Phenotype‐Based Isolation of Antigen‐Specific CD4⁺ T Cells in Autoimmunity: A Study of Celiac Disease

Abstract

The pathogenic immune response in celiac disease (CeD) is orchestrated by phenotypically distinct CD4 + T cells that recognize gluten epitopes in the context of disease-associated HLA-DQ allotypes. Cells with the same distinct phenotype, but with elusive specificities, are increased across multiple autoimmune conditions. Here, whether sorting of T cells based on their distinct phenotype (Tphe cells) yields gluten-reactive cells in CeD is tested. The method′s efficiency is benchmarked by parallel isolation of gluten-reactive T cells (Ttet cells), using HLA-DQ:gluten peptide tetramers. From gut biopsies of 12 untreated HLA-DQ2.5 + CeD patients, Ttet + /Tphe + , Ttet − /Tphe + , and Ttet − /Tphe − cells are sorted for single-cell T-cell receptor (TCR)-sequencing (n = 8) and T-cell clone (TCC)-generation (n = 5). The generated TCCs are TCR sequenced and tested for their reactivity against deamidated gluten. Gluten-reactivity is observed in 91.2% of Ttet + /Tphe + TCCs, 65.3% of Ttet − /Tphe + TCCs and 0% of Ttet − /Tphe − TCCs. TCR sequencing reveals clonal expansion and sequence sharing across patients, features reflecting antigen-driven responses. The feasibility to isolate antigen-specific CD4 + T cells by the sole use of phenotypic markers in CeD outlines a potential avenue for characterizing disease-driving CD4 + T cells in autoimmune conditions.

Intestinal and blood lymphograms as new diagnostic tests for celiac disease

“…The adequacy of flow cytometry to identify gluten-induced T cells was also evidenced by the work of Christophersen et al., who showed that gluten reactive CD4 + T cells could be identified from biopsy based solely on phenotypic markers ( 56 ).…”

“…Accordingly, in the published works there are several cases of patients who respond to deamidated gliadin but they do not to the specific peptide used. The use of restricted sequences (antigen stimulation) may reduce the response to be observed although a cocktail of immunodominant peptides is used ( 56 ). Moreover, the use of antigenic peptides also needs to determine the optimal antigen concentration to obtain maximum responses.…”

Immunopathogenesis and environmental triggers in coeliac disease

“…A newer promising method derives from the extensive phenotypic characterisation of tetramer + cells. This analysis has identified a combination of 7–11 membrane markers that can be used for detecting gliadin-specific CD4 + T cells 25 26. Finally, the peak of IL-2 released in the serum 4 hours after oral gluten challenge in treated CD might be a useful biomarker to detect the recall response of gluten-specific CD4 + T cells residing in the gut 27.…”

Identification of gluten T cell epitopes driving celiac disease

“…The starting point for T cell epitope characterization is access to CeD-relevant TCCs with unknown epitope specificity. In a recent study ( 22 ), we described a method to isolate T cells solely on the basis of distinct phenotypic markers (CD3 + , CD4 + , CD127 − , PD-1 + , CD161 + , ICOS + , CD39 + , and CXCR3 + ) that are hallmarks of the gluten-specific T cells ( 23 ). By generating TCCs from such phenotypically distinct T cells, we found that more than 65% of these TCCs were indeed gliadin/glutenin reactive ( 22 ).…”

“…In a recent study ( 22 ), we described a method to isolate T cells solely on the basis of distinct phenotypic markers (CD3 + , CD4 + , CD127 − , PD-1 + , CD161 + , ICOS + , CD39 + , and CXCR3 + ) that are hallmarks of the gluten-specific T cells ( 23 ). By generating TCCs from such phenotypically distinct T cells, we found that more than 65% of these TCCs were indeed gliadin/glutenin reactive ( 22 ). Here, we identified yet uncharacterized T cell epitopes by testing phenotypically distinct gluten-reactive TCCs with unknown epitope specificity against peptides representing potential HLA-DQ2.5–restricted epitopes identified by knowledge-based strategies.…”