SI.John's
The Effec tof Pre-Existin gAntibody On the Resultof ImmunizationofMiceWithHuma n B Cell
LinesAndTransfcctcdMouseCell Lines
by LewisSamuelsB.Sc. (Hcns.)
AThesisSubmutcdto the Schoolof GraduateStudies
in partial fulfilme nt ofthe req uirements for the degree of
Masterof Scienc e
FacultyOf Medicine Memorial UniversityorNewfoundland
February1995
Newfoundland
1.1
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ABST RACT
HenryandIerne (I9{>8)described an interesting phenomenon.whenSRIK"s werc usedasantige n,thepresenceof IgG antibodyprier toinjcction of antigen led toa suppressed respo nse.Incontrast.pre-existing IgM resuhrd inan increase ofthe primnry immuneresponse. The aimsofthisproject were twofold.to determineifthesame phenom enonisobservedwhen usingeither human Bcellsormouse trnnsfl'Clctl fibrobl asts,HSantigens;tostudy the feasibilityof usingIgM enhancementto improveIhc yieldof antigen-specifichybridomas frommice againstHLA·Drand DR.
Studieswereconducted usingmiceimmunised eitherwithamo use transfectant cell lineexpressing HLA-DP(l2S. 4)orahuman8cell line(RAJ!).Preliminary experiments uncovered an unexpec tedreverse doseresponse~henU."iAcells were used as immunogen. Pre-treatmentofmice withantibodiesto monomorphic IlLA-I>I>
determinantsincreasedand supp ressed theresponsesas predicted; werestati!ll:ically significantwith both Raji andL25A.Ina secondexperime nt,doub tin goftheIgMdose ledtoanunexpecteddiminution of theprimary response againM1..25.4andrevea led that thehalflife oftheadministeredIgM antibody appearedtobelengthe ned.
Experim ent!>we re then conducted to determine thevalueof lg M pretreat ment in the creationof hybridomas specific lorHLA·DP andHLA-DR epitopes. Micewe re
pretreated with varyingamountsof IgMantibody and thenimmunized.Threedays later theirspleenswere removed, cellscounted andfusions performed.Spleensize was found tobeincreased ina stepwisefashionwithincreasingdoses ofISM past a previously describedantigeninduced plateau . Thelowdose of 19M producedspleens pred ominately composed ofBcells,increasingthe IgMdose decreased the8 cellsbutincrea sed the T cells.TheIgM pretreated animals gavea 22.8%yield of specifichybridoma s whereas thecontrolgroup producedan 8%yieldof specific hybridomas. Within the19M pretreatmentgroup, 96%ofthe selected hybrids originatedfrom thelow dosagegroup.
indicating thattheremay exist an optimum pretreatment dose forIgM.Thesepreliminary experimentssupportthc potentialof specificantibodypret reatment inthe modulationof the primaryresponseand the development of antigen-specifichybridcmas.
ACKNOWLEDGEMENTS
1 wouldlike tothank my supervisor. Dr.W.H.Marshall,for hishelpandguidance throughout everyaspect of thisproj ect. I wouldparticularlyliketo thank himlo r allowingmetothinkandplanthese experimentsas bestas IS<lWfit,whiteat theS:1I11e
time keepingmeonthe right track! Next,Iwould like tothank mysupervisory committee.especiallyDr.V .Skancsforhersuggestions andadvice.
I wouldalso liketo expressmy apprec iation toJaneGnmberg,DianneCodner, Doug Coppand Sheila Droverfor theirhelp and advice,particularlywith the technical aspects ofthisstudy. I would also like to thank Norman Lee forhishelpinthe lab, Kns Clements forthe proof readingand HeatherDovefor everything(fromgellingsupplies in as soon as possibleto ensuringI receivedmycheques!).
This work wascarriedout attheImmunology Laboratoriesand theAnimalCare facilitiesatthe HealthSciences Centre.the use andinvaluableadviceby the stafforthese facilitiesisgreatlyappreciated.
I wouldespecially like to thank my parentsfor all theencouragement, suppo rt and guidancetheyhave given rne,notonlyduringmyproject bUIthroughoulmylife.Itis largely due to them thatIhaveaccomplished whatI haveIodate.
FinancialassistancewasprovidedfromTerraNovaBiotechnologyCoUd.and the Faculty of Medicine.MemorialUniversity ofNewfoundland. Their supportmadethis project possible.
iii
TABLE OF CONTENTS
ABSTRAC T .
ACKNOW LEDGE MENTS....
LIST OFTABLES ..
LISTOFFIGURES .
LIST OFABBREVIATIONS.•.. . . . .•.. . . . ..•....•.
CHAPTER I .. . . INTR O DUCTIO N
1.1Human LeukocyteAntigen-DPProjcct
. ...•. ii
. ....xii
••.• •• • ••• J{iv
.... xvi
1.2 Basic PhenomenonShownwithRed Cellsas Antigen 1.3 Review of LiteratureRelating toPrimaryImmune Response
Modulation..• . ..••. 1.3.1 EarlyHisto ry ...
1.3.2 LandmarkStudyby Claudia Henry&Niels Jer ne
1.3.3 Ig MMediated Enhancement .
iv
1.3.3.1 SpecificityCharacteristics 1.3.3.2 Basic Criteria .... 1.3.3.3 In \limversusIn \lilroExperiments
1.3.3.5 RoleofCo mplement . 1.3.3.6 PostulatedMechanisms
J.•14 IgG Mediated Suppression .
1.3.4.1 Specificity Characteristics 1.3.4.2 Abilityof Various Subclasses to
1.3.3.4 TCellComponent II
13
14 16 16
Suppress. . . 17
1.3.4.J Experimentsinvolving F(ab')lFragments
andthe Fe Receptor. 18
1.3.4.4 Density Factor... . . 19
1.3.4.5 In\Ii/roExperiments 19
1.3.4.6 Role ofTCells
.. .
201.3.4.7 Role of Complement 23
1.3.4.8 Memory... . . ... . 24 1.3.4.9 AccessoryCell Involvement 24 1.3.4.10 Postulated Mecha nisms... 26 1.3.5 IgGMediated Enhancement ....•,... . . 29
1.3.5.1 Response to DifferingAntigensand Localizatio n.. .. .... .. . ....••..., 29
. "
. "'
1.J5.2 Density
1.3.5.3 Roleof Complement... .. 1.3.5.4 Mechanisms of I~G PokdiatN
Enhancement .\1
1.3.6 Summary... . ... .\-1
1.3.6.1 IgMMediated Enh:mcement. .\-1 1.3.6 .2 IgGMediatedSuppression... .\-1
1.3.6.3 IgOMediatedEnhance ment CHAPTER2 .. . . . .. ... ....•. AIMSAND OBJECT IVES . .t'i .17 J7 CHAPTER3 . ...•.•.•. •.• •.... . .••.. •.. •... . . . .. ....•. )9
MATERIALSANDMETHODS. ••••• ••• •.•••.••••..•. ..• 39
3.1 Animals.•.. ••.•• ••. .. .• •... . ... .. .. )9
3.2 Antibodies. . .... ... . . .. . .. . . . ... .... 39
3.3 AntibodyPurification. .. •...•• . .• •... .. 41
3.3.1 IgM Purification••...• •... 41
3.3.2 IgO Purification...•. ... 42
3.4 50 S-Gel Electrophoresis... 0
3.5 Antigens.... . . . ... . .•. . . .. . . .. . . ).5.1 HLA Typingof Raji CellLine .•.... 46
3.6 Administration ofAntibody and Antigen ...
3.7 BloodCollection
3.8 SerumCollection and Storage .. . 3.9 Measurementof AntibodyLevels.
3.9.1 Modified Procedure for theCELISA involvingHumanBCells. . • . . •. 3.9.2 Procedure for CELISA involving Fibroblasts
3.10 FlowCytometry 3.11 CreationofHybrido mas..
48
49 49 50
50
54 55 57 3.11.1 Preparationof Reagentsand Media 57 3.11.2 PreparingSpleensfor Fusions ... 58 3.1 1.3 T CellDepletionof the SpleenSuspension 60 3.11.4 Preparation of Myeloma Cellsfor Fusion•... 61 3.11.5 Fusionby Pipctting withPolyethylene Glycol .. . 62 3.11.6 CellPlating.. .. . ... •. ... 62 3.11.7 Hybridoma Feeding . .. . .•. .... 63 3.11.8 Screening ofHybriC:omas.• . .,.. .•••. .. .. 64 3.11.8.A PrimaryScreening ... ••... 64 3. 11.8. B SecondaryScreening. ... ... . .•. 64 3.11.8.C TertiaryScreening .... ...• . . .. •• 67 3. 11.8.0 Quaternary Screening.•. ..•.. .... 70
vii
J.12CellFreeTing•.. .. 3. 1JCellThawing .• • •. .1.12StatisticalAnalysis
CHAPTER4 ...
... .... ....•.• 72
.. . . ... . . .. . . . .. .. i:
.•••.• •. • • .. .• .• • • 7.\
74
RESULTS 74
4.1 TheEffectofAdministeredAntibody on the SubSt.'qLlCnt Serum AntibodyResponse ••. . . . .•. .•••. .. . 74
4.1. 1AntibodySpecificiticsandPurityAn;llyloi lO•• 74 4.1.1.1 Test forCross-ReactivityofNFLD.M6.'iCJand
NFLD.M68CIle 8withMouseMHC:
4.1.1.2 TestforAntibodyHomogeneity 4.1.2 Selection of Antigens
7S 7.
