Salmonella polarises peptide-MHC-II presentation towards an unconventional Type B CD4+ T-cell response.

1.1 MB·English

by  Jackson, Nicola P

Eur.J.Immunol.2013.43:897–906 DOI:10.1002/eji.201242983 Antigenprocessing 897
Salmonella polarises peptide-MHC-II presentation
+
towards an unconventional Type B CD4 T-cell
response
Nicola P. Jackson1, Yu Hui Kang1, Nicolas Lapaque1, Hans Janssen2,
John Trowsdale∗1 and Adrian P. Kelly∗1
1DepartmentofPathology,UniversityofCambridge,Cambridge,UnitedKingdom
2DivisionofCellBiology,TheNetherlandsCancerInstitute,Amsterdam,TheNetherlands
Distinct peptide-MHC-II complexes, recognised by Type A and B CD4+ T-cell subsets,
are generated when antigen is loaded in different intracellular compartments. Conven-
tional Type A T cells recognize their peptide epitope regardless of the route of pro-
cessing, whereas unconventional Type B T cells only recognise exogenously supplied
peptide. Type B T cells are implicated in autoimmune conditions and may break toler-
ance by escaping negative selection. Here we show that Salmonella differentially influ-
encespresentationofantigentoTypeAandBTcells.Infectionofbonemarrow-derived
dendritic cells (BMDCs) with Salmonella enterica serovar Typhimurium (S. Typhimurium)
reduced presentation of antigen to Type A T cells but enhanced presentation of exoge-
nous peptide to Type B T cells. Exposure to S. Typhimurium was sufficient to enhance
TypeBT-cellactivation.SalmonellaTyphimuriuminfectionreducedsurfaceexpressionof
MHC-II, by an invariant chain-independent trafficking mechanism, resulting in accu-
mulation of MHC-II in multi-vesicular bodies. Reduced MHC-II surface expression in
S. Typhimurium-infected BMDCs correlated with reduced antigen presentation to Type
A T cells. Salmonella infection is implicated in reactive arthritis. Therefore, polarisation
ofantigenpresentationtowardsaTypeBresponsebySalmonellamaybeapredisposing
factorinautoimmuneconditionssuchasreactivearthritis.
Keywords: Autoimmunity (cid:2)BacterialInfections (cid:2)CD4Tcells (cid:2)Tolerance
(cid:2)
Additional supporting information may be found in the online version of this article at the
publisher’sweb-site
Introduction invasion of non-phagocytic enterocytes or via M cells overlying
Peyer’s Patches [2]. Alternatively, Salmonella is directly taken
Salmonellaentericaisanintracellularpathogenthatsurvivesand up by DCs that intercalate between intestinal epithelial cells
replicates in phagocytic cells within specialised compartments [3]. Salmonella can disseminate extracellularly or be engulfed
known as Salmonella-containing vacuoles (SCV) [1]. Following by macrophages in the submucosa [2]. Salmonella pathogenic-
oral ingestion, Salmonella crosses the intestinal epithelium by ity islands (SPI) are critically important for virulence. They
Correspondence:Dr. AdrianP.Kelly ∗Theseauthorscontributedequallytothiswork.
e-mail:[email protected]
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898 NicolaP.Jacksonetal. Eur.J.Immunol.2013.43:897–906
encode type III secretion systems (T3SS) that inject bacterial Results
effector proteins into host cells. T3SS-1 is encoded within SPI1
and is required for invasion of host cells, whereas T3SS-2 is MHC-IIaccumulatesinMVBsinSalmonella-infected
encoded by SPI2 and contributes to immune evasion and main- cells
tenance of the SCV by intracellular Salmonella [4]. Salmonella
enterica serovars such as Typhimurium (S. Typhimurium) and MHC-II is specifically removed from the surface of Salmonella-
Enteritidiscauserapid-onsetgastroenteritisinarangeofspecies, infectedcellsandaccumulatesinintracellularvesiclesthatresem-
whereas serovars such as Typhi and Paratyphi cause systemic ble HLA-DM+ LAMP-1+ EEA− peptide-loading compartments
typhoid fever in humans. Salmonella Typhi can establish life- [15,22]. To better define the nature of these compartments,
long infection of the gall bladder in 1–4% of patients. These MHC-IIlocalisationwasassessedinSalmonella-infectedMelJuSo
typhoidcarriersexhibitnormalantibodyresponsestoSalmonella cells,astheendocyticpathwayiswellcharacterisedinthishuman
Typhi antigens but have an impaired cell-mediated immune epithelial-likemelanomacellline[23].CellsurfaceHLA-DRwas
response[5]. labelledwiththemonoclonalantibodyL243andafterinternalisa-
MHC-II molecules play an essential role in the cell-mediated tionwasvisualisedbycryo-immunoelectronmicroscopy.
