Fig. 3.

Insulin B:9-23/IFA treatment induces Tregs that protect from diabetes. a Treg (CD4⁺CD25⁺FOXP3⁺CD127^low) frequency in the PDLN, (b) spleen and (c) blood of NOD mice immunised with B:9-23/IFA (white bars) or PBS/IFA (grey bars) s.c. at 9 weeks of age or left untreated (black bars). Treg frequency in the blood was monitored for 3 consecutive weeks after immunisation (as indicated), whereas in PDLN and spleen Treg frequency was monitored for 2 consecutive weeks as shown. The experiment was repeated at least twice with three to four mice per group (ND, not done). p < 0.05 for PDLN at 12 weeks and p < 0.01 for spleen at 11 weeks of age when B:9-23/IFA is compared with controls. d Total Treg numbers in PDLN and (e) the spleen. The Treg percentage for each individual mouse was multiplied with its respective total spleen or PDLN lymphocyte number. Trypan blue staining was used to exclude dead cells. p < 0.01 for PDLN at 12 weeks, p < 0.05 and p < 0.01 for spleen at 12 and 11 weeks of age respectively (B:9-23/IFA vs controls). Mice analysed were those analysed above (a–c). f Representative flow cytometry plots as indicated in the PDLN and spleen at 12 weeks of age, indicating the CD25⁺FOXP3⁺ cells after gating on CD4⁺CD8⁻CD127^low lymphocytes. g, h CD8-depleted lymphocytes from B:9-23/IFA-treated and protected mice (>35 weeks) were pooled and cultured for 3 days in the presence of B:9-23 (10 μg/ml) and rhIL-2 (50 U/ml). Subsequently, CD4⁺CD25⁺ (Tregs) or CD4⁺CD25⁻ T cells were isolated and adoptively transferred to 8- to 9-week-old new prediabetic NOD animals. The CD4/CD25 profile of the cells prior to purification is shown above (g). h Diabetes incidence in NOD animals after receiving >1 × 10⁶ CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells i.v. Donor cells were derived from mice treated at either 5 or 9 weeks of age; CD25⁺ n = 8, CD25⁻ n = 14; p < 0.05. i Mean percentage and (j) total number of NRP-V7⁺CD8⁺ cells in the spleen after gating on live CD4⁻CD19⁻ lymphocytes from 12-week-old mice immunised at 9 weeks of age with B:9-23/IFA and control. p < 0.05