Su.33. CD40 Is Expressed On a Subpopulation ofCd4hi T-Cells from NOD Mice.Rocky Baker, David Wagner, Kathryn Haskins. Immunology,University of Colorado at Denver and Health SciencesCenter, Denver, CO.

We have reported previously that the costimulatorymolecule CD40 is a marker of autoreactive T-cells found ondiabetogenic T-cell clones and that CD40+ T-cells from theNOD mouse transfer diabetes (PNAS 2002, 99:3782). Char-acterization of CD40+ T-cells in mice has been controversialbecause of a typically low level of expression of CD3 and CD4on the CD40-expressing lymphocyte population. T-cells fromNOD spleen can be found in two subsets on a FSC/SSC dotplot: one that is FSClo/SSClo and another one that is FSCint/SSClo. We have found that a subpopulation of these cells inthe FSClo/SSClo subset is consistently present in NOD mousespleen, lymph nodes, and pancreas, and is characterized byexpression of CD40 and high levels of CD3 and CD4. Wepredict that this population of cells is the origin ofdiabetogenic T-cells as it closely resembles the diabetogenicT-cell clones in our panel, which are also CD40+ and expresshigh levels of CD3 and CD4 on their surface. It is also found inthe spleens of 2.5 TCR-Tg mice. Ligation of CD40 on thediabetogenic T-cell clone BDC-2.5 induces upregulation ofCD69, an early activation marker on the surface of T-cells.Levels of CD40 expression do not vary on T-cell clones inculture upon stimulation with anti-CD3 or cross-linking withanti-CD40. Interestingly, however, we observed upregulationof CD40 on T-cells in the 2.5 TCR-Tg/NOD mouse afterimmunization with a peptide mimotope for BDC-2.5 and alsoon the surface of BDC-2.5 T-cell clones upon adoptivetransfer into a NOD.scid mouse. Manipulation of CD40 levelson T-cells through exposure to antigen may be anotherdemonstration of this molecule’s importance on T-cells.Supported by a research grant from the Juvenile DiabetesResearch 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. WayneSmith.1 1Immunology, Baylor College of Medicine, Houston,TX; 2Obstetrics and Gynaecology, University of Adelaide,Adelaide, SA, Australia; 3Biochemistry and MolecularBiology, Cornell College, Mt. Vernon, IA.

In human obesity, abdominal fat deposition is positivelycorrelated 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 ofsystemic inflammation. Intercellular adhesion molecule-1(ICAM-1) is a transmembrane adhesion molecule involved inleukocyte migration to sites of inflammation. Here weinvestigate adipose tissue production and transcriptionalregulation of ICAM-1 in a mouse model of obesity induced bya 21% milkfat, high cholesterol diet. ICAM-1 in serum andadipose tissue was analysed by ELISA, northern blot, real-

time quantitative PCR, and flow cytometry. After 6 monthson the high fat diet sICAM-1 levels significantly correlatedwith body and abdominal fat pad weights. Northern blotsfrom adipose tissue showed significantly higher levels ofICAM-1 mRNA in males than females. After 3 weeks on thehigh fat diet there was an adipose tissue specific, increase inmRNA for ICAM-1, IL-6 and MCP-1 in male but not femalemice. Analysis of the stromal vascular fraction of maleadipose tissue revealed CD11b negative cells with increasedsurface ICAM-1 and CD34. We also found two populations ofF4/80+, CD11b+, and ICAM-1+ cells, one of which waspositive for CD14 and CD11c and significantly increased inresponse to a high fat diet. Returning mice fed a high fatdiet to a low fat control diet significantly decreases cytokineresponse and macrophage number in adipose tissue. Theseresults indicate that as little as 3 weeks of a high fat dietinduces significant increases in pro-inflammatory factors inadipose tissue of male mice that may represent linksbetween dietary obesity and its associated inflammatory-like complications.

doi:10.1016/j.clim.2006.04.461

Su.35. The Destruction of Islets Lacking NativeInsulin Genes with Mutated B16:A InsulinTransgene By Diabetic NOD Mice.Maki Nakayama, Joshua Beilke, Jean Jasinski, Edwin Liu,Ron Gill, Geogre Eisenbarth. Barbara Davis Center forChildhood Diabetes, UCHSC, Aurora, CO.

Recently we found that NOD mice lacking wild-typeproinsulin genes but with a mutated proinsulin (B16:Ainsulin) transgene are protected from anti-islet autoimmu-nity. This suggests that native insulin, especially the insulinB:9—23 sequence is a key to initiate autoimmune diabetes inNOD mice. However it is unknown whether or not insulinB:9—23 sequences are still essential to destroy islet cells. Toexplore whether pancreatic islets lacking native insulingenes but with mutated B16:A insulin transgene areresistant to the autoimmune destruction, we transplanteddouble insulin knockout islets with B16:A insulin to diabeticNOD mice. Transplantation of double insulin knockout isletscould restore normoglycemia in diabetic NOD mice for 10 to15 days after transplant, but the recipients recurreddiabetes finally. Severe lymphocytic infiltration was ob-served in the islet graft obtained one day after diabetesrecurrence. Consistent with the islet rejection in diabeticNOD mice, diabetogenic splenocytes induced diabetes inirradiated double insulin knockout NOD mice. To seewhether T-cells reacting insulin B:9—23 sequences areinvolved in the destruction, we transplanted double insulinknockout islets with B16:A insulin to insulin B:9—23-specificBDC12-4.1 TCR transgenic rag—/—mice that bear monoclo-nal T-cells reacting to insulin B:9—23. Surprisingly, doubleinsulin knockout islets with B16:A insulin were rejected in adiabetic BDC12-4.1 TCR transgenic mouse with severeinfiltration to islet grafts. This is consistent with the B:9—23 peptide with B16:A mutation being an altered peptideligand for anti insulin B:9—23-reacting T-cells. This suggeststhat replacing native insulin B:9—23 sequences with mutat-

Abstracts S171