Ibitokou S, Brutus L, Vianou B, Oesterholt M, Massougbodji A, Deloron P, Troye-Blomberg M,
Fievet N & Luty AJF.
Journal of Reproductive Immunology, (2013) sous presse
Contexte : Le concept de l’immuno-modulation au cours de la grossesse a été décrit et des réponses
de type TH1 et TH2 permettent le maintien de l’équilibre fœto-maternel. Le 1er et le 3ème trimestre
de la grossesse sont caractérisés par l’implantation de l’œuf et la placentation d’une part, et d’autre part l’accouchement et l’expulsion du nouveau-né [65]. Ces phénomènes requièrent des réponses pro-inflammatoires générées par les cellules de l’immunité pour maintenir l’équilibre physiologique de la grossesse. Les études longitudinales récentes ont montré des changements qualitatifs et quantitatifs importants dans les populations cellulaires en fonction de l’âge gestationnel au cours des grossesses normales [122, 124, 221-223]. A partir des données de l’article précédent, nous avons souhaité rechercher, la composition des cellules immunocompétentes chez les femmes incluses au Bénin et en Tanzanie au cours du projet STOPPAM.
Méthode : L’originalité de cette étude réside dans l’utilisation des données appariées à l’inclusion
et à l’accouchement pour évaluer les différences en fonction de l’âge gestationnel. Parmi les 131 inclusions et 111 accouchements enrôlés au Benin, les données paires inclusion-accouchement de 24 femmes ont permis de valider nos analyses. Dans un premier temps, nous avons comparé dans la cohorte du Bénin, les fréquences cellulaires des femmes non-infectées à l’inclusion (69) par rapport aux femmes non-infectées à l’accouchement (74), ensuite celles des femmes infectées à l’inclusion (62) comparées aux femmes infectées à l’accouchement (37). Dans un second temps, les populations cellulaires qui présentaient des différences significatives dans les deux groupes ont été sélectionnées et introduites dans un modèle d’analyse appariée sur les 24 paires de femmes sélectionnées. Les données de l’étude en Tanzanie ont été utilisées pour compléter et pour la confirmation des données du Benin.
Résultats : Indépendamment de l’infection palustre, les fréquences de lymphocytes T CD4+, T effecteurs et de monocytes exprimant le CD86 diminuaient entre l’inclusion et l’accouchement alors
que celles de lymphocytes T CD8+, mDC exprimant le CD86 sont augmentées dans une analyse non
appariée. Lorsqu’on inclut ces paramètres dans une analyse appariée sur 24 paires de femmes, les
42
Discussion et conclusion : Nos résultats montrent des variations dans les populations
lymphocytaires au cours de la grossesse indépendamment de l’infection palustre. Ces résultats confirment ceux déjà décrits chez les populations non africaines sur la diminution de fréquence de T
CD3+ entre le second et le troisième trimestre de grossesse et apportent des informations
complémentaires sur la diminution des fréquences de lymphocytes T effecteurs au cours de la grossesse chez les populations africaines. Cette étude est une première décrivant les changements de populations cellulaires au cours de la grossesse chez les femmes africaines.
ContentslistsavailableatSciVerseScienceDirect
Journalof
Reproductive
Immunology
jou rn al h om ep a ge :w w w . e l s e v i e r . c o m / l o c a t e /j r e p r i m m
Gestationalage-related
changes
in
theperipheral
bloodcell
composition
ofsub-SaharanAfrican
women
SamadIbitokoua,b,c,LaurentBrutusb,c,BertinVianoua,MaykeOesterholtd,
AchilleMassougbodjia,PhilippeDeloronb,c,MaritaTroye-Blomberge,NadineFievetb,c,
AdrianJ.F.Lutyb,c,d,∗
aCentred’étudeetderecherchesurlepaludismeassociéàlagrossesseetàl’enfance(CERPAGE),FacultédesSciencesdelaSanté,Université d’Abomey-Calavi,Benin
bInstitutdeRecherchepourleDéveloppement,UMR216,Mèreetenfantfaceauxinfectionstropicales,Paris,France cPRESSorbonneParisCité,UniversitéParisDescartes,FacultédePharmacie,France
dDepartmentofMedicalMicrobiology,RadboudUniversityNijmegenMedicalCentre,Nijmegen,TheNetherlands eDepartmentofImmunology,Wenner-GrenInstitute,StockholmUniversity,Stockholm,Sweden
a r t i c l e i n f o
Articlehistory:
Received5November2012
Receivedinrevisedform22January2013 Accepted4March2013
Keywords: Pregnancy
Peripheralbloodmononuclearcells Africanwomen
Longitudinalcohortstudy Lymphocytes
Antigen-presentingcells
a b s t r a c t
Gestationalage-relatedchangesinthecellularcompositionofperipheralbloodhavenot beendescribedinsub-SaharanAfricansettings.Weconductedlongitudinalcohortstudies inBenineseandTanzanianmotherswithquantificationofperipheralbloodmononuclear cell-typesexvivousingflowcytometry.Betweenthesecondtrimesteranddeliverythe frequencyofCD4+Tcellsdeclinedsignificantly,contrastingwithanon-significantincrease
inCD8+ T cells,but no changesinT-regulatory, NKor NKTcellfrequencies. Antigen-
presentingcellprofileswerealsounaltered,althoughnon-significanttrendswereevident. Thesechangesresembleinsomerespectsthosereportedduringpregnanciesindeveloped countries,butdifferinothers.
