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Abstracts / Molecular I

ill wonders never cease? Unraveling the Intricacies of the classprocessing pathway

onathan Yewdell ∗, Alexandre David, Brian Dolan, Xiuju Lu, Jackennink

Laboratory of Viral Diseases, NIAID, Bethesda

In the typical cartoon conception of cell biology, the cytosol isepresented as a homogeneous compartment. This view is beingodified as various cytosolic processes appear to supersede the

aw of mass action due to compartmentalization. I will presentur recent evidence that protein translation is highly compart-entalized (including translation in the nucleus) and that the

losely related process of antigenic peptide generation is compart-entalized to modulate competition between peptides, enhancing

mmunosurveillance of low abundance translation products. A sur-rising consequence of compartmentalized antigen processing ishe intracellular segregation of class I molecules based on their pep-ide cargo. Peptide-specific segregation of Kb is clearly detectedn the Golgi complex, and is maintained for hours after deliveryf class I peptide clusters to the cell surface. The likely func-ion of peptide specific-clustering is to increase the sensitivityf T cell immunosurveillance. I will also present evidence thathe antigen processing machinery has the remarkable ability toistinguish between ostensibly identical antigens based on theirranslation from cellular vs. virus-encoded mRNA. Efficiency mea-urements (i.e., number of substrates degraded per cell surfaceomplex generated) from these experiments shake the foundationsf our quantitative understanding of antigen processing.

oi:10.1016/j.molimm.2012.02.105

ystematically exploring the contribution of individualmmunoproteasome subunits to influenza Cd8+ T cell repertoirend epitope abundance

amien Zanker ∗, Jason Waithman, Weisan Chen

Ludwig Institute for Cancer Research

The immunoproteasome is a specialized piece of protein degra-ation machinery that is reported to enhance MHC class I-restrictedntigen processing and presentation. The immunoproteasome dif-ers from the housekeeping proteasome through the substitution ofhe catalytically active beta1, beta2 and beta5 subunits for LMP2,

ECL-1 and LMP7 respectively, which alters the cleavage speci-city.

To explore the contribution of each individual immunoprotea-ome subunit during Influenza A virus (IAV) CD8+ T cell response,e used biochemical and functional assays to assess the impact

f loss of 1 or 2 subunits on T cell repertoire, epitope abundancend epitope presentation. Polyclonal PR8-specific CD8+ T cell linesere generated from either wild-type, LMP2-/-, LMP7/MECL-1-/- orECL-1-/- mouse strains, and T cell repertoire was assessed with 41

reviously identified epitopes. Immunoproteasome deficient linesere observed to have a distinctly altered T cell repertoire, with

arious immunodominant and sub-dominant responses enhanced,ecreased or absent.

Epitope generation and abundance was explored using naturallyrocessed peptides derived from immunoproteasome subunit defi-ient bone-marrow derived dendritic cells, which were separatedia HPLC. Distinct differences in specific-peptide abundance werebserved for a variety of epitopes, including a dependence on indi-

idual immunoproteasome subunits for certain epitopes. Singlepecificity T cell lines were also used to ascertain the peptide quan-ity and kinetics of presentation of specific epitopes. Our resultshowed that individual immunoproteasome subunits greatly influ-

ology 51 (2012) 5–41 37

enced the antigen processing and presentation of various peptidesleading to different thymic selection of T cell repertoire as well asdramatically skewed CD8+ T cell responses.

Our study is the first systematic in vivo and biochemical char-acterisation of the contribution of individual immunoproteasomesubunits to CD8+ T cell repertoire and peptide generation inan IAV model. The knowledge may help us to induce desiredimmune responses through manipulating the expression of variousimmunoproteasome subunits in future vaccines.

doi:10.1016/j.molimm.2012.02.106

Mannose receptor poly-ubiquitination regulates endosomalrecruitment of P97 and cytosolic antigen translocation forcross-presentation

Zehner Matthias ∗, Chasan Achment, Schütte Verena, Embgen-broich Maria, Quast Thomas, Kolanus Waldemar, Burgdorf Sven

Life and Medical Science Institute, University of Bonn

The mechanisms regulating non-canonical protein trans-port across cellular membranes are poorly understood. Cross-presentation of exogenous antigens on MHC I by dendritic cellsgenerally require antigen translocation from the endosomal com-partment into the cytosol for proteasomal degradation (Burgdorfet al., 2007).

