High Diversity in the TCR Repertoire of GAD65 Autoantigen-Specific Human CD4+ T Cells

Abstract

Autoreactive CD4(+) T cells are an essential feature of type 1 diabetes mellitus. We applied single-cell TCR α- and β-chain sequencing to peripheral blood GAD65-specific CD4(+) T cells, and TCR α-chain next-generation sequencing to bulk memory CD4(+) T cells to provide insight into TCR diversity in autoimmune diabetes mellitus. TCRs obtained for 1650 GAD65-specific CD4(+) T cells isolated from GAD65 proliferation assays and/or GAD65 557I tetramer staining in 6 patients and 10 islet autoantibody-positive children showed large diversity with 1003 different TCRs identified. TRAV and TRBV gene usage was broad, and the TRBV5.1 gene was most prominent within the GAD65 557I tetramer(+) cells. Limited overlap (<5%) was observed between TCRs of GAD65-proliferating and GAD65 557I tetramer(+) CD4(+) T cells. Few TCRs were repeatedly found in GAD65-specific cells at different time points from individual patients, and none was seen in more than one subject. However, single chains were often shared between patients and used in combination with different second chains. Next-generation sequencing revealed a wide frequency range (<0.00001-1.62%) of TCR α-chains corresponding to GAD65-specific T cells. The findings support minor selection of genes and TCRs for GAD65-specific T cells, but fail to provide strong support for TCR-targeted therapies.

Predicting Type 1 Diabetes Using Biomarkers

“…Considering recent developments in T-cell phenotyping methods (24), it is possible that the responsive phenotypes may allow researchers to develop functional assays that stratify the risk of islet autoimmunity in genetically susceptible infants.…”

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8+ T cell receptor alpha repertoire following allogeneic transplantation

“…Quantification and quality control was performed with the RNA 6000 Pico Kit on the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). First-strand cDNA was synthesized by a 5' RACE (template-switching) reverse transcriptase using 200 U SuperScript V R II (Invitrogen, Life Technologies, Carlsbad, CA, USA), as described previously [13,14]. For samples with <250 000 cells for RNA isolation, 50 000 Jurkat cells (human T cell leukaemia, DSMZ no: ACC 282) with a monoclonal T cell receptor (amino acid sequence CAVSDLEPNSSASKIIF) were added prior to cDNA amplification.…”

“…For samples with <250 000 cells for RNA isolation, 50 000 Jurkat cells (human T cell leukaemia, DSMZ no: ACC 282) with a monoclonal T cell receptor (amino acid sequence CAVSDLEPNSSASKIIF) were added prior to cDNA amplification. Further steps were performed as described previously [14]. Purification of cDNA was performed using the MinElute PCR Purification Kit (Qiagen).…”

“…Whole cDNA was amplified in three rounds of PCR, as described previously [14]. The final PCR product was purified using the QIAquick PCR purification Kit (Qiagen).…”

Autoreactive T cells in type 1 diabetes

“…However, those TCRs matching sequences with previously reported autoreactive T cells were highly enriched in the T1D donors. These included autoreactive T cells directed against epitopes of proinsulin C-peptide 19-35 previously identified in the islets of a T1D donor (179) and several GAD65-reactive clones reported in blood of prediabetic individuals and patients (183). These proinsulin and GAD65 TCR sequences were found in the PLN of some T1D donors.…”