Su.33. CD40 Is Expressed On a Subpopulation of Cd4hi T-Cells from NOD Mice. Rocky Baker, David Wagner, Kathryn Haskins. Immunology, University of Colorado at Denver and Health Sciences Center, Denver, CO. We have reported previously that the costimulatory molecule CD40 is a marker of autoreactive T-cells found on diabetogenic T-cell clones and that CD40+ T-cells from the NOD mouse transfer diabetes (PNAS 2002, 99:3782). Char- acterization of CD40+ T-cells in mice has been controversial because of a typically low level of expression of CD3 and CD4 on the CD40-expressing lymphocyte population. T-cells from NOD spleen can be found in two subsets on a FSC/SSC dot plot: one that is FSClo/SSClo and another one that is FSCint/ SSClo. We have found that a subpopulation of these cells in the FSClo/SSClo subset is consistently present in NOD mouse spleen, lymph nodes, and pancreas, and is characterized by expression of CD40 and high levels of CD3 and CD4. We predict that this population of cells is the origin of diabetogenic T-cells as it closely resembles the diabetogenic T-cell clones in our panel, which are also CD40+ and express high levels of CD3 and CD4 on their surface. It is also found in the spleens of 2.5 TCR-Tg mice. Ligation of CD40 on the diabetogenic T-cell clone BDC-2.5 induces upregulation of CD69, an early activation marker on the surface of T-cells. Levels of CD40 expression do not vary on T-cell clones in culture upon stimulation with anti-CD3 or cross-linking with anti-CD40. Interestingly, however, we observed upregulation of CD40 on T-cells in the 2.5 TCR-Tg/NOD mouse after immunization with a peptide mimotope for BDC-2.5 and also on the surface of BDC-2.5 T-cell clones upon adoptive transfer into a NOD.scid mouse. Manipulation of CD40 levels on T-cells through exposure to antigen may be another demonstration of this molecule’s importance on T-cells. Supported by a research grant from the Juvenile Diabetes Research Foundation, JDRF 1-2004-113. doi:10.1016/j.clim.2006.04.460 Su.34. Diet-Induced Increases in ICAM-1, CD11c, and CD34 in a Murine Model. Danett Brake, 1 Tyler Ulland, 3 Rebecca Robker, 2 C. Wayne Smith. 11 Immunology, Baylor College of Medicine, Houston, TX; 2 Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA, Australia; 3 Biochemistry and Molecular Biology, Cornell College, Mt. Vernon, IA. In human obesity, abdominal fat deposition is positively correlated with elevations in the soluble form of ICAM-1 (sICAM-1). Obesity has been linked to cardiovascular dis- ease, hypertension, hypercholesterolemia, insulin resis- tance and metabolic syndrome with elevated markers of systemic inflammation. Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane adhesion molecule involved in leukocyte migration to sites of inflammation. Here we investigate adipose tissue production and transcriptional regulation of ICAM-1 in a mouse model of obesity induced by a 21% milkfat, high cholesterol diet. ICAM-1 in serum and adipose tissue was analysed by ELISA, northern blot, real- time quantitative PCR, and flow cytometry. After 6 months on the high fat diet sICAM-1 levels significantly correlated with body and abdominal fat pad weights. Northern blots from adipose tissue showed significantly higher levels of ICAM-1 mRNA in males than females. After 3 weeks on the high fat diet there was an adipose tissue specific, increase in mRNA for ICAM-1, IL-6 and MCP-1 in male but not female mice. Analysis of the stromal vascular fraction of male adipose tissue revealed CD11b negative cells with increased surface ICAM-1 and CD34. We also found two populations of F4/80+, CD11b+, and ICAM-1+ cells, one of which was positive for CD14 and CD11c and significantly increased in response to a high fat diet. Returning mice fed a high fat diet to a low fat control diet significantly decreases cytokine response and macrophage number in adipose tissue. These results indicate that as little as 3 weeks of a high fat diet induces significant increases in pro-inflammatory factors in adipose tissue of male mice that may represent links between dietary obesity and its associated inflammatory- like complications. doi:10.1016/j.clim.2006.04.461 Su.35. The Destruction of Islets Lacking Native Insulin Genes with Mutated B16:A Insulin Transgene By Diabetic NOD Mice. Maki Nakayama, Joshua Beilke, Jean Jasinski, Edwin Liu, Ron Gill, Geogre Eisenbarth. Barbara Davis Center for Childhood Diabetes, UCHSC, Aurora, CO. Recently we found that NOD mice lacking wild-type proinsulin genes but with a mutated proinsulin (B16:A insulin) transgene are protected from anti-islet autoimmu- nity. This suggests that native insulin, especially the insulin B:9—23 sequence is a key to initiate autoimmune diabetes in NOD mice. However it is unknown whether or not insulin B:9—23 sequences are still essential to destroy islet cells. To explore whether pancreatic islets lacking native insulin genes but with mutated B16:A insulin transgene are resistant to the autoimmune destruction, we transplanted double insulin knockout islets with B16:A insulin to diabetic NOD mice. Transplantation of double insulin knockout islets could restore normoglycemia in diabetic NOD mice for 10 to 15 days after transplant, but the recipients recurred diabetes finally. Severe lymphocytic infiltration was ob- served in the islet graft obtained one day after diabetes recurrence. Consistent with the islet rejection in diabetic NOD mice, diabetogenic splenocytes induced diabetes in irradiated double insulin knockout NOD mice. To see whether T-cells reacting insulin B:9—23 sequences are involved in the destruction, we transplanted double insulin knockout islets with B16:A insulin to insulin B:9—23-specific BDC12-4.1 TCR transgenic rag—/—mice that bear monoclo- nal T-cells reacting to insulin B:9—23. Surprisingly, double insulin knockout islets with B16:A insulin were rejected in a diabetic BDC12-4.1 TCR transgenic mouse with severe infiltration to islet grafts. This is consistent with the B:9— 23 peptide with B16:A mutation being an altered peptide ligand for anti insulin B:9—23-reacting T-cells. This suggests that replacing native insulin B:9—23 sequences with mutat- Abstracts S171