Check GAD antibody positivity with the Diamyd anti-GAD RIA plate*

GAD in GraphsIncidence of diabetes (%)10080604020In Type 1 diabetes patients, GAD65-reactive T cells are primarily memoryT cellsAnti–B7-1[ 3 H] Thymidine604020(%) Inhibition0IFN-γ6040200IL-136040200GAD65 (µg/ml)Viglietta et al (2002) GAD65-reactive T cells areactivated in patients with autoimmune Type 1adiabetes. J Clin Invest 109:895-903.• T cell recall immune responseswere studied by proliferation andcytokine assays using GAD65 andinhibition of the costimulatorymolecule B7.1• T cells from Type 1 diabetics werenot inhibited by anti-B7.1, whilethose from controls were. This indicatesthat the Type 1 diabetic T cellsare of a memory T cell phenotype.Treatment of young NOD mice withrrVV-GAD65 prevents diabetesdevelopmentrVV-GAD65rVV-MJ601rVV-NS1Uninjected010 20 30age (in weeks)Sun et al. (2002). Prevention of autoimmunediabetes by immunogene therapy using recombinantvaccinia virus expressing glutamic aciddecarboxylase.Diabetologia 45:668-676• 3 week old female NOD mice wereinjected with vaccina constructscoding for GAD or control antigens• Mice receiving the GAD65construct were protected fromdiabetes40The GAD65 Stanford PerspectiveHugh McDevittInitial work with GAD65 identified it as one of the first to elicit a spontaneousimmune response in 3-4 week old NOD mice. Next, we used GAD65 to suppress ordelay the diabetic process. Treatment with 4 doses of GAD65 intravenously at 12weeks of age resulted in a large decrease in diabetes incidence at 30 weeks. Otherislet cell proteins, or foreign proteins had no effect.Tcell hybridomas were used toidentify immunodominantpeptide epitopes of GAD 65in NOD mice: GAD65 aminoacids 206-220, 221-235, and286-300.Transgenic mice with T Cell Receptors (TCRs)for the 206 and 286 peptides, and a hybrid TCRtransgenic with the 221α chain and the 286βchain, revealed that in all 3 TCR transgenic NODlines neither insulitis nor Type 1 diabetes developed.T cells in all 3 lines are reactive to cognatepeptide and produce IL-2, IFNγ, IL-10, and someTNFα. The T cells are positively selected in thethymus, but only escape negative selection via theexpression of a second α chain. The 206 and 286TCR transgenic lines on the Cα -/- backgroundhad near complete deletion of T cells. MHC tetrameranalysis of 286 TCR affinity of the 286 TCRshowed that it was of extremely low affinity.T cells were partially negatively selected in thethymus, and underwent further negative selectionin the periphery. Only 10-20% of peripheral CD4 +T cells are 286 tetramer positive, while 30-40% ofCD8 + T cells are tetramer positive. Cell transferexperiments showed that these mice have T cellscapable of partially suppressing, and definitelydelaying the onset of Type 1 diabetes in a standardtransfer system.To date, none of these mice have developed diabetesor insulitis. Crosses with a RIP-HuGAD65high expressing transgenic NOD line has not resultedin diabetes or insulitis in either the 206 or 286lines. Other studies are currently underway.However, it appears that in all 3 TCR transgenicmouse lines,GAD65 specificT cells areprotective.A review ofthe literatureshows thatmost GAD65-specific T cellclones eitherdo not causediabetes orHugh McDevitt, MD, isProfessor of theDepartment ofMicrobiology andImmunology atStanford UniversitySchool of Medicine,with a joint appointmentin the Department of Medicine.Following his discovery of linkage of the majorhistocompatibility complex with specificimmune responses, McDevitt has pursued themechanisms of this association over the pastseveral decades, and most recently has beenstudying the immune response of NOD miceto GAD65.insulitis, or in some cases are protective. (Oneclone, reported by Robert Sherwin, did cause Type1 diabetes.) Further, von Boehmer (J. Exp. Med., inpress) has shown that induction of recessive toleranceto GAD65 with a transgene insuring highlevel GAD65 expression in endosomal compartmentsdoes not change Type 1 diabetes incidence.These results suggest that GAD65 is primarilya protective antigen in Type 1 diabetes in NODmice. Whether it is also a protective antigen inpatients is an open question. Nonetheless, experimentsfrom our and other laboratories suggestthat administration of GAD65 in non-inflammatoryvehicles result in a delay in onset, or adecrease in incidence of NOD diabetes. This raisesthe question of whether GAD65 is a protectiveislet cell antigen because of its unusual locationin ß cells (in vesicles similar to neuronalvesicles), with this presentation resulting in apreferential elicitation of a TH2 response to thisislet cell protein. This increase in TH2 T cells inthe islets may then be a factor in delaying onsetof diabetes and/or decreasing its incidence.page 20 dmccad june 2003