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Abstracts / Molecular I

e show that mice deficient for the immunosubunits beta5i/LMP7nd beta2i/MECL-1 develop early-stage multi-organ autoimmunityollowing irradiation and BM transplantation. Disease symptomsre caused by CD8 T cells and transferrable into immunosubunit-eficient, RAG1-deficient mice. Moreover, using the human Typediabetes genetics consortium (T1DGC) MHC dataset, we identi-

ed two SNPs within the beta5i/LMP7-encoding gene sequences,hat were in strong linkage disequilibrium (LD), as independentenetic risk factors for T1D development in humans. Strikingly,hese SNPs significantly enhanced the risk conferred by HLA hap-otypes that were formerly shown to predispose for T1D. Theseata suggest that inflammation-induced immunosubunit expres-ion in peripheral tissues constitutes a mechanism that preventshe development of CD8 T cell mediated autoimmune diseases.

oi:10.1016/j.molimm.2012.02.084

aploid genetic screening to identify novel regulators of MHClass I antigen presentation

obbert Spaapen a,∗, Vincent Blomen a, Matthijs Raaben b, Thijnrummelkamp a, Jacques Neefjes a

The Netherlands Cancer Institute (NKI-AVL), AmsterdamHarvard Medical School, Boston

MHC molecules are critical directors of the adaptive immuneesponse in many pathological conditions, like cancer, infection,tem cell and organ transplantation, and auto-immune disease. Thexpression of and antigen presentation by MHC class I moleculess tightly controlled, and many components of this pathway areurrently known. A genome-wide survey to determine the criti-al proteins playing a role in MHC class I antigen presentation wasever performed, mainly because genome-wide knockout screen-

ngs were not available in human cells.Recently a near haploid cell line (containing one copy of most

enes) was exploited as a powerful genetic screening tool in humanells. It is therefore relatively easy to generate single gene com-lete knockout cells and perform loss-of-function screens. In suchscreen, random mutagenesis of ∼100 million cells generated

ome cells that could be sorted for a MHC class I low or negativehenotype by flow cytometry. Subsequent deepsequence analysisevealed that some of these cells were knockout for known keyroteins in the MHC class I antigen presentation pathway: tapasinnd �2-microglobuline. Other hit proteins from this screen are cur-ently subjected to validation experiments.

Similarly, we sorted cells using antibodies specific for the MHClass I heavy chain only. Here, we identified cells phenotypicallyaving high levels of this unproper folded molecule at the cellurface. The mutants causing this phenotype are currently beingequenced and expected to be chaperone like proteins involved inhe folding of the MHC class I heavy chains.

In conclusion, using the latest state-of-the-art technology, were currently identifying novel components involved in the MHClass I cell surface expression. Also proteins involved in the properolding of this protein are under investigation. The results will have

ajor implications for the understanding of the MHC class I antigenresentation.

oi:10.1016/j.molimm.2012.02.085

ology 51 (2012) 5–41 31

Differential surface transport of class I alleles is caused by amaturation step in the endoplasmic reticulum

Sebastian Springer ∗, Susanne Fritzsche

Jacobs University, Bremen

We are interested in peptide binding, intracellular transport,and quality control of MHC class I molecules. We follow this with amultifaceted approach, in which our main expertise are biochem-istry with isolated membrane fractions and microscopy.

So far, our focus has been on the ER-Golgi cycle of peptide-receptive class I molecules, the role of calreticulin in quality control,and the mechanism of tapasin in peptide optimization.

Recently, we have investigated the kinetic difference in the sur-face transport of different MHC class I alleles. Using a COPII vesiclebudding assay and a novel technique of arresting ER export, weshow that the kinetic difference lies in a maturation step in the ERthat follows peptide binding but is prior to export from ER exit sites.

doi:10.1016/j.molimm.2012.02.086

Tapasin dependence of MHC class I molecules correlates withtheir conformational flexibility

Sebastian Springer

Jacobs University, Bremen

The surface expression of most MHC class I molecules at leastpartially depends on the endoplasmic reticulum protein, tapasin,which helps them to optimize peptide load. To determine whatmakes a class I molecule dependent on support by tapasin, wehave conducted in silico molecular dynamics (MD) studies andlaboratory experiments to assess the conformational state oftapasin-dependent and -independent class I molecules. We findthat in the absence of peptide, the region around the F pocket of thepeptide binding groove of the tapasin-dependent molecule HLAB*44:02 is in a disordered conformational state and that it is convertedto a conformationally stable state by tapasin. This novel chaperonefunction of tapasin has not been described previously. We demon-strate that the disordered state of B*44:02 is caused by the presenceof two adjacent acidic residues in the bottom of the F pocket of classI, and we suggest that conformational disorder is a common featureof tapasin-dependent class I molecules, making them essentiallyunable to bind peptides on their own.

doi:10.1016/j.molimm.2012.02.087

Dendritic cells sequester antigenic epitopes or their precursorsfor several weeks

Changying Li a, Buckwalter Matthew a, Manish Garg a, SreyashiBasu a, Jiwu Chang b, Pramod Srivastava a,∗

a University of Connecticut, School of Medicineb Tianjin Medical University, Tianjin, China

Dendritic cells (DCs) play an essential role in adaptive immuneresponses. Tissue-resident DCs take up viral or tumor antigens, pro-cess and present them on their MHC molecules, and migrate to thedraining lymph nodes where they engage naive cognate T lympho-cytes. Here, we report that DCs sequester antigenic epitopes in animmunologically active form for weeks after the initial encounterwith the antigen, and long after the intact antigen is degraded

beyond detection. This novel and extraordinary property of DCs isdemonstrable in vitro and in vivo and is mediated through the inter-action of antigen with hsp90 during the initial uptake of antigen bythe DCs. Through such interaction, the DCs create an intracellular