kidney transplanted recipients. We are presently assessingtheir ability to recognise and lyse both autologous HCMV-infected cells and normal donor-derived allogeneic fibro-blasts. Our data support the notion that: 1) HLA-E-restrictedT-cells may represent additional effector cells to limitreplication of HCMV, a virus which can escape the controlexerted by bconventionalQ CTL or NK cells; 2) VMAPRTLIL-specific T-cells might worsen graft rejection or GvHD.

doi:10.1016/j.clim.2006.04.225

OR.70. Characteristics of BDC12-4.1 T-CellResponsiveness to Insulin Peptide B:9-23.Marcella Li, Jean Jasinski, Maki Nakayama, MasakazuKobayashi, Liping Yu, George Eisenbarth, Edwin Liu.Pediatrics, Barbara Davis Center for Childhood Diabetes,Aurora, CO.

Insulin peptide B:9-23 is a major CD4 epitope of insulinfor type 1 diabetes of the NOD mouse. We have created a T-cell receptor (TCR) transgenic mouse from a diabetogenicB:9-23-reactive T-cell clone isolated from the islets of ayoung prediabetic NOD mouse, BDC12-4.1. This mousespontaneously develops diabetes (50% by 30 weeks) onlyon the RAG�/� genotype, and although beta chaintransgenic TCR expression is efficient (95% of all CD4 cellsexpress the Vb2 transgene), the mice are markedlylymphopenic. Diabetic BDC12-4.1 splenocytes can transferdisease to NOD.scid mice, with a rate of transfer of 50% by12 weeks. ELISPOT studies indicate that the T-cells in theBDC12-4.1 can be induced to secrete both IFN-g and IL-4 inresponse to B:9-23 with increasing stimulation to in-vitroconcentrations of peptide as high as 200 ug/ml. In-vivo,immunization of BDC12-4.1 TCR mice results in expansion ofthe Vb2 CD4 T-cell compartment in the spleen, withmarkedly enhanced IFN-g and IL-4 responses by ELISPOT.Activation markers CD69, CD44, and CD62L were examinedin BDC12-4.1 TCR RAG�/� mice. There is constitutiveexpression of CD69, CD44hi, and CD62Lhi on Vb2 T-cells. Insummary, primary BDC12-4.1 T-cells are diabetogenicdespite lymphopenia and show activation/memory markersin the periphery and produce IFN-g and IL-4 upon stimula-tion with the B:9-23 peptide. The availability of an NODTCR transgenic mouse targeting an essential defined CD4islet autoantigen of insulin will facilitate studies to definedisease pathogenesis initiated by targeting of insulin.

doi:10.1016/j.clim.2006.04.226

OR.71. Identification of Auto-Aggressor T-Cells andBreach of Self-Tolerance in Type 1 Diabetes.David Wagner. Medicine, University of Colorado HealthSciences Center, Denver, CO.

Type 1 diabetes, T1D, is a classic autoimmune diseasecharacterized by lymphocyte infiltration of pancreaticislets, loss of insulin production and subsequent hypergly-cemia. We identified a unique auto-aggressor T-cell

subset, CD4+CD40+, present at contained percentages innon-autoimmune controls but expand concurrently withprogressive insulitis in the NOD mouse model of T1D. TheNOD recombinant congenic NOR expresses the identical I-Ag7 MHC haplotype but CD4+CD40+ T-cells do not expandin that strain. Blocking CD40 - CD154 interactions in vivoprevents diabetes and expansion of aggressor T-cells. CD40functionally induces RAG1 and RAG2, the recombinationmachinery altering TCR expression, in peripheral T-cells.Because this occurs outside the thymic safety-net fornegative selection this suggests a mechanism to breachself-tolerance. In fact, we show that CD40-induced,revised T-cells expressing the Va3.2+ molecule are highlydiabetogenic. We find that CD4+CD40+ T-cells are greatlyexpanded in peripheral blood of human T1D, but not innon-autoimmune controls or T2D patients. Expanded levelsof CD4+CD40+ T-cells correlate with HLA-DR3 and DR4expression and occur independently of patientTs age ortime of onset of T1D. CD4+CD40+ T-cells within thedefined memory phenotype, CD45RO+ from T1D but notfrom T2D or control patients are responsive to humanislets, pre-pro-insulin and GAD diabetes associated pep-tides. Together these data describe a unique CD4+effector T-cell subset capable of undergoing TCR revisionto become highly pathogenic in NOD T1D, which translatesinto the human model for disease. This mechanism couldexplain the means to thwart negative selection andpromote autoimmunity. Grant Support: American DiabetesAssociation and Juvenile Diabetes Research Foundation toDHW.

doi:10.1016/j.clim.2006.04.227

OR.72. Enhanced Recognition of Insulin a Chain1-15 By Expanded T-Cell Clones from Human Type 1Diabetic (T1D) Pancreatic Draining Lymph Nodes.Sally Kent, David Hafler. Neurology, Brigham and Women’sHospital and Harvard Medical School, Boston, MA.

Identification of autoantigens triggering pathogenic Tlymphocytes associated with destruction of beta islet cellsis a central question in human T1D. We reported oligoclo-nal T-cell expansion and autoantigen reactivity in T-cellclones derived from the pancreatic draining lymph nodes(PLN) of two long-term human T1D subjects. Expanded T-cell clones from PLN of both long-term diabetics respondedto insulin A1-15 restricted by DRB1*0401. Clones respondedto insulin A1-15 presented by DRB1*0401 expressing EBVtransformed B-cells with proliferation and secretion of IL-13, but not IFNg secretion. In addition, peptide for EC50,in the range of 100-500 AM was required to activate the T-cell clones, indicating lower range avidity. Since insulin A1-15 peptide contains three cysteine residues, we reasonedthat secondary and tertiary structure of the peptide insolution would impede efficient loading of the peptide intothe antigen-binding groove of the HLA Class II molecule.Reduction of insulin A1-15 peptide in solution by either oftwo reducing agents resulted in the T-cell clones respond-ing by proliferation to less peptide (EC50, 50-100 AM) andimportantly, with IFNg secretion. The avidity of this

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