Blockade of CD94/NKG2A Prevents EAE

Abstracts CD4+ proINS 76-90 (SLQPLALEGSLQKRG) specific T cells using soluble fluorescent MHC class II tetramers. Subjects with type 1 diabetes (T1D; n = 10) and healthy controls (n = 8) with high levels of peripheral proINS -specific T cells were characterized by the presence of a “disease-susceptible” polymorphism in the INS-VNTR gene. Conversely, subjects with a “protective” polymorphism in the INS-VNTR gene had nearly undetectable levels of proINS tetramer-positive T cells. Using tetramers to isolate proINS -specific T cells from subjects with both INS-VNTR genotypes, transcript arrays (Affimetrix) followed by quantitative Q-PCR profiling have identified several induced pro-apoptotic genes (FASLG, TNF, TNFSF11), and cytokine (IL-2, IFNg, IL-22, IL1B, IL-10, IL-7) and chemokine (CCL22) pathways in controls, but not diabetic subjects, likely representing a second peripheral mechanism for maintenance of tolerance to self antigens. These results strongly imply a direct relationship between genetic control of autoantigen expression and peripheral autoreactivity, in which proINS genotype restricts the quantity and quality of the potential T cell response. doi:10.1016/j.clim.2010.03.026

OR.10. Altered Regulation of CD46 Expression and Processing in MS Siobhan Ni Choileain1, Nathan Weyand 2, Magdalene So 2, Belinda Weller1, Anne Astier1. 1University of Edinburgh, Edinburgh, United Kingdom; 2University of Arizona, Tucson, AZ The regulator of complement activity CD46 acts as a costimulatory molecule for T cells and promotes their differentiation into Tr1 cells, secreting notably the antiinflammatory cytokine IL-10. We previously demonstrated that CD46 pathway is dysregulated in T cells from patients with multiple sclerosis (MS), as IL-10 secretion is impaired. Herein, we have followed CD46 expression upon T cell activation in cells from a cohort of healthy donors and patients in the RRMS stage. Upon CD46 activation, we first observed the cleavage of its ectodomains by MMPs, which is regulated by the TCR. By homology to Notch, this generates a C-terminal fragment (CTF) that can be further cleaved by the gamma-secretase enzymatic complex. Indeed, loss of expression of both cytoplasmic isoforms occurred after activation, which could be partly restored by addition of a gamma-secretase inhibitor. We did not observe a defect in cleavage of CD46 ectodomain in T cells from patients with MS. However, the loss of CD46 cytoplasmic tails was not observed in T cells from patients. CD46 processing was required for its functions, as demonstrated by the following results: i) addition of gamma-secretase inhibitors to T cell culture inhibited IL-10 production by CD46activated T cells; ii) expression of a pre-cleaved CTF-cyt1 fragment of CD46 resulted into increased IL-10 production; iii) our preliminary data suggest that both tails are found in the nucleus of activated T cells. Altogether, this suggests that an altered CD46 processing might be at least partly responsible for its defective role in MS. doi:10.1016/j.clim.2010.03.027

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OR.11. Citrullinated Fibrinogen Stimulates TNF Release via TLR4 and Citrullinated Fibrinogen Immune Complexes Co-stimulate through TLR4 and the Fc Gamma Receptor Jeremy Sokolove, Xiayan Zhao, William Robinson. Stanford University, Palo Alto, CA Extravasation of fibrin(ogen) is a characteristic pathologic finding within the RA synovium. Citrullinated proteins have been demonstrated at many sites of inflammation and citrullinated fibrin(ogen) has been consistently identified within the RA synovium. The presence of anti-citrulline protein antibodies characterizes RA and has been associated with increased RA severity. The innate immune receptor TLR4 is critical in murine models of RA and has been implicated in human RA. Several studies have demonstrated TLR4 dependent signaling by non-microbial damage associated molecules including fibrin(ogen). In this study we demonstrate a dose dependent ability of native and cFb to stimulate macrophage TNF secretion and confirm that this process is TLR4 (and MyD88) dependent. Notably, cFb displayed approximately ten-fold increased potency over native fibrinogen. In-vitro generated cFb containing IC stimulated TNF release at levels significantly above that seen with cFb alone via a process dependent on the combination of TLR4 and the Fc gamma receptor. This study demonstrates that citrullination of fibrinogen dramatically increases it's potency on TLR4 mediated TNF release and that cFb IC can co-stimulate through TLR4 and the Fc gamma receptor. Given that citrullination is ubiquitous to sites of inflammation, the ability to increase the potency of an innate damage associated molecule via citrullination is highly novel with potentially broad generalization to the field of innate immunity. Additionally, the ability of cFb containing IC to co-stimulate TNF release provides a potential pathogenic mechanism for RA associated autoantibodies in the initiation and propagation of synovial inflammation. doi:10.1016/j.clim.2010.03.028