.n
4.1.3 SelectionofNon-SpecificIgMAntibody ...•. .. 83 4.1.4 Antig~nDoseResponse ... ...• . ... 85 4.l.s In
".0
Baperimerus ... .. .. .. .... ... .... lJO4.1.5.1 PretreatmentbyHLA·I)Pspecifc IgM(NA.D.M65C3)and HLA-OI' specific IgG (NFLD. M68Cl le 8) with Rllji Antigen
4.1.5 .2 PretreatmentbyHLA-DI' Specific IgMandHLA-DPSpecific IgG withHLA-
viii
90
100
101 DP Transfcctant as Antigen ... . . •. 93 4.1.5.3 Pretreatmentby HLA-DPspecific IgMandNon-specifir-[gM with HLA-DP transfccted fibroblastasAntigen•.. .. .. . 96 4.2 Effec tof AdministeredAntibodyonthePopulation of
HybridomasHarvested3DaysAfter Antigen,•. ,,, 100 4,2.1 HLA TypeDeterminationufRaji,
4.2. 1.1 HLATypeDetermination viaCDC Typing Plates.. "
4,2,1.2 HLA-DPDeterminat ion via MonoclonalAntibodies.•,,•••,., ,, .• 101 4.2.2 Splee n DoseResponseofImmunizationswithRaji
Cells" ,. 110
4.2.3 Fusion Experi ments.. ..,,.•, ,.• • ,.,,, .•113 4,2.3,1 SpleenSize ctDay 3... 114 4.2.3.2 PrimaryScreen : Hybridcells against Raji '" ,, .,.•,..117 4.2.3.3 Seconda ryScreen " " ., .. " ,120 4.2,3.4 Tertiary Screen.•, 123 4,2.3,5 QuaternaryScreen,. . . .•.... . 126
CHAPTER 5••" " ... .• •.• •. .•..•.••.•..•• .••• ••••.••129
DISCUSSION .
5.1 In\.71'QExpcrimenlswithMOPISCTransfccrant andHumanB Cell Lines
I:!Q
5.1.1 Reverse Dose ResponseWithTransfectants. 1.10 5.1.2
5.1.3
InViroExperimentswith Raji asAntigen 1.11 In\111) Experiments with L25.4 Cellsas Antigen . IJ:!
5.1.3A Initial Experiment IJ:!
5.1.3 .8 Diminished ResponsewithDoublingof
Antibody .
5.L\.81 AntibodyDosage 5.1.3.82 Non-specificIgM.,
5.1.3.B3 ActualAmountof Antibody and Antigen 1JJ 13.\
U5
Rcceivcd. 5.2 Fusion Experiments .
5.2.1 Choice of Antibody.. 5.2.2 Splecn Analysis..• •...
136 137
. ...• .. . 137 138 5.2.2A SpleenSizeandDose ofAntibody 138 5.2.28 SpleenTCell Composition 139 5.2 .3 SpecificityAnalysis..
5.2.3.1 Thc ScreeningRationale.
140 140 5.2.3.2 Specificity ofResultant Hybridomas 142
5.3 Future Directions 144
LITERATU RE errED 147
,;
4()
6H
'"
JI LIST OF TABLES
Table1:Summary of MonoclonalAntibodies Used With RespecttoClass and Specificity
Table2:HapJotypesof cellpanelusedin the secondary screen.
Table3:Haplotypcs of cells used in the tertiaryscreen... , Table4:Hybridomatesting selectionpatternfor tertiary screen.
Table5:Haplotypesofcellsusedin the quaternaryscreen..
Table6 :ELlSA results from a panelof sixHLA-DP transfcctantstestedagainst NFLD.M65C3andNFLD.M68CIIC8antibodies. ... 112 Table7: ELISAresults from a panel of IgM antibodiestested against 8305 and
8306transfectantcelllines ,. R4
Table8: DP131aminoacid sequencevariationsand their associatedhaplot ypes. 104 Tab le10 : Summary of Row Cytomctry and CElISA resultsintheinvestigation
of Raji's HLA-DPtype by monoclonalantibodies , . . 107 TableII: Summary of possible HLA-DP,61 epltopes of Raji cclllincas aresult
of monoclonal antibody testingbyFlowCytometry and CELISA. .. .•. 109 Table12 : Percentage recovery of spleen cellsafter T cell depiction priorto
fusionwith theSp2/0 partnercell... . J16
Table13:Percent positiveimmunoge nreactivehybridsas seen in the primary 119 Table14:Percentage of positive hybrids as seen in thesecondaryscreen. 122
xii
Table15:Pl:rccnt";e positivehybridsas seenafterthetertiaryscreen. • •.•.•125 Table16:Percentagepositivehybridsas seeninthe quaternaryscreen.• •...•128
xiii
LISTOFFIGURES
FigureI:Elevationby specific 195 an tiblJdy.anddcprc!>5ionbyspecific7S antibody ortheprimary immuneresponseoflniccto asingle intravenous injection of4 X 10' SRBCasmeasuredby('QUntingthe:lobinumberof plaqueforming cellsperspleen. • .• ...••. .••.•. . . • •... . Figure2:Theore ticalexplanationoflgPto I- mcdialcdlnbenccment... .•. ... 15 Figure3:TheoreticalexplanationofIgG mediatedsUI/prcssion. ..• . .. 2S Figure4: TheoreticalexplanationofI'!Gmediatedenhancement.,..., :'.1 Figure5:FAC Sanalysistotes t for cross-reactivityofNFLI>.M65 C) (lgM),
NFLD.M68CI IC8 (lgG)aooDPt39BD9(noe-spccifieIgM)antibodies withmouseMHC••,.••.... ....•...•.• ..•. . .. ...• 77 Figure 6:50S-PAG E analysisofNFLD.M6 5C3 and Nl--l.D. M6OC1I C8
antibod ies.•••..•.• . ...•.•.,..••.. .•.•... .• , .. . • .,. 79 Figure7:CELISAresultsofgmupiofC3Hmiceinjectedwilhdifferingdoses
of Rajicells. •• •••..•.. ....,.. .. . .... ... . ... .... K7 Figure8:CE LISAresultsof groupsof C3H mice injectedwithdifferingdoses
of125.4Cells..".,•.•..•• •,...••... ...•... ... . ..,. 89 Figure 9:CE LISAresu lts ofseraobtaine dalday7ofC3Hmicepretreated wilh
HLA·DP specific IgM (NFL D.M65C 3), HLA·DP specific IgG (NFLD.M68CIIC8jandsterilePBSpriortoadministrationof5x 10"
Rajice lls/animal.. , ,... • •. •.,... ... • .• . . . , .• • .... .•.. 92
xiv
FigureII) : CELlSA resultsofC3HmicepretreatedwithHLA-DP specific IgM (NF L D.M65 C31. HLA-DP spe ci ficIgO(NFLD.M68CIIC8) and PBS priortoadministrationofSl 10" cells/an imalOP nan sfected fibro blasts
(125.4). •• . .....•.... ... ...•... •....•. ... . 95
Figure II:CEUSA resultsof C3Hmicepretreated with HLA·DPspecificIgM.
non-specificIg M andPBS prior10administration of5 l 10' L2SA cells/animal. .•. ..••.. . •••.. . .•••..• .• • ..•.• •... .. 99 Figure 12:FlewCytcmetry results of variousmonoclonalantibodies tested
aga insttheRaji cellline. .. . ...• . . . .•... ... 106
A BSA CDC
CELISA D DMEM E ErA Mic:mpla(es ELISA F FACS FBS
GAM HAT lIT IfLA HLA - DP HRr I,G I,M IIV K L N.M.S 0.0
oro
PBS PEG PIS PLLpvcMicroplates R 50S-PAGE . SRBe TEMED
TN.
V y
USTOF ABBREVIATIONS
Adenine Bovineserum albumin Complement dependent cytotoxicity Cellu larenzymelinkedimmunosorbcntassay Asparticacid
Dulbccco'smodified eaglemedium Glutamicacid
Enzyme - linkedimmunoassayabsorbentmicroplates Enzyme-linkedimmunosorbcntassay Phe nylalanine
Flou mscenceactivated cellsorter Fetalbovine serum Goat anti-mouse
Hypoxanthine-aminoptc rin-Ihymidine Hypo xanthine- thymidi ne Humanleucocyteantigen
One of the sixlocicomprisingtheHLA"system"
Hor seradish peroxidase Immunoglobulin G ImmunoglobulinM Intra venous Lysine Le uc ine Nor malmousesera Optical density Orthophenyle nediamine Phosphatebufferedsaline Polyethyleneglycol I%Penicillinand 1%Streptomycin Poly.I-lysine
Polyvinylchloride microplate s Arginine
Sodi umdodccyl sulphate-polyac rylamidegelelec trophores is Sheepredblood cell
N.N.N' -tetra methyle ihylenediamine TerraNovaBiotechnol ogy Valine
Tyrosine
xvi
CHAPTER I
INTROD UCTION
1.[ Hu manLeukocyte Antigen - DP Project
Oneofthe requirementsfor successfultransplantation is to ensureproper matching of the recipientand donorattheMajorHistocompatibilityComplex (M HC)level.The humanMHCcontainsa group of highlypolymorphicgenes, productsof whicharecalled humanle ukocyte antigens(HLA)molecules and whic hare expressed on a varietyotceu surfaces.There arctwo classes of MHCmolecules.Class I and II. The ClassIIgenes consist of DR,DQ and DPloci.DP isa fullyfunctio ning MHCmolecu le, presenting to Tcells andshowingMHCrestriction.Untilnow theemphasis fortissu e matc h inghas been on the DR and DQ loci.