immune response by presenting antigenic peptides to CD4+ HLA-DR was predominantly detected at the cell surface at
T cells. Immature MHC-II molecules are assembled in the ER 12 h post-infection in both uninfected (data not shown) and
andarecomposedofαandβchainsincomplexwithpreformed Salmonella-infected cells (Fig. 1A). Between 12 and 20 h post-
trimersofinvariantchain(Ii)[6].Iioccupiesthepeptide-binding infection,HLA-DRwasendocytosedanddistributedwithinearly
grooveofMHC-IItopreventprematurepeptidebindingandchap- endosomes, MVBs and at the cell surface in uninfected cells
eronestheMHC-IIcomplexfromtheERtotheendocyticpathway. (Fig.1CandF).InSalmonella-infectedcells,therewasatwofold
Entry into the endocytic pathway is predominantly by clathrin- greater accumulation of HLA-DR in MVBs compared with unin-
mediated endocytosis from the plasma membrane [7], but can fectedcells(Fig.1B,DandE).TheinternalisedMHC-IIwasnot
also be direct from the trans-golgi network [8]. Once inside the significantly associated with the SCV but localised to MVBs that
endosomalcompartments,Iiisdegradedbylysosomalproteases most likely represent conventional MHC-II containing compart-
untilonlyCLIPisleftboundintheMHC-IIpeptide-bindinggroove. ments found in the Salmonella-infected cells. There were fewer
HLA-DMexchangesCLIPforantigenicpeptidesinlateendosomal MVBsinuninfectedcellssuggestingthatSalmonellamayenlarge
compartmentsandmaturepeptide-MHC-II(pMHC-II)complexes thiscompartmentthroughaccumulationofintracellularHLA-DR
arethenexportedtothecellsurface[9].InDCs,ubiquitinationof (datanotshown).SinceSalmonellainfectionresultsinpolyubiqui-
aconservedlysineresidueintheβchaincytoplasmictailregulates tinationofMHC-II,andubiquitinationregulatessortingofMHC-II
surfaceexpressionandtargetingofpMHC-IIintolateendosomal at MVBs [10,15], these results may suggest that Salmonella-
multi-vesicularbodies(MVBs)[10]. inducedubiquitinationofMHC-IIenhancesaccumulationinMVBs
FormationofpMHC-IIconformersfromnativeproteinoccurs topreventrecyclingofmatureMHC-IItothecellsurface.
primarilyinHLA-DM+lateendosomesandgeneratesstablecom-
plexesthatarerecognisedbyconventionalTypeACD4+ Tcells.
In contrast, loading of exogenous peptide can occur throughout MHC-IIdown-regulationbySalmonellarequires
the endosomal pathway or at the cell surface and can generate clathrinbutnotinvariantchain-directedtrafficking
pMHC-IIconformersthatarerecognisedbyconventionalTypeA
and unconventional Type B CD4+ T cells [11]. Type B T cells TodeterminewhetherIi-directedtraffickingofMHC-IIisrequired
only recognise exogenous peptide and not the identical peptide by Salmonella to regulate MHC-II surface expression, we gener-
when processed from protein. As a consequence, Type B T cells atedHeLacelltransfectantsstablyexpressingHLA-DR,butlacking
escapenegativeselectionandareimplicatedinautoimmunecon- endogenous Ii. There was no significant difference in the extent
ditions.IntheNODmousemodel,TypeBinsulin-reactiveTcells ofHLA-DRdown-regulationbySalmonellainHeLacellsexpress-
are pathogenic and trigger diabetes in adoptive transfer experi- ing CIITA (Ii-positive) and HeLa cells transduced with HLA-DR
ments[12].TypeBTcellsconstitute30–50%oftheT-cellreper- (Ii-negative) (Fig. 2A). As expected, HLA-DR dimers that lacked
toire [13], and phenotypically may resemble either Th1 or Th2 the DRβ cytoplasmic tail (DRα-(cid:2) ,β-(cid:2) and DRα,β-(cid:2) ) or
219 223 223
CD4+Tcells[12]. withalysinetoargininemutationintheβchainubiquitinationsite
Salmonella is reported to interfere with MHC-II antigen pro- (DRα,β-K R),werenotdown-regulatedbySalmonella(Fig.2A).
225
cessingandpresentationtoCD4+ Tcells[14–17].Therelevance Endocytosis of pMHC-II is clathrin, AP-2 and dynamin inde-
of these mechanisms in vivo is not clear as CD4+ T-cell prim- pendent [24]. To examine whether HLA-DR down-regulation
inghasalsobeenobservedinmousemodelsofSalmonellainfec- by Salmonella requires AP-2 and clathrin, Ii-negative HeLa cells
tion[18–21].WehavepreviouslyshownthatSalmonellainfection stablyexpressingHLA-DRweretransfectedwithAP-2andclathrin
ofhumanDCsresultsinpolyubiquitination andreducedsurface siRNA oligonucleotides and surface expression of HLA-DR was
expressionofMHC-II[15,22].Inthisstudy,weinvestigatehow assessed by flow cytometry. In the absence of Ii, siRNA knock-
SalmonellainfluencesMHC-IItraffickingandpresentationofanti- downofclathrin,butnotAP-2,reducedHLA-DRdown-regulation
gentoTypeAandBCD4+Tcells. by Salmonella (Fig. 2B, right panel). These data show that
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Eur.J.Immunol.2013.43:897–906 Antigenprocessing 899
Figure1. MHC-IIaccumulatesinMVBsinSalmonella-infectedcells.MelJuSowereinfectedfor20minwithinvasiveGFP-S.Typhimurium(MOI50).
