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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+ CD25hi 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+ CD25hi CD127lo) 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+ CD25hi CD127lo and CD4+ FoxP3+ cells compared to placebo. Moreover, GAD65 stimulation induced a population of CD4hi 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 CD25hi CD127lo cells, but no difference in regulatory T cell function 4 years after GAD-alum treatment. [ABSTRACT FROM AUTHOR]

Extracorporeal photochemotherapy (ECP) has demonstrated immunological effects. The proposed cytotoxic lymphocyte antigen 4 (CTLA-4) involvement, together with forkhead box P3 (FoxP3) and transforming growth factor (TGF)-β are associated with regulatory T cell activity. The aim of the study was to evaluate the regulatory T cell-associated effect of ECP in recent onset type 1 diabetic (T1D) children. Children ( n = 20) with T1D received photopheresis 8-methoxypsoralen + ECP or placebo + shampheresis. Peripheral blood mononuclear cells (PBMC) collected pretreatment (day 1) and post-treatment (day 90) were stimulated with phytohaemagglutinin (PHA) and T1D-associated glutamic acid decarboxylase 65 (GAD65) peptide a.a. 247–279. CTLA-4, sCTLA-4, FoxP3 and TGF-β mRNA transcription was quantified. Photopheresis-treated individuals' relative mRNA expression was generally maintained during the course of the study. Placebo individuals increased in spontaneous CTLA-4 mRNA ( P < 0·05) but decreased in expression after stimulation with GAD65-peptide ( P < 0·05) and PHA ( P < 0·05). Spontaneous TGF-β ( P < 0·05) increased whereas PHA- ( P < 0·01) and GAD65-peptide ( P < 0·01)-induced TGF-β expression decreased in the placebo group, whereas it was maintained in the treated group. Without intervention, expression of CTLA-4 and TGF-β, stimulated with PHA and GAD65 peptide, decreased with time, with a parallel reduction of GAD65-peptide and PHA-stimulated TGF-β expression. These parameters were counteracted by ECP. In conclusion, our results indicate that ECP maintains regulatory T cell-associated activity in recent-onset T1D. [ABSTRACT FROM AUTHOR]