© 2013 Published by Elsevier Ireland Ltd.
1. Introduction
The concept of pregnancy as an immunosuppressive statepermittingthefoetalallografttoimplantandgrow has been well described (Munoz-Suano et al., 2011).
Abbreviations: APC,antigen-presentingcells;ANV,antenatalvisit; BD,Becton–Dickinson;CPDA,citratephosphatedextroseadenine;DC, dendriticcell;EDTA,ethylenediaminetetraaceticacid;FACS,fluorescent activatedcellsorter;GA,gestationalage;HIV,humanimmunodeficiency virus;NK,naturalkillercells;PBMC,peripheralbloodmononuclearcell;P. falciparum,Plasmodiumfalciparum;RDT,rapiddiagnostictest;Teff,effec- torTcells;Treg,regulatoryTcells.
∗ Correspondingauthorat:IRD/UPDUMR216,FacultédePharmacie, 4avenuedel’Observatoire,75006,Paris,France.Tel.:+33170649434; fax:+33153739617.
E-mailaddress:adrian.luty@ird.fr(A.J.F.Luty).
Cell-mediatedimmunityplaysapivotalroleinthetoler- ance of the foetusby the mother’simmunesystem,but discrepancies exist in the results of several studies that haveassessedchangesinthe distributionand activation levelsofdifferentcirculatingcellpopulationsduringpreg- nancy. Thus, studies conducted in developed countries havevariouslyreportedanincreasedfrequencyofperiph- eralbloodCD8+Tcellsinwomenintheirthirdtrimester comparedwithnon-pregnantwomen(Luppietal.,2002a), nochangesinthefrequenciesofCD4+ orCD8+Tcellsat anystageofpregnancy(Kuhnertetal.,1998;Luppietal., 2002b), and a decreased frequency of ‘helper’ (CD4+) T cellsinlatepregnancy(Watanabeetal.,1997).Aprogres- sive gestational age (GA)-related increase in circulating monocytes’activationstatushasalsobeenreported(Luppi etal.,2002b).Datafromrecentlongitudinalstudiesinhigh- income countries have revealed evidence of significant
0165-0378/$–seefrontmatter © 2013 Published by Elsevier Ireland Ltd.
GA-relatedchangesinboththe quantityand thequality ofregulatoryTcells(Treg),dendriticcells(DC)andother celltypes in peripheral blood (Bachy et al., 2008; Della Bellaet al., 2011; Kolte et al., 2011; Leberet al., 2010; Somersetetal.,2004).Whethersimilarchangesoccurdur- ingpregnanciesinsub-SaharanAfricansettingsiscurrently unknown.
