Memory phenotype and clinical diabetogenic potential of CCR4-bearing T cells. (a) Memory/effector phenotype of CCR4-expressing CD4⁺ T cells. To assess and characterize the in vivo immunophenotype of CCR4⁺CD4⁺ T cells, flow cytometry of CD4⁺ T cells from the spleen and panLN’s of 10- to 12-week-old NOD mice (n = 3) was performed. Values for CD25, CD27, CD45RB, CD62L, CD69, and CCR4 expression were determined by FACS analysis. We found that CCR4-expressing CD4⁺ T cells have a memory/effector phenotype. (b) Systemic expansion of pathogenic CCR4-expressing CD4⁺ T cell populations in lymphoid organs. To verify the clinical significance of CCR4-bearing CD4⁺ T cells in vivo, the CCR4-positive pathogenic population was monitored by FACS analysis during disease progression in NOD mice. Six-week-old and 12-week-old prediabetic mice and 16-week-old diabetic mice are shown. Squares mark the CCR4^high population. The CCR4-bearing population was expanded in the secondary lymphoid tissues such as spleen and panLN’s of NOD mice during the diabetic or prediabetic state. The data shown are representative of three separate experiments.