mmun

sh

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locctcn

d

Il

PKN

a

cBb

Bc

ud

piahhcadtmiopWdtatriaapnafip

Abstracts / Molecular I

titutive proteasome (subunit �5c) favors hydrolysis after smallerydrophobic residues.

Additional ligand complex structures with the inhibitor PR-957evealed that its binding to subunit �5c is hampered by a clashf its P1 phenylalanine side chain with Met45 and requires theislocation of the latter. Moreover, investigation of the two-stepeaction mechanism of PR-957 with the �5-subunit of the yeastroteasome indicated that binding of the inhibitor to the activeite occurs only via its electrophilic headgroup and its P1 site andhereby proved the importance of the interactions of the S1 pocketf the proteasome with the P1 site of the ligand.

In conclusion, PR-957 preferentially targets subunit �5i, as itsarge P1 phenyl side chain perfectly fits into the spacious S1 pocketf the chymotrypsin-like active site of the immunoproteasome. Inontrast, the significantly smaller S1 pocket of subunit �5c of theonstitutive proteasome has to undergo conformational changeso accommodate PR-957. With this knowledge specific inhibitoryompounds for either constitutive or immunoproteasomes canow be developed.

oi:10.1016/j.molimm.2012.02.047

mmune self-reactivity triggered by drug-modified humaneukocyte antigen-peptide presentation

atricia Illing a,∗, Julian Vivian b, Nadine Dudek c, Lyudmilaostenko d, Zhenjun Chen d, Mandvi Bharadwaj d, Lars Kjer-ielsen d, Anthony Purcell c, Jamie Rossjohn b, James McCluskey d

Department of Microbiology & Immunology, Department of Bio-hemistry and Molecular Biology and Bio21 Molecular Science andiotechnology Institute, University of MelbourneDepartment of Biochemistry and Molecular Biology, School ofiomedical Sciences, Monash UniversityDepartment of Biochemistry and Molecular Biology and Bio21 Molec-lar Science and Biotechnology Institute, University of MelbourneDepartment of Microbiology & Immunology, University of Melbourne

The basis for strong immunogenetic associations betweenarticular human leukocyte antigen (HLA) class I allotypes and

nflammatory conditions like Behcet’s disease (HLA-B51) andnkylosing spondylitis (HLA-B27) remain mysterious. Recently,owever, even stronger HLA associations are reported in drugypersensitivities to the reverse-transcriptase inhibitor aba-avir (HLA-B*57:01), the gout prophylactic allopurinol (HLA-B58),nd the antiepileptic carbamazepine (HLA-B*15:02), providing aefined disease trigger and suggesting a general mechanism forhese associations. However, little is known about the underlying

echanisms of this process, even for abacavir hypersensitiv-ty syndrome (AHS), a prototypical HLA-associated drug reactionccurring exclusively in individuals with the common histocom-atibility molecule, HLA-B*57:01+ and with a relative risk of >1000.e have previously shown that systemic reactions to abacavir were

riven by drug-specific activation of cytokine-producing, cyto-oxic CD8 + T cells. Recognition of abacavir required the transporterssociated with antigen presentation and tapasin and was fixa-ion sensitive. Abacavir-specific T cell responses were uniquelyestricted by HLA-B*57:01 and not the closely related HLA allotypesncluding HLA-B*57:02, HLA-B*57:03, HLA-B*57:11, HLA-B*58:01nd selected site directed mutants of HLA-B*57:01. These naturalnd mutated forms of HLA-B*57:01 collectively contain polymor-hisms in the C, D, E and F-pocket of the antigen-binding cleft. Weow show a basis for the strong association of HLA-B*57:01 with

bacavir hypersensitivity through perturbed Ag-presentation. Ourndings simultaneously highlight the importance of HLA polymor-hism in the evolution of pharmacogenomics as well as providing

ology 51 (2012) 5–41 19

a novel mechanism for the growing number of HLA-linked hyper-sensitivities that involve small molecule drugs.

doi:10.1016/j.molimm.2012.02.048

Tetraspanins CD82 and CD37 play vital roles in antigen presen-tation and processing in dendritic cells

Eleanor Jones a,∗, Po Ki Ho a, Javier Vega Ramos b, JoseVilladangos b, Mark Wright a

a Department of Immunology, Monash Alfred Medical Research andEducation Precinctb Department of Immunology, The Walter and Eliza Hall Institute ofMedical Research

Tetraspanins are a superfamily of membrane molecules whichform microdomains to organise the cell surface. 33 tetraspaninshave been identified in humans, and while some show functionaloverlap, individual tetraspanins have unique roles in complex cel-lular events such as trafficking, signalling, adhesion and migration.Here we examine the novel phenotype of a new knockout mousemodel of the tetraspanin CD82. This broadly expressed molecule isbest characterized as a metastasis suppressor gene, and is the clos-est phylogenic relative of the tetraspanin CD37. Our data indicatesthat CD82 often opposes the role of CD37 and is pivotal in antigenpresentation.

CD37 is known to negatively regulate MHC/peptide presenta-tion, as CD37KO DCs are hyperstimulatory to T cells in vitro, andthis effect is due to an increase in signal 1 (MHC presentation). Here,however, we show CD82 ablation causes a significant decease in theability of dendritic cells to stimulate T cell proliferation in vitro. Wepresent data to indicate this is not due to uptake, surface expres-sion, costimulation or signal 1, but may be due to maturation andantigen processing.

doi:10.1016/j.molimm.2012.02.049

Chicken TAP genes are polymorphic and co-evolve with thedominantly-expressed class I gene

Anna Sowa a, Michael Harrison a, Clive Tregaskes a, Paul Chappell b,Pietro Roversi b, Susan Lea b, Megan O’Mara c, Rachelle Gaudet d,Jim Kaufman a,∗

a University of Cambridgeb University of Oxfordc University of Queenslandd Harvard University

In most mammals, the MHC class I molecules are polymor-phic and determine the specificity of peptide presentation, whilethe transporter associated with antigen presentation (TAP) het-erodimers are functionally monomorphic. In chickens, there aretwo classical class I genes but only one is expressed at a high level,which results in very strong MHC associations with resistance toparticular infectious pathogens.

We have eluted and sequenced peptides from cells, anddetermined peptide motifs for the dominantly-expressed class Imolecule of seven MHC haplotypes. Using renaturation assays andX-ray crystallography, we have found two kinds of peptide motif,one simple but very fastidious and the other involving remodellingof the peptide binding site along with co-variation between vari-ous peptide positions. These two kinds of binding correlate with cell

surface localisation as well as resistance to Marek’s disease causedby an oncogenic herpesvirus.

We have determined the TAP1 and TAP2 sequences from thesame seven MHC haplotypes, finding that both genes have high

2 mmun

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d

Ec

E

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d

Uf

F

ta(op(spf

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