Research Article Evidence for Contribution of CD4+CD25...

Research ArticleEvidence for Contribution of CD4+CD25+ Regulatory T Cellsin Maintaining Immune Tolerance to HumanFactor IX following Perinatal Adenovirus Vector Delivery

Megha S Nivsarkar1 Suzanne M K Buckley1 Alan L Parker2 Dany Perocheau1

Tristan R McKay3 Ahad A Rahim4 Steven J Howe5 and Simon N Waddington16

1Gene Transfer Technology Group University College London 86-96 Chenies Mews London WC1E 6HXZ UK2Institute of Cancer amp Genetics Cardiff University School of Medicine Heath Park Cardiff CF14 4XN UK3Stem Cell Group Cardiovascular amp Cell Sciences Research Institute St Georgersquos University of London Cranmer TerraceLondon SW17 0RE UK4Department of Pharmacology School of Pharmacy University College London 29-39 Brunswick Square London WC1N 1AX UK5Molecular and Cellular Immunology UCL Institute of Child Health 30 Guilford Street London WC1N 1EH UK6Antiviral Gene Therapy Research Unit Faculty of Health Sciences University of the Witwatersrand Johannesburg South Africa

Correspondence should be addressed to Simon N Waddington swaddingtonuclacuk

Received 26 November 2014 Accepted 12 January 2015

Academic Editor Nejat K Egilmez

Copyright copy 2015 Megha S Nivsarkar et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Following fetal or neonatal gene transfer in mice and other species immune tolerance of the transgenic protein is frequentlyobserved however the underlying mechanisms remain largely undefined In this study fetal and neonatal BALBc mice receivedadenovirus vector to deliver human factor IX (hFIX) cDNA The long-term tolerance of hFIX was robust in the face of immunechallenge with hFIX protein and adjuvant but was eliminated by simultaneous administration of anti-CD25+ antibody Naiveirradiated BALBcmice which had received lymphocytes from donors immunised with hFIX developed anti-hFIX antibodies uponimmune challenge Cotransplantation with CD4+CD25+ cells isolated from neonatally tolerized donors decreased the antibodyresponse In contrast cotransplantation with CD4+CD25minus cells isolated from the same donors increased the antibody responseThese data provide evidence that immune tolerance following perinatal gene transfer is maintained by a CD4+CD25+ regulatorypopulation

1 Introduction

Fetal or neonatal gene transfer has been proposed as apotential therapeutic strategy with several advantages overadult gene transfer including increased likelihood of immunetolerization towards the transgenic protein [1 2] Absence ofimmune response after fetal or neonatal gene or stem celldelivery has been demonstrated in several studies [2ndash5] someof which have provided evidence for immune tolerance Wehave previously observed prolonged expression of humanfactor IX (hFIX) after adenovirus-mediated gene transfer to

fetal mice of the MF1 outbred strain There was transient andminimal production of anti-hFIX antibodies after immunechallenge by repeated injection of purified hFIX protein andhFIX-adenovirus in adulthood [6]

To interrogate the immunemechanisms after fetal or neo-natal gene transfer more extensively we used inbred BALBcmice These mice were chosen since they express relativelyhigh levels of hFIX after neonatal retroviral gene transfer [4]exhibit a dominant Th2 response [7] and unlike outbredMF1 mice can be used for adoptive transfer experimentsAdenovirus serotype 5 vector was used throughout

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 397879 6 pageshttpdxdoiorg1011552015397879

2 Journal of Immunology Research

2 Materials and Methods

Adenoviral vectors with CMV driving human factor FIX[6] (AdhFIX) and firefly luciferase [8] (AdLuc) genes wereprepared Vectors were titered by 293-cell plaque assays E16fetal mice received intravenous injection via the vitellinevessels [6] P0 neonates received injections under hypother-mic anaesthesia via the superficial temporal vein [9] Adultsreceived injections via the lateral tail vein Fetuses neonatesand adults received 1 times 109 1 times 109 and 3 times 1010 vectorgenomes per mouse Citrated blood was collected for plasmafor hFIX assay and for serum for antibody analysis Plasmaconcentrations of hFIX protein were determined using theAsserachrom FIX ELISA kit with pooled human plasmaas assay standard Antibodies against hFIX and adenoviruswere detected as previously described [6] Anti-CD25 IL2Rrat IgG1 antibody was produced by PC61 hybridoma cells(American Type Culture Collection Manassas USA) usinga capillary cell culture system as per manufacturerrsquos proto-col (CellMax Artificial Capillary Module Cellco Inc MDUSA) Antibody was purified and concentrated using affinitychromatography (Mab Trap Amersham Biosciences UKLimited) For vector genome quantitation 100 ng extractedDNAwas subjected to quantitative PCR (ABI Prism 7900HTApplied Biosystems Ltd Warrington UK) using hexon spe-cific primers (51015840CGCGGTGCGGCTGGTG31015840 and 51015840TGG-CGCATCCCATTCTCC31015840) normalized against a standardcurve ranging from 101 to 107 adenovirus particles Foradoptive transfer experiments recipients received sublethalirradiation (400 cGy) 24 h before cell transfer and hFIXimmunization Lymphocytes were harvested from inguinalcaudal and mesenteric lymph nodes and combined withsplenocytes CD4+CD25+ and CD4+CD25minus populationswere purified using magnetic beads (MACS Cell SeparationMiltenyi Biotec Surrey UK) Purity was confirmed by FACSanalysis From sensitized or naive pooled donors 3 times 107lymphocytes were injected intravenously into recipients 1 times104 or 1 times 105 CD4+CD25+ or CD4+CD25minus cells werecotransplanted both concentrations were equally effectivefor the two transplanted populations therefore antibodydata was pooled for subsequent analysis and presentationLuciferase expression in organ homogenates was assayedusing the Luciferase Assay System (Promega WI USA)and luminometer (Lucy1 Anthos Germany) as previouslydescribed [10]

