purified and sequenced using the Illumina paired-end 250 bp MiSeq platform at the Norwegian Sequencing Centre (NSC), at Oslo University Hospital. The resulting paired-end sequencing reads were processed in a multistep pipeline using selected steps of the pRESTO toolkit (Vander Heiden et al, 2014) according to (Risnes et al, 2018). In short, high-purity reads (average Phred score >30) were deconvoluted using barcode identifiers and collapsed, and only the top three for each well were retained for further analysis.…”

“…Total RNA was extracted using RNAclean XP beads (Agencourt) following the manufacture's protocol. A modified SMART protocol (Quigley et al, 2011) was used in first-strand cDNA synthesis, described in detail elsewhere (Risnes et al, 2018). In brief, TCRα and TCRβ genes were amplified in two rounds of semi-nested PCR reactions.…”

“…The plates were spun-down and snap frozen immediately after sorting and stored at -80ºC until cDNA synthesis. Paired TCRα and TCRβ sequences were obtained after three nested PCR with multiplexed primers covering all TCRα and TCRβ V genes, as described before (Risnes et al, 2018). In brief, cDNA plates were stored at -20ºC, and each of the three nested PCR steps was carried out in a total volume of 10 µL using 1 µL cDNA/PCR template and KAPA HiFi HotStart ReadyMix (Kapa Biosystems).…”

Affinity-engineered human antibodies detect celiac disease gluten pMHC complexes and inhibit T-cell activation

“…Having confirmed that cell-surface pMHC was specifically detected both on in vitro peptideloaded cells and on patient-derived APCs loaded in vivo, we wondered if we could block the interaction between T cells and APCs. We focused on the DQ2.5-glia-α2 epitope, as it is the most extensively characterized gliadin T-cell epitope to which all patients mount a T-cell response (29).…”