HLA-Liganden für die aktiv personalisierte Immuntherapie von Leberkrebs und Gliomen

Primäre Lebertumore sind aufgrund ihrer hohen Mortalitätsraten und ihrer steigenden Inzidenzen ein ernstzunehmendes Problem. Mit übermäßigem Alkoholkonsum, Leberzirrhose und Infektionserkrankungen wie Hepatitis-B und -C als Hauptrisikofaktoren wird sich das in naher Zukunft nicht ändern. Durch das begrenzte Repertoire an Behandlungsmöglichkeiten stellt sich die Frage nach neuen Therapieansätzen. Durch die in den Fokus der Forschung gerückte Krebsimmuntherapie bietet sich eine Vielzahl an neuen Ansatzpunkten. Die Impfung mit tumorassoziierten und tumorspezifischen HLA-Liganden steht im Mittelpunkt dieser Arbeit. Um einen solchen Ansatz zu verfolgen, müssen zunächst Erkenntnisse über das Ligandom dieser Tumore gewonnen werden. Zu diesem Zweck wurde gesundes Gewebe sowie Tumorgewebe von insgesamt 21 Patienten untersucht. Die Isolation der HLA-Liganden von diesen Geweben und deren massenspektrometrische Analyse lieferte über 15.000 unterschiedliche Peptide. Mit diesen Daten konnten 31 patientenübergreifende Antigene identifiziert werden. Außerdem wurden Möglichkeiten demonstriert, wie eine solche Impfung individuell auf einen Patienten abgestimmt werden kann. Durch den Einsatz von next-generation-sequencing ist es möglich nach HLA-Liganden zu suchen, deren Aminosäuresequenz durch Mutationen in den zugrundeliegenden Genen verändert sind. Solche Peptide als Ziel einer Immuntherapie bieten Vorteile gegenüber tumorassoziierten Antigenen. Leider konnte im Rahmen dieser Arbeit kein solches Neoepitop nachgewiesen werden. Gliome stellen ähnlich wie Lebertumore eine schwer heilbare Tumorerkrankung dar. Auch hier besteht dringender Bedarf an neuen Möglichkeiten zur Behandlung der Patienten. Die Mutation R132H im Gen der IDH 1 stellt jedoch ein interessantes Ziel für eine Immuntherapie dar. Bis zu 70% der Grad II- und III- Gliome tragen diese Mutation. Da die Mutation sehr früh bei der Entstehung eines Glioms auftritt und vermutlich einen entscheidenden Schritt hierbei darstellt, sind die meisten Zellen eines solchen Tumors davon betroffen. Die Präsentation auf HLA-Molekülen und die Erkennung durch das Immunsystem im Patienten macht sie zu einem idealen Ziel einer Impfung. Es wurden mehrere Tumorproben von Gliompatienten sowie einige Gliom-Zelllinien untersucht. Zu diesem Zweck wurde eine gezielte massenspektrometrische Methode entwickelt und etabliert, um die Sensitivität des Massenspektrometers zu erhöhen. Es konnten jedoch nur wildtypische Peptide der Isocitratdehydrogenase 1 identifiziert werden. Des Weiteren wurden für insgesamt vier Patienten individuelle Cocktails für eine Impfung mit HLA-Liganden vorgeschlagen. Diese Vorschläge basierten auf der Analyse von Tumorgewebe dieser Patienten. Dabei wurden sowohl Ligandom-, als auch Exom- und Transcriptom Daten in Betracht gezogen

TAPBPR bridges UDP-glucose : glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway

Funding Wellcome: Senior Research Fellowship 104647, Andreas Neerincx, Louise H Boyle Royal Society: University Research Fellowship, UF100371, Janet E Deane Cancer Research UK: Programme Grant, C7056A, Andy van Hateren, Tim Elliott Deutsche Forschungsgemeinschaft: SFB 685, Nico Trautwein, Stefan Stevanović Wellcome: PhD studentship, 089563, Clemens Hermann Wellcome: Strategic Award 100140, Robin Antrobus Wellcome: Programme grant, WT094847MA, Huan Cao Acknowledgements We are extremely grateful to Peter Cresswell and Najla Arshad (Yale University School of Medicine, New Haven, CT) for valuable advice, tapasin and TAP-specific antibody reagents, and the recombinant calreticulin proteins. We thank John Trowsdale (University of Cambridge, UK) for his mentorship and critical reading of this manuscript, and Jim Kaufman (University of Cambridge, UK) for useful discussions. We also thank Yi Cao (Cranfield University, UK) for MATLAB programming for densitometry analysis, and Mark Vickers and Sadie Henderson (Scottish National Blood Transfusion Services, UK) for permitting the use of and assistance with the Amersham WB system. The reagent ARP7099 FEC peptide pool was obtained from the Centre for AIDS Reagents, National Institute for Biological Standards and Control (NIBSC), and was donated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.Peer reviewedPublisher PD

TAPBPR alters MHC class I peptide presentation by functioning as a peptide exchange catalyst.