CellsurfaceMHC-IIwaslabelled(L243)at12hpost-infectionandthencellswerefixed(A)orfurtherincubateduntil20hpost-infectionbefore
fixation(B,C,EandF).Cellsectionswereprocessedforcryo-immunoelectronmicroscopyandHLA-DRlocalisationwasvisualisedwithProtein
A-gold(10nm).(D)Graphrepresentsaverageamountofgold(HLA-DR)/MVBineachcellanalysed.Averageamountofgold/MVBwascalculated
foratleast15cellsperconditionandcomparisonofdistributionswasassessedbyunpairedtwo-tailedt-test.Boxedareasfrom(B)and(C)are
magnifiedtwofoldin(E)and(F),respectively.HistogramsshowsurfaceHLA-DRmeasuredbyflowcytometryininfectedanduninfectedMelJuSo
attimepointsindicated.RefertoSupportingInformationFig.1Aforgatingstrategy.Dataarerepresentativeoftwoindependentexperiments.
down-regulation of pMHC-II surface expression by Salmonella BMDCactivationandmaturation[25](datanotshown).Infection
requires clathrin but was independent of Ii-directed trafficking ofBMDCswithWTSalmonellareducedsurfaceexpressionofI-Ak
andAP-2. andI-Ek.I-AkandI-Ekdown-regulationwasnotdetectedfollowing
infectionwith(cid:2)ssaVSalmonella(Fig.3A),asobservedpreviously
forHLA-DR[15].ThisindicatedthattheSPI2effectorsystemwas
Salmonelladown-regulatesmurineMHC-IIsurface
expressionandantigenpresentationtoCD4+ Tcells also required to regulate MHC-II surface expression in murine
cells.ComparableI-AandI-Edown-regulationwasalsoseenfor
To examine the effect of Salmonella on T-cell presentation, we thebanddhaplotypes(datanotshown).MHC-IIdown-regulation
first exposed murine BMDCs to GFP-expressing Salmonella and wasnotdetectedineitherhumanmonocyte-derivedmacrophages,
examinedsurfaceI-AandI-Eexpression.ExposuretoSalmonella or a murine macrophage cell line, RAW264.7-CIITA (Support-
increasedoverallI-AkandI-Eksurfaceexpression,consistentwith ing Information Fig. 2). The reason for this is unknown but
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900 NicolaP.Jacksonetal. Eur.J.Immunol.2013.43:897–906
Figure2. MHC-IIdown-regulationbySalmonellarequiresclathrinbut
notinvariantchain-directedtrafficking.(A)HeLacellsstablyexpress-
ingHLA-DRWT(DRα,β)andcytoplasmictailmutantsweregenerated.
HLA-DR surface expression was assessed by flow cytometry at 20 h
post-infection with invasive GFP-S. Typhimurium and compared with
HeLa-CIITA(Iipositive)cells.RefertoSupportingInformationFig.1A
andBforgatingstrategyandrepresentativeflowcytometrydata.Graph
shows percent of normal HLA-DR surface expression in uninfected
(GFP-negative)cellscombinedfromatleastfourindependentexper- Figure3. SalmonelladownregulatesI-AandI-Esurfaceexpressionand
iments. (B) HeLa cells stably expressing HLA-DR WT (DRα,β)(Ii neg- presentation of antigen to CD4+ T cells. (A) BMDCs were infected
ative) were transfected with AP-2, clathrin or control siRNAs. Cells with opsonised GFP-S. Typhimurium (MOI 10) then I-Ak (OX6) and
wereinfectedwithinvasiveGFP-S.Typhimuriumafter5daysofAP-2 I-Ek (14.4.4s)surfaceexpressionwascomparedininfected(GFPpos-
orclathrindepletionandsurfaceHLA-DRwasassessedasdescribedin itive) and uninfected (GFP negative) CD11c/CD11b+ BMDCs by flow
(A).WesternblotshowsAP-2andclathrindepletionfromrepresenta- cytometry. Refer to Supporting Information Fig. 1A for gating strat-
tivecelllysatesafter5daysofsiRNAtreatment.Theloadingcontrolis egy.Histograms(upperpanels)showI-Eksurfaceexpressionininfected
β-actin.GraphshowspercentofnormalsurfaceHLA-DRexpressionin anduninfectedBMDCsfromarepresentativeofatleastfourindepen-
uninfected(GFPnegative)cellscombinedfromfourindependentexper- dentexperiments.Graphs(lowerpanels)showpercentofnormal(GFP
iments.Comparisonofdistributionswasperformedbyunpaired(A)or negative)I-AkorI-Eksurfaceexpressioncombinedfromfourindepen-
paired(B)two-tailedt-tests. dentpreparationsofBMDCsinfectedwithWTorSPI2-deficient((cid:2)ssaV)
S.Typhimurium.(B)BMDCs(intriplicate)wereuninfectedorinfected
with opsonised WT, HKWT or (cid:2)ssaV S. Typhimurium (MOI 10). From
mayreflectfunctionaldifferencesbetweenDCsandmacrophages
20hpost-infection,cellswereincubatedwithHELproteinandType
[26]. ACD4+ Thybridomacells(3A9)ataratioof5Tcells:1BMDC.After
To assess antigen presentation in the context of Salmonella 24h,culturesupernatantswereharvestedandT-cellactivationwas
infection we analysed I-Ak-dependent presentation of the model quantifiedbyIL-2ELISA.Graphshowspercentofnormalmean(unin-
fected)I-Ak-dependentHELpresentationtoTypeATcellscombined
antigen hen egg lysozyme (HEL) by BMDCs to a CD4+ T-cell fromatleastfourindependentexperiments.Antigenpresentationin
hybridoma expressing aHEL-specific TCR(3A9). T-cellhybrido- uninfectedBMDCsisshownasadashedline.Comparisonofdistribu-
tionswasperformedbypairedtwo-tailedt-tests.