3 Results

31 Longevity of Expression and Immune Challenge Wemea-sured hFIX expression and anti-hFIX antibodies followingintravascular injection of AdhFIX into fetal (119899 = 20) neona-tal (119899 = 21) (Figure 1(a)) and adult (119899 = 4) (Figure 1(b))BALBc mice After perinatal delivery hFIX concentrationswere greater than 2 120583gmL at 48 hours but declined toa stable concentration of approximately 50 ngmL beyond150 days some mice (asymp15) lost expression None devel-oped significant anti-hFIX antibodies In contrast followingadult delivery hFIX concentrations were between 01 and

06 120583gmL at 48 hours and declined to zero within 7 daysHigh concentrations of anti-hFIX antibodies were detectedfrom day 14 onwards

As a stronger test of immune tolerance to hFIX adultmice which had received AdhFIX in utero received twosubcutaneous injections (119899 = 9) of purified hFIX plus MPL-TDM adjuvant (at 140 and 175 days of age) (four receivedan additional challenge of AdhFIX at 275 days (Figure 1(c))to test tolerization to vector described below) Eight miceshowedno significant decrease in hFIX expression or increasein anti-hFIX antibodies after each challenge however onemouse which had no hFIX expression at the time ofchallenge developed high titer anti-hFIX antibodies Thissupports the observation of others that continual antigenexpression is required for maintenance of tolerance followingperinatal gene transfer [4] All naive adult controls developedhigh anti-hFIX antibody titers following challenge (119899 = 4data not shown)

32 Depletion of CD4+CD25+ Cells The mechanism ofimmune tolerance was investigated Six months after fetalAdhFIX injection mice still expressing hFIX (range 24ndash148 ngmL) received three intravenous injections of anti-CD25+ antibody (119899 = 7) to deplete CD25+ regulatory Tcells controls received rat IgG antibody (119899 = 7) (1mgmouse)(0 4 and 29 days) Subsequent to each antibody injectionmice received a subcutaneous injection of a mix of hFIX(1 gmouse) ovalbumin (1 gmouse) and adjuvant (Freundrsquoscomplete adjuvant and then 2x Freundrsquos incomplete adju-vant) Blood was collected 9 days after the third immunechallenge All mice that received the anti-CD25+ antibodydeveloped high anti-hFIX titres unlike those that receivedcontrol rat IgG (Figure 1(e)) These results provide evidencethat CD4+CD25+ regulatory T cells contribute to maintain-ing tolerance after perinatal gene transfer To confirm that thedepleting antibody was antigen-specific in breaking immunetolerance to hFIX rather than acting as a global immunestimulus anti-ovalbumin antibody titres were measuredUnexpectedly mice receiving depleting antibody developed areduced immune response to ovalbumin (Figure 1(f)) SincePC61 also binds to recently activated cells which expressthe IL-2 receptor 120572 those reactive to ovalbumin may beinactivated or deleted

33 Adoptive Transfer Experiments The mechanism of im-mune tolerance was interrogated further A cohort of micestill expressing hFIX more than 250 days after neonatalAdhFIX injection received three subcutaneous immunisa-tions with hFIX and Freundrsquos adjuvant immune tolerancewas confirmed by hFIX expression and absence of antibodiesA second cohort of naive mice was sensitised by subcuta-neous immunisation three times with hFIX and Freundrsquosadjuvant Irradiated BALBc recipients received lymphocytesfrom sensitized mice (119899 = 4) Alternatively recipients re-ceived cotransplantation of sensitised lymphocytes pluseither CD4+CD25+ (119899 = 9) or CD4+CD25minus (119899 = 7) lym-phocytes from tolerized mice (119899 = 9) Two recipients re-ceived whole lymphocyte population from naive donors

