78 pages

Mechanisms of NKT cell-mediated DC licensing and cross-priming [Elektronische Ressource] / Verena Semmling. Mathematisch-Naturwissenschaftliche Fakultät

rheinische_friedrich-wilhelms-universitat_bonn - Verena Semmling

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Publié par	rheinische_friedrich-wilhelms-universitat_bonn
Publié le	01 janvier 2012
Nombre de lectures	18
Poids de l'ouvrage	7 Mo

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MechanismsofNKTcell-‐mediatedDClicensing
andcross-‐priming
Dissertation
zur
Erlang ungdesDoktorgrades(Dr.rat.er.) n
der
Mathematisch -‐NaturwissenschaftlichenFakultät
der
RheinischenFriedrich-‐Wilhelms-‐UniversitätBonn
vorgelegtvon
VerenaSemmling
aus
Zell/Mosel
Bonn,März2011AngefertigtmitGenehmigungderMathematis-‐cNaturh wissenschaftlichenFakultät der
RheinischenFriedrich-‐Wilhelms -‐UniversitätBonn
1.Gutachter : Prof. Dr.ChristianKurts
2. : Prof.DrPer .cyKnolle
TagderPromotion:22.11.2011
Erscheinungsjahr20121 TableofContent s
2 INTRODUCTION ...............................................................................5
2.1 C ROSS-‐PRESENTATION5 ..........................................
2.1.1 Genealrmchanismes ................................. 5
2.1.2 Celtypescapableofcross -‐presentation... 6
2.2 DC LICENSING 7 .....................................................
2.2.1 Requirementforhelp ................................................................ 7
+2.2.2 ClassicallicensingviaCD4 Thelpercells .................................. 7
2.2.3 AlternativeNKTcellmediatedlicensing.... 8
2.3 C HEMOKINES ................................................................ 11....................
2.3.1 Genealrpropetiers .................................. 11
2.3.2 ThechemokinereceptorCCR4anditsligands........................ 12
2.3.3 Chemokinesasregulatorsofimmuneresponses.................... 13
2.4 AIMSOFTHISSTUDY14 ...........................................
3 MATERIALSANDMETHOD S...........................15
3.1 M ATERIALS 15 .......................................................
3.1.1 Equipment ............................................................................................................... 15
3.1.2 Software.................. 16
3.1.3 Consumables........... 17
3.1.4 Chemicalsandreagents .......................................................................................... 18
3.1.5 Buffers,mediaandsolutions................... 20
3.1.6 Antibodies ............................................... 21
3.1.7 Mousestrains.......................................................................... 23
3.2 M ETHODS 24 ........................................................
3.2.1 Experimentaltreatmentofmice............. 24
3.2.2 Isolationandtransferofprimarymurinecells ........................................................ 24
3.2.3 Invitrocross -‐primingassay .................................................... 25
+3.2.4 CFSEproliferationassayofCD8 Tcells .................................................................. 26
3.2.5 Invivocytotoxicityassay......................... 27
3.2.6 Flowcytometry ....................................... 27
3.2.7 Geneationrofmixedbonemarowrchimaser ......................................................... 28
3.2.8 Immunohistochemistry ........................................................... 28
+3.2.9 InvitroanalysisofCD8 TcellrecruitmentbyDCs.................. 30
3.2.10 Transwellcell -‐migrationassay ............................................................................. 30
3.2.11 Real-‐ timereverse -‐transcriptionPCR..... 30
3.2.12 Statisticala nalysis ................................. 31
4 RESULTS........................................................................................32
4.1 NKT CELL-‐MEDIATED DC LICENSING................................32 ......................
+4.1.1 CognateNKTcel-‐ mediatedlicensingisindependentofCD4 help ......................... 32
4.1.2 NKTcellmediatedcross-‐ primingisregulatedbyCCR4........... 33
4.2 ACTIVATED NKT CELLSINDUCE CCL17EXPRESSIONINSPLENIC DC S 34 ...........4.2.1 CharacterizationofCCL17-‐ pr oducingcellsinthespleen ........................................ 33
4.2.2 MechanismofCCL17inductionbyNKTcels ................................ ........................... 34
4.3 EFFECTOF CCL17 ON DC SAND NKT CELLS ............................................................................35
4.3.1 CCL17doesnotalterthecross -‐pr imingabilityofDCs ............................................ 35
4.3.2 CCL17doesnotincreaseDCorNKTcelrecruitment ................................ .............. 36
4.3.3 CCL17positivelyregulatesitsownproduction ........................ 36
