Figure 1. Analysis of autoantigen reactive CD8⁺ T cells by T cell libraries.

Libraries were prepared from CD8+CD45RO+ cells sorted from the peripheral blood as described from 29 patients with T1D and 15 HC. Each well was expanded with irradiated allogeneic PBMC+IL-2, IL-7, and IL-15 as described in Materials and Methods. After 10 days the wells were washed and stimulated with K562 cells that had been pulsed with 6 diabetes peptides, peptides from EBV/flu, or treated with DMSO alone. IFNγ levels in the supernatants were measured by ELISA after 6 days. Data from a representative patient and HC subject are shown in Supplemental Figure 2B. The frequencies of positive wells that were above the threshold, which was mean+3SD of DMSO wells were calculated for each subject. There was a significantly greater proportion of positive wells from CD45RO+ cells from patients with T1D reactive with islet antigens (A)(*p=0.028, t-test with Welch’s correction) compared to HC, but not to peptides from EBV/flu (B). CD45RA+CD8+ cells were sorted from 25 and 13 of the T1D patients and HC respectively. (C,D) There was not a significant difference between the frequency of islet antigen-reactive (C) or EBV/flu reactive cells (D). The relationship between frequency of CD45RO+ CD8+ islet antigen-reactive cells and diabetes duration (E) or age (F) are shown (p=ns, Spearman corr). The orange and green dots represent samples from concordant identical triplets and twins respectively.