Journal of Immunology Research 3

005

1

435

3

225

Days after injection

1

0 50 100 150 200 250 3000

2

hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

Xan

tibod

ies (120583

gm

L)

(a)

0

0

02

04

06

10 20 30 40 50

500

150

250

350

450


hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

X an

tibod

ies (120583

gm

L)

(b)

0

05

10

15

30

0

60

90

500

1000

Days after injection0 50 100 150

hFIX hFIX AdhFIX

200 250 300

hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

X an

tibod

ies (120583

gm

L)

(c)


Ant

i-ade

novi

rus a

ntib

odie

s

0

04

08

12

16 S1-3

C1-3

hFIX hFIX AdhFIX

0 50 100 150 200 250 300

(rel

ativ

e abs

orba

nce)

(d)

Serum dilution110000110001100A

nti-h

FIX

antib

odie

s (re

lativ

e abs

orba

nce)

0

1

2

3

4 P lt 001 anti-CD25 versus rat IgG

(e)

Serum dilution110000110001100

Ant

i-ova

lbum

in an

tibod

ies

0

1

2

3


(rel

ativ

e abs

orba

nce)

(f)

Figure 1 Measurement of concentrations of hFIX and anti-hFIX antibodies following perinatal and adult AdhFIX injection (a)Concentrations of hFIX (red upper graph) and anti-hFIX antibodies (black lower graph) following fetal (119899 = 20 small squares) and neonatal(119899 = 21 large diamonds) injection of AdhFIX (b) Concentrations of hFIX (red upper graph) and anti-hFIX antibodies (black lower graph)following adult injection of AdhFIX (c) Concentrations of hFIX (red upper graph) and anti-hFIX antibodies (black lower graph) in miceinjected in utero with AdhFIX Two immune challenges with hFIX plus adjuvant and one AdhFIX were administered at 119905 = 140 175 and 275(labelled arrows) (d) Concentration of anti-adenovirus antibody in naive controls (C1-3) positive control mice which received hFIX plusadjuvant (S1-3) andmice having received AdhFIX in utero (plotted lines) Mice having received AdhFIX in utero received immune challengeswith hFIX plus adjuvant and one AdhFIX was administered at 119905 = 140 175 and 275 (labelled arrows) (e) Anti-hFIX antibodies in seriallydiluted plasma of mice tolerized to hFIX by fetal AdhFIX Mice received three injections of anti-CD25+ depleting antibody (black closedcircles and unbroken line) or rat IgG negative control (blue open circles and dotted line) simultaneously with hFIX plus ovalbumin plusadjuvant Nine days after the final challenge sera were collected for analysis (f) Anti-ovalbumin antibodies in serially diluted plasma of micetolerized to hFIX by fetal AdhFIX Mice received three injections of anti-CD25+ depleting antibody (black closed circles and unbroken line)or rat IgG negative control (blue open circles and dotted line) simultaneously with hFIX plus ovalbumin plus adjuvant Nine days after thefinal challenge sera were collected for analysis


00

10

20

30

40

Day 7

sensitised versus

sensitised versus

Day 17 Day 24

Ant

i-hFI

X an

tibod

ies

(rel

ativ

e abs

orba

nce a

t 11

00)

+Sensitized

+CD

25+

+CD

25minus

+N

aive

+N

aive

+Sensitized

+CD

25+

+CD

25minus

+Sensitized

+CD

25+

+CD

25minus

+N

aive

P = 0037

CD25+

P = 0040

CD25minus

CD25minus

P lt 0001CD25+ versus

(a)

Heart Liver Spleen Thymus

1 times 107

1 times 106

1 times 105

1 times 104

Vect

or g

enom

es100

ng D

NA

(b)

Luci

fera

se ac

tivity

(RLU

mg

prot

ein

s)

Heartheart

Thymusthymus

Liver Spleen

1 times 104

1 times 103

1 times 102

1 times 101

1 times 100

1 times 10minus1

1 times 10minus2

1 times 10minus3

P lt 001

P lt 001

minusve minusve

(c)

Figure 2 Adoptive transfer of regulatory cell populations tissue distribution of vector and transgene expression (a) Anti-hFIX antibodyconcentrations in serum diluted 1100 following adoptive transfer of lymphocytes from hFIX-sensitised mice (black bar) cotransplantationof sensitised cells plus CD4+CD25+ cells from neonatally tolerized mice (blue bar) or plus CD4+CD25minus cells from neonatally tolerized mice(red bar) or adoptive transfer of lymphocytes from naive donors (grey bars) Recipients underwent sublethal irradiation adoptive transferand hFIX-immunisation 24 hours later and blood collection on days 7 17 and 24 (b) Quantitative PCR analysis of vector genome distributionand (c) luciferase expression six weeks after neonatal injection of AdLuc

