(A) Insulin reactivity of 2H6 TCR transgenic T cells. Splenocytes from 3 randomly selected 2H6 TCR transgenic mice (1 from line 22 and 2 from line 50) were used in the proliferation assay. Cells (10⁵ per well) were cultured in Click’s medium for 72 hours (in triplicate). ³H-thymidine was added for the last 16–18 hours. (B) The reactivity to insulin B chain peptide 12–25 or 9–23 was also analyzed using the same protocol as in A. Results are illustrated as Δ cpm = proliferation with peptide – proliferation with medium. (C and D) TGF-β (C) and IFN-γ (D) production from cells of transgene-negative and -positive NOD mice. Splenocytes (10⁵ cells per well) from both types of mice were cultured in Click’s medium in the presence or absence of anti-CD3 (1:100 dilution of 2C11 hybridoma supernatant) for 48 hours (in triplicate). The culture supernatants were collected. IFN-γ and TGF-β contents were measured with an IFN-γ ELISA kit (BD Biosciences) and a TGF-β ELISA kit (R&D Systems). (E) Expression of CTLA-4 and FoxP3 in transgene-positive and -negative NOD mice. Splenocytes (10⁶) from both types of mice were stained for intracellular CTLA-4 and FoxP3 expression with or without TCR stimulation (anti-CD3). Monoclonal antibodies against CTLA-4 and FoxP3 were purchased from BD Biosciences and eBioscience, respectively. The staining was carried out according to the manufacturers’ protocols. The numbers in the top right quadrants represent the percentage of positive cells among the total cells analyzed.