Identification of Candidate Tolerogenic CD8⁺ T Cell Epitopes for Therapy of Type 1 Diabetes in the NOD Mouse Model

Figure 1

(a) Proliferation assay of PLN cells. LN cells were mixed with mitomycin C-treated spleen cells plus peptide for 48 h before pulsing with [³H]-thymidine and scintillation counting. Shown is data from 1 of 5 assays. “Fold stimulation” (-axis) is the average thymidine incorporation at a given peptide concentration divided by average incorporation into wells pulsed with Flu NP_147–155. (b) IFN- expression of LN cells. Expression of IFN- was assayed as described in “Section 2”. In brief, PLN were isolated from NOD mice (3 weeks, striped bars; 13 weeks, white bars; and diabetic mice, black bars), pulsed with peptides for 48 h, and supernatants were analyzed by ELISA for IFN production. Shown is data from 1 of 2 assays. (c) RMA-S stabilization assay. Plotted are the ratios of MFI values of peptide-pulsed RMA-S cells to nonpulsed cells stained with either anti- or . For each peptide as indicated the stabilization assay was performed >3 times. (d) Tetramer analysis of NOD spleen cells. Tetramers were used together with anti-CD8 to stain spleen cells of different aged NOD mice (as indicated) as described in “Section 2.” Plotted are tetramer positive cells as a percentage of CD8 cells. (e) Example of tetramer analysis of NOD spleen cells. As example of the data used to generate (d), Anti-Flu NP_147–155 (top panels), IGRP_206–214, DβH_233–241, and ZnT8_282–290 (bottom panels) tetramers were used together with anti-CD8 to stain spleen cells of 13-week-old NOD mice as described in “Section 2.” DβH tetramer positive cells were 0.42% of CD8⁺ cells, IGRP tetramer positive cells were 0.66% of CD8⁺ cells, and ZnT8 positive cells were 0.69% of CD8⁺ cells.

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