In a collaborativeprojectthatis presently unde rway, the aim has beento produce monoclonalantibodies that reactwith polymorphic determinants on HLA·DPmolecules for uscintissue matching. The productio nof antibodiesto thediffe ri ng epitopeshas proventobea difficultundertakin g,anditwas suggested thai it mightbepossible to make use of the manipulation of preexisting antibodylevels to improve the immunisation schedulesand consequently to increase the yieldof hybridom as specific forepttopes of theDP molecules.
1.2 BasicPhenomenonShownwithRed Cells as Antigen
Aninte resting occurrencehasbeendocume ntedin the literature:whcnsheep red bloodcells (S R BC) areusedasan antigen , the presenceofspecificIgG antibody in the blood at thetime of injectionof antigencan cause theanimal's response tobesuppressed (Heym an,I990;Heyman, 1988:Heymaneral.1988:Quintanact el,1987).Porcmmplc one study reported more than99%of theimmune responsewas suppressed in sucha manner(Sinclair,1978).On the otherhand, pre-existing speci fic IgM(macroglobulin)<It the timeof antigeninj ectionresult sinan increaseintheprimary immunerespo nse(Coulic and Snick,1985;Dennert,1971;Henryand Jeme.19 68; Heymanetet.1982;Heymanand Wigzell,19 85;Heymanetal.1988;Powclleta!.1982;Reiteraet.1986). Apractical exampleof the regulating capacityof pre-existingantibodie s exists inhumans asshown bythe injectionofIgGanti-rhesusD antibodies in to Rh negativemothersatthe time of delivery oftheir Rh+babies. Thistreatmenthassignifica ntly reduced thefrequencyof iso-im munisationagains t the RhesusD antigenand hence hasdecreasedhaemol y tic disea se ofthenewborn (Clarkeetal.1963).It waslater shown by Pollackthat antibody- media tedimmunesuppressionmechanismswere responsibleforthiseffect asopposedto simple clearance ofthe antigen antibody(Pollacketei.1971).
1.3 Review ofLiterature Relating to PrimaryImmu ne Response Modulation
1.3. 1Earl yHistory
To understandhowthisline of investi gationfirststarted one can go backto the early 1900' s ,In1909, Theobald Smithdemons tratedthat injection of part iallyneutralized diphtheri ato xin-a nt itoxinmixturesinduced an titoxinformationin experimentalan imals but thatimmunity didnotresult from mixture s containi nga sufficient excess of antitoxin (S mith,1909).The resultsof theseexperimentswere ex tendedto the studyof add itional antigenssuc hastetan usto xoid (IT), sheepredbloodcells and bacteriop hages (Uh rand Baumann .1960).One ofthemany questionsla terraised from Smith's experime ntwas: Doprc-exis tingantibody levelshaveanyeffec t on theresult of immunisation?
Itwas not until the1960's tha tsignifica ntwork andresultsstartedto appea r in Ihe literature concerningthis question. Investigatorsbegan 10theorize and provetha t pre- existingantibodies couldre gulate the primaryimmuneresponseinbothenhanc ingand suppressive ways.
1.3.2 Land markSt udyby Claudia Henry&NielsIern e
Cla udiaHenryand Niels Jeme,in 1968,investigated theideaofpa ssi vely ad ministere dantibod yhavinganeffecton theprimaryimmune response.The st udy used
inexcessof1000animals .purifiedantibodiesfroma varie tyofmethods,a varietyof antigenandantibodydosesanda varietyofimmuniza tiontimeschedules.Thefocusof theexperimentsWa5 10determinetheeffect ofprc< ltisring75and 195antibodiesonthe resultof immunizationof micewith5RBCon theprimaryimmuneresponseasmeasured byplaque assays. The resultsclearlyshowedthatall7Spreparationssuppressed the responsewhileall 195pre parations enhancedtheresponse.'tbeIgMmediatedincrease wasa fifteenfoldenhance mentinthenumberof pla q ueformingcells(Henry andJemc . 1968).However,in earlie rstudics(Mollerand Wigzell,1965;Pearlman,1967), itwa s shown thatlgMgivenathighdoses (comparableto thehigh dosesofthe Henry and Ierne experiment)couldbe suppressive.TheHen ryand Je me experiment(Figure I)includ ed a broad spectrumofdosesofIgMantibodyandall levelsofdosage weresuccessfulin providing an enhancingeffect.Thc Henry andJemestudyclearlydemonstratedforthe firsttime.theenhancingeffectofpre-existingIgMantibody andthesuppressiveeffectof IgG antibody.
....
-
+19 5
"
eof.! Q V
24' 1fi)154Ql m s u MOLARCONCENTRATIONOF ANTIBODY
rieur.:1:Ekva t ionhyspe elflc19 53ll1ibllrJy. anddep ressionbyspecific75antibodyofthe primaryimmuneresponsent'miceto asinglelntraveeous injection of4 XlOSSRBCasmeasured bycoun t ingthetotal numberofplaqueformingcellsperspleen.Theopensymbols(including 11k!triangl es)representthe averageresponsesof groups of micethat received 195 antibody,the Slliidctrcks repre sentthe average responsesof groupsthat received75antibody.Eachpointis Ilk!meannumber ofplaqueformingcellsper sp leenof20- 50miceafterdeductingthe number of had groundplaqueformingcensshown(HenryandJern e.I968).
1.3.3 IBMMediated Enhancement
Currentknowledgeontheeffectofpre-e xistingIgG and IgMantibody isquite detailedbutunfortunately notcomplete.Theexactmechanismandl ormechanismsofthc)C antibodiesisstillunknown. Thereis.however.a consensusinlheliteraturethatIgM an tibodyhas an enhancingeffecton theprimary response. Inthe case oftheIgG antibody the re isaparadoxicaleffect;insomeincidences theimm uneresponsehasbeen show n 10beenhancedwhileinothersit has beensuppre ssed.
1.3.3.1 SpecificityCharacteristics
FromtheHe nryandJemc(1968)studyitwassho wnthatantibod ies of theIgM classcanha veanenhancingeffecton theprimary imm u ne response.Itisnowknown that whetherparticulate(Wason.I973)or soluble(Enriq uesandKlaus.1984) antigen.. are used ,theenhancementbyIgMisstillobservab le.
ColJissonetst.(1983)further investigatedthespeci ficityoflgMenhancementby simul:.aneouslyinjecti ngtwo antigenstogetherwiththeIgMantibodydirectedonlyagainst one ofthem . Onlythe response10the antigenagainstwhichtheIgM wasdirectedwa~
enhanced.This wasshownbothin anerythrocytesystemwithox andhumanerythrocytes andin apro tein systemwithovaalbumi n(OA)and 2,4-dinitrophenyl-bovineserum al bumin(DNP-BSA). This specificityhas been further characterizedasbeing
antigen/panicle specific butnot epitopespecific.Heymanetsl.(1982) used the factthat SRIJC andgoal redbloodcells(GRBC)cross-react at both theTand B cell levels.After IgM-anti-SRnC monoclonalantibody administration,GRBCor SRBCwere usedas the immunogen.Theythen assayedfor plaque forming cellsagainst GRBC and SRBC.It' wasfoundthatthemonoclonal antibodiesthat cross-reactedwith GRBe enhancedboth the anti-GRBC and theanti-SRBC response.However,monoclonalantibodies usedthat did not cross-reactwith GRBC didnotgive anyenhancement when GRBewasthe immunogen.Theseresults suggestthatthe 'lgMantibodies enhance viabindingto the erythrocytes,therebyallowing otherepitopeson thesame erythrocyteto displayenhanced immunogenicproperties' (Heymanetst.1982). Since theseexperiments.the effects has been clearlycharacterized asbeingantigen/particle specific but not epltopespecific.
Usinga malariavaccine.Harte and his group investigatedwhether specific monoclonal IgM antibodiescouldbe usedtoenhance the responsetoablood-stagemurine malaria vacci ne,Thegroup found that small amountsofa monoclonal anti-parasite IgM couldspectflcilt y enhanceboth primary responseand memory cells in responseto vaccination. These results weresuppo rted bysurvival after infection,In offspring ofthe murinemother,wherevaccination wasineffectiveforup to eight weeksdue to the presenceofmaternalIgG. itwaspossibleto overcomethe inhibitionof thematernal antibodieswhen theIgM monoclonalwas administeredbefore thevaccine, This was described asbeingmadepossiblebytheincreased concentrationof antigeninthespleen as measuredbythe injectionofradiolabellcd vaccinemeasured24 hoursafter injection
in control mice [Harteetal.1983).
