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0 Abstracts / Molecular I

llelic polymorphism and moderate sequence diversity, with vari-tion in positions expected for peptide-binding. We have analysedeptide translocation in three MHC haplotypes, showing thathicken TAPs can specify translocation at three peptide positions,atching or exceeding the specificity of the single dominantly-

xpressed class I molecule. We have used structures of bacterialBC transporters to model the TAP heterodimer, located theositions of the residues polymorphic in rats and chickens, and

dentified the residues apparently responsible for the specificeptide-binding.

These results show that co-evolution between class I and TAPenes can explain the presence of a single dominantly-expressedlass I molecule in many common chicken MHC haplotypes, androvide the basis for mutagenesis experiments to map the peptide-inding site in detail. The salient features discovered in the chickenHC can be found in many if not most non-mammalian verte-

rates. Comparison with the MHC organisation of humans andypical mammals suggests that a large inversion brought the classII region in between the class I and class II regions and thus sepa-ated the antigen processing genes from the class I genes, breakinghe co-evolutionary relationships and allowing a multigene familyf well-expressed class I genes. We propose that such co-evolutionn the primordial MHC was responsible for the appearance of thentigen presentation pathways and receptor-ligand interactions athe birth of the adaptive immune system.

oi:10.1016/j.molimm.2012.02.050

ffects of proteasome catalytic subunit deficiencies on CD8 Tells thymic selection

leanor Kincaid ∗, Kenneth Rock

University of Massachusetts Medical School

In the thymus, immature T cells must undergo both positivend negative selection, in which cells with either too low or tooigh affinity for peptide-MHC complexes are culled. The nature ofhe peptide-MHC complexes on which cells are selected, and the

echanism that allows cells with the same TCR to survive bothositive and negative selection continue to be a puzzle. We usedice lacking three (beta1i, beta2i and beta5i) or four (beta1i, beta2i,

eta5i, and beta5t) of the proteasome catalytic subunits expressedn the thymus to investigate the effects of altered peptide reper-oires during CD8 T-cell development. Using TCR transgenic strainsH-Y, OT-I and P14), bone marrow chimeras and the CD8 lineageeporter Runx3d-YFP, we have found defects in both positive andegative selection in our triply and quadruply deficient mice.

oi:10.1016/j.molimm.2012.02.051

biquitin-receptor CXCR4 promotes cross presentation throughacilitated uptake of poly-ubiquitylated antigens

rédéric Ebstein, Andrea Lehmann, Peter-Michael Kloetzel ∗

Charité-Universitätsmedizin Berlin, Institut für Biochemie

Dendritic cells (DC) as professional antigen-presenting cellsake up antigens shed from infected and/or damaged cellsnd present them to CD8 + T cells for immune stimulationtermed cross-presentation). Here, we have studied the rolef the recently discovered ubiquitin receptor CXCR4 in crossresentation. Expressing the Ub-tagged human cytomegalovirus

HCMV)-derived pp65 antigen in HeLa cells as antigen donorstrongly improved cross-presentation of the immuno-dominantp65 (495–503) epitope peptide. Consequently, we studied theunctional significance of substrate ubiquitination in donor cells

ology 51 (2012) 5–41

by investigating the CXCR4-dependence of pp65 (495–503) cross-presentation. By loading DC with different pp65 antigenic forms,our experiments demonstrated that RNAi-mediated knockdown ofthe Ub-receptor CXCR4 in DC strongly impaired cross-presentationof pp65 (495–503) when DC were fed with poly-ubiquitinatedpp65. In contrast, no effect was observed when recombinant non-ubiquitinated pp65 was offered to DC. Our experiments thusdemonstrate that poly-ubiquitin can serve as signal for proteinuptake and that poly-ubiquitinated proteins taken up via CXCR4are a major antigenic source for cross-presentation by DC.

doi:10.1016/j.molimm.2012.02.052

Oxidant-damage of viral proteins by NADPH-oxidase Nox4-induced radical production drives MHC class I antigenprocessing

Elke Krüger ∗, Annika Warnatsch, Theresa Bergann, FredericEbstein, Peter-Michael Kloetzel

