Do Dendritic Cells Participate in the Immune Response to T-independent Antigens Type 2?

S58 response to a variety of cellular insults, including growth factor or nutrient withdrawal, organelle damage and misfolded proteins. Autophagy is rapidly induced in T lymphocytes following antigenic stimulation and blockade of autophagic signaling greatly reduces T cell clonal expansion, suggesting that autophagy is primarily involved in promoting T cell survival. In contrast, a recently identified negative feedback loop involving FADD and caspase-8, limits the level of autophagy in T cells. Failure to activate caspase-8 during T cell mitogenesis leads to hyperactive autophagy and cellular death through a programmed necrotic mechanism. These findings suggest that crosstalk between these cellular processes is essential for T cell activation and homeostasis.

doi:10.1016/j.clim.2010.03.176

T.65. Role of Host and Tumor CD200 Expression in Control of Local and Distant (metastatic) Growth of Breast Cancer in Mice Reg Gorczynski 1, Nuray Erin 2, Kai Yu 1, Ismat Khatri 1. 1 University Health Network, Toronto, ON, Canada; 2Akdeniz University, Antalya, Turkey Previously, we explored the regulation of expression of the novel molecule CD200 on EMT6 mouse breast cancer cells following tumor challenge in control or immunocompromised female recipients, observing that levels of CD200 expression on tumor cells isolated from the primary implantation site increased markedly during growth in immunocompetent mice, while persistent low levels of expression were observed in NOD-SCID.IL-2γr−/− mice. Similar low levels of tumor CD200 expression were seen in mice with generalized overexpression of a CD200 transgene (CD200tg mice). We nevertheless observed faster local tumor growth in both CD200tg and NOD.SCID mice, with decreased evidence for a host immune reaction in lymph nodes draining the tumor (DLN). We now report investigations into the importance of CD200 expression (by the host and/or tumor cells) in breast cancer growth using limiting dilution cloning of tumor cells from DLN (vs contralateral lymph nodes, CLN), evidence for CD200-mediated regulation of metastasis in this breast cancer model. Mammary fat pad injection of control EMT6 tumor cells in normal mice led to fewer (∼ 10-fold) DLN metastatic colonies than when tumor cells were implanted into CD200tg mice. In contrast, CD200tg–EMT6 tumor cells produced relatively equal numbers of metastatic colonies in both control and CD200tg mice. Neutralization of expressed CD200 by noncytotoxic F(ab′)2 anti-CD200mAbs decreased EMT6 tumor metastasis at the same time as increasing detection of cytotoxic antitumor immune cells in DLN. Independent studies confirmed that increased number of macroscopic lung metastases were also seen using 4THMpc breast cancer cells injected orthotopically into CD200tg (vs control) mice.

doi:10.1016/j.clim.2010.03.177

Abstracts

T.66. Efalizumab Down-regulates CD25 Expression on FOXP3+ Regulatory T Cells and Exacerbates the Autoimmunity in Primary Sjogren's Syndrome Dat Tran 1, Lolita Bebris 2, Ethan Shevach 3, Gabor Illei 2. 1 University of Texas Medical School at Houston, Houston, TX; 2NIDCR/NIH, Bethesda, MD; 3NIAID/NIH, Bethesda, MD Rationale: FOXP3+ regulatory T cells (Tregs) are central to the prevention of autoimmunity. Tregs expressed high level of CD25 and require IL-2 signaling for their function. Since CD25 is up-regulated with activation and LFA-1 is required, we hypothesize that efalizumab treatment would result in down-regulation of CD25 on Tregs and potential exacerbation of autoimmunity. Methods: Subjects with Sjogren's syndrome were enrolled in a phase II clinical trial for weekly injection of efalizumab (1 mg/kg) up to 25 weeks. Peripheral bloods were analyzed for Tregs by flow cytometry. Clinical conditions were measured by objective improvement in salivary and lacrimal flows and minor salivary gland (MSG) inflammation. RESULTS: All three subjects had progressive down-regulation of CD11a and CD18 on Tregs (CD4+FOXP3+), which correlated with dramatic decrease in CD25. The frequency and level of FOXP3 in the Tregs were not affected. There was no clinical improvement but instead repeat MSG biopsy showed significant increase in inflammation. Two subjects developed antidsDNA; one of whom developed multiple autoantibodies and lupus-like syndrome which resolved after efalizumab discontinuation. There was also significant increase in serum IgG. Conclusions: Efalizumab exacerbates Sjogren's syndrome and induces significant increase in local inflammation and systemic autoimmunity. Inhibition of LFA-1 interaction on Tregs negatively affected their CD25 without altering their FOXP3. These results suggest that Tregs are continually interacting with their target cells via LFA-1 and disruption of this interaction with efalizumab inhibits their activation, expression of CD25, and suppressor function. This study cautions the potential detrimental effect of efalizumab on Tregs and autoimmunity. doi:10.1016/j.clim.2010.03.178

T.67. Do Dendritic Cells Participate in the Immune Response to T-independent Antigens Type 2? Dmitriy Khochenkov, Marina Gavrilova, Ekaterina Sidorova. Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, Moscow, Russian Federation Dendritic cells (DCs) belong to the most effective antigenpresenting cells. Their role in the presentation of thymusdepending antigens is well known. At the same time, the participation of DC in the immune response to T-independent antigens type 2 (TI-2 antigens) is almost not studied. The ability of DC to initiate the immune response to TI-2 antigens-α (1→3) dextran (Dex) and Streptococcus pneumoniae polysaccharide type 3 (SIII) is now investigated. Mouse bone marrow derived DC were generated by culturing of precursors with GM-CSF and then DCs were charged by TI-2 antigens. The pulse induced DCs

