title: Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets.
abstract: Presentation of peptides on major histocompatibility complex class I (MHC I) is essential for the establishment and maintenance of self-tolerance, priming of antigen-specific CD8(+) T cells and the exertion of several T-cell effector functions. Cytosolic proteasomes continuously degrade proteins into peptides, which are actively transported across the endoplasmic reticulum (ER) membrane by the transporter associated with antigen processing (TAP). In the ER lumen antigenic peptides are loaded onto MHC I, which is displayed on the cell surface. Here we describe an innovative flow cytometric approach to monitor time-resolved ER compartmentalization of antigenic peptides. This assay allows the analysis of distinct primary human immune cell subsets at reporter peptide concentrations of 1 nM. Thus, this ultrasensitive method for the first time permits quantification of TAP activity under close to physiological conditions in scarce primary cell subsets such as antigen cross-presenting dendritic cells.
authors: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany.
authors: Hanna
authors: H
authors: Fischbach
authors: Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz-Centre for Infection Research and the Hannover Medical School, Feodor-Lynen Str. 7-9, 30625 Hannover, Germany.
authors: Marius
authors: M
authors: Döring
authors: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany.
authors: Daphne
authors: D
authors: Nikles
authors: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany.
authors: Elisa
authors: E
authors: Lehnert
authors: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany.
authors: Christoph
authors: C
authors: Baldauf
authors: Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz-Centre for Infection Research and the Hannover Medical School, Feodor-Lynen Str. 7-9, 30625 Hannover, Germany.
authors: Ulrich
authors: U
authors: Kalinke
authors: 1] Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany [2] Cluster of Excellence Frankfurt - Macromolecular Complexes, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany.
authors: Robert
authors: R
authors: Tampé
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism*; D002451:Cell Compartmentation; D003199:Computer Systems; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D007242:Herpesvirus 1, Bovine / Q000378:metabolism; D006801:Humans; D016131:Lymphocyte Subsets / Q000276:immunology*; D021381:Protein Transport; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D000941:Antigens; D014764:Viral Proteins; C477327:transporter associated with antigen processing (TAP)
references: J Biol Chem. 2008 May 9;283(19):13428-36
references: 18321854
references: PLoS Pathog. 2008 May;4(5):e1000080
references: 18516302
references: Annu Rev Cell Dev Biol. 2008;24:343-68
references: 18729726
references: Curr Opin Cell Biol. 2008 Dec;20(6):624-31
references: 18926908
references: J Biol Chem. 2009 Dec 4;284(49):33740-9
references: 19808685
references: Nat Rev Immunol. 2010 Jun;10(6):403-14
references: 20498667
references: Nat Chem Biol. 2010 Aug;6(8):572-80
references: 20644544
references: J Immunol. 2010 Sep 15;185(6):3426-35
references: 20729332
references: J Cell Sci. 2010 Dec 15;123(Pt 24):4271-9
references: 21098634
references: Mol Biotechnol. 2011 Mar;47(3):262-9
references: 20938757
references: Mol Immunol. 2011 Mar;48(6-7):835-45
references: 21292324
references: Nat Rev Immunol. 2011 Dec;11(12):823-36
references: 22076556
references: J Mol Biol. 1999 Dec 17;294(5):1203-13
references: 10600378
references: Mol Immunol. 2013 Sep;55(2):139-42
references: 23141382
references: J Exp Med. 2013 May 6;210(5):1035-47
references: 23569327
references: Cold Spring Harb Perspect Biol. 2013 Aug;5(8):a013359
references: 23751184
references: J Clin Invest. 2013 Sep;123(9):3766-76
references: 23908113
references: Nat Commun. 2014;5:5419
references: 25377891
references: Biochim Biophys Acta. 2015 Mar;1850(3):449-60
references: 24923865
references: J Mol Biol. 2015 Mar 13;427(5):1102-18
references: 25224907
references: Immunity. 2000 Jul;13(1):117-27
references: 10933400
references: J Biol Chem. 2003 Aug 8;278(32):29686-92
references: 12777379
references: Physiology (Bethesda). 2004 Aug;19:216-24
references: 15304636
references: Immunogenetics. 1985;21(3):235-46
references: 3872841
references: Science. 1993 Aug 6;261(5122):769-71
references: 8342042
references: Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9130-4
references: 8415666
references: EMBO J. 1996 Jul 1;15(13):3247-55
references: 8670825
references: Immunity. 1997 May;6(5):623-32
references: 9175840
references: Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):8976-81
references: 9256420
references: Lancet. 1964 Feb 1;1(7327):238-40
references: 14086209
references: Cancer. 1965 Apr;18:522-9
references: 14278051
references: J Immunol. 2007 Jan 15;178(2):657-62
references: 17202325
references: Physiol Rev. 2007 Oct;87(4):1377-408
references: 17928587