Institute of Biochemistry, Charité University medicine Berlin

Peptide generation by the ubiquitin proteasome system is therate limiting factor in major histocompatibility complex class Irestricted antigen presentation. The dynamics of this process intarget cells remains however poorly defined. Here, we identifiedthe NADPH-Oxidase Nox4 to be essential for the efficient presenta-tion of viral epitopes as early as two hours post infection. Infectionof target cells with either human cytomegalovirus (HCMV) orinfluenza A virus resulted in the activation of Nox4. Althoughthe majority of antigenic peptides originate from so called defec-tive ribosomal products (DRiPs), both investigated viral epitopes,pp65495–503 and M158-66 deriving from structural proteins ofthe virion, were demonstrated to be presented in a translation-independent manner. The rapid production of radicals by Nox4resulted in oxidant-damage of the HCMV tegument protein pp65or influenza A matrix protein M1. Concomitantly, virus infectionalso induced their UBE2L6-dependent ubiquitin conjugation, theirsubsequent immunoproteasome-mediated degradation and thegeneration of virus-specific epitopes. Thus, radical production byNox4 in target cells represents a mechanism that gives the immunesystem access to structural proteins from the virus particle inde-pendent of their de novo synthesis and before immune escapemechanisms of viruses can be initiated.

doi:10.1016/j.molimm.2012.02.053

Direct targeting of invariant chain to the MHC II loading com-partment

Ana Kucera ∗, Oddmund bakke, Tone F. Gregers

Institute of Molecular Biosciences/Centre for Immune Regula-tion/University of Oslo, Norway

The MHC II molecules associate with their chaperone; Invari-ant chain (Ii) already in the Endoplasmic Reticulum. Ii has beenassigned many functions; however, the most vital one is probablyits ability to facilitate MHC II trafficking to the MHC II peptide load-ing compartments (MIICs). This function depends on two leucinebased sorting signals within the Ii cytoplasmic tail which are knownto act as binding sites for the adaptor proteins AP-1/AP-2 andclathrin. Two clathrin dependent traffcking pathways to MIICs havebeen described; either direct targeting of MHC II to the endosomal

pathway from the Trans Golgi Network (TGN), or indirect via theplasma membrane (PM) followed by rapid internalization to endo-somes. Most studies on Ii-MHC II trafficking have been performedin model cell lines without any implications for MHC II antigen

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Abstracts / Molecular I

resentation. Thus it is not known wether the efficiency of antigenresentation depends on the Ii-MHC II trafficking pathways. In thistudy we have constructed different Ii trafficking mutants wherehe AP-1/AP-2 binding motif of Ii is replaced with an AP-3 bind-ng one, directly targetting Ii-MHC II to late endosomes/lysosomes.

e are therefore now able to study the two pathways indepen-ently, with regard to MHC II trafficking, peptide loading, half lifend antigen presentation.

oi:10.1016/j.molimm.2012.02.054

dentifying novel ubiquitin E3 ligases in the MHC class I antigenresentation pathway

aul Lehner a,∗, Florencia Cano a, Marian Burr a, Richard Timms a,essica Boname a, Felix Randow b, Lidia Duncan a

Cambridge Institute for Medical Research, University of CambridgeMedical Research Council Laboratory of Molecular Biology, Cam-ridge

Cell surface MHC I molecules occupy a central position in bothhe innate and adaptive immune system, providing key ligands forhe different receptor families on cytotoxic T-lymphocytes (CTL)s well as Natural Killer (NK) cells. Cell surface expression of MHCmust therefore be finely tuned, as small changes in class I levelsffect susceptibility to NK cell killing, making the precise regulationf MHC I expression essential for cell survival. Using a number ofifferent screening technologies we have identified novel genes inhe MHC class I antigen presentation pathway, including two newigases involved in MHC I antigen regulation. MEX-3 is a novel RNA-inding ubiquitin E3 ligase responsible for the post-transcriptional,llotype-specific regulation of MHC I. Expression of this ligase isncreased upon NK cell activation and affects the threshold of killingy these cells. We find that MEX-3 binds and induces the RING-ependent degradation of HLA-A2 mRNA through its 3′UTR. Sincehe RING domain of MEX-3 is required for HLA-A2 mRNA degra-ation, but not HLA-A2 cell surface downregulation, our findingsncover a novel mechanism for HLA-A-specific regulation that pro-ide a direct link between ubiquitination and mRNA decay. Athe post-translational level, misfolded MHC class I molecules arene of the few known endogenous ERAD substrates and providen important prototype to study the glycoprotein ERAD pathway.o identify the E3 ligase regulating the degradation of misfoldedHC I we developed a flow cytometry based functional siRNA

