Antigen Specific T Cells in Lupus Recognize U1-70 and Produce IL-17 by Tetramer Staining

Abstract response characteristic of the disease; perhaps similar mechanisms occur in other autoimmune diseases. doi:10.1016/j.clim.2010.03.032

OR.16. Antigen Specific T Cells in Lupus Recognize U1-70 and Produce IL-17 by Tetramer Staining Nicole Kattah, Evan Newell, Alvina Chu, Michael Kattah, Mark Davis. Paul Utz. Stanford University, Stanford, CA Antigen-specific CD4+ T cells have been implicated in the autoimmune disease lupus, yet little is known about the function of these T cells or the precise peptide antigens that they recognize. We generated MHC class II tetramers of I-Ek containing peptides from the spliceosomal protein U1-70 that specifically stain distinct CD4+ T cell populations in MRL/lpr mice. Using these tetramer reagents, we demonstrate that CD4+ T cells specific for U1-70(131-150):I-Ek associate with disease, correlate with anti-U1-70 autoantibody production, express RORgt, and produce IL-17 in an I-Ek-dependent manner. These studies represent the first reported tetramers for studying antigen-specific T cells in a mouse model of lupus, and demonstrate an antigen-specific source of IL-17 in disease. doi:10.1016/j.clim.2010.03.033

Genomic and Genetic Approaches to Dissect Disease Pathways Friday, June 25 2:45-4:45 pm OR.17. Whole Transcriptome Sequencing in SLE: Deep Sequencing of RNA from SLE Blood Reveals Interferon-regulated Alternative Splicing Events Donna Thibault Flesher1, Christina Chaivorapol1, Kristen Wolslegel1, Alexander Abbas1, Stephen Kingsmore 2, John Rinn 3, Marco Sorani1, Thomas Wu 2, Timothy Behrens1. 1 Genentech, Inc., South San Francisco, CA; 2National Center for Genome Resources, Santa Fe, NM; 3Broad Institute, Cambridge, MA Whole transcriptome sequencing of RNA (RNA-seq) using massively parallel sequencing platforms allows for the simultaneous detection of transcript abundance, alternative splicing events, genetic variation in protein coding domains, and expression of transcripts not present on microarrays such as pseudogenes, non-coding RNAs, and novel genes. The coordinate overexpression of IFN-regulated transcripts in blood from subsets of SLE patients has been well characterized using gene expression microarrays. We performed deep sequencing of cDNA from peripheral blood mononuclear cells of five SLE patients with high expression of IFN-regulated genes (IFN Signature Metric (ISM) positive) and five ISM negative patients on the Illumina platform. Short reads were aligned to the human genome and to a database of large intergenic non-coding RNAs (lincRNAs). The expression of N 13,000 known genes was detected using this method and

S9 1,718 genes were differentially expressed between ISM negative and ISM positive patients. RNA-seq was more sensitive and had a larger dynamic range than Affymetrix microarrays. The expression of several lincRNAs correlated with ISM, demonstrating the ability of this method to detect non-coding RNAs. The relative expression level of known isoforms of each gene was estimated using an expectation/ maximization algorithm. Of the 11,158 genes that were not differentially expressed at the gene level, 1,423 (13%) demonstrated differential isoform expression in ISM negative vs. ISM positive cases, suggesting that a large percentage of transcripts undergo IFN-induced alternative splicing. Our data suggest that the regulation of non-coding RNA expression and alternative splicing by IFN may represent novel mechanisms of pathogenesis in SLE. doi:10.1016/j.clim.2010.03.034

OR.18. The First Transcriptional Map of the Human Major Histocompatibility Complex Reveals New Transcripts and Haplotype-specific Pattern of Expression Claire Vandiedonck1, Martin Taylor 2, Helen Lockstone1, Katharine Plant1, Jennifer Taylor1, Benjamin Fairfax1, Caroline Durrant1, John Broxholme1, Julian Knight1. 1 Oxford University, Oxford, United Kingdom; 2EMBL, Hinxton, Cambridge, United Kingdom As the most gene dense and polymorphic region in the human genome, the human Major Histocompatibility Complex (MHC) has been a paradigm for genomics. It is also the topranking region for the number of associations with immune and non-immune diseases. Recently, it was entirely re-sequenced for frequent haplotypes associated with common autoimmune diseases. Here, using an original hybrid microarray, including both tiling and junction probes, we draw the first strandspecific transcription map of the MHC in the context of three common haplotypes strongly associated with autoimmune disease, HLA-A3-B7-Cw7-DR15, HLA-A1-B8-Cw7-DR3 and HLAA26-B18-Cw5-DR3-DQ2, carried by the homozygous lymphoblastoid cell lines PGF, COX and QBL. In total, 6% of the MHC is transcribed with an average density of one transcriptional active region (TAR) per 3kb, including new TARs in intergenic regions. Up to 11% of the TARs are haplotype-specific. The distributions of differentially expressed probes and of polymorphisms between haplotypes are significantly correlated, arguing for a role of cis-regulatory variants. We identified 96 differentially expressed genes and 305 differentially spliced exons between haplotypes. These patterns were validated by RT-PCR and, for the top gene, confirmed in an independent cohort of 96 healthy volunteers. We also observed that alternative splicing is significantly more extensive in the MHC than in the rest of the genome, but was independent of its immune related function. This study marks a new step towards the identification of regulatory variants involved in the control of MHC associated phenotypes. doi:10.1016/j.clim.2010.03.035