1.3.3.2 BasicCriteria
ItwasDennen (1971)thatfirstdescribed thelocaliZ<1tion of the antigenhcin~
enhancedinthe spleen bypretreatmentwithspecificIgMantibody. Dcnncrt IabeI1c:d antigenwith'ICr and injected itafte rIgMantibodyadministration.After ten minutesthe animals were sacri ficedand the rad ioactivityofthe spleensassayed,Comparedto control animals whichreceived noIgMantibody, there wasalen to twentyfold increase in antigen uptakewhen animalsweretreatedwith the specificIgM antibody.IfIgMworks by trappingtheantigen-antibodycomplexes thenthere shouldalsobelocaliza tionfound inotherorgans,such asthe liver.However,the concentrationsof antigenin the liver was not enhanced bythe pretreatmentofIgM antibody(Dennert.1971)ThisfindiJig maybe exptalredbythe factthattheblood content and, thus,thebackground ofantigeninthe liveristoo hightoseefurther IgMinduced concentrationofantigen. DennentcMOOa variety of rimeintervals betweenIgM and antigenadministrationtodcterminea noptimum schedule. Itwas seen thatadministrationof antibody one to two hours before antigen injectionwould resultinsufficient enhancemen t (Dennert,J971).From Henry and Jerne (1968)it wasestablished thatstimulatoractivityofIgMdepends on theamountof administered IgMantibody as wellasthe antigen dose. However, inDennen's experiment. augmentationbyIgM could onlybeobservedwhen antigendoses wereat suboptimallevels(Oennert, 1973). This is incontrasttoHenryandIerne'sexperiment
in which they demonstratedalinearrelationship withno evidence foraplateau or levelling off.Thus, from these experimentsit wasestablished thatcertain criteria must beoptimizedforthe enhancement10beobserved;specifically,the dose of antibody,and an optimum time frame of antibody administration.Dennert alsoprovided a clue as to themechanism involved,showing thatthe antigen localizationin the spleenwas increased bythe enhancingIgM antibody.
1.3.3.3 In VivoversusIn l1troExperiments
Thein wvoinvestigation of theresponseto heterologouserythrocytesin the mouse by Hcnry and Jeme(i968) was also followedup by JohnSchraderin a series of experiments jhar examinedthe effectof specific IgM antibod ieson theinvitroprimary response to sheeperythrocytesbymouse spleencells.Schrader'sclaim was thai in such a system the amountof antigenandantibody to which a given population of immunocompetentcellsis exposedmay be controlledwith much greate rprecision than in thcinWKI situations,wherecompartmentalizationof bothantigenand antibodymay complicatcinterpretationsat many levels (Schrader,1973). Schrader(1973)inreplicating Henry' s andIerne's1968 cxperiment,used antibodypreparations of lgMshownbyHenry andJemc (1968) 10beenhancing; thesehadmarked suppressive propert iesinvitro,while thein wvoexperimentsperformedwiththesame antibody replicated Henry and Jerne's (1968)results.The antibody was prepared exactlyas statedinthe originalexperiment.
The resultsdemonstrateda paradox icaleffect ofthein vimandin vitroactivitiesof
passivelyadministered IgM.
In1973 William Wason also investigated theeffectsof pre-existing antibodyinin vitroandin vi100 experimentaldesigns. His experiment comparedthe effect of antibody givenbefore and after antigenbothin viwandinvitro. The inlit ro experiments allowed for "control"over variablessuch as an accurate quantificationof the antibody halflife.
andeliminationofthepossibilityoftheIgMalteringantigendistributionatthetissuelevel or affecting the selectionof circulatingcellsinto the spleen. The half life of IgM antibodyinthe mousecirculation has been reported as oneday (Brittonct.11.19(8 ) whereas the halflifein vitrowasfoundtobegreater thansixdays (Wason,1973). His designallowedhimto testthehypothesis thatthe IgM antibodycausedaugmentationof theimmuneresponseinvivo.by alteringantigendistributionandthusincreasingthe amount of effectiveimmunogeninthe spleen. Injected IgM anti-SRBC antibody was foundto havea dualeffect onthe primaryimmuneresponse in mice.Theresponse 10 SRBCwas either augmentedor suppressed depending on whetherthe IgM antibody was givenbeforeor afterthe immunizing dose of SRBC.Intheinvitro experiments, IgM anti-SRBCantibodysuppressedtheresponse of cultured mouse spleencells 10 SRBC under allthe conditionsthat were tried. Therefore,the findingswereconsistent withthe hypothesis that IgM antibodyincreases theresponseinvilVbyalteringantigen distribution ,a conclusionwhich had beensuggestedbyDennen's work(Dennett, 1973).
10
1.3.3.4 T CellComponent
AnothercharacteristicofIgMmediatedenhancement isitsdependenc yon T-cells.
Dennert,in testingtoseeifanti·SRBC IgMco uld substit uteforthe helperfunction of T cells,optedto testits action inthymectomizedmice. These mice werethymecto mized atthe ageof3-4 weeks.irradiated and protectedwithsyngeneicfetallivercells.
Experiments wereconducted.asbefore , withSRBCas theantige n. Whenlow or high doses ofantigenwere administeredandthe plaque assays performed,there was no detectable stimulationbyIgM. Dennen also conductedinvitroexperiments.WhenT cell deficientspleencells were used, and lowdoses of IgMwere given,therewasa small (but notsignificant) improvementofthe SRBC response.ThisIgM enhancementwaslower thanthat found inexperiments in which8 cells weremixed withsensitized Tcells (Dennen, 1973).Collissonetst.in1983were also interestedin the Tcellcomponent.
In experiments conducted withnude mice whichlackfunctionallyactive mature T cells, there wasno potentiationoftheprimaryresponse (Collissonetal.1983),confirming the results described earlierby Dennett.
As previouslymentioned,non-specificIgM administered prior to theantigendocs not produceanyenhancement. However,thereisone study that showedenhancementof anti-horsered blood cell responseby monoclonalanti·SRBCIgMantibodywhich fail sto bindtothehorse red bloodcells(Heymanet st.1982).Itwasthe beliefof Collissonet .11.thattheremay exist across-reactivityatthe Tcelllevel,to explainthistype of result.
II
They chosetwo proteinsthatdonot cross-reactat theT·helpercell level.bovine serum albumin(BSA)andova albumin (OA),for furtherexperiments.Inadoptivetransfersof spleen cellsfrom mice injectedwith2,4-din itropheny1(DNP)-OAalone, BSAaloneor11 mixture of the two,into sublethallyirradiatedmice, theeffect oftreatment with IgM anti- 2.4,6-trinitrophen}'J(TN P)wasstudied.SinceTNPand DNPcross-reac t. IgM with TNI' specificity wasusedto modify responses against DNrcoupledmolec ules. A distinc t specificitywasobserved .Only inthecasewhen anti-FN p IgMantibody and DNI'-USA (antigen)weregiven did thenaive mice.whichrecei ved T cells specific\0nSA, show an observableincreaseintheantibodyresponse to BSA and DNP (remarkably.1fourfold enhancement). Thisexperiment alsoincludedgroupswherethe effect of anti-hapten antibody on the pure anti-proteinresponse againstnenccnjugatedUSA or OA was studied.
No effect was observedineitherofthesccases(Cotllsson ct,11.1983).Basedon previous workby this group theauthors were able toshowthc existence ofIgMFcrec eptorson murine Tcells(Lamo n er31.1976). Fun hermore.removalof thisdistinct T cell population(lgM -Fe-recep torpositi veTcells) fromaprimed T-cellpopulationreducedthe helper Tcellactivityinapassivetransfersystem(Andersonctet.1979),JanewaycfIII.
(1971) have shown that onesubpo pulationofT helper cellsrequire s the presence ofU cells 10function.Itispossiblethat suchantibodydependentThelpe rcells(unciion by binding IgMcomplexesvia IgM Fereceptor s.These findingssupport theclaim thatIgM Fc recepto rson helperTcells play afunctio nal role in regula tion of antibody responses inWKJ(Collissonet al. 1983).
12
1.3.3.S Role ofComplement
In1988Heyman and colleaguesconductedexperimentstostudytheimportance of complement facton onthe abilityoflgMtoinduceenhancement.In thesestudiesthree differentapproaches wereused. Inthe first,a mutantIgMantibodyunable to activate complementwas used. Inthe second,mice genetically deficient in complementwere utilized. Finally ,mice depleted ofcomplement by treatment with cobravenomfactor wereproduced. Inthe first approac h,twomonoclonalIgM-anti-TN P antibodieswere used.Oneof theantibodiescontaineda point mutationintheIlchain constant region,
therefore, could notactivate co mplement. The normal IgM antibodyenhancedthe
responsein a dose-dependentfashion,whereas,themutantIgM antibody didnot produce anysig nificantenhancement(Heymanct al.1988). Whencomplementdeficient mice were used.itwas shownthatIgMcanproduce an enhancedresponse inCS-deficientAKR mice, thus.demonstratinglhat at least thelate acting complementcomponents,CS-C9, are not critical (Heyman etIll.1988).In thethird case,miceweretreatedwhhcobra venom factorand then injected withpoIyclonallgM·anti-5RBC.IIwaspreviously shown
thatin";10doses ofcobra venom factorcausestransientdepletionofplasmaC3toleu
than SlJL ofnormal levels(Pepys,1975). After a periodof time the mice werethen injected withthe antigen,SRBC.Thistreatment abolishedthe six-fold enhancingeffect onthedirect plaqueforming cellresponseof ISM seen incontrol mice immunizedthe sameway.Thesestudiesclearlyindicatethatthe enhancingeffectoftheIgMantibody isdependentuponcomplementfactors,prob,'lblythose acting beforeC5.