Our understanding of the antigen presentation pathway has recently been enhanced with the identification that the tapasin-related protein TAPBPR is a second major histocompatibility complex (MHC) class I-specific chaperone. We sought to determine whether, like tapasin, TAPBPR can also influence MHC class I peptide selection by functioning as a peptide exchange catalyst. We show that TAPBPR can catalyse the dissociation of peptides from peptide-MHC I complexes, enhance the loading of peptide-receptive MHC I molecules, and discriminate between peptides based on affinity in vitro. In cells, the depletion of TAPBPR increased the diversity of peptides presented on MHC I molecules, suggesting that TAPBPR is involved in restricting peptide presentation. Our results suggest TAPBPR binds to MHC I in a peptide-receptive state and, like tapasin, works to enhance peptide optimisation. It is now clear there are two MHC class I specific peptide editors, tapasin and TAPBPR, intimately involved in controlling peptide presentation to the immune system.This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.755

Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors

BACKGROUND: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells. METHODS: Here, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM. RESULTS: Comparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8(+) T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors. CONCLUSIONS: These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion

Janus kinase 2 inhibition by pacritinib as potential therapeutic target for liver fibrosis

anus kinase 2 (JAK2) signaling is increased in human and experimental liver fibrosis with portal hypertension. JAK2 inhibitors, such as pacritinib, are already in advanced clinical development for other indications and might also be effective in liver fibrosis. Here, we investigated the antifibrotic role of the JAK2 inhibitor pacritinib on activated hepatic stellate cells (HSCs) in vitro and in two animal models of liver fibrosis in vivo.Jonel Trebicka is supported by the German Research Foundation project ID 403224013–SFB 1382 (A09); by the German Federal Ministry of Education and Research (BMBF) for the DEEP‐HCC project; by the Hessian Ministry of Higher Education, Research, and the Arts (HMWK) for the ENABLE cluster project; and by Eurostars (Grant ID 12350). The MICROB‐PREDICT (project ID 825694), DECISION (project ID 847949), GALAXY (project ID 668031), LIVERHOPE (project ID 731875), and IHMCSA (project ID 964590) projects have received funding from the European Union's Horizon 2020 research and innovation program. The manuscript reflects only the authors' views, and the European Commission is not responsible for any use that may be made of the information it contains. The funders had no influence on study design, data collection and analysis, decision to publish, or preparation of the manuscript

Tapasin-related protein restricts the MHC class I peptide repertoire by enhancing peptide exchange

Recently we have found the tapasin-related protein TAPBPR is an additional MHC I chaperone of unknown function in the antigen presentation system. In contrast to tapasin, TAPBPR is not an integral component of the peptide loading complex and is not required for peptide loading. Furthermore, we have found TAPBPR and tapasin bind in a similar orientation to the same face of MHC I.This raised the possibility that, like tapasin, TAPBPR could functionas an MHC I peptide “editor”.Here we demonstrate that TAPBPR expression has a significanteffect in shaping the peptide repertoire presented by MHC I. Usingin vitro peptide exchange assays we demonstrate that TAPBPRis able to increase peptide association and peptide dissociationfor both HLA-A*02 and -B*08. Therefore, like tapasin, TAPBPR enhances peptide exchange on MHC I. Interestingly, the luminal portion of TAPBPR alone, without the need for a leucine zipper,is sufficient to promote MHC I peptide exchange, suggesting that TAPBPR might have a higher affinity for MHC I than tapasin. Todetermine the in vivo effect of TAPBPR expression on the MHC Ipeptidome, we isolated pMHC I from WT and TAPBPR knockout cells, eluted the bound peptides and determined their amino acid sequence by mass spectrometry. This revealed a significantly broader repertoire of peptides presented on MHC I in the absence of TAPBPR (up to a 9.5 fold increase in the total number of pMHC), despite the fact that cell surface levels of MHC I were unaffected byTAPBPR depletion. This is in contrast to tapasin depletion where a significant decrease in the total number of pMHC was observed