Following subcutaneous immunisation with hFIX and Fre-undrsquos adjuvant blood was collected on days 7 17 and 24 foranti-hFIX analysis (Figure 2(a)) At all times recipients ofsensitised cells produced substantially more anti-hFIX anti-body than those which had received naive cells Importantlycotransplantation of CD4+CD25+ cells from tolerized micesignificantly decreased antibody production (general linearmodel Tukey pairwise comparison 119875 = 0037) whereascotransplantation of CD4+CD25minus cells from tolerized micesignificantly increased antibody production (119875 = 0040)Thisprovides further evidence that after perinatal gene transfer

a CD4+CD25+ population downregulates immune reactivityto hFIX as measured by anti-hFIX antibody productionThis cell population appears to mediate tolerance in agreater number of gene therapy protocols than previouslyanticipated For example CD4+CD25+ regulatory T cellshave been implicated in immune tolerance following livergene transfer using adenoassociated virus vector [11] Sincecotransplantation of CD4+CD25minus cells did not abolish anti-body development in recipients the role of other regulatorycell populations or deletional tolerance cannot be ruledout Indeed others provide evidence for deletional tolerance


following injection of hFIX retrovirus into neonatal mice[12] Nevertheless recently Gaensler and colleagues providedelegant demonstration for a role of CD4+CD25+ regulatoryT cells in maintaining immune tolerance following neonatalintraperitoneal injection of adenoassociated virus vector[13]

34 Biodistribution of Vector Genomes and Gene Expres-sion Six to twelve weeks after neonatal AdLuc luciferaseexpression and vector genomes were quantitated in differentorgans Although fewer vector genomes were present inspleen (19880 plusmn 4600 vg100 ng DNA) and thymus (45072 plusmn14693 vg100 ng DNA) compared with those in other tissues(Figure 2(b)) significant luciferase expression was detected(Studentrsquos 119905-test 119875 lt 001) (Figure 2(c)) Therefore a role forcentral tolerance cannot be ruled out

35 Evidence for Tolerization to Vector Detection of anti-adenovirus antibodies was performed on four adult micefrom the experiment shown in Figure 1(d) (ie they receivedAdhFIX in utero two challenges of hFIX plus adjuvant on140 and 175 days and AdhFIX on 275 days) Only one ofthese mice developed anti-adenovirus antibodies after adultAdhFIX challenge as did all the naive adult mice receivingAdhFIX challenge (S1-3) whereas antibodies remained unde-tectable in the remaining three like in the nonchallengedcontrols (C1-3) (Figure 1(d)) This provides evidence forimmune tolerization to vector components This may beattributable to the use of first E1E3-deleted adenovirus whichmight lead to continuous expression of small amounts of viralproteins particularly since vector genomes can be detectedin thymuses from adult mice injected neonatally with AdLuc(Figure 2(b)) Alternatively it may arise from cotolerizationfrom simultaneous initial exposure to hFIX and adenovirusantigens

4 Discussion

Lifelong gene expression of various transgenes in mice hasbeen achieved following perinatal delivery of vectors derivedfrom adenovirus [6] gamma retrovirus [4] lentivirus [14]and adenoassociated virus [15 16] These studies have ledto improvements in the vector and expression cassette andprovidemotivation for childhood or even infant gene therapyfor early onset genetic diseases However understanding ofthe immune response to perinatal gene therapy remains poorHere we provide evidence for a contribution of regulatorytolerance to transgenic proteins following fetal and neonataldelivery in BALBc mice This supports recent observationsof regulatory tolerance following AAV vector delivery [13]One unexpected finding was evidence for immune toleranceto vector components Although this might permit multiplepostnatal administrations of the same vector it might alsorender the recipient vulnerable to infection by the archetypalvirus clearly an undesirable outcome One possible explana-tion for tolerization is residual and continuous expression ofviral proteinsThis provides strong justification for removal of

all viral sequences from vectors designed for long-term treat-ment of genetic diseases For example third generation HIV-based vectors contain approximately 18 kb of viral geneticmaterial This situation contrasts with recent observationsby Carlon and colleagues where perinatal delivery of AAVvector to murine airways benefitted from immune ignorance(rather than tolerance) to the capsid proteins permitting asecond administration at a later time point [17]

In these studies it is important to consider the develop-mental stage of the mouse immune system in late gestationrelative to the immune system of the fetal and newbornhuman Holladay and Smialowicz have provided an excellentcomparison of the developing mouse and human immunesystem By the end of the third trimester the human B cellsexpress surface IgG lymphocyte precursors are present inthe thymus thymocytes subsequently become responsive tomitogens and functional natural killer cells arise In contrastin the mouse lymphocyte precursors in the thymus aredetected at around 11 days of gestation whereas B cell sur-face IgG and thymocytes responsiveness to mitogen occursaround 17 days of gestation and NK cells arise after birth [18]In humans mature peripheral ab T cells first appear towardsthe end of the first trimester whereas inmice they only appearat the end of gestation [19] However the situation is morecomplex in that human serum IgM and IgG concentrationsdo not reach adult concentrations until 1-2 years of age and4ndash6 years of age respectively Moreover before 2 years ofage babies fail to mount humoral immune responses to someantigens [18] An additional complication is that recently ithas been shown that in humans as in mice and birds theimmune system is layered whereby hematopoiesis occurs inwaves specifically an early one which gives rise to fetal T cellsand a later one that gives rise to different adult T cells Thefetal and adult T cells possess different functional propertiesand gene expression patterns [20]

