Investigation Title Transcription profiling of human single B cells isolated from patients deficient for IL-1R-associated kinase (IRAK)-4, myeloid differentiation factor 88 (MyD88) and UNC-93B Comment[Submitted Name] IRAK-4- and MyD88-dependent pathways are essential for the removal of developing autoreactive B cells in humans Experimental Design unknown_experiment_design_type transcription profiling by array Experimental Design Term Source REF EFO Comment[AEMIAMESCORE] 4 Comment[SecondaryAccession] GSE13300 Comment[ArrayExpressReleaseDate] 2008-11-13 Comment[ArrayExpressAccession] E-GEOD-13300 Comment[MAGETAB TimeStamp_Version] 2010-07-30 09:11:03 Last Changed Rev: 13058 Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Person Last Name Isnardi Person First Name Isabelle Person Mid Initials Person Email isabelle.isnardi@gmail.com Person Phone Person Fax Person Address Hospital for Special Surgery, 535E 70th Street, New York, 10028, USA Person Affiliation Hospital for Special Surgery Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-11-13 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description Most autoreactive B cells are normally counterselected during early B cell development. To determine whether Toll-like receptors (TLRs) regulate the removal of autoreactive B lymphocytes, we tested the reactivity of recombinant antibodies from single B cells isolated from patients deficient for IL-1R-associated kinase (IRAK)-4, myeloid differentiation factor 88 (MyD88) and UNC-93B. Indeed, all TLRs except TLR3 require IRAK-4 and MyD88 to signal and UNC-93B-deficient cells are unresponsive to TLR3, TLR7, TLR8 and TLR9. All patients suffered from defective central and peripheral B cell tolerance checkpoints resulting in the accumulation of large numbers of autoreactive mature naïve B cells in their blood. Hence, TLR7, TLR8, and TLR9 may prevent the recruitment of developing autoreactive B cells in healthy donors. Paradoxically, IRAK-4-, MyD88- and UNC-93B-deficient patients did not display autoreactive antibodies in their serum nor developed autoimmune diseases, suggesting that IRAK-4, MyD88 and UNC-93B pathway blockade may thwart autoimmunity in humans. Experiment Overall Design: RNA was extracted from 105-3.105 batch sorted new emigrant and mature naïve B cells isolated from donors using the Absolutely RNA microprep kit (Stratagene). 100-200 ng of RNA was obtained per sample, and the quality of the purified RNA was assessed by the Bioanalyzer from Agilent. Using the Ovation biotin system kit from Nugen, 30-50ng of RNA was amplified and labeled to produce cDNA. Labeled cDNA was hybridized on chips containing the whole human genome (Human Genome U133 2.0 from Affymetrix). Raw data from new emigrant (1 healthy donor) and mature naive (4 healthy donors) B cells were analyzed in order to determine the expression of some molecules involved in the TLR pathway in these B cell population in humans. Protocol Name P-G13300-7 P-G13300-1 P-G13300-4 P-G13300-9 P-G13300-5 P-G13300-3 P-G13300-6 P-G13300-8 P-G13300-2 P-AFFY-6 Affymetrix:Protocol:ExpressionStat Protocol Type specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction labeling labeling feature_extraction bioassay_data_transformation Protocol Description Peripheral B cells were purified from the blood of the donor by negative selection using RosetteSepTM procedure (StemCell Technologies, Vancouver, BC, Canada). Enriched B cells were stained with fluorescein isothiocyanate (FITC) anti-human CD27, phycoerythrin (PE) anti-human CD10, anti-human IgM biotin, and allophycocyanin (APC) anti-human CD19. Biotinylated antibodies were revealed using streptavidin-PECy7. Single CD19+CD10-IgM+CD27- mature naive from the donor were sorted on a FACSVantage (Becton Deckinson). Peripheral B cells were purified from the blood of the donor by negative selection using RosetteSepTM procedure (StemCell Technologies, Vancouver, BC, Canada). Enriched B cells were stained with fluorescein isothiocyanate (FITC) anti-human CD27, phycoerythrin (PE) anti-human CD10, anti-human IgM biotin, and allophycocyanin (APC) anti-human CD19. Biotinylated antibodies were revealed using streptavidin-PECy7. Single CD19+CD10+IgM+CD27- new emigrant from the donor were sorted on a FACSVantage (Becton Deckinson). Peripheral B cells were purified from the blood of the donor by negative selection using RosetteSepTM procedure (StemCell Technologies, Vancouver, BC, Canada). Enriched B cells were stained with fluorescein isothiocyanate (FITC) anti-human CD27, phycoerythrin (PE) anti-human CD10, anti-human IgM biotin, and allophycocyanin (APC) anti-human CD19. Biotinylated antibodies were revealed using streptavidin-PECy7. Single CD19+CD10-IgM+CD27- mature naive from the donor were sorted on a FACSVantage (Becton Deckinson). Total RNA was extracted from 250,000 cells and purified using the AbsolutelyRNA microprep kit from Stratagene, by following the manufacturer’s protocol. Prior to use in amplification studies, the size integrity profile of all RNA samples was determined with an Agilent Bioanalyzer. All RNA preparation provided (A260/280) ratios greater than 1.8. RNA concentration was determined by Nanodrop spectrophotometry. Total RNA was extracted from 720,000 cells and purified using the AbsolutelyRNA microprep kit from Stratagene, by following the manufacturer’s protocol. Prior to use in amplification studies, the size integrity profile of all RNA samples was determined with an Agilent Bioanalyzer. All RNA preparation provided (A260/280) ratios greater than 1.8. RNA concentration was determined by Nanodrop spectrophotometry. Total RNA was extracted from 230,000 cells and purified using the AbsolutelyRNA microprep kit from Stratagene, by following the manufacturer’s protocol. Prior to use in amplification studies, the size integrity profile of all RNA samples was determined with an Agilent Bioanalyzer. All RNA preparation provided (A260/280) ratios greater than 1.8. RNA concentration was determined by Nanodrop spectrophotometry. Total RNA was extracted from 150,000 cells and purified using the AbsolutelyRNA microprep kit from Stratagene, by following the manufacturer’s protocol. Prior to use in amplification studies, the size integrity profile of all RNA samples was determined with an Agilent Bioanalyzer. All RNA preparation provided (A260/280) ratios greater than 1.8. RNA concentration was determined by Nanodrop spectrophotometry. Total RNA was extracted from 100,000 cells and purified using the 50 ng total RNA samples were amplified and labeled with biotin using the Ovation Biotin system (NuGEN Technologies, Inc., San Carlos, CA). Amplification of starting total RNA yielded on average 7 μg of amplified single stranded cDNA. This material was then fragmented and labeled with biotin. 50 ng total RNA samples were amplified and labeled with biotin using the Ovation Biotin system (NuGEN Technologies, Inc., San Carlos, CA). Amplification of starting total RNA yielded on average 7 μg of amplified single stranded cDNA. This material was then fragmented and labeled with biotin. Title: Affymetrix CEL analysis. Description: Title: Affymetrix CHP Analysis (ExpressionStat). Description: Protocol Parameters Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF The MGED Ontology SDRF File E-GEOD-13300.sdrf.txt Term Source Name ncbitax The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version