In this study, we demonstrated for the first time that suchtranslocation is controlled by the endocytic receptor and regulatedby ubiquitination. Antigens internalized by the mannose receptor(MR), a receptor that targets its ligands specifically towards cross-presentation (Burgdorf et al., 2007), were translocated into thecytosol only after attachment of a poly-ubiquitin chain to its cytoso-lic region. Furthermore, we identify TSG101 as a central regulator ofMR ubiquitination and antigen translocation.By the establishmentof a novel method, which allowed a flow-cytometrical analysisof individual endosomes, we could demonstrate that MR poly-ubiquitination mediated the recruitment of p97, a member of theER-associated degradation machinery that provided the drivingforce for antigen translocation towards the endosomal membrane(Jarosch et al., 2002).

References

Burgdorf, S., Kautz, A., Bohnert, V., Knolle, P.A., Kurts, C., 2007. Distinct pathwaysof antigen uptake and intracellular routing in CD4 and CD8 T cell activation.Science 316 (5824), 612–616.

Jarosch, E., Geiss-Friedlander, R., Meusser, B., Walter, J., Sommer, T., 2002. Proteindislocation from the endoplasmic reticulum–pulling out the suspect. Traffic 3(8), 530.

doi:10.1016/j.molimm.2012.02.107

Cross-presentation: How to get there – or how to get the ER

Christoph Kreer, Judith Rauen, Matthias Zehner ∗, Sven Burgdorf

Life and Medical Sciences Institute, University of Bonn

Antigen cross-presentation enables dendritic cells (DCs) topresent extracellular antigens on major histocompatibility com-plex (MHC) I molecules, a process that plays an important role inthe induction of immune responses against viruses and tumors andin the induction of peripheral tolerance. In order to allow intracel-

lular processing for cross-presentation, internalized antigens aretargeted by distinct endocytic receptors toward specific endoso-mal compartments, where they are protected from rapid lysosomaldegradation. From these compartments, antigens are processed for

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oading onto MHC I molecules. Such processing generally includesntigen transport into the cytoplasm, a process that is regulatedy members of the ER-associated degradation (ERAD) machinery.fter proteasomal degradation in the cytoplasm, antigen-derivedeptides have been shown to be re-imported into the same endo-omal compartment by endosomal transporter associated withntigen processing, another ER protein, which is recruited towardhe endosomes after DC maturation.

In our review, we highlight the recent advances on the molecu-ar mechanisms of cross-presentation. We focus on the necessity ofuch antigen storage compartments and point out important paral-els to MHC I-restricted presentation of endogenous antigens. Weiscuss the composition of such endosomes and the targeting ofxtracellular antigens into this compartment by specific endocyticeceptors. Finally, we highlight recent advances on the recruitmentf the cross-presentation machinery, like the members of the MHCloading complex and the ERAD machinery, from the ER toward

hese storage compartments, a process that can be induced by anti-en encounter or by activation of the dendritic cell after contactith endotoxins.

oi:10.1016/j.molimm.2012.02.108

echanism of superior cross-presentation of synthetic longeptides in comparison with protein for therapeutic cancer vac-ination

sther D. Quakkelaar a, Rodney A. Rosalia a, Selina Khan a,aap Oostendorp a, Sjoerd H. van der Burg a, Ferry Ossendorp a,ornelis J.M. Melief a,b,∗

Leiden University Medical Center, Leiden, The NetherlandsImmune System Activation Pharma, Leiden, The Netherlands

Previous work from our laboratory has demonstrated that syn-hetic long peptides (SLP) in comparison with recombinant proteinsnduce superior CD8 CTL responses in mice following variousmmunization schemes. In vivo this was shown to involve preferen-ial processing and presentation of long peptides (25–35 AA long)y CD11c+ dendritic cells (DC) in comparison with protein. ExactHC class I binding peptides, in contrast, were presented in vivo by

ll cell types expressing MHC class I, including B and T cells, presum-bly by exogenous loading of MHC class I with such short peptides.herapeutic activity following immunization with SLP was supe-ior over both short peptide and protein immunization in a mousePV16-induced tumor model. Based on these results we success-

ully treated established pre-malignant lesions in women causedy HPV16 using a vaccine containing 13 overlapping SLP coveringhe entire E6/E7 oncogenic protein sequences.

