Combinatorial detection of autoreactive CD8+ T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop

Abstract

Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting.

Autoreactive CD8+ T cell exhaustion distinguishes subjects with slow type 1 diabetes progression

“…We identified antigen-specific CD8 + T cells using a modified combinatorial Tmr staining approach based on the method described by Newell et al (26) (Supplemental Figure 1). In verifying Tmr staining by CyTOF, we found that the intensity of Tmr staining was generally greater for the virus-specific pool than for the islet-specific pool (Supplemental Figure 1D and Figure 1A) and that both the Tmr + frequencies and the intensities of markers were highly reproducible (Supplemental Figure 2), as previously described with flow cytometric Tmr analysis (11)(12)(13)(14)(15).…”

“…Ample evidence indicates that islet-specific CD8 + T cells are a significant driver of β cell destruction in T1D (34). However, across numerous studies involving individuals with T1D, the frequencies of islet-specific CD8 + T cells in peripheral blood have been neither consistently altered nor strongly correlated with disease progression (11)(12)(13)(14)(15)35). Here, through an unbiased and multidimensional approach, we demonstrate that islet-specific CD8 + T cells of HCs and T1D subjects comprise 3 dominant phenotypes that display characteristics of transitional memory or exhausted memory cells.…”

“…In spite of the complex phenotypes and heterogeneity within autoreactive CD8 + T cells, approaches to analyze these rare T cell subsets in T1D typically utilize a small number of singleparameter values (11)(12)(13)(14)(15)27). To address this issue, we generated a high-content CyTOF panel incorporating HLA class I tetramers to identify antigen-specific CD8 + T cells and additional markers of differentiation, activation, and exhaustion (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI126595DS1).…”

“…The character of the immune response probably plays a role in the rate of functional β cell loss following T1D diagnosis, as treatment with immune-modulating therapies results in shortterm preservation of β cell function in some individuals (6). CD8 + T cells in particular influence T1D susceptibility and progression as clearly shown in mouse models (7)(8)(9)(10), and islet-specific CD8 + T cells are detectable in the peripheral blood of individuals with T1D (11)(12)(13)(14)(15). Within the pancreas, CD8 + T cells are the most abun-dant lymphocyte of insulitic islets (16), and both polyclonal and islet-specific CD8 + T cells are more prevalent in the pancreas of individuals with T1D than in at-risk or healthy controls (HCs) (15,(17)(18)(19).…”

Slow progressors to type 1 diabetes lose islet autoantibodies over time, have few islet antigen-specific CD8+ T cells and exhibit a distinct CD95hi B cell phenotype

“…Tetramers Fluorochrome-labelled peptide-HLA-A2 tetramers were assembled from monomers from NIH Tetramer Core Facility (Atlanta, GA, USA) and streptavidin Qdots (Thermo Fisher Scientific, Waltham, MA, USA). Tetramer staining was performed as described previously [19]. Briefly, thawed PBMCs were treated with 50 nmol/l dasatinib for 15 min at 37°C, then pelleted and resuspended in tetramer Qdot master mix for 15 min at 37°C (details in ESM Table 2).…”