Regulatory T cell phenotype and function 4 years after GAD–alum treatment in children with type 1 diabetes

Glutamic acid decarboxylase (GAD)₆₅ formulated with aluminium hydroxide (GAD‐alum) was effective in preserving insulin secretion in a Phase II clinical trial in children and adolescents with recent‐onset type 1 diabetes. In addition, GAD‐alum treated patients increased CD4⁺CD25^hi forkhead box protein 3⁺ (FoxP3⁺) cell numbers in response to in‐vitro GAD₆₅ stimulation. We have carried out a 4‐year follow‐up study of 59 of the original 70 patients to investigate long‐term effects on the frequency and function of regulatory T cells after GAD‐alum treatment. Peripheral blood mononuclear cells were stimulated in vitro with GAD₆₅ for 7 days and expression of regulatory T cell markers was measured by flow cytometry. Regulatory T cells (CD4⁺CD25^hiCD127^lo) and effector T cells (CD4⁺CD25^–CD127⁺) were further sorted, expanded and used in suppression assays to assess regulatory T cell function after GAD‐alum treatment. GAD‐alum‐treated patients displayed higher frequencies of in‐vitro GAD₆₅‐induced CD4⁺CD25⁺CD127⁺ as well as CD4⁺CD25^hiCD127^lo and CD4⁺FoxP3⁺ cells compared to placebo. Moreover, GAD₆₅ stimulation induced a population of CD4^hi cells consisting mainly of CD25⁺CD127⁺, which was specific of GAD‐alum‐treated patients (16 of 25 versus one of 25 in placebo). Assessment of suppressive function in expanded regulatory T cells revealed no difference between GAD‐alum‐ and placebo‐treated individuals. Regulatory T cell frequency did not correlate with C‐peptide secretion throughout the study. In conclusion, GAD‐alum treatment induced both GAD₆₅‐reactive CD25⁺CD127⁺ and CD25^hiCD127^lo cells, but no difference in regulatory T cell function 4 years after GAD‐alum treatment.