Immunology of Diabetes Society T-Cell Workshop: HLA class I tetramer-directed epitope validation initiative T-Cell Workshop Report-HLA Class I Tetramer Validation Initiative.

BACKGROUND: Identification of T-cell reactivity to ß-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). METHODS: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545). We also compared the readout of TMrs and PMrs with a CD8+ T-cell interferon-γ enzyme-linked immunospot assay. RESULTS: Despite the minute frequencies of autoreactive cells detected by TMrs/PMrs, most (73-77%) T1D patients had responses to one or more of the epitopes used. All four epitopes were recognized by T1D patients, with a prevalence ranging from 5 to 25%. TMrs and PMrs detected more positive responses to the ß-cell epitopes than CD8+ T-cell interferon-γ enzyme-linked immunospot. However, concordance between positive responses to TMrs and PMrs was limited. CONCLUSIONS: Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.

Immunology of Diabetes Society T-Cell Workshop: HLA class II tetramer-directed epitope validation initiative.

BACKGROUND: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study. METHODS: A tetramer-based assay was used in five participating centres to measure T-cell reactivities to DR0401-restricted epitopes. Three participating centres concurrently performed ELISPOT or immunoblot assays. Each centre used blind-coded, centrally distributed peptide and tetramer reagents. RESULTS: All participating centres detected responses to auto-antigens and the positive control antigen, and in some cases cloned the corresponding T cells. However, response rates varied among centres. In total, 74% of patients were positive for at least one islet epitope. The most commonly recognized epitope was GAD270-285. Only a minority of the patients tested by tetramer and ELISPOT were concordant for both assays. CONCLUSIONS: This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course.