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. 2017 Aug 25;66(12):3051–3060. doi: 10.2337/db17-0666

Figure 1.

Figure 1

Identification of InsB:IAg7 tetramer–binding cells in PBMCs from NOD and B6.g7 mice, with in vitro and in vivo validation of the InsB:DQ8 tetramer reagent. A: Quantification of InsB:IAg7 tetramer–binding cells from pancreatic lymph nodes and PBMCs isolated from 0.5 mL blood per mouse of 4- to 15-week-old NOD and age-matched B6.g7 mice. B: The percentage of CD44+ cells of InsB:IAg7 tetramer–binding cells within PBMCs from panel A (n = 6–11 mice/group [biological replicate], data are pooled from three independent experiments [technical replicate] for panels A and B). C: Representative flow cytometry plots showing dual InsB:DQ8 tetramer staining of CD4+ T cells from the secondary lymphoid organs of naïve (left panel) and InsB10–23 immunized (right panels) NOD.DQ8.Ab0 transgenic mice (data are representative of three independent experiments [technical replicates]). D: Quantification of tetramer+ cells shown in panel C from the secondary lymphoid organs of NOD.DQ8.Ab0 transgenic naïve mice (n = 4–6 [biological replicates]) and mice immunized (n = 9–13) with InsB10–23 peptide emulsified in complete Freund’s adjuvant (CFA). Cells were stained with InsB10–23:DQ8 tetramers in APC and PE (data are pooled from three independent experiments [technical replicates]). E: Representative flow cytometry plots showing dual tetramer staining within peripheral blood CD4+ T-cell (top) and no dual staining of CD8α+ T-cell compartments (bottom) from DR4+/DQ8+ human subjects after 14 days of in vitro stimulation with modified InsB10–23 or HA306–318 (data are representative of four independent experiments [technical replicate], n = 8 T1D patients for each tetramer [biological replicate]). Three patients are shown: T1D PBMC (a)—male, age 14, T1D duration of 3.5 years; T1D PBMC (b)—female, age 6, T1D duration of 2.6 years; and T1D PBMC (c)—male, age 34, T1D duration of 32 years. The solid lines in panels A, B, and D represent the mean ± SEM. The Mann-Whitney U test was used to determine statistical significance for nonparametric data. *P < 0.05, ***P < 0.0001. PE, R-phycoerythrin.