13
1.3.3.6 PostulatedMechanisms
Of the postulatedmechanismsofIgM enhancement,one hypothesis suggests!lull antigen-antibody-C3bcomplexesaretrapped illthe spleen.possiblyonthe follicular dendritic cells.which carry complementreceptors (Dennert.1971.197J: Heinenc.'f..I.
1985). Supporting evidence comes from studiesshowing failure of IgM pctenuarion when IgM is injected afterthe antigenindicatingIhat the immunecomplexes are in some way responsible for the enhancement.in addition10evidenceprovided by thein111m experiments(Schrader, l97 3). The trapped immune complexes thencan capturen cells carryingantigen-specificreceptors and C3b receptors(secFigure 2,page 15).
Crosslinked C3b isknown to stimulateactivatedBcellsto proliferateandsecrete immunoglobulin(Erdei eta/.1985).Additional help may be provided byantigen-specific Thelpercells that are activated more efficientlydue toincreased ccncenuutionsof antigen ana, possibly,by IL·I secretion by thefolliculardendriticcells(Heymanet11/.198M;
Heyman,I990b).
14
Figure2:Theoretical explanationofIgM-mediated enhancement.Hypothesis:IgM•antigen complexesactivatecomplement and stimulate B cells viacrosslinklngofcomplementandantigen receptors.Inaddition,increased antigen localization(viacomplementreceptors)onfollicular dendrith:cellsin germinalcentres may playan importantrole[Heyman,1990b).
15
1.3.4 (gO Mediated Suppression
Heyman and Wigzellwiththe aid of a simple enzyme-linked immunosorbent ass..'\Y (ELISA),were able to showthat mice pre-treated withIgO antibodyand then administeredantigenshowed suppressedlevels of bothIgO andIgM antibodylevelsin the primary response (HeymanandWigzell.1 985).
1.3.4.1 Specificity Characteristics
IgO antibodies can specificallysuppressthe humoral immuneresponse against particulateas wellas solubleantigens (HenryandJerne,1968; Bruggemannand Rajewsky,1982; Heyman andWigzell, 1984). Studies have shownthat theIgG suppressionis non-cpitopespecific butis antigen/particlespecific(Bruggemannand Rajewsky,1982).IgO antibodiesbindingto one epitope suppressthe antibodyresponse to allepitopes of theparticularantigenused (Heyman,1990b).
Wiersmaet st.(1989)studied the abilityof IgO10mod ulate the immune response against TNP-SRDC andacontrolantigen,horsered bloodcells(HRBC)withtheuseof a polyc1onalantibodyandpanel of nineIgO TNP·s pecificmonoclonalantibodies. Rot h antigens(TNP-SRBCandHRBCtogether)wereinjected into miceone totwo hours after administrationof the antibody.Theplaque for ming cell responses againstSRBCand HRBC werethen determined . Five of tbe nineIgO TNPspecificmonoclonals andthe
16
IgG anti·TNPspecific polyc1onalantibody were able to suppresstheresponseto SRBC whereas therewasno significantsuppression observed ontheHRBCresponse (Wiersma nsl:1989). Wiersma'sgroup also examinedtheantigenbinding propertiesof the variousantibodies used.The antigenbindingcapacity was tested in threedifferent assays :bindingtoSRBC<oupledTNPin an ELISA;ammoniumsulphate precipitationby the Farrtechnique; hemagglutination.II was found that the antibodiesthat wereunable to suppressthe responsein"100gavesignificantlyweakerbindingresultswith SRBC as compared to the antibodies that were suppressive (Wiersmaclal.1989). Thus, suppression is antigen specific, ron-epitope specific,and theantigenbinding ability (affinity) ofthe IgGantibodyappears tobeof importance.
1.3.4.2 Ability of VariousSubclassestoSuppress
Ithasbeenshownbya numberof investigaton thatallsubclasses of IgO canbe suppressive (Bruggemann and Rajewsky,I982; Heyman and wlgzell, 1984).
Bruggemann'sandRajewsky' s mainaimwps to studytherole of antibodyclass in feedback inhibitton.Feedbackinhibitionhasbeenone of the proposedmechanismofIgG suppression andit implies thattheIgO antibody is abletodirecta negative signal orable toblock the productivephase oftheprimary response. Bindingofantigen-antibody complexesto Fc receptors mayplayacrucialrolesince Fe receptorsof various isotype specificitieshave beenfound onTand B lymphocytesand onmaerophages (UnkeJesset al.1981). ItwasBruggemann's and Rajewsky'sbeliefthatthesereceptorscould well
17
confer isotype specificity to feedback inhibition. They foundthat suppression was equally achieved withIgGuIgG1. ,IgG)andIgGl~'Thus, they concluded that antibody feedback inhibition isnot a function of a particular IgG subclass.and that the antibodyneeds no property otherthan its affinity to the antigen(Bruggemann and Rajewsky,1982).
1.3.4 .3 Experiments involving F(ab') lFragments and theFcReceptor
Asearlyas (969,investigatorshad been curious as to theroleof the Fefragment of antibody in the signalling process of an antibodyresponse. Sinclairwasspecifically interestedin ascertainingwhether binding of antibodyto antigen.and the maskingor antigenicdeterminants.are sufficientto induce the suppressionof the immune response.
In Sinclair'sexperiment he was able to remove the Fe portion of the IgG antibody.In this way the bindingof the antibody to antigenwasnot altered.TheresultingP(ab')1 portionwas testedfor itsabilitytoinhibit the primary immune response.He found that the F(ab'hportionswere 100 to 1000 times less suppressivethan completeantibody (Sinelair, 19 69). Theseresultswere later confirmed by others,Heyman (1990) and Gordon and Murgita (1975). Heyman (1989) again investigated the role of the Feregions ofIgG antibodiesin IgG mediated suppression using antibodiesblocking cellularFe receptors. Heyman found thatwhen she used theseantibodiesit reversed theabilityof the IgG antibodies to induce suppression (Heyman,1989).From these experimentsit may be concluded that interactionsbetweenFe regionsand membrane bound, or possibly secreted Fe receptors,are crucial eventsin IgO mediatedsuppression.
18
1.3.4.4 Density Factor
Ithas beenfound thatthehigher the densityof lgG antibod ies bound tothespecific:
antigen,themore efficientthe suppression ofthe responsebytheparticular180 antibody (Quintanaetaf.1987).Two factors involvedinthesuppression. therefore ,areaffinity and the numberofepitopes recognizedbyanadministeredIgOpreparation. Thismay explainwhy someresearchershavefound thatpolyclonalIgG isusuallymoresuppressive thanmonoclonallgG (Bruggemannand Rajewsky,1982;Wiersmaetaf.1989). Some researchers have commentedonthis. suggesting that theremaybeathreshold density dividing monoclonalIgG antibodiesinto thosethat can suppressandthosethat do not suppress (W iersmaetal.19 89).When mixturesofdifferent monoclonalIgG antibodies are used thesuppressiveeffectoftheindividual monoclonals is additive(Heymanand Wigzell.1984). Ithas beensuggested thatthelinkbetweenthe suppressiveeffectand densityof IgGontheantigenscanbeexplained byincreasedbindingto Fereceptorsby thehigh-densitycomplexes (Heyman.1990).Aninterestingexperiment to test thiswould betoseeif adivalent antibody (one withtwospecificities) wouldgivemore5Uppre ssion ascomparedtoamonoclonal antibody.
1.3.4.5 In UtroExperiments
Anumber of researchershave confirmed thatIgG induced suppression is
19
observableinvitro.(Abrah amsetsl.1973:Kappleretet.1973;HoffmannetIll. 1974).
In one such experimentspleen cells were cultured with SRBC aloneor with SRBCand anti-5RBCIgGantibody;anumber of different ratiosofantigen to antibodywere used.
The plaquefonningcell kineticsweremeasuredoveranumber of days. The results clearly showed that culture s containing antigen-antibodycomplexesgivenat the beginning of the culture suppressed the appearanceof plaque formingceltsat all subsequentdays of cellculturing(Oberbam scheid t andxolsch.I978). Thedegreeofsuppressio n depended directlyupontheconcentrationofantibod iesinthe complex .These experiments indicate thatantibod y-antigencomplexesin vitrocan efficiently inhibit theinductio n of antibody formation.
1.3.4 .6 Roleof T Cells
Oberbamscheidtand Kelsch(1978)alsoexa mined the role of T cellsinIgG mediatedsuppressionwith the aid of nude mouse (nu/nuBalb/c )spleen cellsinanin vitro setof experiments.Itwas first establishedthatspleen cellsfroM nu/nuRaiblemicecould respond (produceantibodies) tosheepredbloodcellsor to TNP-SRBCin the presence of supernatant containingthymus-replacing factor obtainedfromconca navalinAactivated normalspleencells. Itwasfound that this responsecouldbe blocked ifanti-SRBCIgG antibodywasadded to the mouse spleencellculture. Thedegree of suppression was .simila r tothat foundwhen nonnal spleencells were used.With this particulardesignthe researchers were confidentthat the Tcells were not directlyinvolved in suppression.but
20
thepossible effectofa suppressive T cell factorin thesupernatantof the concanavalinA activatedspleen cellswas notincluded.Atthistime, concanavalinAactivated Tcells were consideredtobesuppressive.Totestthis further, the effectofantigen-antibody complexes on nu/nuRaible spleencellsinthepresenceofantigenand lipopolysaccharide (I.PS).anon-T cellstimulator, was studied. Againit wasshownthat the cultures containingtheantigen-antibodycomple xeshada significantlysuppressed response.The researcherswerethenconcernedwith thepossibilitythatanti-Th y-1.2 sensitive immature T cellsmightmediateantigen-antibody inducedsuppression.Thesuppression of the anti- SRBCresponse inculturesofanti-Th y-1.2 treatednormalor nu/nuBalb/cspleencells supplemented withthymus replacingfactororLPSwasthenexamined. Once again, immunecomple xes suppressed theimmune responseshowingclearly thatthe suppression ofIgG antibodyisnot dependentuponthe presenceofT cells,(Oberbamscheidtand Ko lsch,1978).1be datasuggeststhatantibody- antigencomple xesdirectlyblock Bcells (ortheiraccessto antigen)insuch amannerthatthey cannotbeaffectedbymaturation signals, such asLPSorthymus-replacingfactor.