OR.12. Blockade of CD94/NKG2A Prevents EAE Jianmei Leavenworth 2, Carola Schellack Wenander1, Hye-Jung Kim 2, Linrong Lu 3, Pieter Spee1, Harvey Cantor 2. 1 Novo Nordisk A/S, Måløv, Denmark; 2Dana-Farber Cancer Institute, Boston, MA; 3Zhejiang University School of Medecine, Hangzhou, China NK cells are important members of the innate immune system that have been implicated in anti-microbial and antitumor defense as well as in maintenance of tolerance and control of autoimmune diseases. The activity of NK cells is regulated by a delicate balance of both activating and inhibitory receptors. CD94/NKG2A is an inhibitory receptor expressed in mice mainly on NK and a subset of CD8+ T cells recognizing the non-classical class I molecule Qa-1. Targeting CD94/NKG2A receptor by blocking its interaction with Qa-1 by a monoclonal antibody is a new therapeutic approach to increase NK cell activity in the treatment of

S8 autoimmune diseases. Anti-NKG2A F(ab’)2 treatment diminishes progression of experimental autoimmune encephalomyelitis in C57BL/6 mice. Analysis of the underlying mechanism reveals an anti-NKG2A-dependent decrease of T cell infiltration and reduced microglia activation in the central nervous system. Finally, anti-NKG2A F(ab’)2 reduced CD4+ T cell responses and skews the proportion of IL-17- and IFN-γ-producing CD4+ T cells towards the protective IL-4and IL-10-secreting CD4+ T cell subpopulation. doi:10.1016/j.clim.2010.03.029

OR.13. Inhibition of Autoimmune Demyelinating Disease by Neuroantigen-specific, Autoreactive CD8+ T Cells Sterling Ortega, Andrew Tyler, Nathan York, Jason Mendoza, Mihail Firan, Andrew Benagh, Todd Eagar, Nitin Karandikar. UT Southwestern Medical Center, Dallas, TX The role of neuroantigen-specific CD8+ T cells during immune-mediated demyelination has turned out to be both intriguing and paradoxical. In the murine model of multiple sclerosis (MS), experimental autoimmune encephalomeylitis (EAE), we have observed that MOG35-55-specific CD8+ T cells were able to suppress disease. This novel and unexpected function appeared to require in vivo Class I-mediated presentation of cognate antigen as MOG-reactive CD8+ T cells were unable to suppress disease in β2m-/- or Tap-/mice. These cells exhibited a unique regulatory immunophenotype as they were Foxp3-, IFN-γ+, perforin+ and IL17-. IFN-γ and perforin production was required as the adoptive transfer of IFN-γ or perforin-deficient CD8+ T cells failed to protect recipient mice from disease. In vivo trafficking experiments did not reveal significant CNS trafficking of adoptively transferred CD8+ T cells, suggesting a peripheral mechanism of action. These regulatory CD8+ T cells were able to kill MOG-loaded CD4+ T cells as well as CD4-depleted APC in vivo, suggesting a cytotoxic/suppressor mechanism. Regulation of disease was associated with modulation of APC function as well as decreased MOG-specific CD4+ T cell responses. Therefore, cytotoxic autoreactive CD8+ T cells are important players in EAE regulation and understanding this arm of the adaptive immune system offers a promising therapeutic strategy for MS. Supported, in part, by grants from the NIH/NIAID and the National MS Society. doi:10.1016/j.clim.2010.03.030

