Cross presentation pathways

Transcription

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2 Cross presentation pathways Cytosolic pathway Endocytic pathway ER TAP1/2 phagosome proteasome ribosome

3 Macrophages MHC class II-restricted antigen presentation to CD4+ T cells particulate antigen Dendritic cells MHC class I and IIrestricted presentation T CD4+ and CD8+ T cells

4 Macrophages, neutrophils Innate immunity: Inflammation Phagocytes Dendritic cells Adaptative immunty: Antigen presentation

5 Functional organisation of the endocytic and phagocytic pathways ROS neutral ph low early endosomes late endosomes acidic high degradation lysosomes

6 ER-phagosomes ER phagosomes ER-phagosome mixt compartment N phagolysosome Desjardins and coll, Cell, 2002 Grinstein and coll, Cell, 2005

7 antigen phagosome ERAD? MHC class I proteasome TAP calreticulin tapasin ERP57 calreticulin tapasin ERP57 TAP MHC class I ER Guermonprez et al, Nature 2003

8 - ERAD (Sec61/p97) is required for antigen export to the cytosol and cross presentation (P. Cresswell) - IRAP: endosomal MHC class I processing (P. van Endert) - TLR-mediated TAP recruitment to endosomes (C. Kurts) -ER SNAREs and Rabs control phagocytosis and cross presentation (our unpublished results)

9 A working model for cross presentation: TAP1/2 proteasome polyubiquitination tapasin calreticulin ERP57 MHC class I IRAP pathogen, apoptotic cell ERAD? Sec61 p97

10 respiratory burst in neutrophils: role of NADPH oxidase ph DeCoursey, Science s STKE, 2004

11 ph regulation in macrophages and neutrophils Phagosomal ph macrophages acidification-dependent protease activation Phagosomal ph Time in minutes neutrophils alkalinisation-dependent protease activation+ros Time in minutes NADPH oxidase H+ ATPase

12 Single cell phagosomal assays phagocytosis Y Y Y subpopulations DHR FluoProbe 647 FITC FACs analysis (cells) PHAGOSOMAL ph and OXYDATION Single phagosome functional assays ova-coated latex bead phagocytosis cell lysis (detergent or not) Y Y Y Y Y YYY abs FACs analysis (beads) PHAGOSOMAL DEGRADATION, RECRUITMENT OF MB PROTEINS

13 the ph in DC-phagosomes is extremely high

14 increased ph acidification in phagosomes from gp91-deficient mice

15 NADPH oxidase V-ATPase O- H+ ph proteolysis antigen presentation Savina et al, Cell, 2006

16 8 7 6 neutrophils 5 Phagosomal ph Time in minutes macrophages NADPH oxidase H+ ATPase Time in minutes dendritic cells Time in minutes

17 Phagocytic pathway in MO and DCs macrophages gp91phox-ko DCs DCs ph ph neutral low alkaline low acidic high neutral int degradation degradation

18 antigen cross presentation is compromised in NOX2-defective DCs

19 cytb558 PAG15 Lamp1 PAG10

20 A population of NOX2- positive lysosome-related organelles Rab27a lysosome-related organelles V-ATPase NOX2 Lamp2+ «inhibitory lysosomes» Jancic, Savina et al, NCB, 2007

21 DC sub-populations in the spleen: CD8+ DCs CD8- DCs - Produce IL-12 - Cross present antigen to CD8+ T cells - Phagocytose apoptotic cells - Present antigen to CD4+ T cells

22 phagosomal ph CD8+ DCs CD8- DCs Phagosomal ph CD8+ DCs gp91phox -/- CD8- DCs gp91phox -/ Time in minutes (chase) Time in minutes (chase) phagosome acidification in CD8+ and CD8- DCs phagosomal ph CD8- CD8+ Savina et al, Immunity, No treat DPI 10 オ M

23 NOX2 subunits Steady state Activated NOX2 gp91 p22 gp91 p22 p60 Rac p47 p67 p60 Rac p67 p47

24 gp91phox and p47phox in CD8+ and - DCS gp91phox p47phox Overlay Phase contrast CD8+ CD8-

25 Phagocytic pathway in CD8+ and - DCs neutral CD8- and gp91-/- CD8+ ph low alkalne CD8+ ph low acidic high neutral int degradation degradation

26 regulation of phagosome function in DCs gp91 p22 Rac1 Rac2 CD8- rac p40 gp91 p22 p67 p47 CD8+ V-ATPase lysosomes Savina et al, Immunity, 2009

27 Phagosomes in DCs -Export to the cytosol? -Loading in phagosomes? -Regulation by TLRs? ER-phagosome Nox2 N lipid bodies V-ATPase