Abstracts activation. It was demonstrated by the increase in the number of CD80 and CD86 positive cells. Uptake of FITC-labeled Dex was examined by flow cytometry. At FITC-Dex concentration of 80– 100 μg/ml, the number of DCs binding antigen reached “plato.” DC charged by TI-2 antigens were mixed with normal mouse splenocytes and cultivated in the RPMI 1640 medium for 4 days. The numbers of antibody- and immunoglobulin-forming cells were determined by ELISPOT method. The mixtures of splenocytes and naive DC not charged by the antigens were used as control. It was shown that the increase in the numbers of AFC and IFC under the influence of naive DC did not exceed 20%. On the contrary, the addition of DC pulsed by the antigens increased specific immune response more that twice. The data obtained point to the direct interactions of DC with TI-2 antigens. Pulsed DCs present TI-2 antigens to mouse splenocytes and induce specific and polyclonal B-cell activation, i.e., possess immunostimulating activity. doi:10.1016/j.clim.2010.03.179

T.68. Loss of the Soluble CTLA-4 Splice Variant Compromises Immune Regulation Kay Fischer 1, Peilin Zheng 1, Daniel Rainbow 2, Linda Wicker 2, Stephan Kissler 1. 1University of Wuerzburg, Wuerzburg, Germany; 2University of Cambridge, Cambridge, United Kingdom CTLA4 gene variation associates with multiple autoimmune disorders, including type 1 diabetes. The CTLA4 susceptibility allele was found to generate decreased levels of mRNA encoding soluble CTLA-4 (sCTLA-4) relative to the full-length isoform, the functional consequence of which is as yet unknown. To model the disease-associated splicing variation of CTLA4, we generated nonobese diabetic (NOD) mice in which sCTLA-4 mRNA is silenced by RNAi. We found that loss of sCTLA-4 impairs the function of regulatory T cells, accelerates spontaneous diabetes, and makes transgenic mice prone to sudden lethal inflammation following cyclophosphamide challenge. Our results demonstrate that sCTLA-4 participates in immune regulation by potentiating the function of regulatory T cells and provide an explanation for the association of CTLA4 variation with autoimmunity in humans. doi:10.1016/j.clim.2010.03.180

T.69. EGFR Inhibition Ameliorates Murine Collagen-induced Arthritis Christina Swanson, William H. Robinson. Stanford University, Stanford, CA Rheumatoid arthritis (RA) is an autoimmune synovitis affecting approximately 0.5% of the population worldwide. In addition to T cells, B cells, and macrophages, cells such as synovial fibroblasts (RASF) and endothelial cells (EC) promote inflammation in the synovium through the production of cytokines, chemokines and nitric oxide (NO). At the same

S59 time, increased osteoclastogenesis causes bone destruction. Previously, others found that RA serum and synovial fluid contained increased concentrations of epidermal growth factor receptor (EGFR) ligands such as EGF and demonstrated EGFR staining on RASF and EC in the synovium. Thus, we hypothesized that erlotinib (Tarceva), a specific EGFR tyrosine kinase inhibitor, may ameliorate murine collagen-induced arthritis. Erlotinib is currently prescribed to treat non-small cell lung cancer and pancreatic cancer. Here, we demonstrate that erlotinib reduces the severity of established murine collageninduced arthritis (CIA) by acting on RASF, EC, osteoblasts, and osteoclasts. In RASF, EGF synergizes with IL-β and TNF to increase IL-6, IL-8, and stromelysin-1 production. Concurrent treatment of RASF with erlotinib reduces the production of these molecules. Similarly, erlotinib reduces EGF-induced EC production of IL-6, IL-8, and NO. Erlotinib decreases bone resorption directly by inhibiting osteoclast development from bone marrow cells and indirectly by acting on osteoblasts. Osteoblasts produce RANKL and osteoprotegerin (OPG), the balance of which control osteoclast-mediated bone erosion. Here, we show that EGF reduces osteoblast OPG production, which is restored by erlotinib treatment. Together, these results suggest that erlotinib treatment could be beneficial in the treatment of RA. doi:10.1016/j.clim.2010.03.181

T.70. Donor Antigen Specific Regulatory T Cell Function and Outcome in Kidney Transplant Recipients Martin Gasser 1, Igor Tsaur 2, Kai Lopau 1, Christoph Germer 1, Anil Chandraker 3, Ana Maria Waaga Gasser 1. 1University of Wuerzburg, Wuerzburg, Germany; 2University of Frankfurt, Frankfurt, Germany; 3Brigham and Women's Hospital, Boston, MA Chronic allograft dysfunction remains a major impediment to long-term allograft survival. Many different factors have been shown to contribute to deteriorating graft dysfunction, including an ongoing alloimmune response sometimes called chronic rejection. Out of 623 patients transplanted at a single center we selected 107 patients who were mismatched with their donors for one of a number of relevant HLA DR antigens. Patients were categorized into groups according to immunosuppression treatment and then further divided into those with stable or deteriorating graft function (chronic allograft dysfunction or CAD). A total of 37 representative T cell lines were generated from these patients. In patients with CAD, only those with biopsy proven chronic allograft nephropathy were selected. Although cell lines generated from both stable CsA- and Tac-treated patients had much higher percentages of CD4+CD25+ regulatory T cells (Tregs) and Treg-associated gene expression compared to the CAD groups, the Tac-treated stable patients also had a considerably higher Treg population compared with the CsAtreated group. Stable patients were associated with better longterm graft function and T cells from these patients showed increased expression of IL-4 and IL-10 and were also able to regulate the in vitro proliferation and production of IFN-γ by CAD cell lines to mismatched HLA-DR-derived peptides in an IL10-dependent fashion. CAD cell lines produced significantly