No abstract

Free radicals contribute to Type 1 diabetes (T1D) autoimmune responses. We recently demonstrated that superoxide-deficient CD4 T cells exhibited increased effector responses and diabetogenicity, but the redox-dependent mechanism(s) mediating T cell activation were unclear. We hypothesized that during T1D progression, CD4 T cells with a reduced cell surface state would exhibit a concomitant increase in pro-inflammatory cytokine and chemokine responses. To test this, alexa fluor 647-conjugated maleimide (ALM)-labeling of cell surface reduced thiols on diabetogenic mouse and human CD4 T cells was performed. We observed an increase in ALM percentage and gMFI from peripheral Non-obese diabetic (NOD) mouse CD4 T cells during progression to overt diabetes. Cognate autoantigen stimulation elicited a 2- and 20-fold increase in CD4 ALM T cell percentage and gMFI, respectively. In addition to serving as a T cell activation marker, ALM gMFI of reduced CD4 T cell surface thiols was enhanced (1.2-fold) with Th1 polarization and blunted (12.2-fold) following Treg polarization in contrast to Th0 conditions. Cell surface reduced thiols from human CD4 T cells with recent-onset T1D exhibited elevated ALM percentage (1.6-fold, p < 0.05) and gMFI (1.7-fold, p = 0.1503) in comparison to age-matched healthy controls following polyclonal stimulation. Mirroring the increase in ALM with T1D individuals, PBMCs (n=12) also synthesized increased IFNγ (1.2-fold, p=0.0031), TNFα (1.3-fold, p=0.0022), and CXCL10 (1.2-fold, p=0.0145) in contrast to healthy controls (n=7). Our studies point to the exciting potential that oxidation of cell surface thiols on diabetogenic CD4 T cells could abrogate effector responses and correlate with CD4 T cell activation.

Type 1 diabetes (T1D) is an autoimmune disease culminating in the destruction of insulin-producing pancreatic cells. There is a need for the development of novel antigen-specific strategies to delay cell destruction, including combinatorial strategies that do not elicit systemic immunosuppression. Gamma-aminobutyric acid (GABA) is expressed by immune cells, β-cells, and gut bacteria and is immunomodulatory. Glutamic-acid decarboxylase 65 (GAD65), which catalyzes GABA from glutamate, is a T1D autoantigen. To test the efficacy of combinatorial GABA treatment with or without GAD65-immunization to dampen autoimmune responses, we enrolled recent-onset children with T1D in a one-year clinical trial (ClinicalTrials.gov NCT02002130) and examined T cell responses. We isolated peripheral blood mononuclear cells and evaluated cytokine responses following polyclonal activation and GAD65 rechallenge. Both GABA alone and GABA/GAD65-alum treatment inhibited Th1 cytokine responses over the 12-month study with both polyclonal and GAD65 restimulation. We also investigated whether patients with HLA-DR3-DQ2 and HLA-DR4-DQ8, the two highest-risk human leukocyte antigen (HLA) haplotypes in T1D, exhibited differences in response to GABA alone and GABA/GAD65-alum. HLA-DR4-DQ8 patients possessed a Th1-skewed response compared to HLA-DR3-DQ2 patients. We show that GABA and GABA/GAD65-alum present an attractive immunomodulatory treatment for children with T1D and that HLA haplotypes should be considered.