Regulation and Programming of Antibody Effector Function through IgG Glycosylation The Harvard ...

191 Pages·2014·6.48 MB·English

Regulation and Programming of Antibody Effector
Function through IgG Glycosylation
Citation
Mahan, Alison Emilia. 2014. Regulation and Programming of Antibody Effector Function through
IgG Glycosylation. Doctoral dissertation, Harvard University.
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©2014 -AlisonEmiliaMahan
Allrightsreserved.
Dissertationadvisor: ProfessorGalitAlter AlisonEmiliaMahan
Regulation and Programming of Antibody Effector Function
through IgG Glycosylation
Abstract
Antibodiesarethedefiningcharacteristicofthehumoralimmuneresponse.Their
functionsarediverse,includingdirectneutralizationofpathogensandrecruitment
ofotherimmunemoleculesorcells. Whilemostsuccessfulvaccinesinducepro-
tectiveneutralizingantibodyresponses,effectivevaccine-elicitedneutralizingan-
tibodies against some pathogens, including HIV, HCV, malaria, and TB, remain
elusive. Thus, researchershavebeguntofocusonhowvaccinescanelicitstrong
non-neutralizingantibodyfunctions,includingrecruitmentofinnateimmunefac-
torsforantibody-dependentcellularcytotoxicity,complementdeposition,andanti-
body-dependentphagocytosis.Theantibody’sconstantregion(Fc)mediatesmost
effectorfunctionsthroughisotypeandsubclassselectionoralterationofthestruc-
ture of the Fc-attached N-glycan, which controls function with exquisite speci-
ficity.Glycanmodificationsarenaturallyinducedduringinflammatoryconditions
suchasautoimmunediseaseandnaturalinfectionhowever,thespecificsignalsthat
regulateFc-glycosylationremainunknown.
Thisdissertationsoughttounderstandhowantibodyglycosylationisregulated
andhowitcanbeprogrammedthroughvaccination.Todothis,wefirstdeveloped
a technique to analyze antibody glycan structures both of bulk Fc and antigen-
specificantibodies. Usingthistechnique,weobservedsignificantmodulationof
antibodyglycansduringviralinfectionaswellasinvaccine-elicitedantibodies.To
iii
Dissertationadvisor: ProfessorGalitAlter AlisonEmiliaMahan
identifyspecificsignalsimportantforalteringtheantibodyglycan,wetranscrip-
tionallyprofiledstimulatedBcellsandidentifiedasetofinnateandadaptivestim-
ulithatregulatethegenesresponsibleforantibodyglycosylation. Theresultsde-
scribed in this dissertation begin to define the specific mechanism(s) by which
infectionandvaccinationmodulateantibodyglycosylationtoelicitfunctionalan-
tibodiesthatcanultimatelyprovideeffectiveandsustainedprotectionfrominfec-
tion.
iv
Contents
1 Introduction 1
1.1 Theimmunesystem . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1 Theinnateresponse . . . . . . . . . . . . . . . . . . . 2
1.1.2 Theadaptiveresponse . . . . . . . . . . . . . . . . . . 4
1.2 Thehumoralimmuneresponse . . . . . . . . . . . . . . . . . . 5
1.2.1 Bcelldevelopment . . . . . . . . . . . . . . . . . . . . 5
1.2.2 Antigen-specificactivationofBcells . . . . . . . . . . . 6
1.2.3 Structureanddiversityofantibodies . . . . . . . . . . 8
1.2.4 FunctionsofIgG . . . . . . . . . . . . . . . . . . . . . 10
1.2.5 Fc-mediatedantibodyfunctions . . . . . . . . . . . . . 12
1.2.6 ProtectiveIgGinvaccination . . . . . . . . . . . . . . 14
1.2.7 RegulationofFc-mediatedantibodyfunctions . . . . . 15
1.3 N-linkedglycosylation . . . . . . . . . . . . . . . . . . . . . . 15
1.3.1 Nomenclatureandstructure . . . . . . . . . . . . . . . 16
1.3.2 N-glycosylationintheERandGolgi . . . . . . . . . . . 18
1.4 IgGglycosylation . . . . . . . . . . . . . . . . . . . . . . . . . 22
1.4.1 Glycosylationoftherapeuticmonoclonalantibodies . . 22
1.4.2 NaturalmodulationofIgGglycosylation . . . . . . . . 25
1.5 RegulationofN-glycosylation . . . . . . . . . . . . . . . . . . 26
1.5.1 NaturalregulationofN-glycosylation . . . . . . . . . . 27
1.5.2 ArtificialmodulationofIgGN-glycosylation . . . . . . 27
v
1.5.3 MechanismsofN-glycosylationregulation . . . . . . . 28
1.6 Dissertationobjectives . . . . . . . . . . . . . . . . . . . . . . 30
2 HighthroughputanalysisofIgGFcglycosylationbycapil-
laryelectrophoresis 32
2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.3.1 Analysisofglycansbycapillaryelectrophoresisishighly
sensitiveandreproducible . . . . . . . . . . . . . . . . 50
2.3.2 CapillaryelectrophoresisiscomparabletoHPLC . . . . 52
2.3.3 FcandFabarereliablyseparatedbyIdeSdigestion . . . 54
2.3.4 IgGFcandFabhavedifferentglycosylationprofiles . . . 57
2.3.5 IgGglycanprofilesdifferamonghumans,rhesusandmice 60
2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3 GlycosylationofIgGismodifiedinHIVinfection 66
3.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.3.1 BulkIgGFcglycosylationismodifiedinHIVinfection . 77