mas do not require co-stimulation and therefore pMHC-II levels
shoulddirectlycorrelatewiththeextentofantigenpresentation.
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Eur.J.Immunol.2013.43:897–906 Antigenprocessing 901
BMDCswereusedbecausetheycanbegeneratedinlargequan- gestedthatdirectinfectionmaynotberequiredandthatasoluble
titiesandtheyresemblethemyeloidCD11b+ DCspresentinthe factor produced by infected BMDCs could be influencing neigh-
sub-epithelialdomeofmurinePeyer’spatcheswhereSalmonella bouringcells.Toscreenforpotentialsolublefactorsproducedby
internaliseearlyafteroralinfectioninvivo[3,27]. Salmonella-infected BMDCs, culture supernatant was harvested
Incubation of BMDCs with exogenous HEL protein resulted from infected BMDCs at 20 h post-infection and incubated with
in dose-dependent HEL-specific T-cell activation, as measured freshBMDCs,TypeBThybridomacellsandHEL peptide.
46–61
by IL-2 production (data not shown). After infection of BMDCs Incubation of fresh BMDCs with culture supernatant from
with Salmonella a reduction in T-cell activation was observed Salmonella-infected BMDCs was sufficient to enhance presenta-
(Fig.3B),inlinewithpreviousobservationsusingexogenousanti- tionofexogenouspeptidetoTypeBT cells(Fig.5A).Clearance
gen[14,28].ThereductioninT-cellactivationwasSPI2depen- ofthesupernatantusinga0.45μmfilterpriortoincubationwith
dent,althoughtheeffectwassubtle.AMOIoftenbacteriatoone freshBMDCs(Fig.5A)orseparationofinfectedBMDCsfromfresh
BMDCwasusedasthisdoesnotinducesignificantNOproduction BMDCsandTcellsusing0.45μmtranswells(SupportingInforma-
bytheBMDCs(confirmedbyGriessassay;datanotshown)[14]. tionFig.4)abrogatedtheeffect.Thissuggestedthatwhilstacom-
Infection with an equal number of heat-killed (HK) Salmonella ponent of culture supernatant from Salmonella-infected BMDCs
hadnoinfluenceonT-cellactivationconfirmingthatviablebac- can influence uninfected BMDCs in trans, the factor responsible
teria are required to inhibit antigen presentation in the absence wasnotsmallerthan0.45μm.
ofNO.Thesedatashowthatdown-regulationofMHC-IIsurface Salmonella are rod-shaped bacteria, 0.5–1.5 μm in diameter
expressionbySalmonellacorrelateswithreducedpresentationof and 2–5 μm in length. Clearance of culture supernatant using
antigentoCD4+Tcells. a 0.45 μm filter would therefore remove any intact Salmonella
present. To determine whether direct exposure of BMDCs to
Salmonella was sufficient to enhance presentation of exogenous
Salmonellaenhancespresentationofexogenous
peptide to Type B T cells, BMDCs were infected with GFP-
peptidetoTypeBCD4+ Tcells
expressing Salmonella and then sorted at 20 h post-infection to
separateinfectedfromexposedbutuninfectedpopulations. Pre-
TodeterminewhetherSalmonellaalsoinfluencedpresentationof
sentation of HEL peptide to Type B T hybridoma cells was
antigen to Type B CD4+ T cells, we compared I-Ak-dependent 46–61
compared for BMDCs that were unexposed, exposed but unin-
presentation ofexogenous HELprotein and HEL peptide by
46–61 fected,orexposedandinfectedwithSalmonella,respectively.
BMDCstoaTypeAT-cellhybridoma(3A9)andaTypeBT-cell
Exposure of BMDCs to Salmonella was sufficient to enhance
hybridoma(11A10)withidenticalpeptidespecificity.
presentation of exogenous peptide to Type B T cells (Fig. 5B).