+4.4 EFFECTOF CCL17 ON CD8 T CELLS .....................................................................................37
+4.4.1 CCL17actsdire ctlyonC D8 Tcells ................................ .......................................... 37
+4.4.2 αGalCer-‐ treatmentimprovesthemigrationofnaïveCD8 TcellstowardsCCL17 .38
+4.4.3 CD8 TcellsaccumulateinsplenicTcellzonesfollowing αGalCer-‐ injection .......... 39
4.4.4 MechanismofCCR4induction ................................ ................................................ 39
4.5 SYNERGISTICEFFECTOFCLASSICALANDALTERNATIVE DC LICENSING ...........................................42
4.5.1 CombinationofclassicalandalternativeDClicensingboostscross -‐pr iming .......... 42
5 DISCUSSION ................................................................ 44 ..................................................
5.1 ROLEOF CCL17 AND CCR4 INNKT CELL -‐MEDIATEDCROSS -‐PRIMINGINTHESPLEEN ....................44
5.1.1 AdjuvanteffectofNKTcellactivationoncross -‐pr iming ......................................... 44
ressioninthespleen ............................................................................... 455.1.2 CCL17 exp
5.1.3 EffectofCCL17onthecross -‐pr imingabilityofDCs ................................................ 49
+5.1.4 CCL17-‐resp onsivenessofCD8 Tcells ...................................... 50
5.1.5 Chemokine -‐de pendentregulat ionofcellularinteractionsinDClicensing.............. 52
5.2 SYNERGISTICEFFECTOFCLASSICALAND NKT CELL -‐MEDIATED DC LICENSING ................................55
5.2.1 DistinctregulationofclassicalandNKTcell -‐me diatedcross -‐priming .................... 55
+5.2.2 SynegisticreffectofclassicalandNKTcell -‐me diatedcross -‐primingonCD8 Tcell
resp onses ............................................................................................................................ 55
6 SUMMARY ................................ 59 ....................................................
7 REFERENCES................................ 60..................
8 ABBREVEATIONS68...........5
2 Introduction
2.1 Cross- presentation
2.1.1 Generalmechanisms
+CD8 TcellsrespondtotheirspecificantigeninthecontextofMajorHis-‐tCompato ibility
(MHC)-‐ Class-‐Imolecules,which areexpressed byallnucleated cellsand allow foraconstitutive
+display of endogenous antigens. However, activation of naïve CD8 Tcelslrequires more than
solely antigen -‐recognition, referred to as signal 11(). The second prerequisite is that T cells
receive additional costimulatory signals that are provided by dendritic cells (DCs) under
+inflammatory conditions. These additional signals confer cytolytic capacity to the CD8T cell,
which can then subsequently destroy infected or altered body cells that display the specific T
cellantigen.
+As naïve CD8 T cells depend on both antigen and costimulatory signals for their activation,
+there hastobeawaytoinduceCD8 TcellresponsesifDCsarenotinfectedoraltered
themselves. A second pathway of antigen processing termed cross -‐presentation allows the
presentation of internalized antigens by professional antigenpr-‐esenting cells on MHC-‐class -‐I.
This pathway is a crucial extension to the classical pathway in preventing the immune escape
+ Tcellactivationduetoaof viruses that do not infect DCs and thereby might circumvent CD8
lack of synchronous presentation of costimulatory molecules and viral antigens in the context
of MHC-‐ class -‐I. Bevan et al. first described the cro-‐prssiming in 19762() after they immunized
+mice with allogenic cells and examined the MHC-‐restriction of the induced CD8Tcell
+response. The immunization generated CD8 T cells that were restricted to both donor and,
surprisingly, host MH-‐Cclass -‐I molecules. The latter was only possible if the host cells acquired
+donor cellular antigens and processed them on MHC-‐class -‐I to CD8 Tcells.Thisantigen
+presentation pathway has since been termed cross -‐presentation. When it results in CD8Tcell
immunity it is referred to as “cross-‐priming” whereas the induction of tolerance is referred to
as“cross-‐ tolerance”.
The cellular mechanisms enabling the crosspr-‐esentation path way are not yet fully
understood. It is evident that cross-‐presentation requires that ingested antigenscircumvent
lysosomal degradationand MHC -‐class -‐II-‐loading and are insteadprocessedbytheMHC -‐class -‐I-‐6
loading machinery that istypically locatedein entdh oplasmic reticulum (ER). However, recent
studies suggest thatloadingof soluble and particulate antigonenMHC-‐ class -‐I moleculesdoes
not necessarily occur in the ER, but rather in specialized endocytic and phagocytic
compartments (3).Burgdorf et al . (4) described that certain antigens, which are taken up via
distinct endocytic recept