We now report studies on in vitro processing of long peptidesor MHC class I presentation by mouse DC and human DC subtypes.

ouse DC processed a long OVA peptide much more efficiently forresentation to OT-1Kb-restricted CD8 T cells than OVA protein. Theechanism of access to MHC class I loading appeared to be differ-

nt. Whereas fluorescently labeled protein ended up in endosomalompartments, labeled long peptide caused diffuse staining, sug-esting direct entrance in the cytoplasm followed by proteasomalntry. Although the human DC considered to be superior in cross-resentation is the CD141+ subset (equivalent to the mouse CD8�C), we observed that human monocyte-derived DC were superior

o pDC and CD141+ DC in processing and presenting a long HIVag peptide to HIV-specific CD8 CTLs. We conclude that the cross-

resentation mechanism of long peptides is different from that ofroteins and accomplished by a different subset of DC.

Based on this and our previous work, we state that all robustherapeutic vaccination schedules should induce both CD4 T helper

ology 51 (2012) 5–41

1 cells and CD8 CTL and must consider proper T cell polarization, aswell as optimal antigen processing and DC targeting. Optimal vac-cination schemes may involve DNA priming/ SLP boosting, shouldinvolve an optimal TLR ligand and should avoid schemes in whichantigenic competition, e.g. by viral vector sequences, can playa role.

doi:10.1016/j.molimm.2012.02.123

Interaction of HLA-DM and HLA-DQ2 in antigen presentation:Implications for celiac disease association

Tieying Hou 1, Shuo-Wang Qiao 4, Xi Jin 2, John Sidney 3, LudvigSollid 4, Henriette Macmillan 1, Michael Strohman 1, Taejin Yoon 1,Elizabeth D. Mellins 1,∗

1 Department of Pediatrics, Program in Immunology, Stanford Univer-sity, Stanford, CA 94305, United States2 Department of Chemistry, Stanford University, Stanford, CA 94305,United States3 La Jolla Institute for Allergy & Immunology, La Jolla, CA 92037, UnitedStates4 Institute of Immunology, University of Oslo, 0027 Oslo, Norway

The association of the HLA-DQ2 (DQ2) allele with celiac diseasehas been appreciated for a long time. The explanation for this asso-ciation is still incompletely understood. We previously found thatDQ2 is a poor substrate for HLA-DM (DM). To further character-ize DQ2-DM interaction, we introduced point mutations into DQ2on the proposed DQ2/DM interface in order to restore the sensi-tivity of DQ2 to DM. The effects of mutations were investigatedby measuring the peptide dissociation and exchange rate in vitro,CLIP and DQ2 expression on cell surface and the presentation of�-gliadin 57–73 Q65E epitope to T cell hybridomas. We found thatthe �+53G mutant restored DQ2 sensitivity to DM, likely due toimproved interaction with DM. As DM determines the immun-odominant and cryptic fate of peptide epitopes, we hypothesizedthat reduced interaction with DM might lead to the presentationof otherwise cryptic DQ2-restricted gliadin epitopes. Using T2 cellstransfected with DQ2 with or without greater than physiologicallevels of DM, we investigated the contribution of DM to the pre-sentation of different, processing-dependent gliadin epitopes (�I,�II, �I, �VII). Cells lacking DM efficiently presented all four epi-topes to gliadin-specific T cells. Interestingly, cells expressing DMwere significantly compromised in their ability to display the aboveepitopes despite the higher level of surface DQ2 in DM+ cells. Thesame finding was observed when murine, DQ2-restricted T cellhybridomas were used. We conclude that all four gliadin peptideswere DM-suppressed epitopes. We hypothesize that in vivo theinefficient interaction between DQ2 and DM influences the fate ofthese epitopes, changing them from cryptic to immunodominant,and inducing a gliadin-specific immune response in CD patientsexpressing the DQ2 allele.

doi:10.1016/j.molimm.2012.02.115

Combined functional and kinetic measurements to address therole of saposins in loading CD1d molecules with iNKT cell ago-nists

Mariolina Salio 1,∗, Hemza Ghadbane 1, Dawn Shepherd 1, JeremyCypen 1, Michael Aichinger 1, Peter Jervis 2, Gurdyal S. Besra 2,Peter Cresswell 3, Vincenzo Cerundolo 1

1 MRC Human Immunology Unit, Weatherall Institute of MolecularMedicine, University of Oxford, UK2 School of Biosciences, University of Birmingham, B15 2TT, UK