TheaimofHo ffman and Kappler(1978)was10investigatetheinhibitory action of tgGantibodyinrelationtothe Fereceptor. Theyfoundthatinhibitoryantigen. antibodycomplexesdid noteffectfheacti vity ofT helpercellsorantigen reactiveBcells.
They took advantage of factthat SRBC and burrored blood cells (BRBC) cross-react at the T-cell level,but,notattheB cell level. SRBCprimedhelper Tcells stimulated fo rmation of antibody\0bothSRBCandBRBe.Theywere abletoshewthat non-cross
21
reactingantibodyagainstSRBC inhibited the production of antibodyto SRBC butdidnOI diminishthehelpereffect of SRBCprimedT cells on BRBe rea ctiveI: cells.
Conversely,TNP-reac\iveB cells(respondingtoTNPcoupledSRBCandBRBC) failed toproduce antibodyto TNP-SRBC in thepresenceof antibodyto SRBC,but,remain responsiveto TNP-BRBC insuch conditions, Theresultsindicate thatneitherT cellsnor B cellswere immediate targets ofthesuppressive antibody,Hoffman andKapplerWere then curioustosee if theprocessof cooperation between T andB cellswas altered.TIley testedthree types of antibodiesto SRBCin experimentsinvestigating the inhibitionof a anti-SRBCresponseinspleencellcultures:IgG rabbit anti-SR BC,IgGrabbit SRne- F(abhandchickenanti-SRBC (Fe portionunableto recognizemammaliancells). All three inhibited the productionofan anti·SRBCplaque formingcellresponseathigh concentrations. However , onlyintactrabbit antibody was inhibitoryalsoatalow concentration.Theaddition of serumcontaining tumournecrosis factor(p reviouslyshown toreplace thehelpersignalandsupport the production ofantibodyin identicalspleen culturecells depletedof T cells) reversedthesuppressive effcetat low concentrations hut not athigh concentrationsofrabbitami-SRBCantibody. Theinhibitory effect of increased concentrationsof rabbitanti-SRBC-F(abhantibody or of chickenanti-SRBC was not reversedby the "tumournecrosis serum".Thus,these results support thebeliefthat antibodycan inhibitthecooperationofTand Bcells.Thisinhibitoryeffectwasdescribed as being"reversiblebyreplacingthe signalwhichresultsfrom thatcooperation". Th us, the ideas that T helper cellactivityis unaffected andthatT -Bcellcooperarlo nwas disturbedbythe inhibitoryantibodyweresupported (Hoffmanand Kapplcr,1978).
22
1.3.4.7 Role ofCompJement
Heymanc/al.(l988b)investigated the role of complement in IgG mediated suppression. In oneexperiment they chose two different IgO anti-TNPantibodies.one that could activateand onethat was unable toactivatecomplement. Bothantibodies bound equallywell tothe antige n,sheepredblood cells coupled with TNP.To avoid detecting suppression caused by the blocking of antigenic determinants by immunoglobulins, the researchers chose to measuretheanti·SRBCresponseas opposed to the anti-TNPresponse.Micewere giventhe antibody preparationone,two hours priorto administrationof antigenandplaque assays were performedonday five.The non-complement activatingantibodysuppressed up to 94%ofthe SRBCantibody response. However.thisdegree of suppressionwas inthe samerange as thatofthe complementactivatingantibody. These researchers alsopresenteddata showingthat polyclonalIgOanti-SRBCantibodieswere as efficient as suppressors inmicedepleted of more than 90% of their serumC3 by treatmentwithcobravenomfactor,asthey were in normal mice. Theresponseto sheepred bloodcellsin AKRmice. geneticallyCS deficient. was alsoefficientlysuppressedbyIgO antibodies.These data clearlysupport thebelief thai complementfactorsare norinvolvedinIgG mediatedsuppressionof the primary immune response (HeymanaaJ.1988b).
23
1.3.4.8 Memory
Heyman and Wigzell (1985) conductedexperiments to determineifIgG mediated suppression of the primary response alsoaffectedthe secondaryresponse,i.c .suppre ssed the production of memory. Mice were either primed with SRBCaloneorwith polyclonat IgGanti~SRBCantibodies followed by SRBC.atadoseknownto give optimal suppression. Thissuppressionwas confirmedoneweeklater by adirect plaqueassay usinga sampleofthe animals.After24-90days. spleeneells from otherprimed animals weretransferred toirradiated syngeneichosts together withantigen. The seco ndary response was measured by directand indirectplaqueassays.BothIgG-SR BC andSRBC primedcellsgave ahigher responsethan did unprimcdcells.However,compared with groups primed with SRBC alone, the responsewas greatlysuppressedinthe IgOtreated groups. Thisexperiment ledthe invest igatorsto conclude that the supp ressionof the primary antibodyresponse,caused bypassivelyadministeredIgG.is alsoextended to the secondary response whichshowed inhibited inductionof memory (Heymanand wigzcll , 1985).
1.3.4.9 Aeccssory Cell Invo lvement
B cell involvement has also been reportedinthe literature. Ithaslongbeen recognizedthat derivatives of C3 playa vital rolein phagocytosis(Ehlenbergcrand Nussenzweig, 1977)As mentionedbefore, no decrease in suppression was obscrved in
24
animalsdepletedof complement. Kolsch ' s groupconductedinvilTOekperimen tsin which macrop hageswere depleted fromthecultures. These macrophage depletedcultures shewed the same degree of suppression asnon-depleted cultures,(Kelschel el.1980).
Another supportingpiece evidencecomes fromin vitroexperimentswherethe importa nce ofthe Fefragment ofrabbitanti-mou sekappachain antibodiesinpreventing the activationof murineBcells was studied. Intact rabbit anti-mousekappachain antibodiestotallyfailed to induce tritiated thymidine incorporationoverathreelogl~
concentratio nrange. However,F(ab')2fragmentspreparedfromthese same antibodies led toa 'marked' incorporation oftheradioactivelabel. A mixtureof theintact antl- kappa antibodies andthe F(ab 'h fragments derivedthere fromagain prevented activation.
Theresearchers then went on toinvestiga teFeinvolvement. Thisideawas studied using goatanti-moosep chain~ficantibodies which.evenwhenintact.produced significant 8cellactivation.Thestimulatorycapacitycouldbeincreased at any time testedbyusing goatF(ab'hof anti·mou se".chain specificantibodies.'Theresearchers concluded thatthe immunoglobulinreceptorofmurineBcells can serveasatriggering receptorprovided tl\ata negative signal mediatedby cross-linkage involvingthe B cellsFcrecepto ris avoided(Tony andSchimpl.1980).
Accessory cell smay alsobeinvolvedin suppression. "[Increa sed)...FeR·
mediated phagocytosi sofimmunecomplexes may explain the requireme ntfor FeR binding,the antigenspecificityandthenon-epitope-specif ichy"(Heyman.l990b), Asit
2S
stands. definitive dataon theeffectofaccessoryand Bcellinvolvement i$ still needed.
however.themostplausible explanation. asof now,may lie in the combinationof the two (Heyman,1990b).
1.3.4.10 Postulated Mechanisms
Thcliterature containsa numberof theories on the mechanismofIgO mediated suppression.Theycanbesummarizedinto three main theories.Thefirst proposes that suppressionisthe resultof simpleblocking of antige nicdeterm inants byIgO antibodies.