BALBc mice produce relatively small litters and fre-quently cannibalise their litters following maternal surgeryFor reasons of cost and to minimise animal wastage weswitched from fetal to neonatal gene transfer for adoptive Tcell transfer experiments as neonatal death following genetransfer is very rare Nevertheless it is important to recognisethat both qualitative and quantitative differences in immunefunction between E16 fetal and P0 newborn mouse exist

Over the course of the studywe switched fromMPL-TDMadjuvant to Freundrsquos adjuvant MPL-TDM is considered topose less of a risk of abscess formation and is therefore a betterchoice when complying with the 3Rs principle of refiningexperiments to minimise animal pain and distress Howeverwe chose to use Freundrsquos adjuvant for experiments involvingregulatory T cell depletion and adoptive T cell transfer asFreundrsquos adjuvant was considered likely to provide the mostpotent challenge [21]

Ex vivo luciferase expression correlated poorly with vec-tor copy number This may be explained by the observationthat hemoglobin interferes substantially with ex vivo lumi-nometry This is particularly apparent in the spleen whichretains a particularly high concentration of red blood cellsand hemoglobin even when perfused [22] An additionalexplanation may be that the CMV promoter which drives


luciferase expression in this vector may vary in activity indifferent tissue types

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Megha S Nivsarkar and Suzanne M K Buckley contributedequally to this work Megha S Nivsarkar prepared vectorMegha S Nivsarkar and Suzanne M K Buckley performedELISA and adoptive transferMegha SNivsarkar SuzanneMK Buckley and Simon NWaddington performed preclinicaldelivery Alan L Parker and Steven J Howe performed quan-titative PCR Ahad A Rahim Tristan R McKay and SimonN Waddington planned experiments and interpreted dataMegha S Nivsarkar and Simon N Waddington prepared thepaper

Acknowledgments

The authors thank Professor Ted Tuddenham for his helpfuldiscussion and review of the data Simon N WaddingtonSuzanne M K Buckley and Tristan R McKay are fundedby the European Research Council grant ldquoSomabiordquo SimonN Waddington Ahad A Rahim and Tristan R McKay arefunded by NC3Rs Grant NCL0017801 Ahad A Rahim wasfunded by MRC Grant G1000709

References

[1] S N Waddington M G Kramer R Hernandez-Alcoceba etal ldquoIn utero gene therapy current challenges and perspectivesrdquoMolecular Therapy vol 11 no 5 pp 661ndash676 2005

[2] N D Tran C D Porada G Almeida-Porada H A GlimpW French Anderson and E D Zanjani ldquoInduction of stableprenatal tolerance to 120573-galactosidase by in utero gene transferinto preimmune sheep fetusesrdquo Blood vol 97 no 11 pp 3417ndash3423 2001

[3] S Hayashi W H Peranteau A F Shaaban and A W FlakeldquoComplete allogeneic hematopoietic chimerism achieved by acombined strategy of in utero hematopoietic stem cell trans-plantation and postnatal donor lymphocyte infusionrdquo Bloodvol 100 no 3 pp 804ndash812 2002

[4] J Zhang L XuM E Haskins and K P Ponder ldquoNeonatal genetransfer with a retroviral vector results in tolerance to humanfactor IX in mice and dogsrdquo Blood vol 103 no 1 pp 143ndash1512004

[5] S N Waddington M S Nivsarkar A R Mistry et al ldquoPerma-nent phenotypic correction of Hemophilia B in immunocom-petent mice by prenatal gene therapyrdquo Blood vol 104 no 9 pp2714ndash2721 2004

[6] S N Waddington S M K Buckley M Nivsarkar et al ldquoInutero gene transfer of human factor IX to fetal mice can inducepostnatal tolerance of the exogenous clotting factorrdquo Blood vol101 no 4 pp 1359ndash1366 2003

[7] F Zhang Z Liang N Matsuki et al ldquoA murine locus onchromosome 18 controls NKT cell homeostasis and Th cell

differentiationrdquo The Journal of Immunology vol 171 no 9 pp4613ndash4620 2003

[8] S N Waddington J H McVey D Bhella et al ldquoAdenovirusserotype 5 hexon mediates liver gene transferrdquo Cell vol 132 no3 pp 397ndash409 2008