Inlinewithpreviouspublications,incubationofBMDCswith
There was no significant difference in the extent of Type B
exogenous HEL protein or HEL peptide resulted in dose-
46–61 T-cellactivationbetweensortedBMDCsthatwereexposedto,or
dependent HEL-specific Type A T-cell activation, whereas only
infected with Salmonella. There was a consistent trend towards
incubationwithexogenousHEL peptideresultedinequivalent
46–61 reducedpresentationintheinfectedBMDCs,althoughtheeffect
activationofTypeBTcells(Fig.4A,opencircles)[11].Infectionof
wassubtle(Fig.5B).Notably,presentationofpeptidetoTypeB
BMDCswithWTSalmonellainhibitedpresentationofbothexoge-
TcellswasenhancedbyHKSalmonellaiftheMOIwasincreased
nousHELproteinandHEL peptidetoTypeATcells.Intrigu-
46–61 (Fig. 5C). This suggested that whilst viable bacteria contribute
ingly,WTSalmonellainfectioncausedadramaticincreaseinthe
more significantly to the enhanced presentation of exogenous
presentationofexogenousHEL peptidetoTypeBTcells,but
46–61 HEL peptide to Type B T cells observed, viability is not
46–61
hadlittleeffectonpresentationofHELprotein(Fig.4A).Unlike
essential.
inhibition of Type A T-cell activation by Salmonella, enhanced
presentation ofHEL peptidetoTypeBTcellswasnotSPI2
46–61
(ssaV)dependent(Fig.4B).Furthermore,infectionwithanequal
Discussion
numberofHKSalmonellahadnoeffectonTypeAT-cellactiva-
tionbutsubtlyincreasedTypeBT-cellactivation(Fig.4B).These
We show that Salmonella infection influences MHC-II antigen
datashowthatSalmonellainfluencedantigenpresentationinsev-
presentation to CD4+ T cells by two distinct mechanisms. Intra-
eraldistinctways.MostdramaticallySalmonellainfectionresulted
cellularreplicationofSalmonellaresultedinreducedexpressionof
inelevatedpresentationofexogenouspeptidetoTypeBTcells.
pMHC-IIcomplexesatthecellsurfaceandalteredpresentationof
Thiswasassociatedwithareductioninpresentationofpeptideor
antigen to CD4+ T cells. Most importantly, exposure of BMDCs
proteinantigentoTypeATcells.
to Salmonella resulted in enhanced presentation of exogenous
peptidetoTypeBCD4+ Tcells,whichhavebeenlinkedtoauto-
ExposuretoSalmonellaissufficienttoenhance immunediseaseprogression[12].
presentationofexogenouspeptidetoTypeBTcells We first examined the influence of intracellular Salmonella
on MHC-II trafficking and localisation. Using Ii-negative HeLa
At the MOI used in the above experiments (MOI = 10), only cells, we showed that down-regulation of MHC-II by Salmonella
10–20% of the BMDCs were infected with Salmonella. This sug- wasindependentofIi-directedtraffickingandAP-2,butrequired
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902 NicolaP.Jacksonetal. Eur.J.Immunol.2013.43:897–906
Figure4. SalmonellainfectionenhancespresentationofexogenouspeptidetoTypeBTcells.BMDCs(intriplicate)wereinfectedwithopsonised
WT(AandB),SPI2-deficient((cid:2)ssaV)(B)orHKWT(B)GFP-S.Typhimurium(MOI10).From20hpost-infection,cellswereincubatedwithHELprotein
orHEL46–61peptideand3A9(TypeA)or11A10(TypeB)Thybridomacellsataratioof5Tcells:1BMDC.After24h,culturesupernatantswere
harvestedandT-cellactivationwasquantifiedbyIL-2ELISA.(A)GraphsshowmeanIL-2concentrationfromarepresentativeofatleastfour
independentexperiments.ErrorbarsrepresentSD.(B)Graphsshowpercentofnormal(uninfected)I-Ak-dependentHEL46–61presentationtoType
AorBTcellscombinedfromatleastthreeindependentexperiments.AntigenpresentationinuninfectedBMDCsisshownasadashedline.
Comparisonofdistributionswasperformedbypairedtwo-tailedt-tests.
clathrin. As AP-2 is the principal adaptor protein required for immunity [5], rapid priming of CD4+ T cells in mouse mod-
formation of clathrin-coated pits at the plasma membrane [29], els of Salmonella infection was reported to elicit effective Th1
itisunlikelythatMHC-IIdown-regulationbySalmonellarequires responses [21]. Regardless, priming of Type B T-cell responses
the formation of clathrin-coated pits. Distinct clathrin coats are does not imply any perturbation in the Th1/Th2 balance and
also present at the cytoplasmic face of MVBs and are proposed couldoccurinthepresenceorabsenceofeffectiveanti-Salmonella
to concentrate cargo for subsequent incorporation into luminal responses. Taken together, these data suggest that Salmonella
vesicles [29]. In addition, sorting of pMHC-II into luminal vesi- infectionpolarisesantigenpresentationbystabilisingorenhanc-
cles at MVBs is regulated by ubiquitination [30]. Therefore, the ing formation of the pMHC-II conformer recognised by Type B
requirementforclathrinbySalmonellamayberelatedtoclathrin- T cells, leading to increased Type B T-cell activation. In heavily
dependentsortingofubiquitinatedcargoatMVBs. infected BMDCs, this polarisation may be further promoted
We next confirmed that Salmonella down-regulated surface by reduced presentation of pMHC-II conformers recognised by
expression of I-A and I-E in BMDCs, similar to what has been TypeATcells.
