Structural Characterization of an Unusual Binding Mode of a Myelin-specific T Cell Receptor

Abstracts activity was evaluated 5 days a week during 5 hours, and mice were also filmed to report any case of seizure. Mice were then sacrificed to analyse brain pathology. Both mice injected with patients' PBMC and one mouse injected with patients' CD34+ cells engrafted. All 3 mice injected with RE patient cells and engraftment demonstrated abnormal EEG activity associated with seizures on video. No EEG abnormalities or seizures have been observed for control mice. The brain of sacrificed mice injected with RE cells all showed presence of strong gliosis, while brain T-cell infiltration was only observed in one mice injected with patient 1 PBMCs. These results suggest that injection of cells of RE patients in NOD/SCID/IL2rgnull mice is sufficient to induce seizures and gliosis, which are characteristic of RE. Nevertheless, while engrafted T cells seem involved in the occurrence of seizures, T cell infiltration is not necessary for seizures to occur. This new pathologic humanized mice model can be useful to elucidate the mechanisms of the disease and can serve for the development of new clinical treatments. doi:10.1016/j.clim.2010.03.215

T.116. Structural Characterization of an Unusual Binding Mode of a Myelin-specific T Cell Receptor Dhruv Sethi, David Schubert, Kathrin Wilbuer, Kai Wucherpfennig. Dana-Farber Cancer Institute, Boston, MA We have previously reported the crystal structure of a TCR–MHC complex from a patient with relapsing–remitting MS. The complex structure was highly unusual as the TCR was not centered over the peptide/MHC surface. Rather, Ob.1A12 TCR only contacted the N-terminal half of the MHC-bound myelin basic protein (MBP) peptide. In the present study, we sought to examine whether altered binding modes would also be observed for other MBPspecific T cells. TCR Hy.1B11 had previously been isolated from a relapsing–remitting MS patient. This TCR is also specific for MBP85–99, but bound to HLA-DQ1. Crystals of the Hy1b11-MBP85–99/HLA-DQ1 complex were obtained by hanging drop vapor diffusion and the structure was determined to a resolution of 2.55 Å. The TCR was diagonally oriented over the MHC as in other TCR–MHC complexes. There was, however, a tilt of the TCR of 15° towards DQ1 α-helix relative to the HA1.7–HA-DR1 complex which has a typical binding mode. As a result of the tilt, the germ line-encoded TCR loops CDRα1 and α2 do not engage the β helix of DQ1. This is highly unusual as germ line CDRs α1, α2, β1, and β2 are known to be involved in MHC restriction and, in the majority of the TCR-peptide/ MHC complexes, all four engage the MHC. Additionally, the CDRβ3 does not engage the peptide. This structure shows another unusual binding mode by a TCR from a MS patient. The unusual interaction with the peptide–MHC complex may have allowed this TCR to escape negative selection in the thymus. doi:10.1016/j.clim.2010.03.216

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T.117. Impact of HLA-DR Upon T cell Response to Gliadin Eric Marietta, David Luckey, Chella David, Joseph Murray. Mayo Clinic, Rochester, MN Background: DQ2 and DQ8 restricted CD4+ T cells in the small intestine of celiac patients contribute significantly to their aberrant response to gliadin. HLA transgenic mice that express DQ2 and DQ8 have been generated and used as models of gliadin sensitivity. However, epidemiological studies have identified the haplotypes, DQ2DR3 and DQ8DR4, as being the genetically predisposing elements for celiac disease. Objective: To determine if HLA-DR3 and HLA-DR4 molecules play a role in the response to gliadin. Methods: HLA transgenic mice that express DQ2, DQ8, DR3, or DR4 alone were compared with mice that express DQ2 and DR3 together and another line that expressed DQ8 and DR4 together. Mice were then injected subcutaneously with different gliadin-derived peptides to determine if the presence of DR3 and DR4 affected the T cell response to gliadin in the respective DQ2 and DQ8 transgenic mice. Results: The combination of DQ2 and DR3 together led to a significantly greater (3-fold) T cell response to the native (amidated) form of a potent immunodominant peptide derived from gliadin that is 33 amino acids long than in single transgenic mice. Approximately 2 mice were in each group, and there were 3 separate injection evaluations. Preliminary studies with the deamidated form of the 33mer indicate a similar pattern. Conclusion: Individually, HLADQ2 and HLA-DR3 contribute to the overall T cell response against a parenteral injection of gliadin; however, the combined presence of both DQ2 and DR3 leads to an amplified T cell response against immunodominant epitopes of gliadin. doi:10.1016/j.clim.2010.03.217

T.118. Human Astrocytes Promote Survival, Activation, and Pro-inflammatory Responses of B Cells: Implications for Multiple Sclerosis Antonia Kobert 1, Nathalie Lebeurrier 1, Ichiro Nakashima 2, Aja Rieger 1, Philippe Saikali 1, Caroline Lambert 1, Lama Fawaz 1, Jack Antel 1, Yasuto Itoyama 2, Amit Bar-Or 1. 1 McGill University, Montreal, QC, Canada; 2Tohoku University School of Medicine, Sendai, Japan Activated astrocytes in the inflamed MS CNS abnormally express factors (including BAFF, IL-6, and IL-15) which may contribute to a B cell fostering environment, although relevance to the MS disease process is unknown. Human CD19+ B cells were studied in coculture/transwell with human fetal astrocytes, astrocyte-conditioned media, or combinations of BAFF, IL-6, and IL-15. Astrocytes were either unstimulated or activated by transient exposure to proinflammatory cytokines (IFNγ + IL-1β). B cell readouts included survival and CD86/MHCI/MHCII expression (FACS), IgG and LTα secretion (ELISA), and APC function (allogeneic T-cell proliferation). Activated astrocytes (in coculture or transwell) significantly promoted B cell survival (P b 0.001) and surface expression of CD86 (P b 0.001), MHC class I (P b 0.05), and MHC class II (P b 0.01). Similar effects were observed with soluble products from activated astrocytes,