[9] A A Rahim A M S Wong K Hoefer et al ldquoIntravenousadministration of AAV29 to the fetal and neonatal mouseleads to differential targeting of CNS cell types and extensivetransduction of the nervous systemrdquo FASEB Journal vol 25 no10 pp 3505ndash3518 2011

[10] SM K Buckley S J Howe A A Rahim et al ldquoLuciferin detec-tion after intranasal vector delivery is improved by intranasalrather than intraperitoneal luciferin administrationrdquo HumanGene Therapy vol 19 no 10 pp 1050ndash1056 2008

[11] O Cao E Dobrzynski L Wang et al ldquoInduction and role ofregulatory CD4+CD25+ T cells in tolerance to the transgeneproduct following hepatic in vivo gene transferrdquo Blood vol 110no 4 pp 1132ndash1140 2007

[12] L Xu M Mei M E Haskins et al ldquoImmune response afterneonatal transfer of a human factor IX-expressing retroviralvector in dogs cats and micerdquo Thrombosis Research vol 120no 2 pp 269ndash280 2007

[13] Y Shi R Falahati J Zhang L Flebbe-Rehwaldt and K M LGaensler ldquoRole of antigen-specific regulatory CD4+ CD25+ Tcells in tolerance induction after neonatal IP administration ofAAV-hFIXrdquo Gene Therapy vol 20 no 10 pp 987ndash996 2013

[14] N J Ward S M K Buckley S N Waddington et al ldquoCodonoptimization of human factor VIII cDNAs leads to high-levelexpressionrdquo Blood vol 117 no 3 pp 798ndash807 2011

[15] R J Chandler and C P Venditti ldquoAdenovirus-mediated genedelivery rescues a neonatal lethal murine model of mut0methylmalonic acidemiardquo Human Gene Therapy vol 19 no 1pp 53ndash60 2008

[16] K D Foust X Wang V L McGovern et al ldquoRescue of thespinal muscular atrophy phenotype in a mouse model by earlypostnatal delivery of SMNrdquoNature Biotechnology vol 28 no 3pp 271ndash274 2010

[17] M S Carlon D Vidovic J Dooley et al ldquoImmunologicalignorance allows long-term gene expression following perinatalrAAV-mediated gene transfer to murine airwaysrdquoHuman GeneTherapy vol 25 no 6 pp 517ndash528 2014

[18] S D Holladay and R J Smialowicz ldquoDevelopment of themurine and human immune system differential effects ofimmunotoxicants depend on time of exposurerdquo EnvironmentalHealth Perspectives vol 108 no 3 pp 463ndash473 2000

[19] J E Mold and J M McCune ldquoImmunological toleranceduring fetal development from mouse to manrdquo Advances inImmunology vol 115 pp 73ndash111 2012

[20] J E Mold S Venkatasubrahmanyam T D Burt et al ldquoFetaland adult hematopoietic stem cells give rise to distinct T celllineages in humansrdquo Science vol 330 no 6011 pp 1695ndash16992010

[21] H F Stills Jr ldquoAdjuvants and antibody production dispellingthe myths associated with Freundrsquos complete and other adju-vantsrdquo ILAR Journal vol 46 no 3 pp 280ndash293 2005

[22] M Colin S Moritz H Schneider J Capeau C Coutelle andM C Brahimi-Horn ldquoHaemoglobin interferes with the ex vivoluciferase luminescence assay consequence for detection ofluciferase reporter gene expression in vivordquo Gene Therapy vol7 no 15 pp 1333ndash1336 2000

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


2 Materials and Methods

Adenoviral vectors with CMV driving human factor FIX[6] (AdhFIX) and firefly luciferase [8] (AdLuc) genes wereprepared Vectors were titered by 293-cell plaque assays E16fetal mice received intravenous injection via the vitellinevessels [6] P0 neonates received injections under hypother-mic anaesthesia via the superficial temporal vein [9] Adultsreceived injections via the lateral tail vein Fetuses neonatesand adults received 1 times 109 1 times 109 and 3 times 1010 vectorgenomes per mouse Citrated blood was collected for plasmafor hFIX assay and for serum for antibody analysis Plasmaconcentrations of hFIX protein were determined using theAsserachrom FIX ELISA kit with pooled human plasmaas assay standard Antibodies against hFIX and adenoviruswere detected as previously described [6] Anti-CD25 IL2Rrat IgG1 antibody was produced by PC61 hybridoma cells(American Type Culture Collection Manassas USA) usinga capillary cell culture system as per manufacturerrsquos proto-col (CellMax Artificial Capillary Module Cellco Inc MDUSA) Antibody was purified and concentrated using affinitychromatography (Mab Trap Amersham Biosciences UKLimited) For vector genome quantitation 100 ng extractedDNAwas subjected to quantitative PCR (ABI Prism 7900HTApplied Biosystems Ltd Warrington UK) using hexon spe-cific primers (51015840CGCGGTGCGGCTGGTG31015840 and 51015840TGG-CGCATCCCATTCTCC31015840) normalized against a standardcurve ranging from 101 to 107 adenovirus particles Foradoptive transfer experiments recipients received sublethalirradiation (400 cGy) 24 h before cell transfer and hFIXimmunization Lymphocytes were harvested from inguinalcaudal and mesenteric lymph nodes and combined withsplenocytes CD4+CD25+ and CD4+CD25minus populationswere purified using magnetic beads (MACS Cell SeparationMiltenyi Biotec Surrey UK) Purity was confirmed by FACSanalysis From sensitized or naive pooled donors 3 times 107lymphocytes were injected intravenously into recipients 1 times104 or 1 times 105 CD4+CD25+ or CD4+CD25minus cells werecotransplanted both concentrations were equally effectivefor the two transplanted populations therefore antibodydata was pooled for subsequent analysis and presentationLuciferase expression in organ homogenates was assayedusing the Luciferase Assay System (Promega WI USA)and luminometer (Lucy1 Anthos Germany) as previouslydescribed [10]

