TIP mice were bred with NOD.FoxP3.GFP reporter mice. Frequency of Foxp3-expressing insulin-specific CD4⁺ T cells was determined by enumerating GFP⁺ insulin tetramer⁺ CD4⁺ T cells. (A) Representative FACS plots showing GFP reporter expression on insulin tetramer⁺ CD4⁺ T cells and (B) frequency of GFP⁺ insulin tetramer⁺ CD4⁺ T cells enriched from peripheral lymphoid organs of indicated mice. Each symbol in the scatter plot (mean ± SEM) represents data from an individual mouse. (C) Incidence of diabetes in 8- to 9-week-old NOD.Rag^–/– recipient mice after coinjection of splenocytes from diabetic NOD mice along with splenocytes from either nontransgenic NOD mice (n = 3 recipients) or TIP mice (n = 5 recipients) at an equal ratio (2 × 10⁷ cells of each per recipient) (P = values were not significant). Insulin_10–23 tetramer⁺ CD4⁺ T cells were enriched from PLNs of TIP mice, and intracellular FoxP3 expression was analyzed by flow cytometry. Representative FACS plots (D) showing FoxP3 expression on insulin tetramer⁺ cells in cohort 1–3 of TIP mice. Cumulative data from 3–4 independent experiments (E) showing number and proportion of CD4+ tetramer+ Foxp3+ Treg subset (top) and CD4+ tetramer+ Foxp3- T effector subset (bottom). Each symbol in the scatter plots (E) represents data from pancreatic lymph nodes (PLNs) pooled from 3 mice (Mean±SEM). Values in FACS plots (A and D) shown in italics indicate percentage and values in parentheses indicate absolute numbers. Incidence of diabetes (F) in 8- to 9-week-old NOD.Rag^–/– recipient mice after coinjection of splenocytes from diabetic NOD mice alone or along with cells from PLN of 12- to 14-week-old nontransgenic NOD mice or TIP mice (cohort 3) at an equal ratio (2 × 10⁷ cells of each per recipient, n = 4 recipients per group, P = values were not significant). *P < 0.05. Data analysed using 2-tailed unpaired t test (B) or One-way ANOVA with Tukey’s multiple comparisons test (E). Survival curves (C and F) compared using log-rank test.