High titers of autoantibodies against glutamic acid decarboxylase 65 (GAD65) are commonly observed in patients suffering from type 1 diabetes (T1D) as well as Stiff Person syndrome (SPS), a disorder that affects the central nervous system, and a variant of SPS, progressive encephalomyelitis with rigidity and myoclonus (PERM). of B cells, high titer anti-GAD65 autoantibodies were generated but these had no effect on the incidence or severity of disease. In addition, GAD65-specific CD4+ T cells isolated from the brain were activated and produced IFN-. These findings suggest that GAD65-reactive CD4+ T cells alone mediate a lethal encephalomyelitis-like disease that may serve as a useful model to study GAD65-mediated diseases of the CNS. INTRODUCTION Glutamic acid decarboxylase (GAD) catalyzes the conversion of glutamic acid to -aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system (CNS). There are two different isoforms of GAD, GAD65 and GAD67 that are generated HKI-272 from two different genes. GAD is expressed in the pancreas and central nervous system and has been implicated as a target antigen in Type 1 Diabetes (T1D) (1, 2), Stiff- Person Syndrome (SPS), a rare autoimmune disorder thought to occur because of an impairment of GABA production, and a variant of SPS, progressive encephalomyelitis with rigidity and myoclonus (PERM) (3, 4). Autoantibodies against GAD are a hallmark of T1D, with 80% of new-onset T1D patients showing detectable levels of anti-GAD antibodies prior to clinical onset of disease (5). High titers of anti-GAD antibodies are also detected in SPS patients suggesting a prominent role of the GAD antigen in this disease. In fact, there is some suggestion that the anti-GAD antibodies may inhibit the function of GABA, leading to the neurological symptoms observed in these patients (6, 7). While there is considerable evidence for the presence of anti-GAD antibodies in SPS and PERM, far less is known about the role and relative importance of GAD reactive T cells in these diseases. We had previously generated a number of GAD65-reactive CD4+ T cell hybridomas or clones from NOD mice either immunized with a series of peptides comprising the major immunogenic GAD65 epitopes (8, 9) or left untreated (10). The TCRs were cloned and expressed using a retroviral-mediated stem cell gene transfer system (referred to herein as retrogenic [Rg] mice) in which sublethally irradiated NOD.mice were reconstituted with NOD.bone marrow transduced with retrovirus containing a self-cleaving 2A-peptide linked TCR and a green fluorescent protein (GFP) in the same vector (11C14). Our previous studies have shown that T cells expressing GAD65-specific TCRs did not mediate diabetes nor cause insulitis (8, 9), consistent with other studies (15). In the present study, we describe the Rabbit Polyclonal to ALK surprising finding that three of these GAD65-reactive clonotypes HKI-272 (4B5, PA19.9G11 and PA17.9G7) induced a lethal encephalomyelitis-like disease and ataxia in Rg mice. In addition to detailing these observations, we also addressed the following questions: (1) Do the GAD65 reactive T cells infiltrate the CNS and cause inflammation? (2) In the presence of B cells, are GAD65 antibodies generated and do these affect the disease phenotype? (3) Is there a link between T cell pathogenesis, GAD65 reactivity and cytokine secretion in the inflammation observed in the CNS? MATERIALS AND METHODS Mice NOD.and mice were obtained from The Jackson Laboratory and bred in-house. B6g7 mice were a gift from C. Benoist and D. Mathis (Harvard Medical School, Boston, MA). All mice were bred and housed at the St. Jude Animal Resources Center (Memphis, TN) in a Helicobacter-free SPF facility following state, national and institutional mandates. The St. Jude Animal Resources Center is accredited by the American Association for HKI-272 the Accreditation of Laboratory Animal Care. All animal experiments followed animal protocols approved by the St. Jude Institutional Animal Care and Use Committee. TCR retroviral constructs All TCRs were generated as 2A-linked single ORFs using recombinant PCR and cloned into an MSCV-based retroviral vector with a green fluorescent protein (GFP) marker as previously described (11, 12, 14). Details of cloning strategies and primer sequences are available upon request (gro.edujts@bal.ilangiv). The PA21.14H4, PA19.9G11 and.