2. Materialsandmethods
2.1. Studydesign,locationandpopulation
Thestudydescribedherecomprisedcellularimmuno- logicalassessmentsconductedinsub-groupsofpregnant women who were participating in a longitudinal study entitled “Strategies to Prevent Pregnancy Associated Malaria”(STOPPAM).TheSTOPPAMstudywasconducted inparallel intwo studysitesin Beninand Tanzaniaand itsoveralldesignhasbeendescribedin detailelsewhere (Huynh et al., 2011; Minja et al., 2012). Malaria, pri- marily due to infection with Plasmodium falciparum, is endemic in the areas of both sites, displaying seasonal peaks,buttransmissionishigherintheBeninsite.After givingwritteninformedconsent,∼1000pregnantwomen at≤24weeks’GAwereincludedbothintheareaofComé, located in the Mono province 70km west of Cotonou, Benin, and in Korogwe, located ∼100km inland from thecoastinthe TangaRegionof north-easternTanzania. Clinicalandparasitologicaldataweresystematicallycol- lectedatinclusionandatsubsequentscheduledante-natal visits(ANV),whenultrasoundexaminationswerealsoper- formedallowingaccuratedeterminationofGA.Datawere alsocollectedatnon-scheduled(‘emergency’)visitswhen- ever women presented at the clinicsbecause of illness. Thesub-groupswestudiedherecomprised,firstly,women selectedsequentially atinclusionwhopresentedwithP. falciparum infection as determined by the combination of rapid diagnostic tests (RDT) and microscopic exami- nation of peripheralblood smears.Using a case–control approach,wesubsequentlyselected,ascloselyintimeas possible,anuninfected‘control’womanmatchedforage, gravidityandgestationalagetothecorrespondingprevi- ouslyselected‘case’.Asecondsub-groupwasselectedat deliverycomprisingwomenwithP.falciparuminfections identified by RDTand examinationof placental impres- sionsmears,and uninfected women inwhom both RDT andsmearwerenegativeatdelivery.Atalltimes,women inwhominfectionwithP.falciparumwasidentifiedbyRDT weregivenanti-malarialtreatmentthesamedayaccord- ingtonationalguidelines.Womenwhowereseropositive for HIV or with no HIV diagnosiswere not included in the present analyses. Ethics approval for the STOPPAM study was obtained from the ethics committees of the HealthScienceFacultyoftheUniversityofAbomey-Calavi, Benin,and the NationalInstitute of MedicalResearchof Tanzania.
2.2. Bloodcollectionandcellpreparation
Peripheralvenousbloodsampleswerecollectedfrom selectedwomenatinclusionandatdeliveryinvacutainers
Fig. 1. Comparison of FoxP3 expression levels in Treg (CD4+CD25+CD127−) and Teff (CD4+CD25+CD127+) in 107 pregnant Beninese at inclusion and in 87 at delivery. Box-plots represent medians with 75thand 25th percentiles andwhiskers for 90thand 10th percentiles. p-values were determined by the non-parametric Mann–Whitney Utest. Significantdifferences aredepictedby ***for p<0.001.
(BD Biosciences, France) containing citrate phosphate dextrose adenine (CPDA)anticoagulant. Forthe detailed immunologicalstudiesdescribedhere,samplesweretaken on two occasions: (i) at inclusion and (ii) a maximum of 8hpriortodelivery,fromatotal,respectively,of 131 and111Beninesewomenandfrom38and27Tanzanian women,allofwhomhaduncomplicatedvaginaldeliver- ies.Thebloodsamplesweretransportedtothestudysites’ research laboratories and processed for immunological assessmentswithinfourhoursofcollection.Haemograms were obtained using an automated Sysmex KX-21N analyser according to the manufacturer’s instructions. Peripheral blood mononuclear cells (PBMC) were iso- latedusingLeucoseptubes(Greiner-Bio,France)according to themanufacturer’s procedureand weresubsequently used for immunophenotyping by flow cytometry. The procedures, including gating strategies, used for flow cytometry-basedquantificationofcellsexvivohavebeen describedindetailelsewhere(Ibitokouetal.,2012).Briefly, PBMC were stained with appropriate combinations of standard cell-surface- and cytoplasmic marker-specific monoclonal antibody reagents to allow identification of (i)T(subsets,anti-CD3-APC/anti-CD4-PerCP/anti-CD8- FITC/anti-CD25-FITC/anti-CD127-PE/anti-FoxP3-APC) and B lymphocytes (anti-CD19-FITC), (ii) NK & NK T (anti- CD3-APC/anti-CD56-PE)cells,(iii)monocytes(anti-CD14- FITC) and (iv) dendritic cells (DC, subsets, anti-BDCA1- phycoerythrin (PE)/anti-BDCA2-PE), as well as estimat- ing the activation status of antigen-presenting cells (APC: monocytes, DC, B cells) through measurement of HLA-DR (anti-HLA-DR-PerCP) and CD86 (anti-CD86- APC) expression. Of note, we used a definition of Treg (CD4+CD25+CD127−)herethatclearlydefines cellpopu- lationswiththehighestexpressionlevelofFoxP3,thereby distinguishingitfromthepopulationwedefinedaseffec- torTcells(Teff,CD4+CD25+CD127+,Fig.1).AFACSCalibur four-colourflowcytometer(BDBiosciences,France)run- ningCellQuestProsoftwarewasusedthroughout.
