Heterogeneity of circulating CD8 T-cells specific to islet, neo-antigen and virus in patients with type 1 diabetes mellitus

Sandra Laban,Bart O Roep,Anna Vilanova,Jos Pool,Nicola Pezzotti,Joris Wesselius,Boudewijn P F Lelieveldt,Vincent Van Unen,Thomas Höllt,Jessica S Suwandi,Francesco Dieli

doi:10.1371/journal.pone.0200818

Abstract

Auto-reactive CD8 T-cells play an important role in the destruction of pancreatic β-cells resulting in type 1 diabetes (T1D). However, the phenotype of these auto-reactive cytolytic CD8 T-cells has not yet been extensively described. We used high-dimensional mass cytometry to phenotype autoantigen- (pre-proinsulin), neoantigen- (insulin-DRIP) and virus- (cytomegalovirus) reactive CD8 T-cells in peripheral blood mononuclear cells (PBMCs) of T1D patients. A panel of 33 monoclonal antibodies was designed to further characterise these cells at the single-cell level. HLA-A2 class I tetramers were used for the detection of antigen-specific CD8 T-cells. Using a novel Hierarchical Stochastic Neighbor Embedding (HSNE) tool (implemented in Cytosplore), we identified 42 clusters within the CD8 T-cell compartment of three T1D patients and revealed profound heterogeneity between individuals, as each patient displayed a distinct cluster distribution. Single-cell analysis of pre-proinsulin, insulin-DRIP and cytomegalovirus-specific CD8 T-cells showed that the detected specificities were heterogeneous between and within patients. These findings emphasize the challenge to define the obscure nature of auto-reactive CD8 T-cells.

Highlights

A hallmark of autoimmune type 1 diabetes (T1D) is the destruction of pancreatic β-cells
Several studies have demonstrated the critical role of auto-reactive CD8 T-cells in the disease pathogenesis [1,2,3,4]. β cell-specific CD8 T-cells are present in the blood of T1D patients, in very low frequencies
To confirm the detection of epitope-specific cells, we spiked a CD8 T-cell clone with reactivity against PPI15-24, INS-DRIP1–9 or CMV-pp65495-503 in peripheral blood mononuclear cells (PBMCs) of a healthy HLA-A2 negative patient followed by staining and acquiring with the mass cytometer (S1B Fig)

Summary

Introduction

A hallmark of autoimmune type 1 diabetes (T1D) is the destruction of pancreatic β-cells. Several studies have demonstrated the critical role of auto-reactive CD8 T-cells in the disease pathogenesis [1,2,3,4]. Β cell-specific CD8 T-cells are present in the blood of T1D patients, in very low frequencies. Several HLA-A2 restricted islet epitopes have been associated with T1D, including pre-proinsulin (PPI), GAD65, IA-2, IGRP and ZnT8 [5]. We recently discovered a novel nonconventional self-epitope derived from an alternative open reading frame. Heterogeneity in the adaptive CD8 T-cell compartment of insulin mRNA [6]. CD8 T-cells directed against this defective ribosomal product (DRIP) destroyed human β-cells in vitro

Objectives

Methods

Results

Conclusion