28 Institut Curie (U932) Acknowledgements ashen mice Miguel Seabra Cell and Molecular Biology, Division of Biomedical Sciences, Imperial College, London gp91-/- mice Azzaq Belaaouaj Departments of Medicine and Molecular Microbiology School of Medicine, Washington University St Louis, MO, USA Michel Mallat Hôpital de la Salpêtriere, Paris, France Gaetan Gavazzi and Karl Krause University of Geneva, Switzerland Jero Calafat (The Netherlands Cancer Institut) Marianne Pocidalo and Jamel el Benna (Xavier Bichat Hospital, Paris) Ariel Savina Ignacio Cebrian Eik Hoffmann Carolina Jancic Adriana Mantegazza Pierre Guermonprez Ana Rodriguez Armelle Regnault Graça Raposo Pablo Vargas Ana-Maria Lennon-Duménil Ivan Cruz Moura Philippe Benaroch

29 regulation of phagosome function in DCs gp91 p22 CD8- rac p40 CD8+ gp91 p22 p67 p47

30 NOX2 subunits Steady state Activated NOX2 gp91 p22 gp91 p22 p60 Rac p47 p67 p60 Rac p67 p47

31 Scramble Rac2 # 1 Rac2 # 2 Phagosomal DHR oxidation (Arbitrary units) Phagosomal ph (15' pulse + 30' chase) Rac2 expression (RT-QPCR) m RNA level (Fold of GAPDH) Scramble Rac 2 # 1 Rac 2 # 2 0 Phagosomal ROS Rac2 KD in DCs using shrna Phagsomal ph Scramble Scramble + DPI + DPI Rac2 # 1 Rac2 # 2

32 Rac2 KD by shrna inhibits X presentation T cell proliferation (%) Scramble Rac2 # 1 Rac2 # Scramble Rac2 # 1 Rac2 # OVA:BSA coated beads Peptide concentration (ng/ml)

33 Metchnikoff: «Phagocytes kill bacteria» bacteria phagosome Killing of the bacteria Clearance of apoptotic bodies

34 Rac2-KO CD8+ DCs gp91phox p47phox Overlay WT Rac2-/-

35 Cross presentation pathways MHC I pathway MHC II pathway ER phagosome TAP1/2 proteasome ribosome

36 Phagocytic pathway in CD8+ gp91-/- DCs neutral CD8- and gp91-/- CD8+ ph low CD8+ ph alkalne low acidic high neutral int degradation degradation

37 Rac2-KO CD8+ DCs, steady state wt Rac2-/- gp91phox p47phox gp91phox p47phox CD8- DCs CD8+ DCs

38 T cell proliferation (%) wt wt CD8+ DCs wt CD8- DCs T cell proliferation (%) CD8+ DCs cross present OVA more efficiently than CD8- NOX2-/- gp91phox-/- CD8+ DCs gp91phox-/- CD8- DCs 0 0,006 0,018 0,05 0,16 0,5 OVA concentration (mg/ml) 0 0,006 0,018 0,05 0,16 0,5 OVA concentartion (mg/ml) T cell proliferation (%) wt CD8+ DCs wt CD8- DCs T cell proliferation (%) gp91phox-/- CD8+ DCs gp91phox-/- CD8- DCs Peptide concentration (ng/ml) Peptide concentration (ng/ml)

39 Rac1 CD8+ DCs CD8- DCs Rac2 Rac2 CD8+ DCs Rac 1 and 2 subcellular distribution in CD8+ and - DCs CD8- DCs

40 regulation of phagosome function in DCs Rac1 CD8+ CD8- gp91 p40 p22 rac p67 p47 Rac2 V-ATPase lysosomes

41 T cell proliferation (%) wt CD8+ DCs Rac2-/- CD8+ DCs wt CD8+ DCs Rac2-/- CD8+ DCs 0 0,006 0,018 0,05 0,16 0, OVA concentration (mg/ml) Peptide concentration (ng/ml) ph, xpresentation and V-TAPase expression in Rac2 KO DCs 8 wt CD8+ DCs Rac2 -/- CD8+ DCs Vo-ATPase wt Rac2 -/- Phagosomal ph α-actin 6

42 Villadangos JA, Schnorrer P Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo. Nat Rev Immunol. 7:

43 Single cell functional phagosomal assays PHAGOSOMAL OXYDATION phagocytosis DHR FluoProbe 647 PHAGOSOMAL ph FITC FluoProbe 647 phagocytosis oxidation acidification FACs analysis (cells) FACs analysis (cells) PHAGOSOMAL ANALYSIS IN SUB-POPULATIONS OF DENDRITIC CELLS : analysis of phagosomal function in complex cell populations without sorting Y Y Y Y Y Y Y Y