observedinhumancells[15,22].Thisvalidatedtheuseofmurine Mere exposure to HK Salmonella or supernatant from
T-cellreagentstoassessantigenpresentationfollowingSalmonella Salmonella-infected BMDCs was sufficient to enhance presenta-
infectionofBMDCs.SalmonellainfectionofmurineDCshasbeen tion of exogenous peptide to Type B T cells. This phenotype is
reportedtoinhibitpresentationofantigentoCD4+Tcells[14,28]. unlikelytobecausedbysolubleTLRligandssuchasLPSandflag-
HerewecomparedtheinfluenceofSalmonellaonpresentationof ellin,astheseareshedbySalmonellainsignificantamounts[33]
antigen to Type A and B CD4+ T-cell subsets. In contrast to the andtheeffectwaslostwhentheculturesupernatantwasfiltered
suppressiveeffectofSalmonellainfectiononpresentationofboth orwheninfectedBMDCswerespatiallyseparatedfromuninfected
exogenousproteinandpeptideantigentoTypeATcells,presen- BMDCsandTcells.Itisunlikelytobeduetosecretionofasolu-
tation of peptide to Type B T cells was significantly enhanced. blecytokineasthiswouldalsoremaininthefilteredsupernatants.
Salmonella infection did not significantly alter presentation of DirectcontactbetweenSalmonellaandBMDCsisrequired.Weare
exogenousproteinantigentoTypeBTcells.Thiscontrastswith currentlyattemptingtoidentifythebacterialcomponentsrespon-
therecentdataofStrongandUnanueshowingthatTLRligands sibleforthiseffect.
havenoeffectonthepresentationofpeptideviatheTypeBcon- The potential for Type B T-cell activation to lead to auto-
formerinsplenicDCs[31].Thismayreflectdifferencesinantigen immune disease is established [12]. Infection of the gastroin-
handlingbetweenDCsubsetsasreportedbyLovitchetal.forpre- testinaltractwithSalmonella,aswellasYersinia,Campylobacter
sentationofnativeantigentoTypeBTcellsusingLPS-stimulated andShigella[34],isfrequentlyassociatedwithreactivearthritis
BMDCs[32]. in humans [35] and mice [36]. Salmonella infection in humans
The relevance of reduced Type A presentation in relation to caused incidence rates of between 6 and 30% (with variable
immunity to Salmonella infection in vivo is not clear. Whilst severity),reflectingthepropensityofdifferentSalmonellaspecies
chronic typhoid carriers exhibit impaired humoral and cellular to induce arthritis. The enhanced activation of Type B T cells
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Eur.J.Immunol.2013.43:897–906 Antigenprocessing 903
Figure 5. Exposure to Salmonella is suffi-
cient to enhance presentation of exoge-
nous peptide to Type B T cells. BMDCs
(intriplicate)wereinfectedwithopsonised
WT, SPI2-deficient ((cid:2)ssaV) or HKWT GFP-
S. Typhimurium (MOI 10, unless specified
(C)).Forantigenpresentation,BMDCswere
incubatedwithHEL46–61peptideand11A10
(Type B) T hybridoma cells at a ratio of
5Tcells:1BMDC.After24h,culturesuper-
natantswereharvestedandT-cellactiva-
tionwasquantifiedbyIL-2ELISA.(A)At20h
post-infection, culture supernatant was
harvestedandincubatedwithfreshBMDCs,
HEL46–61peptideand11A10(TypeB)Tcells.
Whereindicated,culturesupernatantwas
filtered(0.45μm)priortoincubationwith
fresh BMDCs. (B) At 20 h post-infection,
GFP-S. Typhimurium-infected BMDCs were
sorted from the exposed but uninfected
population. Refer to Supporting Informa-
tion Fig. 1A for representative gating
strategy. Presentation of 0.5 μM HEL46–61
peptideto11A10(TypeB)Tcellswascom-
pared for BMDCs that were unexposed,
exposed but uninfected, or infected with
S. Typhimurium. Comparison of distribu-
tions was performed by unpaired two-
tailed t-tests. (A–C) Data shown are the
mean+SDandarerepresentativeofone
out of at least four independent experi-
ments.
observed with Salmonella infection is not limited to pathogen-
Materials and methods
specificTcellsastheantigensusedintheseexperimentswerenot
derived from Salmonella. Therefore, it is possible that infected
Antibodies
DCs in vivo may incidentally present self-peptide-associated
TypeBconformers,leadingtoactivationofpotentiallyautoreac-
AntibodieswerefromThermoScientific:rabbitanti-mouseIgG-Fc
tiveTypeBTcells.Processessuchasinflammationmayincrease
RPE; Dako: rabbit anti-mouse Igs/HRP; BD Transduction Labo-
thesupplyofexogenouspeptideandtherebyfacilitatethegener-
ratories:mouseanti-humanAP-50(611350),mouseanti-human
ationofTypeBpMHC-IIconformers.Infact,severalimmunecell
clathrinheavychain(610499);Sigma:mouseanti-humanβ-actin
types,includingneutrophils[37]andDCs[38,39],areknownto
(AC-74); eBioscience: anti-mouse CD11c PE-Cy5 (N418), anti-
generateexogenousantigenicpeptides.InType1diabetes,related
mouseI-Ed/kPE(14.4.4s);GeneTex:anti-mouseI-AkR-PE(OX-6).
mechanismsarethoughttogeneratepeptidesfromtheinsulinB
Theanti-HLA-DRantibodywasfromcloneL243andisspecificfor
chain, which when presented on MHC-II are specifically recog-
peptide-loadedHLA-DR.
nisedbydiabetogenicTypeBTcells,leadingtodisease[12].