These determinantsarethenhidden fromimmunocompetentcells(Hoffman and Kappler.1978;Moller,1985). This mechanism is independent of the Fe compone ntofthe IgG antibodyandis contradictedbythelite rature cited in thisreview which documents the need fortheFe region. Thesecond theoryhas beentermed the'periphera l theory' (RyderandSchwartz,1969) andcontendsthat the eliminationof antige n ismed iatedby increased phagocy tosisoftheIgO-antigen complexes.However,comp lementis known to increase phagccytosiaof immunecomplexes(Egwang and Befus, 1984) and thefinding thatimmunosuppression isindependent ofcomplementactivation, therefo re, arguesagainst thistheory. The thirdhypothesis,the'centraltheory'. proposesthatthe immune complexescross-linkFe and antige n recepto rs onBcells thereby inducing an offsignal to thecells(Oberbamscheidtand Kelsch ,1978; O'Garraet al.1987;Ph illipsand Parker, 1985;Wiersma et at.1989 ;Heyman,1990b). The biochemicalbasisofthe result of the crosslinkingofFe andantigen receptorsis not completely understood.Inmanycell
26
types, bindingof ligandstotheir receptors causes anearly increase infreeintracellular calciumand a rapiddegradationofphosphatidylinosito l bisphosphate to inosito l trisphosphateandI,2-diacylglycerol (Berridge,1984).Oneparticularstudyexa minedthe effectofcrossJinking ofsurfaceim munoglobulinandR:receptorsandthedegradationof phosphatidylinosilolbisphosphateandcalciumcationconcentta tion,twoearlybiochemical responsesinactivated8cells. Theresul ts indicatedthattherapi ddegrndationof phosphatidylinositolbisphosphatewasabrogatedbuttheincreaseincalciumcatio n concentration remainedthesame(BijsterboschandKlaus,1985). Thesefindingsmay explain and/or provide a frameworkfor understan dingthe modeofactionofthe Fe regions of theIgGandtheFereceptorsin regulatingtheact ivation of Bcellsinconce rt wilhtheirantigen recepto rs(see Fig ureJ). It isquite possible.howe ver.thatmorethan one mechanism maybeoperating.
Figu re3: TheoreticalexplanationoflgGm~d iat~dsuppressio n.Cnlssll nklngofacriticalnumber of Fereceptors and antigenreceptorsontheBcellksu[L~ina negativesignalwhichcannothe reversedbycomplementrecepto rhinding(Heyman. I99Oh).
28
1.3.5 IgGMediated Enhancement
Enhancement induced by passivelyadministered 19G is thoughtto mimic the action of naturalIg G antib odies inanormal primaryresponse and/orthe actionofantibodies producedin aprima ry responseonthe secondary immune response(VanRoo ijen,1990).
The complexi tyof the eventsassociatedwith passive antibodyadministratio n is furt her increasedby the observationthat, under certaincircumstances theprimaryresponse itself isenhanced ratherthansuppressed,with pretreatment of IgG antibody(Co ulieand Van Sn ic k,1985; Parkusetsl.1982; Wiersm aet al.1990;Wiersma,l 99 2), This is incontrast to the prev iousrepo rts describingexperimentsinwhichpretreatedIg G mediated suppression. Unfort unately, to date,research andthusthe literaturedonot conta ina complete de seription ofthis phenomenon. Antigencharacteristics suchas particulate vers us solub le,mayplay arole.forexample,the same TNP·spedfi cIgG monoclonal antibodiesthat suppressedtheantibody responseto partic ulate TNp·SRBChave opposite effectsonthe response to TNP-KLH,a solubleantigen(Heyman, l99Ob).Otherfac tors postulated as beingimportantare densi tyofepltopes,andthe roleofcomplement.
1.3.5,I Respo nsetoDifferingAntigensandLocalizatio n
In studying the specificenhance mentofthe antibodyresponsestoprote in antigens byIgG amibodiesinliw, Wiersma inj ectedan tibodybefore and after antigenhadbeen clea redfromcircula tion.andattemptedto dete rmine iftheantibod y acts by increasing
29
antigen uptake(wlersma,t992).Theresponse 10ke yhole limpel haemocyanin (KLH)- TNPantigenwasenhancedbyaTNP specific IgGmonoclonal ;antibody.High andlow concentrationsofantibodyandantigen wereusedandallproducing:anenhancedresponse.
Wiersma thentriedotherantigenssuchasBSA-TNP .OA-TNP,IT-TNP anddiphtheria toxoid(01)-TNPin similaresperimenrs.Ofthese.onlythe response10BSA·TNPwas enhancedby thespecificIgO.In order 10enhancetheresponse 10KLH·TNPand BSA- TNP.theIgO antibodyhad 10beinjected whiletheantigenWlUcirculating intheblood stream. Thesedata suggestedthatIgGmediatedstimulation actsbyconcentrating circulating antigenintothesecondary lymphoidorgans (Wiersma,1992).therebygiving a more efficient presentation ofantigen10theBcells.
1.3.5.2 Density
Wiersma (1992)fo und thatIgO antibody caused optimumenhancementwhenit recognizeda low number of epilopeson the administeredantigen.Thus,as seen before.
densityof epitopesalso seems tohave animpactonthe primaryresponse.
1.3.5.3 Role of Complement
Therole ofcomplement alsowas investigatedbyvariousresearchers.Wiersma etsl.(1990) examinedtheimportanceof complementtoIgOmediated enhancementby immunizing mice withTNP-KLHafterinjectingthemeitherwitha complement-activating
30
TNP-specific monoclo nal lgGantibody,or with a variantofthis antibody which was non- complement activating.Both the nonnalandmu tant antibodiesenhancedthe anti-KLH response, although thenormalantibodygave a stronger enhancement. Thus,indica ting thaithe classicalpathwayis likelyto be of someimportan ce, In another experiment,a complement-activatingIgGantibodyenhancedthe antibody responsein mice depletedof C3 bytreatm ent withcobra venom factor.Ana turally occurringIgGnon-complem ent activating'r'Np-specifi c antibody, in the sameexpe riment,was also able10enhance the responseto TNP-BSA.These findingsdemonstratethattheability10activatecomplement is not an absoluterequiremenlfor theability toenhance (Wiersmaeta1.1990),Ithas been earlier shown that thehaemolytic capacity of antibodies mainly requiresclassical pathwayCactivation, indicating thatthe classical pathway is the main means by where IgO activates (Bindonetet.1988),Thedata demonstratingthatC-activatedIgO is an equallyefficientenhance r ofthe antibodyresponsein normal mice as in mice depletedof theirC3,also suggests thaithe alternative pathway isunlikely to playarole.Although these miceha ve low traces of C3 in circulation itis not believedthat there is enough for completely normalenhancement(Wiersmaetst.1990), Thus, from these studies the requirement to activate complementis not essentialto theIgG mediatedenhancement suggestingthat there may be anothercooperati ngfactor orfactorsinvolved in the enhancement process. Interestingly,these studiesdo not address C4b,since italsobinds C3b receptorsandwoul dbeactivated inthe absence of C3,
1.:15.4 Mechanismsof IgG Mediated Enhancement
31
Although the inve stigators whoproposed the vari ousmec hanisms have demonstra tedthatco mplem e ntmaynotbenece ssaryfor enhancement,involvementof complementis stillamajorpart of the speculation.Itmay betheantige nch.1ractcristics, suchassize,and theden sityof epitepesrecognizedbytheIgGantibody thatmight influencetheinte ra ctionbetweentheimmune complexes,complementreceptorsandtile Fereceptors(Wiersma,1992).Aspreviously mentioned.evidenceio-date implics tbal IgGsuppressionactsbycross-linkingmembraneimm unoglobu li nandFc recepto r(type two) ontheBcell,withacriticalsize of the antigen needed forobtaininganinh ibitory cross-link (Heyman,I990b). Some researchersfavoura model of IgOme dimed enhance me nt where IgG-2.nt igencom plexes arecap tured by folliculardendriticcells.
whichcarry FcRas wellas comple mentreceptors(He inenctsl.1985 ;Wiersm acl af.
1990).Boththeabi lityoftbeIgG antibodyto bind totheFeR .aswell asthe ab ility to activatecomplementart essentialprope rtiesinthismodel.This modelwouldalso elpbin why non-complement activati ng monoclonal(gGantibodi es gaveslightlylessstimubt ion as comparedto complement activatingIgO monoclonalantibodies(Ca rtertfet.1988).
Bcells mayalsobeinvolved .Enhancementmaybeinparidueto thestimulationof B cellsbycomplementfragmentsviatheir com plementrecepto rs(Melc herstfal.1985).
Heyman(I99Ob)hasproposedthattheenha ncingeffectdepe ndsontheabilityof IgG antibodyto bind bothfeRandoom plemcnt receptors (sec Figure4), thus causing efficient localization on follic ulardend riticcell s,whichcanybothtypes of rece p tors.In addition to this,thecomplexescouldstimulateBcellsdirectlyvia complementrec eptors onthese cells.
32
B("~11
antrqen receptor , / FeR ...'---I-- -+--->...I_
/ '
.\.--._~
no ellecl
Figure4:Theoretica l expla nation ofIgGmediatedenhancement.IgG solubleantigen complete s cannot inducethe criticalnumberofFereceptor'antigenreceptorcrosslinking on tileB cellsurfac e to induceanegativesignal(Hey man,199Ob).
33
1.3.6 Summary
1.3.6.1 IBM Mediated Enhancement
Theenhancingeffectonthepri maryimmunerespo nseby pretreatmentwith specificIgM antibody has been welldocumented.Theeffect canbedemonstrated with both particulate and solubleant igens. With respect tospeci ficity.therespo nse canbe described as antigen specific butnotepitopespecific. Atime fac torexists, such that administration ofspecific ISM antibody onetotwohour s priorto antigenresultsin optimal enhancem ent.IgMantibody is toableenhanceantigenlocalization inthespleen.
