A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation — ASN Events
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  3. A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation

A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation (#113)

Shilpi Chandra 1 , James Gray 2 , William B Kiosses 1 , Archana Khurana 1 , Kaori Hitomi 1 , Catherine Crosby 1 , Zheng Fu 3 , Meng Zhao 1 , Natacha Veerapen 4 , Stewart K. Richardson 5 , Steven A. Porcelli 6 , Gurdyal Besra 4 , Amy R. Howell 5 , Sonia Sharma 2 7 , Bjoern Peters 8 , Mitchell Kronenberg 1 9
  1. Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, LA Jolla, CA, United States
  2. The Functional Genomics Center, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States
  3. Bioinformatics Core, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States
  4. School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK
  5. Department of Chemistry, University of Connecticut, Storrs, CT, USA
  6. Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, United States
  7. Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States
  8. Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States
  9. Division of Biological Sciences, University of California, San Diego, La Jolla, CA, United States

The pathways governing lipid antigen uptake and presentation by CD1 proteins remain incompletely defined. We have carried out a whole genome siRNA screen in a macrophage cell line for genes that affect the presentation of a potent glycosphingolipid antigen, GalGalCer, to iNKT cells. This antigen requires internalization and lysosomal carbohydrate antigen processing to remove the terminal galactose. After several rounds of validation, functional classification and gene expression analysis, we identified a set of 57 genes that lead to decreased CD1d antigen presentation. The majority of the genes we identified, including members of the HOPS and ESCRT complexes, did not perturb surface CD1d expression, but we demonstrate instead that they effected the formation of surface CD1d complexes with the stimulating glycolipid, either by altering antigen localization, CD1d traffic, or both. Interestingly, our data show that Abcc1 and several other ABC family transporters are involved in lipid antigen presentation by CD1d. Mice deficient for Abcc1 had reduced iNKT cell responses to glycolipid lipid antigens, and decreased survival following infection with Streptococcus pneumoniae, which has an antigen that stimulates iNKT cells. Although the CD1d and MHC-II antigen presentation pathways both depend on antigen loading in the lysosome, the majority of genes did not affect MHC-II antigen presentation, indicating a clear distinction between these two pathways.

Supported by NIH grants AI 71922, AI 105215 and U01 GM111849