Figure 2. Preservation of Treg cell functions and reduced Teff cell priming in the PLN at the time of disease onset.

(A.) Flow cytometric analysis of CD62L expression on CD4⁺ Foxp3⁺ cells in the PLN of 6-week old and new-onset diabetic NOD mice. The graph on the right is a summary of four independent experiments comparing CD62L expression on CD4⁺CD25⁺ cells in pancreatic LN of young prediabetic (n=9) and newly diabetic (n=11) female NOD mice. (B.) CD4⁺CD62L⁺CD25^- cells from BDC2.5 T cell receptor transgenic mice were purified by FACS, labeled with CFSE and transferred to 6-week old NOD mice and to mice within three days of diabetes onset. The movement dynamics of transferred Treg cells in explanted PLN were monitored using two-photon laser scanning microscopy. The normalized average CFSE fluorescent intensity over a 30-minute imaging period is illustrated in the “heat maps” shown. The large aggregates of strong fluorescent intensity represented by the yellow-red-white colors indicates restricted movement of cell clusters due to antigen recognition and the lower intensity represented by the blue-purple-black colors indicates random movement in the absence of antigen recognition. (C.) In Vivo Proliferation of transferred CD4⁺CD62L⁺CD25^- cells from a BDC2.5 T cell receptor transgenic mice in PLN of 6-week old or new-onset diabetic mice were determined by CFSE dilution assay. Two representative histograms are shown. (D.) Proliferation of endogenous T cells in the PLN of 6-week-old and new-onset diabetic NOD mice was determined by co-staining PLN sections for the mitotic marker Ki67 (green), anti-CD4 (blue), and anti-Foxp3 (red). Representative micrographs are shown (left) and the average numbers of Foxp3⁺ cells and Foxp3^- Ki67⁺ cells in three randomly selected objective fields in the T cell zones of the PLN are summarized (right, mean±sd, n=3 mice). Results in each panel represent two to four independent experiments.