44 Single phagosome functional analysis PHAGOSOMAL DEGRADATION ova-coated latex bead phagocytosis degradation cell lysis (detergent) Y Y Y Y Y YYY anti-ova abs FACs analysis (beads) PHAGO FACs latex bead phagocytosis phagosome maturation cell disruption (no detergent) Y Y Y Y Y YYY anti-phagosomes marker abs FACs analysis (phagosomes) PHAGO XP ova-coated latex bead phagocytosis cross presentation cell disruption (no detergent) + fixation-pemeabilisation Y Y Y Y Y YYY Anti-Kb+ova peptide ab FACs analysis (phagosomes)

45 tissues lymph nodes QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. tissues From Mellman and Steinman

46 ROS production (Arbitrary Units) CD8+ wt DCs CD8- wt DCs CD8+ Rac2 -/- DCs CD8- Rac2 -/- DCs Total and phagosomal NOX2 activity in CD8+ and CD8- DCs PMA Time in minutes total phagosomal Phagosomal ROS generation wt DCs wt DCs 10µM DPI CD8+ DCs CD8- DCs Rac2 -/- DCs

47 Phagocytic pathway in DCs alkalne ph low early endosomes late endosomes neutral degradation intermediat lysosomes

48 Small rac GTPases in CD8 + and - DCs Rac Rac1 CD8+CD8- Tot Rac Rac1 Protein expression (%) Protein expression (%) α-actin CD8+ DCs CD8 DCs CD8+ DCs CD8 DCs

49 - CD8+ DCs cross present antigens - activated, but not resting, CD8- DCs also cross present antigens - pdcs produce IFNa - migrating DCs transfer antigen to LN-resident DCs Specialization of DC-sub populations relies on the adaptation of their membrane transport pathways to particular functions

50 Phagosomal ph in human DCs and macrophages 9 DCs phagosomal ph 8,5 8 7,5 7 6,5 6 5,5 5 4,5 MO Time (hs.)

51 Phagosomal ph in human monocyte-derived DC p H 9,5 9,3 9,1 8,9 8,7 8,5 8,3 8,1 7,9 7,7 7,5 7,3 7,1 6,9 6,7 * ** Time (hs) ** pulse: 20 min. chase: 0-4 hs. * p< 0,05 ** p< 0,01 Wilcoxon test fluorescence intensity (FL-1: FITC)

52 ph regulation in macrophages and neutrophils Phagosomal ph macrophages acidification-dependent protease activation Phagosomal ph Time in minutes neutrophils alkalinisation-dependent protease activation+ros Time in minutes

53 Phagosomal ph in DCs from CGD patients 10 9,5 control DC patients DC 9 phagosomal ph 8,5 8 7,5 7 6,5 6 5, Time (min)

54 NADPH oxidase O- H+ V-ATPase ph proteolysis antigen presentation

55 immature DCs (peripheral tissues) activated and mature DCs (lymph nodes) lymph T antigen uptake and processing T tolerance priming T T T T T T T T T T tumor microrganism infected cell CTL tumor infiltration and destruction CTL CTL CTL

56 CD8- gp91 p22 Rac1/2 control the localization of NOX2 CD8+ p40 gp91 p22 p67 p47 Rac1/2 V-ATPase lysosomes

57 ** 1,000,000 MHC class II molecules/dc peptide loaded class II molecules/dc 1/10,000-1/100,000 MHC molecules are loaded with a paricular peptide ** each DC-T cell interaction must be -long enough to optimize the chances that the TCR «finds» the right ligand -short enough to optimize the chance that each T cell «finds» the right DC

58 finding each other ** antigen bearing DCs arrive to a LN there are 100,000-1,000,000 DCs: 1 DC/ ,000 bears antigen ** T cell clones represent cells there are 1,000,000 T cells in one LN 1/20,000-1/50,000 T cells specific for a particular peptide

59 ER-phagosome fusion -ER-phagosome fusion -Export to the cytosol phagosomes ER endosomes N lysosomes

60 is dendritic cell s endocytic pathway that different? incomplete assembly of the V-ATPase export to the cytosol recruitment of ER resident proteins low levels of proteases protease inhibitors

61 Ph regulation in macrophages and neutrophils macrophages Phagosomal ph acidification-dependent protease activation Phagosomal ph neutrophils alkalinisation-dependent protease activation Time in minutes

62 Phagosome-cytosol transport phagosomes - DC-specific - Size-dependent - Selective export into the cyosol Rodriguez et al, NCB, 1999