This study is the first to show that exposure of BMDCs to
Salmonella enhances the presentation of exogenously supplied Plasmidconstructs
peptides to Type B T cells. It suggests a mechanism by which
Salmonellainfectioncouldleadtoabreakdowninimmunological pCMV8.91, pMD-G and pHRSin-cPPT-SGW lentiviral constructs
tolerance. Further studies will be required to identify the factor were provided by Paul Lehner (Cambridge, UK). The HLA-DR3
responsibleforthisalterationinpeptidepresentationandtoeval- sequences [15] were cloned into the BamHI and NotI sites of
uatetheroleofTypeBTcellsininfectionandautoimmunity. pHRSin-cPPT-SGWaftereGFPwasexcised.
(cid:3)C 2013WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.eji-journal.eu
904 NicolaP.Jacksonetal. Eur.J.Immunol.2013.43:897–906
Cellculture,lentiviraltransductionandsiRNA described previously [15]. MelJuSo, RAW264.7-CIITAand HeLa
transfection cellswereinfectedbySPI1-invasionasdescribedpreviously[15].
BMDCs and monocyte-derived macrophage were infected with
HEK293-T, MelJuSo, RAW264.7-CIITA, HeLa and HeLa-CIITA stationaryphaseSalmonella(pre-opsonisedin20%normalmouse
cells were maintained in DMEM, 10% FCS. 3A9 and 11A10 serum (PAA Laboratories) or autologous human serum, respec-
T-cellhybridomasweremaintainedinDMEM,5%FCS.Monocyte- tively,for30min)for60minataMOIof10:1.Wherespecified,
derivedmacrophagesandserumwerepreparedfromPBMCsiso- SalmonellawereHKat65◦Cfor45minpriortoopsonisation.For
lated from Buffy Coats (British National Transfusion Service) flow cytometry, cells were harvested by scraping and incubated
using Lymphoprep (Axis-Shield). Serum was filtered and heat- withappropriateantibodies,inFACSbuffer(PBS,5%FCS)at4◦C.
inactivated.Monocytesweredifferentiatedintomacrophagesfor BMDCswereincubatedwithMouseFcBlock(BDBiosciences)for
7daysinRPMI-1640(Sigma),3%autologousserum,50ng/mL 5minat4◦Cpriortoantibodyaddition.Afterwashing,cellswere
M-CSF (Peprotech). For lentivirus production, HEK293-T cells fixed in 1% paraformaldehyde and analysed using an FACScan
were transfected with pCMV8.91, pMD-G and pHRSin-cPPT- FlowCytometerandSummitsoftware(BDBiosciences).MHC-II
SGW using polyethylenimine (Sigma Aldrich, UK) [40]. After surfaceexpressionwascalculatedasmeanfluorescenceofinfected
48 h, lentivirus-containing supernatants were filtered (0.2 μm) cells (GFP positive)/mean fluorescence of uninfected cells (GFP
and applied to HeLa cells. Transduced cells were sorted using negative)×100.
a MoFlo flow cytometer (Cytomation). For siRNA transfection,
HeLa were seeded in 6-well plates and transfected with siRNA
oligonucleotides using Oligofectamine (Invitrogen). Cells were
Westernblot
reseeded at 48 h post-transfection before a second transfection
with the same oligonucleotide. siRNA oligonucleotides for AP-2
Cells were harvested using Cell Dissociation Buffer (Sigma) and
andclathrinheavychainwerefromQiagen[15].
lysedfor30minat4◦CinPBS,1%NonidetP-40substitute,50mM
Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, protease inhibitor
BMDCpreparationandantigenpresentation mixture(RocheDiagnostics),and5mMIodoacetamide.Samples
were boiled in SDS-PAGE loading buffer before protein separa-
Miceweremaintainedaccordingtoinstitutionalguidelinesatthe tionbySDS-PAGEandtransfertoImmobilon-PPVDFmembrane
UniversityofCambridge.BMwasharvestedfromfemurs/tibiasof (Millipore). Membranes were probed with antibodies and anal-
8–12weekfemaleC3H/HeNCrlmice(CharlesRiver)andpassed ysed by rapid immuno-detection using ECL. Membranes were
througha70μmstrainerinIMDM.BMcellswereseededin9cm blockedwith5%non-fatmilkpowder/0.05%Tween-20forsub-
plates at 1 × 106 cells/mL in IMDM, 10% FCS, 2 mM Ultra- sequentdetections.
glutamine (Lonza), 10 ng/mL IL-4 (Peprotech), 20 ng/mL GM-
CSF(Peprotech)andpenicillin/streptomycin(PAALaboratories)
for ∼30 min to adhere macrophage-precursors. Non-adherent
BM cells were reseeded in 6-well plates for the differentiation Cryo-immunoelectronmicroscopy
intoBMDCs,withmedia/cytokinereplacementondays3and5.