creen using beta2m-depleted cells expressing GFP-tagged HLA-2. This approach identified an essential role for the ER-resident3 ligase HRD1 together with the E2 ubiquitin-conjugating enzymeBE2J1 in the degradation of endogenous misfolded MHC I. Theiffering requirements for ER luminal chaperones and integral com-onents of the HRD1 complex for degradation of non-glycosylated,oluble, tailless and lysine-deficient forms of MHC class I will beresented. The recent description of insertional mutagenesis in theear-haploid KBM7 cell line offers a further approach for screening

n human cells. We have used this system to probe the regulation ofHC class I molecules by viral ubiquitin E3 ligases and will present

ndings of a novel gene required for MHC class I regulation by theseiral gene products.

oi:10.1016/j.molimm.2012.02.055

ology 51 (2012) 5–41 21

Opposite effects of short-tail and long-tail class I Myosins onAntigen Processing and Presentation

Ana-Maria Lennon-Duménil ∗, jheimmy Diaz, Paolo Pierobon,Maria-Isabel Yuseff, Danielle Lankar

Institut Curie, Paris

Engagement of the B Cell Receptor (BCR) by surface-tetheredantigens (Ag) leads to formation of a synapse that promotes Aguptake and presentation onto MHCII molecules. We have recentlyhighlighted the membrane trafficking events and associated molec-ular mechanisms required for efficient Ag extraction and processingat the B cell synapse. MHCII-containing lysosomes are recruited atthe synapse and locally undergo exocytosis, a process that relieson the SNARE protein Vamp-7. Lysosome secretion allows theextracellular release of proteases, whose activities promote theextraction of the immobilized Ag. (Yuseff et al. Immunity, 2011).We here show that local re-organization of cortical actin by type IMyosins, which link the cortex to the plasma membrane, is requiredfor lysosome exocytosis and Ag extraction. Remarkably, while theshort-tail Myosin IC is recruited at the synapse and facilitates vesi-cle secretion and Ag uptake, the long-tail Myosin IE negativelyregulates both processes. Interestingly, these differential effects ofclass I Myosins result from their opposite functions on the actincortex organization at the synapse. The B cell synapse thereforeemerges as a highly specialized site where tightly regulated exo-cytic and endocytic events take place thanks to the local shapingof the membrane-cytoskeleton interface by class I Myosins, ulti-mately leading to Ag extraction and processing.

doi:10.1016/j.molimm.2012.02.056

Human mucosal associated invariant T cells: A unique, lung-resident T cell subset with features of both innate and adaptiveimmunity: Use of an shRNA library to define the requirementsfor MR1-dependent antigen processing and presentation

David Lewinsohn a,∗, Melanie Harriff a, Luis Moita b, MarielleGold a, Sue Smyk-Pearson a, Yvonne Eberling a, Lynne Swarbrick a,Elizabeth Canfield a, Steven Langley a, Phillip Streeter a

a Oregon Health & Sciences Universityb Institute of Molecular Medicine

Human mucosal associated invariant T (MAIT) cells are a uniqueT cell subset that expresses the semi-invariant T cell receptorV�7.2 and are restricted by the MHC-Ib molecule MR1. How-ever, the extent to which MAIT cells are innate and their capacityto adapt and mature is unknown. To determine if human MAITcells are innate we evaluated the intrinsic function of V�7.2 + Tcells from the thymus, cord blood, and peripheral blood. Antigen-inexperienced MAIT cells displayed a naive phenotype but hadintrinsic effector capacity. As evidenced by the presence of sjTREC,Mtb-reactive thymocytes had undergone limited replication, andhence were of thymic origin. In evaluating the capacity of MAITcells to adapt following thymic egress, we found that Mtb-reactiveMAIT cells in peripheral blood displayed a memory phenotype andexpanded to high frequency suggesting that once in the periph-ery they responded to antigenic stimulation. Finally, we find thatMAITs reflect roughly 50% of airway-resident CD8 + T cells. ThusMAIT cells, an evolutionarily conserved T cell subset that detectsa variety of intracellular infections, share features of innate andadaptive immunity.

At present, the mechanisms by which Mtb-derived ligands areprocessed and presented remain unclear. By analogy with CD1d-restricted NKT cells, it is possible that Mtb infection leads to analtered endogenous ligand, or that Mtb-derived ligands are directly