3 Results

31 Longevity of Expression and Immune Challenge Wemea-sured hFIX expression and anti-hFIX antibodies followingintravascular injection of AdhFIX into fetal (119899 = 20) neona-tal (119899 = 21) (Figure 1(a)) and adult (119899 = 4) (Figure 1(b))BALBc mice After perinatal delivery hFIX concentrationswere greater than 2 120583gmL at 48 hours but declined toa stable concentration of approximately 50 ngmL beyond150 days some mice (asymp15) lost expression None devel-oped significant anti-hFIX antibodies In contrast followingadult delivery hFIX concentrations were between 01 and

06 120583gmL at 48 hours and declined to zero within 7 daysHigh concentrations of anti-hFIX antibodies were detectedfrom day 14 onwards

As a stronger test of immune tolerance to hFIX adultmice which had received AdhFIX in utero received twosubcutaneous injections (119899 = 9) of purified hFIX plus MPL-TDM adjuvant (at 140 and 175 days of age) (four receivedan additional challenge of AdhFIX at 275 days (Figure 1(c))to test tolerization to vector described below) Eight miceshowedno significant decrease in hFIX expression or increasein anti-hFIX antibodies after each challenge however onemouse which had no hFIX expression at the time ofchallenge developed high titer anti-hFIX antibodies Thissupports the observation of others that continual antigenexpression is required for maintenance of tolerance followingperinatal gene transfer [4] All naive adult controls developedhigh anti-hFIX antibody titers following challenge (119899 = 4data not shown)

32 Depletion of CD4+CD25+ Cells The mechanism ofimmune tolerance was investigated Six months after fetalAdhFIX injection mice still expressing hFIX (range 24ndash148 ngmL) received three intravenous injections of anti-CD25+ antibody (119899 = 7) to deplete CD25+ regulatory Tcells controls received rat IgG antibody (119899 = 7) (1mgmouse)(0 4 and 29 days) Subsequent to each antibody injectionmice received a subcutaneous injection of a mix of hFIX(1 gmouse) ovalbumin (1 gmouse) and adjuvant (Freundrsquoscomplete adjuvant and then 2x Freundrsquos incomplete adju-vant) Blood was collected 9 days after the third immunechallenge All mice that received the anti-CD25+ antibodydeveloped high anti-hFIX titres unlike those that receivedcontrol rat IgG (Figure 1(e)) These results provide evidencethat CD4+CD25+ regulatory T cells contribute to maintain-ing tolerance after perinatal gene transfer To confirm that thedepleting antibody was antigen-specific in breaking immunetolerance to hFIX rather than acting as a global immunestimulus anti-ovalbumin antibody titres were measuredUnexpectedly mice receiving depleting antibody developed areduced immune response to ovalbumin (Figure 1(f)) SincePC61 also binds to recently activated cells which expressthe IL-2 receptor 120572 those reactive to ovalbumin may beinactivated or deleted

33 Adoptive Transfer Experiments The mechanism of im-mune tolerance was interrogated further A cohort of micestill expressing hFIX more than 250 days after neonatalAdhFIX injection received three subcutaneous immunisa-tions with hFIX and Freundrsquos adjuvant immune tolerancewas confirmed by hFIX expression and absence of antibodiesA second cohort of naive mice was sensitised by subcuta-neous immunisation three times with hFIX and Freundrsquosadjuvant Irradiated BALBc recipients received lymphocytesfrom sensitized mice (119899 = 4) Alternatively recipients re-ceived cotransplantation of sensitised lymphocytes pluseither CD4+CD25+ (119899 = 9) or CD4+CD25minus (119899 = 7) lym-phocytes from tolerized mice (119899 = 9) Two recipients re-ceived whole lymphocyte population from naive donors


005

1

435

3

225


1

0 50 100 150 200 250 3000

2

hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

Xan

tibod

ies (120583

gm

L)

(a)