OR.14. Efficient Activation of Self-reactive T Cells from MS Patients with Altered Synapse Formation Susana Gordo1, David Schubert1, Santosh Vardhana 2, Nilufer Seth1, Jason Pyrdol1, Khadir Raddassi 3, David Hafler 3, Michael Dustin 2, Kai Wucherpfennig1. 1Dana-Farber Cancer Institute, Boston, MA; 2Skirball Institute of Biomolecular Medicine - NYU School of Medicine, New York, NY; 3 Brigham and Women's Hospital, Boston, MA In multiple sclerosis (MS) the immune system attacks the myelin sheath in the CNS. The CD4 T-cell clones Ob.1A12 and

Abstracts Ob.2F3 were isolated from a patient with relapsing remitting MS and recognize an epitope of myelin basic protein presented by HLA-DR2 with low affinity. Imaging of T-cell/ B-cell conjugates by confocal microscopy shows that the pattern of interacting molecules in the immunological synapse (IS) formed between the self-reactive clones Ob.1A12 and Ob.2F3 and the APC differs substantially from anti-microbial T-cell clones (specific for an influenza peptide). Both self-reactive and anti-microbial T-cell clones show significant accumulation of the adhesion molecule LFA-1. However, recruitment of T-cell receptors (TCRs) at the center of the contact area is only seen for antimicrobial but not the self-reactive clones. The recruitment of co-receptors (CD4) and co-stimulatory molecules (CD2) is also impaired. Nevertheless, substantial recruitment of ZAP-70 at the interface is observed with both sets of T-cell clones. Furthermore, rapid calcium flux upon interaction with APC is observed in both populations, but sustained calcium flux is reduced in the self-reactive clones. CD3 down-regulation and CD69 up-regulation patterns, on the contrary, do not significantly differ between antiviral and self-reactive clones. These data suggest that the self-antigen specific Tcell clones can be efficiently activated despite defects in IS formation suggesting mechanisms that compensate for low affinity TCR binding to self-peptide/MHC ligands. doi:10.1016/j.clim.2010.03.031

OR.15. Regulatory Role of T Cell TrkA Receptor in Human Autoimmune Disease: NGF/TrkA System Disrupts Peripheral Tolerance Siba Raychaudhuri, Smriti Raychaudhuri. University California-Davis/VAMC Sacramento, Davis, CA Neurotropic and other Nerve Growth Factor (NGF) activities in the nervous have been well defined. We have shown earlier that NGF levels are increased in synovial fluid (SF) from psoriatic arthritis (PsA) and rheumatoid arthritis (RA) patients, but not in SF from osteoarthritis (OA) patients. Here we report that significantly higher levels of NGF is spontaneously produced by synovial fibroblasts (FLS) from PsA and RA patients but not by FLS from OA patients. In addition, we observed that the high affinity NGF receptor (TrkA) is upregulated on activated memory T cells present in the SF from PsA (18 ± 6.2 %) and RA (4.5 ± 1.3 %) patients whereas SF from OA did not show any detectable HLA-DR +TrkA+CD3+ T cells. Regulatory role of NGF and NGFreceptor (NGF-R) in human T cell biology remain largely unknown. Moving to studies with activated peripheral blood T cells, we noticed that NGF induces T cell proliferation via TrkA and that T cell activation with CD3/28 upregulates T cell expression of TrkA. Finally, we show that NGF induces phosphorylation of Akt (p-Akt) in the activated T cells and decreases the sensitivity of these cells to apoptosis induction by TNF- α. These findings lead us to propose a model in which NGF produced by fibroblasts at arthritic sites of autoimmune origin signals TrkA to prolong survival of activated T cells and hence promotes to sustain the T cell mediated inflammatory