2.3. Statisticalanalyses
Data analyses wereperformed using STATA/MP 11.2 (StataCorp,CollegeStation,TX,USA)andPrism5.0(Graph padInc,CA,USA).
In order to assess GA-related changes in the PBMC composition,wecomparedthemedianvaluesofPBMCfre- quenciesorofantigen-presentingcells’expressionofacti- vationmarkers(HLA-DR/CD86)betweenasetofpregnant womenof≤24weeks’GAandasecondsetofwomenat deliveryusingthenon-parametricMann–WhitneyUtest. First,we compared parameters of interest separately in groupsof P. falciparum-infected and uninfected women. WeselectedthoseparametersthatshowedsignificantGA- relatedchangesin bothinfectedand uninfected women foruseinasecondroundofanalysisforwhichwegrouped datafromallwomentogether(infectedwithuninfected) to compare the selected parameters between unpaired samplesat inclusion and delivery. In orderto avoid the problemsofmultipletesting,weusedBonferronicorrec- tionbydividingthenominalalphalevelbythenumberof comparisonsandrejectingthenullhypothesisifthepvalue waslessthanorequaltothecorrectedsignificancelevel. Thecorrectedpvalueforcomparisonoftheseunpaireddata wastherefore0.002(0.05/23).
In order to validate the findings from analyses of unpairedsamples, wesoughttocircumventthe issueof individualbiasinherenttothelatter.Forthispurposewe comparedPBMC(forthesixparameters thathadshown significant GA-related changes in unpaired samples) in pairedsamplescollectedatinclusionanddeliveryfroma sub-groupof 24Beninese women.Sincepaired samples are,by their verynature, notindependent,we used the non-parametricWilcoxonsigned-rank test todetermine thesignificanceofdifferences,andagainusedBonferroni correction(0.05/6,p≤0.008)inordertoavoidbiasdueto multipletesting.
3. Results
The sub-groups of women included in the analy- ses here were drawn from the larger cohorts of the
STOPPAMstudy.Inordertoexcludethepossibilityofselec- tionbias,wethereforefirstcomparedselectedparameters – essentially demographic in nature, but including the prevalence of anaemia – in the sub-groupsof Beninese womenwiththesameparametersinthewholecohortat thetwotime-points.Table1illustratestheresultsofthose comparisons,revealingthatmediangestationalageswere significantly longer in the delivery sub-groupcompared withthewholecohort,butthatallothervariablestested weresimilar.Inordertoexcludeotherpotentialsourcesof bias,wealsocomparedthebasicclinicalandhaematolog- icalcharacteristicsofthesub-groupsofBeninesewomen segregatedaccordingtothepresenceorabsenceofinfec- tionwithP.falciparum(Table2).Atinclusion,aspreviously reported (Ibitokou et al., 2012), a higher proportion of those withP. falciparuminfectionswereanaemic,whilst the median axillary temperature was also significantly higherintheinfectedgroup,eventhoughonlyoneofthe infectedwomenpresentedwithafever(38.0◦C).Atdeliv- erynoneoftheparametersmeasureddifferedaccordingto thewomen’sinfectionstatus.
OurownrecentdatahaveshownthatP.falciparumalters the composition of PBMC in infected versus uninfected women(Ibitokouetal.,2012).Wethereforefirstcompared the frequenciesofcelltypesatinclusionand atdelivery within groupsofBeninese andTanzanian womensegre- gated accordingtothe presence orabsence ofinfection. IntheBeninesewomentherewasasignificantdeclinein the frequencyof CD4+ Tcellswithaparallel increasein the frequencyof CD8+ Tcellsfrom inclusion(mean GA: 18 weeks) to delivery, regardless of their infectionsta- tus(Fig.2AandB).ThedeclineinCD4+Tcells’frequency wasparalleledbyadeclineinthefrequencyofeffectorT cells(Teff,CD4+CD25+CD127+)andaconsequentincrease in the regulatory T cell (Treg, CD4+CD25+CD127−)/Teff ratio overtime concurrent with stable Treg frequencies (Fig. 2C–F). The relative level of expression of FoxP3 (R Treg), however, as a marker of the activation status of Treg,declinedsignificantly(Fig.2EandF).Thefrequency ofnaturalkiller(NK,CD56+)cellswasunchanged,whilst thatofNKT(CD3+CD56+)cellsincreasedsignificantly,but in theuninfectedgrouponly(datanotshown).Amongst
Table1
Characteristicsofsub-groupscomparedwiththewholecohortinBeninwomenatinclusion(A)andatdelivery(B).