0

0

02

04

06

10 20 30 40 50

500

150

250

350

450


hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

X an

tibod

ies (120583

gm

L)

(b)

0

05

10

15

30

0

60

90

500

1000


hFIX hFIX AdhFIX

200 250 300

hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

X an

tibod

ies (120583

gm

L)

(c)


Ant

i-ade

novi

rus a

ntib

odie

s

0

04

08

12

16 S1-3

C1-3

hFIX hFIX AdhFIX

0 50 100 150 200 250 300

(rel

ativ

e abs

orba

nce)

(d)


nti-h

FIX

antib

odie

s (re

lativ

e abs

orba

nce)

0

1

2

3


(e)


Ant

i-ova

lbum

in an

tibod

ies

0

1

2

3


(rel

ativ

e abs

orba

nce)

(f)



00

10

20

30

40

Day 7

sensitised versus

sensitised versus

Day 17 Day 24

Ant

i-hFI

X an

tibod

ies

(rel

ativ

e abs

orba

nce a

t 11

00)

+Sensitized

+CD

25+

+CD

25minus

+N

aive

+N

aive

+Sensitized

+CD

25+

+CD

25minus

+Sensitized

+CD

25+

+CD

25minus

+N

aive

P = 0037

CD25+

P = 0040

CD25minus

CD25minus


(a)


1 times 107

1 times 106

1 times 105

1 times 104

Vect

or g

enom

es100

ng D

NA

(b)

Luci

fera

se ac

tivity

(RLU

mg

prot

ein

s)

Heartheart

Thymusthymus

Liver Spleen

1 times 104

1 times 103

1 times 102

1 times 101

1 times 100

1 times 10minus1

1 times 10minus2

1 times 10minus3

P lt 001

P lt 001

minusve minusve

(c)








4 Discussion













Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















005

1

435

3

225


1

0 50 100 150 200 250 3000

2

hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

Xan

tibod

ies (120583

gm

L)

(a)

0

0

02

04

06

10 20 30 40 50

500

150

250

350

450


hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

X an

tibod

ies (120583

gm

L)

(b)

0

05

10

15

30

0

60

90

500

1000


hFIX hFIX AdhFIX

200 250 300

hFIX

conc

entr

atio

n (120583

gm

L)

Ant

i-hFI

X an

tibod

ies (120583

gm

L)

(c)


Ant

i-ade

novi

rus a

ntib

odie

s

0

04

08

12

16 S1-3

C1-3

hFIX hFIX AdhFIX

0 50 100 150 200 250 300

(rel

ativ

e abs

orba

nce)

(d)


nti-h

FIX

antib

odie

s (re

lativ

e abs

orba

nce)

0

1

2

3


(e)


Ant

i-ova

lbum

in an

tibod

ies

0

1

2

3


(rel

ativ

e abs

orba

nce)

(f)



00

10

20

30

40

Day 7

sensitised versus

sensitised versus

Day 17 Day 24

Ant

i-hFI

X an

tibod

ies

(rel

ativ

e abs

orba

nce a

t 11

00)

+Sensitized

+CD

25+

+CD

25minus

+N

aive

+N

aive

+Sensitized

+CD

25+

+CD

25minus

+Sensitized

+CD

25+

+CD

25minus

+N

aive

P = 0037

CD25+

P = 0040

CD25minus

CD25minus


(a)


1 times 107

1 times 106

1 times 105

1 times 104

Vect

or g

enom

es100

ng D

NA

(b)

Luci

fera

se ac

tivity

(RLU

mg

prot

ein

s)

Heartheart

Thymusthymus

Liver Spleen

1 times 104

1 times 103

1 times 102

1 times 101

1 times 100

1 times 10minus1

1 times 10minus2

1 times 10minus3

P lt 001

P lt 001

minusve minusve

(c)








4 Discussion













Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

10

20

30

40

Day 7

sensitised versus

sensitised versus

Day 17 Day 24

Ant

i-hFI

X an

tibod

ies

(rel

ativ

e abs

orba

nce a

t 11

00)

+Sensitized

+CD

25+

+CD

25minus

+N

aive

+N

aive

+Sensitized

+CD

25+

+CD

25minus

+Sensitized

+CD

25+

+CD

25minus

+N

aive

P = 0037

CD25+

P = 0040

CD25minus

CD25minus


(a)


1 times 107

1 times 106

1 times 105

1 times 104

Vect

or g

enom

es100

ng D

NA

(b)

Luci

fera

se ac

tivity

(RLU

mg

prot

ein

s)

Heartheart

Thymusthymus

Liver Spleen

1 times 104

1 times 103

1 times 102

1 times 101

1 times 100

1 times 10minus1

1 times 10minus2

1 times 10minus3

P lt 001

P lt 001

minusve minusve

(c)








4 Discussion













Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















4 Discussion













Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Evidence for Contribution of CD4+CD25...

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