The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear， partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure， inferred from expanded TCR clonotypes， and essential phenotypic properties in their transcriptomes. To test this， we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4 memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides... More
The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear， partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure， inferred from expanded TCR clonotypes， and essential phenotypic properties in their transcriptomes. To test this， we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4 memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells， especially from individual T1D subjects， consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo， demonstrating clonotype stability. Notably， we found no clonotype sharing between subjects， indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides， implicating this molecule as a trigger for CD4 T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar， profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4 memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood.