Day 7 BMDCs were harvested by gentle scraping on ice. Differ- MelJuSocellswereinfectedwithGFP-Salmonellain9cmplates.
entiatedBMDCswereroutinely50–60%CD11c/CD11b+,CD80hi, At 12 h post-infection, cells were washed and surface HLA-DR
CD86lo andMHC-IIlo,asassessedbyflowcytometry.Forantigen was labelled (L243 antibody) at 4◦C. After 20 min, cells were
presentation,BMDCswereseededat3×104cells/wellin96-well- washed and returned to 37◦C. After 0 and 7 h endocytosis,
flat-bottomedplatesmorethan6hpriortoSalmonellainfection. cellswerefixedin0.2%glutaraldehyde/2%paraformaldehydefor
For sorts, DCs were seeded at 5 × 106 cells/9 cm plate prior to 2hatRT.Fixedcellswereharvested,embeddedingelatin,and
infection.At20hpost-infection,cellswerewashedwithPBS,then cryo-sectionedusingaLeicaFCS[41].Ultrathinsections(50nm)
antigen (HEL protein (Sigma) or HEL peptide (Cambridge werecutat–120◦CusingaCryo-immunoknife(Diatome,Switzer-
46–51
Bioscience))andTcellswereadded.T-cellhybridomaswerepre- land) and cells were labelled with 10 nm Protein A gold parti-
washedwithDMEM,5%FCSandwereaddedata5:1Tcell:DC cles(CellBiology,MedicalSchool,UtrechtUniversity)inPBS/1%
ratio(refertoSupportingInformationFig.3fortitration).Culture BSA. Images were collected using a Philips/FEI CM10 electron
supernatants were harvested after 24 h, frozen at −80 ◦C and microscope.
then IL-2 quantified by ELISA using mouse IL-2 Ready-SET-Go!
kits(eBioscience).
Statisticalanalysis
Salmonellastrains,infectionandflowcytometry
Statistical analysis was performed using Graphpad Prism where
Salmonella Typhimurium 12023 (ATCC) WT and (cid:2)ssaV strains p=0.01–0.05(*),p=0.001–0.01(**)andp<0.001(***)was
that constitutively express GFP from pFVP25.1 were grown as consideredsignificant.
(cid:3)C 2013WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.eji-journal.eu
Eur.J.Immunol.2013.43:897–906 Antigenprocessing 905
generatedwithintheisletsofLangerhansinautoimmunediabetes.Nat.
Immunol.2010.11:350–354.
13 Peterson,D.A.,DiPaolo,R.J.,Kanagawa,O.andUnanue,E.R.,Quan-
titativeanalysisoftheT-cellrepertoirethatescapesnegativeselection.
Acknowledgments: ThisworkwassupportedbytheRoyalSoci-
Immunity1999.11:453–462.
etyofNewZealandRutherfordFoundationwithadditionalsup-
14 Cheminay,C.,Mohlenbrink,A.andHensel,M.,IntracellularSalmonella
port from the Wellcome Trust and the National Institute for
inhibit antigen presentation by dendritic cells. J. Immunol. 2005. 174:
HealthResearch(NIHR)CambridgeBiomedicalResearchCentre. 2892–2899.
WethankDr.EmilUnanue(Washington,USA)forprovidingthe
15 Lapaque,N.,Hutchinson,J.L.,Jones,D.C.,Meresse,S.,Holden,D.W.,
3A9and11A10T-cellhybridomas,PaulLehner(Cambridge,UK) Trowsdale,J.andKelly,A.P.,Salmonellaregulatespolyubiquitination
forthepCMV8.91,pMD-GandpHRSin-cPPT-SGWlentiviralcon- andsurfaceexpressionofMHCclassIIantigens.Proc.Natl.Acad.Sci.USA
structs,KarindePunderandNicolevanderWelforassistancewith 2009.106:14052–14057.
cryo-immunoelectron microscopy, Nigel Miller for FACS sorting, 16 vanderVelden,A.W.,Copass,M.K.andStarnbach,M.N.,Salmonella
andSarahGibbs,StephenNewlandandPaolaZacconeforassis- inhibitT-cellproliferationbyadirect,contact-dependentimmunosup-
pressiveeffect.Proc.Natl.Acad.Sci.USA2005.102:17769–17774.
tancewithBMDCpreparation.
17 Bueno,S.M.,Riquelme,S.,Riedel,C.A.andKalergis,A.M.,Mechanisms
usedbyvirulentSalmonellatoimpairdendriticcellfunctionandevade
ConflictofInterest: Theauthorsdeclarenofinancialorcommer-
adaptiveimmunity.Immunology2012.137:28–36.
cialconflictsofinterest.
18 McSorley, S. J., Asch, S., Costalonga, M., Reinhardt, R. L. and Jenk-
ins, M. K.,TrackingSalmonella-specificCD4Tcellsinvivorevealsa
localmucosalresponsetoadisseminatedinfection.Immunity2002.16:
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