Extensive characterization of islet-reactive CD8 T cells in type 1 diabetes by mass cytometry (CyTOF)

In type 1 diabetes (T1D), insulin-secreting beta cells of the pancreatic islets are targets of autoimmune destruction by T lymphocytes, resulting in insulin deficiency and dysregulation of glucose metabolism. CD8 T cells reactive to islet antigens have been implicated in this process, and phenotypic changes in CD8 T cells have been observed in the transition to clinical diabetes. While efforts have led to identification of peptide antigens targeted by autoreactive CD8 T cells, less has been done to characterize the phenotype and function of this population. We developed a 25+ marker mass cytometry (CyTOF) panel to identify and extensively characterize CD8 T cells reactive to pooled class I (HLA-A2) tetramer loaded with peptides of viral or islet antigens in the same sample. Results were confirmed with several smaller flow panels. In PBMCs of three healthy controls and four T1D patients, we found virus-specific CD8 T cells had a more memory- and exhausted-like phenotype compared to total CD8 T cells, including increased T effector memory (CD45RO+CCR7−), KLRG1, CD57, TIGIT, and PD1, and decreased naive T cells (CD45RO−CCR7+). In contrast, islet-specific CD8 T cells (detectable in three T1D patients) did not vary significantly from total CD8 T cells. Given the differences in phenotype between viral and total CD8 T cells, we successfully used unsupervised clustering analysis software to reveal a distinct virus-specific CD8 T cell population. This approach of using pooled tetramer for detection of total viral and autoreactive CD8 T cells in the same sample, in combination with extensive phenotyping by mass cytometry will be useful in monitoring important phenotypic changes in this population during disease progression and in response to therapy.