In vimdemonst rationofenhancementwas not replicatedin vitro. In fact.invi tro expe ri mentsresulted in a suppressionofthe primary respon se. ISMenhancement is furtherdependent uponTcellhelpandupo n complementfactors,speci fically uose:lCting before 0. Increased antigenlocalization on follicular dendritic cells bycomplement receptorsisbelievedto playarole.Themostplaus iblecellularmechanismis stimulation ofBcells viacrosslin kingofcomplementand antigenreceptors byIgM•antigen complexes,
1.3.6.2 IgOMediated Suppress ion
Ithasbeenclea rl y shown thatmicepretreatedwithspecific IgG antibodyeahibit
34
suppressedlevelsofboth IgG andIgM antibody levels inthe primaryresponse. IgO antibodies canspecificallysuppressthe response against both particulateand soluble antigens. This suppressionis non-epitopc specific butis antigen specific. IgO antibodies binding to oneepitape suppress the responseto all epitopes ofthat same antigen. All subclasses ofIgG canbe suppressive. Other factorsaffectingsuppressionincludethe affinityof the antibody and the numberof cpitopes recognizedby an administeredIgG preparation;increasesin either havethe effect ofa greatersuppression.F(ab')lfragments of specific IgO antibody werefoundtobesignificantlyless suppressivecompared to complete antibody.Thus,Fe receptorsare probablyrequiredfor suppression.In vitro experimentst.avc alsobeen ableto demonstrateIgGinduced suppression.Erperiments have alsoshownthatthe suppression by IgG is notdependent upon the presenceof T cells noris complementneeded.T helpercell activity is unaffectedinantibodysuppressed mice.Suppressionof the primaryresponseis also extended to the secondaryresponse, shownby the inhibited induction ofmemory. Of the proposedmechanisms for IgG mediatedsuppression.thecentral theory seems to fit best withtheabove findings. This theorycontendsthat immunecomplexescrosslink the Fc and antigenreceptorson the B celltherebydelivering a negativesignaltothe celtwhichcannotbereversed orovercome by binding of complement receptors.
1.J.6.3 IgG MediatedEnhancement
The fieldof modulationof immuneresponses bypre-existing antibodyhasbeen
35
considerably complicatedbythe finding, incertainexperiments withsoluble;lntittcn,that specificIgG cansometimesenhancethe response.This contradicts an earlierconceptthat IgMenhanced, whileIgGsuppressedtheimmune response. Although someexperiments havedemcnstn-cd that complement appears nottobeimportantto IgG-mcdi"tcd enhancement.paradoxically,the proposedmechanisms ofthe IgGmediatedenhancement viewcomplementas playinga vitalrole. Themost widelyacceptedmechanismisthat this suppression resultsfrom the antibody-antigencomplexesnot being able toinduce the thresholdnumber ofFeandantigen receptorcrossfinkson thesurfaceof theBcell.
36
CHAPTE R2
AIMSAND OBJECTIVES
2,I ResearchGroupObjectives
One overallobjective of our researchgroupis tomake mousemonoclonal antibodies to polymorphicdeterminantsonHLA·DPmolecules. Asthisis by nomeans easy orstraightforwardwe havebeenlooking forways toimprovethecreation of hybridomas fromimmunised mice. Amongseveralprojects withthisaim is the present one,namelyto see if antibodyenhancementcanbeusedto improvethehybridoma technology and to explorethepossibility that pre-existingIgGproduced, forexample,by repeated immunisations might actually reduce thesubsequent immuneresponse to class IIHLAmolecules.
2.2 Specific Thesis Objectives
The particular aims of the experiments.reportedin this thesis,were:
I)Todetermineifthe same phenomena are observedwhen usingeither humanBcells or mouse fibroblasts transfectcd withHLA·DP,as antigens;namelythat aspecific IgM
37
antibodycouldenhance and conver sely,anIgG antibodycouldsuppress, the primary immunerespon se10HLA-DP antigens.
2)Tostudy thefeasibilityof usingIgMenhance menttoimprovethe yieldof HLA·I)P and HI.A-ORspecifichybridoma s frommiceby decreasing the timenecessary10produce hybridomas or by increasing the numberof hybrido mas specific for HLA-DP and HI.A- DR.
38
CHAPTER 3
MATERIALSAND METHODS
3. 1 Animals
Balb/candC3H mice obtained fromthe breedingfacilities of CharlesRiver Canada,Inc.(188 Lasalle,St-Constant,Quebec,Canada)and FIhybridmice resulting fmm a cross betweenBalb/cm~!,'sand C3H femaleswere used.The Fl hybrid mice were bred bytheAnimal Care Unit personnelattheHealthSciencesCentre.Memorial Universityof Newfoundland. All animalswereapproximately 6-8week old females unlessotherwisestated. The micewere housed instandard clear plasticcages and allowed food andwater atwill. Animals werekeptat theAnimalCare facility in the Health SciencesCentre.
3,2 Antibodies
The two primarymonoclonal antibodies used to manipulatethe prc-existing antibodylevels were NFLD. M65C3 and NFLD,M68CIICItMonoclonalDPI39BD9was usedas aIgM non-specificantibody. NFLD,M67 was usedin theassays asa positive control.
39
Table I:Sum ma ry of MonoclonalAntibodiesUsed With Respect to Class and Specificity
ANTIBODY ISOTY PE SPECIFICITY
NF LD.M65 C3 10M IILA-DP.U""lIline. -n:,,, 1I<
wilhancpilt>re 1i\Ulldtln.1I nt' IYr<:s.l'",t ymll'l'hi<:wilh
trtln~Ii.'eI.'1lfihrul>lllsls
NFLD.M68CIICB IgOll• fIIA-OP.B"clIlinc~-!'Cud.•
w;lhll ncl';l<lf'I'f"unJtlll.lI l>1' lyr""'"nUIrlln~ti.'eIL'llIi",, ~lIaML'ell
line.
DPI39 BD9 10M MIlu....llnl i"t1dy .<It "'. "' ~ "inllt"
hlllTUlnlllA
NFLD.M67 IgOI III.A ·DP.BLoclllillt:s-reIId~
wilbllnc l'iblf'Cfuu nll,1(1l1l1 01' IYl"l" lInJtn n1lf""I...J fihr..lll",bI
Monoclonal antibodiesweresecretedbythehybrid cellsintoDulbecco' sModified Eagle Medium (DME M)containing, 10%hea t inactivatedFeta lBovineSerum (PBS) suppleme nted with I% sodium pyruvate, 1%L-Glutamine, I% Penicillinand
40
Streptomyci n,(GibcoBRl, CanadianLife Technologies,Burlington,Ontario, Canada). All hybridcelllines wcre grown at 37°C,in a5%COlincubator. These monoclonals wereob tainedfrom, andha ve beenexaminedfor the irrespectivespecificities previously in thisla borato ry,
3.3 AntibodyPurification
IgMandIgOhybridomace lllineswereallowed to overgrow in approp riatemedia fo r34weeks.The antibodycontainingmedia were first centrifugedat3400rpm for 30 minutesandthen concentra tedat 4(1Cwithan AmiconUltrafiltration Cell,Mod el402 (ArniconInc,USA).
3.3.1 IgM Purification
Inthecase of theIg M antibod ies,the concentratedantibodycontaining culture supernatantwas applied to an Anti-Mo use IBM(I{chainspecific)•Agarose columnand purified accordingly(SigmaChemica lCompa ny,USA). Thecolumnwasequilib rated in a.DlM sodium phosphatebuffer, pH7.2containing0.5Msodium chloride. The supernatant was appliedslow lyand re-cycled atleast threetimes. The colu mnwas strippedby washing with0. 1Mglyci ne,O.15Msodiumchloride,pH2.4.Onernilhlitre fractionswere collectedin 1mlofTris·HC lbuffer , pH9.0 ,onice; this procedure immedia telyre-adju ststhe collectedfractionsto a neutral pH. The Bio-Rad Econo
41
Syste m, Model ES-I (Bio-Rad, USA) was used in order10monitor column washing and theelutedfractionsfor protein.Fractionswerethen pooledand measuredlorproteinby a Ou Series60Beck man Spectrophotomete r, Nucleic AcidSort-PucModule(Beckman Instrume nts, USA). The sample was the n dialysed llSi n ~ SPECT RA/POR Molecularporo us MembraneTubing 2 (molecular weightcut off :1.2000 . 140( 0) (Spectrum,USA) against O. 15 MPBS at4''Cfor24hours, withfreq uentchangesofbuffer.
The sampleswereagainmeasured forprotein as beforeandconcentrated with 01 Cen trip rep- IOConcentrator(Amicon Inc"USA), filteredsterilewitha 22101m Milhporc filte runit(Millex-GS, Millipore,USA ) andagain measuredfor protein. The purified antibodywas storedsterile at4nC,Aftereluting, thecolumn wasre-cquilibrmed as before andstoredwith0.05%sodiumazide inanequi libratingbuffer,at 4nC.
3.3.2 IgG Purification
TheIgG antibod ieswere purifiedwith theusc oftheAffi-GclProtein AMAPS II Kit [Bic-Rad,USA).Binding buffe r and elutio n buffers were preparedusing Am -Gel Protein A MAPSII Binding Buffer and Affi-GelProtein AMAPSIIElution Buffer (llio- Rad, USA). The sample wasappliedto the column, diluted one to onewith Binding Bufferand re-cycled atleast 3 times. Afterelution ofthe antibody,the columnwas wa shed withAffi-Gel ProteinA MAPS IIRegencrationBufferand storedin 0.05 % sodiumazidein O.ISMPBSat 4°C.TheBio-Rad EconoSystem, ModelF~')·J(Bio-Rad, USA) was used in order to monitortheeluted fractionsfor protein andtoensu re the
42
