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

GADGAD in GraphsGAD ELISPOT outperforms proliferationassays for detection of GADreactiveT cellsStimulation IndexIL-5 (pg/ml)121086420300250200150100500Control Type 2 Type 1*(n=23) (n=12) (n=31)Kotani et al (2002) Detection of GAD65 reactiveT cells in Type 1 diabetes by immunoglobulinfreeELISPOT assays. Diabetes Care 25:1390-1397• Cellular immune responses toß-cell autoantigens were studiedby proliferation and ELISPOT inPMBCs of Type 1, Type 2 diabetespatients and healthy controls• GAD65-specific proliferation wasdetected in 32% Type 1 patients,while IFN-γ ELISPOT GAD65-specificactivity was detected in 66% patientsImmunization with a GAD65/IL-4plasmid DNA vaccine prevents diabetesin NOD miceCMedium*untreatedHELGADGAD/IL-4GAD65 HSP60 CPHTisch et al. (2001) Antigen-specific mediatedsuppression of cell autoimmunity by plasmidDNA vaccination.J Immunol 166:2122-2132p=.003• Enhanced secretion of IL-5 anddecreased secretion of IFN-γ by Tcells from NOD mice immunized at12 wks old with pDNA encodingGAD65-IgFc and IL-4 and restimulatedwith GAD65.• Vaccination caused a shift in cytokineresponse to GAD65 stimulation*Human T Cells RecognizingGAD65 in Type 1a DiabetesODavid Hafler, Jack Sadie, David Breakstone and Sally Kentur laboratory has beeninterested in the functionof autoreactive T cells inhumans with autoimmunedisease and in particular,Multiple Sclerosis (MS) formany years. The T cell response is regulated in partthrough two signaling events. The first signal isthrough the T cell receptor by peptide processedfrom an antigen in the context MajorHistocompatibility Complex (MHC) proteins onan antigen-presenting cell (APC) and the second isthrough costimulation proteins, CD28 and CTLA-4by B7-1 and B7-2 proteins on APCs. We and othershad previously found that myelin basic protein(MBP) reactive T cells in patients with MS werecostimulation-independent (second signal) as comparedto T cells from normal individuals. This indicatesthat the autoreactive T cells behaved morelike T cells in a memory response and that the patienthas had T cells reactive to MBP for some time.This also suggests a method for differentiating patientT cell responses from those of controls andways of intervening in the autodestructive immuneresponse by the T cells.These data prompted us to examine this issuein Type 1 diabetes. Insulin-dependent Type 1a diabetesis an autoimmune disease mediated by Tlymphocytes recognizing pancreatic islet cell antigens.Glutamic acid decarboxylase 65 (GAD65)appears to be an important autoantigen in the disease.We found that in patients with new-onsetType 1a diabetes, GAD65-reactive T cells werestrikingly less dependent on CD28 and B7-1 costimulationto enter into cell cycle and proliferatethan were equivalent cells derived from healthycontrols. B7-2 appears to be the primary costimulatorymolecule engaging CD28 in T cell activationof GAD65-reactive T cells, and its engagementwith CTLA-4 appears to deliver a negativesignal. We hypothesize that these autoreactive Tcells have been activated in vivo and have differentiatedinto memory cells, suggesting a pathogenicrole in Type 1 diabetes. These findings stronglyindicate that the activation state of antigen-specificcells plays a role in the autoimmune processand selected costimulatory molecules may representthe target of future therapies.We are currently utilizing GAD65 for other studiesin Type 1a diabetes. These include studies examiningthe quantitation and phenotype of GAD65reactive T cells from controls and Type 1 diabeticswith a GAD peptide-loaded tetramer (HLADR*0401 loaded with GAD p555-567) and monitoringpotentially destructive GAD65 T cell autoreactiveresponses in long-term Type 1a diabetics receivingislet transplants. We are enthusiastic to continueto utilize GAD65 as a means of examining autoreactiveT cellresponses inhuman Type 1adiabetes.David Hafler, MD. is Professor of Neurology(Neuroscience) at Brigham and Women’sHospital and Harvard Medical School inBoston, MA., and the head of the MolecularImmunology Laboratory. Hafler’s main clinicaland research interests are in human autoimmunediseases: Multiple Sclerosis, Type 1diabetes and rheumatoid arthritis. His goalsare to understand the nature of self-recognitionby T cells, to understand how that immuneresponse leads to autoimmune disease andhow one can alter this response to developnovel therapiesSally Kent, Ph.D., is an Instructor in Neurologyand Associate Immunologist at Brigham andWomen’s Hospital and Harvard MedicalSchool in Boston, MA. Kent has specialized inNKT cell function in Type 1a diabetics by examiningperipheral blood and pancreatic draininglymph nodes. In Type 1a diabetes, Kenthas focused on GAD65 T cell reactivity as ameasure of autoreactivity and memory T cellresponses in controls and patients and inpatients undergoing islet cell transplantation.page 28 dmccad june 2003