Variables Wholecohort Sub-group pa
n n
(A)Inclusion
Gestationalageinweeks 975 17(6) 102 17.8(4.7) 0.06
Gravidity 1029 3.0(3.0) 107 3.0(4.0) 0.11
Ageinyears 1015 25.0(9.0) 107 25.0(10.0) 0.17
%possessingabednet 1029 32.1 107 28.0 0.39
%withhaemoglobin<11g/dl 1022 60.6 106 67.9 0.14
(B)Delivery
Gestationalageinweeks 629 39.7(1.6) 87 40.0(1.8) 0.03
Gravidity 635 3.0(3.0) 87 3.0(3.0) 0.61
Ageinyears 623 26.0(9.0) 86 26.5(7.0) 0.48
%possessingabednet 635 30.2 87 35.6 0.30
%withhaemoglobin<11g/dl 582 46.4 82 40.2 0.29
Valuesaremedians(interquartileranges).
Table2
Clinicalandhaematologicalcharacteristicsofsub-groupsofBeninesewomenatinclusionanddelivery.
Variables Inclusion(n=131) Delivery(n=111)
Uninfected(n=69) Infected(n=62) pa Uninfected(n=74) Infected(n=37) pa
Systolicpressure(cmHg) 10.0(2.0) 10.0(1.0) 0.88 11.0(1.0) 11.0(1.0) 0.25 Diastolicpressure(cmHg) 6.0(1.0) 6.0(1.0) 0.71 7.0(2.0) 8.0(2.0) 0.13 Axillarytemperature(◦C) 36.8(0.5) 37.0(0.6) <0.01 36.8(0.5) 36.8(0.5) 0.63 Haemogram %Lymphocytes 32.0(11.0) 32.0(11.0) 0.55 32.0(12.0) 33.5(15.0) 0.85 %Basophils 0.0(0) 0.0(0) 0.34 0.0(0) 0.0(0) 0.36 %Eosinophils 0.0(1.0) 0.0(2.0) 0.08 0.0(1.0) 0.0(0.0) 0.73 %Neutrophils 60.0(11.0) 60.0(12.0) 0.29 60.0(13.0) 61.0(12.5) 0.88 %Monocytes 6.0(2.0) 6.0(3.0) 0.07 7.0(1.0) 6.0(2.0) 0.06 %withvomiting 5.8 3.7 0.60 2.7 2.9 0.96 %withheadache 21.7 17.0 0.51 2.7 8.6 0.17 %withanaemia 55.1 80.3 <0.01 32.4 45.9 0.16
Valuesaremedians(interquartileranges)
aComparisonsmadeusingthenon-parametricMann–WhitneyUtestorChi-squaredtestforproportions.
antigen-presenting cell (APC) populations, GA-related declinesinthe frequenciesofbothmyeloidand plasma- cytoiddendriticcells(mDC,pDC)aswellasofBcellswere alsoevident,butthesechangeswerenotconsistentlysig- nificantacrosstheuninfectedandinfectedgroups,whilst monocytefrequencieswereunchangedfrominclusionto deliveryirrespectiveofinfectionstatus(Fig.3A–D).Inthe Tanzaniandatasetnotallchangeswereconsistentacross theuninfectedandinfectedgroups,butsimilarGA-related changesas seenin Beninese women in the frequencies of CD4+ and CD8+ T cells were evident, as well as in thoseofmDCandBcells,whilstthefrequencyofmono- cytesincreased (data notshown). Separate comparisons of the activation status of different cell types revealed significantly increased levels of expression of CD86 on mDCovertimeinbothuninfectedandinfectedBeninese women (Fig. 4A and B), but a contrasting decrease in CD86expressiononmonocytes (Fig.4AandB) thatwas alsoevidentinTanzanians(datanotshown).Lastly,there werenon-significant trends for GA-related decreases in theexpressionofHLA-DRonpDCinbothuninfectedand infectedBeninesewomen(Fig.4CandD).
