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

Summary G lutamic 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 CD 4 + CD 25 hi forkhead box protein 3 + ( FoxP 3 + ) cell numbers in response to in‐vitro   GAD 65 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 GAD 65 for 7 days and expression of regulatory T cell markers was measured by flow cytometry. Regulatory T cells ( CD 4 + CD 25 hi CD 127 lo ) and effector T cells ( CD 4 + CD 25 – CD 127 + ) 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   GAD 65 ‐induced CD 4 + CD 25 + CD 127 + as well as CD 4 + CD 25 hi CD 127 lo and CD 4 + FoxP 3 + cells compared to placebo. Moreover, GAD 65 stimulation induced a population of CD 4 hi cells consisting mainly of CD 25 + CD 127 + , 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 GAD 65 ‐reactive CD 25 + CD 127 + and CD 25 hi CD 127 lo cells, but no difference in regulatory T cell function 4 years after GAD ‐alum treatment.