3.3.2 BulkFcglycosylationchangesoverthecourseofHIVin-
fection . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.3.3 Glycosylationchangesareassociatedwithinflammatory
cytokinesinHIVinfection . . . . . . . . . . . . . . . . 82
3.3.4 Bulk IgG glycosylation is associated with specific anti-
bodyeffectorfunctions . . . . . . . . . . . . . . . . . 87
3.3.5 Antigen-specificantibodieshavedistinctglycosylationpro-
files . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
vi
4 Antigen-specific IgG glycosylation is directed by HIV Vac-
cination 97
4.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.3.1 BulkIgGFc-glycosylationishighlyvariableindifferent
regions . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.3.2 Vaccinationelicitsglycosylationpatternsindependentof
bulkglycosylation . . . . . . . . . . . . . . . . . . . . 108
4.3.3 Vaccine-inducedantibodyglycosylationisindependent
ofregionalbulkglycosylationdifferences . . . . . . . . 110
4.3.4 Vaccineselicitdistinctglycosylationprofiles . . . . . . . 110
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
4.5 Additionaldata . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4.5.1 HIVriskfactorisapotentialdriverofIgGFc-glycosylation115
5 Glycosyltransferaseexpressionisalteredbyinnateandadap-
tivesignals 121
5.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5.3.1 Glycosyltransferases associated with inflammatory IgG
glycosylationareregulatedinacoordinatedmanner . . 126
5.3.2 Glycosyltransferasesassociatedwitheffectorfunctional-
ityareresistanttochangeafterinnatestimulation . . . . 128
5.3.3 BCRengagementdown-regulatesexpressionofglycosyl-
transferases . . . . . . . . . . . . . . . . . . . . . . . . 128
5.3.4 T-cellhelpisamodulatorofinnateimmunesignals. . . . 129
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6 Conclusions 134
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6.1 Dissertationsummary . . . . . . . . . . . . . . . . . . . . . . 135
6.1.1 Inflammatorysignalsareassociatedwithchangesinbulk
IgGglycosylationduringnaturalinfection . . . . . . . . 135
6.1.2 Glycosylationisregulatedatanantigen-specificlevel . . 136
6.1.3 Polyclonalantigen-specificglycosylationisassociatedwith
particularantibodyfunctions . . . . . . . . . . . . . . 137
6.1.4 Innateandadaptivesignalscanalterglycosyltransferase
expressioninBcells . . . . . . . . . . . . . . . . . . . 138
6.2 AhypotheticalmodelofIgGglycosylation . . . . . . . . . . . . 139
6.3 Futuredirections . . . . . . . . . . . . . . . . . . . . . . . . . 142
References 149
Colophon 177
viii
Listing of figures
1.1 Immunoglobulinstructureanddiversity . . . . . . . . . . . . . 9
1.2 Symbolicrepresentationsofglycansubunits . . . . . . . . . . . 17
1.3 N-Glycosylationstepsintheendoplasmicreticulum . . . . . . . 19
1.4 N-glycantrimmingtoproducecomplexstructures . . . . . . . . 21
1.5 N-GlycanstructureofIgG . . . . . . . . . . . . . . . . . . . . 23
2.1 Sensitivityofcapillaryelectrophoresis . . . . . . . . . . . . . . 51
2.2 Reproducibilityofcapillaryelectrophoresis . . . . . . . . . . . 53
2.3 ComparisonofcapillaryelectrophoresisandHPLC . . . . . . . 55
2.4 DigestionandfractionationofIgG . . . . . . . . . . . . . . . . 56
2.5 GlycosylationofFab,Fc,andintactIgG . . . . . . . . . . . . . 59
2.6 SpeciesbasedvariationinIgGglycosylation . . . . . . . . . . . 62
3.1 FcGlycosylationinHIVinfection . . . . . . . . . . . . . . . . 78
3.2 BulkFcglycosylationinacuteversuschronicHIVinfection . . . 81
3.3 AssociationofbulkFcglycanwithage,sex,andviralload . . . . 83
3.4 AssociationofFcglycanwithplasmacytokines . . . . . . . . . 85
3.5 Associationofglycosylationwithantibodyfunction . . . . . . . 88
3.6 Glycosylationofantigen-specificantibodies . . . . . . . . . . . 90
3.7 Associationofantigen-specificIgGglycosylationandfunction . 92
3.8 SummaryofbulkFc-glycanstructuresinHIVinfection . . . . . 94
4.1 GeographicaldifferencesinbulkIgGglycosylation . . . . . . . 107
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4.2 Glycosylationofadenovirusvectoredvaccine-elicitedIgGs . . . 109
4.3 VaccinespecificIgGglycosylation . . . . . . . . . . . . . . . . 112
4.4 BulkFcglycosylationinRV144andVAX003participants . . . . 116
4.5 RV144-andVAX003-specificIgGglycosylation . . . . . . . . . 119
5.1 TranscriptionofglycosyltransferasesinTLR-stimulatedBcells . 127
5.2 TranscriptionalanalysisofBcellswithBCRcrosslinking . . . . 130
5.3 TranscriptionalanalysisofBcellswithCD40stimulation . . . . 131
6.1 HypotheticalmodelofIgGglycosylation . . . . . . . . . . . . . 140
6.2 miRNAexpressioninstimulatedBcells . . . . . . . . . . . . . 144
6.3 Geneexpressioninantigen-specificBcells . . . . . . . . . . . . 145
6.4 CpGislandsinglycosyltransferasegenes . . . . . . . . . . . . . 147
x