We sought to confirm the findings described above firstlythroughanalysesoftheeightparameters(%CD4+T cells,%CD8+Tcells,%Teff,RTreg,%mDC,CD86expression onmDCand onmonocytes,HLA-DRexpressiononpDC)
that displayedchangesrelatedtoGA,bycomparingdata from107Beninesewomenatinclusionwith87atdelivery regardlessoftheirinfectionstatus.Table3showsthat,in allcases,thedifferencesbetweenimmunologicalparame- tersatinclusionversusdeliverywerestronglysignificant and6/8werebelowthelevel(p≤0.002)setbycorrection formultipletesting.
Inafinalstep,weanalysedaseparatesetofdatafrom another24Beninesewomenforwhomrelevant,i.e.paired sampleswereavailableatinclusionanddelivery,inorderto validatetheGA-relatedchangesindicatedbytheanalyses ofunpaired data(Table3).Theseanalysesconfirmedthe followingchangesinPBMCprofilesbetweeninclusionand delivery:(i)adeclineinthefrequenciesofCD4+Tcellsand of Teff;(ii)anincreasein thefrequencyofCD8+ Tcells; (iii)reducedexpressionoftheactivationmarkerCD86on monocytes (Table3).After adjustmentformultiple tests onlythereductionsinthefrequenciesofCD4+Tcellsand ofTeffremainedsignificant(Table3).
4. Discussion
Ourdatarevealsignificantgestationalage-relatedalter- ations betweenthe secondtrimester anddeliveryin the PBMC composition of African women with uncompli- catedpregnancies.Themoststrikingofthesealterations
Table3
Comparisonofselectedperipheralbloodmononuclearcell(PBMC)profilesbetweeninclusionanddeliveryinunpairedandpairedgroupsofBeninese women.
Variables Unpaired Paired
Inclusion(n=107) Delivery(n=87) p Inclusion(n=24) Delivery(n=24) p
%CD4 63.0(10.4) 57.9(13.3) <0.0001 63.8(9.1) 58.9(9.4) 0.005 %CD8 27.1(9.0) 31.3(12.3) 0.0011 28.4(9.4) 31.8(11.1) 0.042 %Teff 4.7(5.4) 1.8(1.8) <0.0001 5.6(8.3) 2.4(3.0) 0.008 RTreg(FoxP3) 2.2(3.0) 1.6(0.7) <0.0001 2.0(2.7) 1.7(0.7) 0.159 %mDC 0.3(0.2) 0.3(0.2) 0.025 – – – %mDC–CD86hi 0.9(1.3) 1.3(2.0) 0.0013 0.5(1.5) 1.2(2.1) 0.133 pDCHLA-DR(MFI) 54.3(34.2) 46.9(29.8) 0.0121 – – – MonocyteCD86(MFI) 6.9(3.8) 4.9(3.6) 0.0033 6.7(6.0) 5.3(3.2) 0.022
InclusionanddeliverygroupsincludewomenwithandwithoutP.falciparuminfections;valuesaremedians(interquartileranges)comparedusingthe Mann–WhitneyUtest/Wilcoxonsigned-ranktestforunpaired/paireddatarespectively;afterBonferronicorrection,pvalues≤0.002(unpairedcomparisons included23variables)or≤0.008(pairedcomparisons,sixvariables)areconsideredsignificant(pvaluesinboldface);MFI:meanfluorescenceintensity;R Treg:ratioofFoxP3expressioninTregvs.naïveTcells(CD4+CD25−CD127+).
Fig.2.Peripheralbloodcompositionchangesaccordingtogestationalage.FrequenciesexvivoofdifferentPBMClymphocytepopulationscomparedat inclusionanddeliveryrespectivelyin69and47uninfectedBeninesewomen(leftpanels)andin62and37P.falciparum-infectedBeninesewomen(right panels).Box-plotsrepresentmedianswith75thand25thpercentilesandwhiskersfor90thand10thpercentiles.RTreg:ratioofFoxP3expressionin TregvsnaïveTcells(CD4+CD25−CD127+).p-valuesweredeterminedbythenon-parametricMann–WhitneyUtest.Significantdifferencesaredepictedby *ifp<0.05,**ifp<0.01and***ifp<0.001.
concernedreciprocal decreasesandincreasesin, respec- tively,thefrequenciesofCD4+and CD8+Tlymphocytes, although the latter did not reach statistical significance