~A " ?" : ~ U bm 1tt e~e SCh'~'6'f ,

....

STUDY OF A foIONOClONAf .ANTI80DYTO

HUHA~

^B'CEllS

BY

. '

©

HARY SHEr/A lEWIS DROVER RT, MT

,

~A " ?" : ~ U bm 1tt e~e SCh'~'6'f ,

G.raduate' ·

. . 'St udi e s in partial fUlfmment of,the r-equtr-enent s for the degree of

Master of Sciencer

De~artmentof Medic in e Memorial University of Newfoundla-nd

February'1986

St. John's Newfoun'dland

" .

':."J' .~

s.

Permission has been gran ted

•to\the Nati o nal,Li br a r y of

•Canada to microfilm thi s the-s is and to lend or sell / c.o~ies·~f thef11 l1\.

"The autho r fcopydgtit.o....ner I

nae reserved other

pub

rrc

ee ron rights, and ne i th e r the th e s i s nd' exte nsive extracts from' it may be pri nted or otherwi se reproduced wit ho ut his/her written permission.'

Ltautorisation.a'-fltAa'ccord lSe

!Ii la Biblioth~que,natio na le du Ca nada de rnicrofi l me r cet te th ~ s e et do _prlltor ou do vendee eee exemplairos,du film.

L'auto ur (tit~ha i re dudro it d'auteu t') se _rl!se rve Jes aut res droitsde pUb li c a t io n , -n i' la'thOs e fli~de 'long s ex e eetee .u e ee'rr e -et ne

.d o ive n t ·@tre irnp r i mAs ou

aut"rement,reproduits eans son "", autorj.satio nacrtee ,

, .

ISBN B-315w33613w7

..

. \

~.

~'.

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11 ABSTRACT

• The purpose of tbts wo r k wa s to ch aracterize.and d'ete r-min e the specif icltY.,of a.aou s e monoc lo na l antibody (NF•.LD.M~) whi~h^was d~rlved ^{from.a fusion between}SP2/0.A.g14"

and spl e en ce l ls fromaBalb/c mo us ethat ha dbeenhyper'1m~u . niz e~wi t ha-~ellS "" aChro nic.l.~~p~a.tIC..le u ~ e m 1 C pe..tie~t..•

"<,.. :",'

Clo ni ng was-d cne by li mit in g dilution and positive,clones .~ were selec ted

,by'

sc~een~ng'ona ~ane l of Vi'abl,ecells ustng~_...

thec'ellu la r enzyme~1inked i~munosorbe.nt~ssay--(CE LlSA). This -"~,":,...

assay.was shownto ee lAoresP~Cl'f1c a~d ^{sensitive than e.n} EllSAthatused

~lu t~r'al·de hYde. f1xed C~·l1S .

^.

. I

Two sources of the ant)bOdY {-p uri fied IgGl from ascites' fl uid and s-iJ-pe....-n:atant from~vergr-own-iif1Tilres)-a'ppearell-to-b-e--·:-

, .

." . ^{. " .}

^"

1de nti c a l in their s er-otcct

.

ca I.pattern onsever

^.

et 8-c';'11 lines. spe ctttctty te s t ing using the 'CELI SA and s e v er a l di

.

ff e ren t cel l,

^{. .}

types reveal edthat NFl O.HI-rec ogni ze d some^~ B- cells, but failed to react with·any'o f the T-cells te s t e d. A

. .

.Fre quen cy Distributionpl,o.t.Of the data s~ow e d thatKFlD .Hl reacted wlJh.the cells in a bimoda l fashioncompared to the

•'{; )O •

nor m4" dis tr i buti on obse rve d withthe monomo r phIc monoclonal

-~n·dJY,dY .

KEI anti -h.

F~rthe r~ore ~hen KFlD, .Hl , ~n 't i bod; \lta~

!"~'p'hi~sed·a's.a percent of the HEI,ant1,="Ja f~ WI $ found that

,"::..:-, : , ,,' ."'{ . ,",j' ,',. . , " .

<\~H the '9,R\ 'p'Osl~tve,cells pr odu c e~ v-alu e s gr~a t 'r,than 501

; \t e re as ..4 neg~ ~ ~e cells gave values le ss than 301. UsIn g

"'. "

..

.

^'~'... ios as a cutoff point·'.a correlationanalys is wasdone i ll

the

to.

.

^.

.

CELIS "" result s' for "4.2 cell lines.The r-value obtained for DR4 an4NFlO.M l·was.1 with a p value of 2 li 10.1

°.

^In

addit i on·sign1fl.cant r

Ya l ue ~

^were ?bt ai ne d f.T1l'ORw53 an d DQw3 which are in linkage disequil fbr fumwithDR4. ,

~ ' . O ~I! -df ml!nSi ona l el~ectr~Ph~·r~s.~~

^{of't'h l!}

Immunop rec1p~',~at -

^.

ed molecules froma DR4ce l l produced a banding patternthat ,1"'''

, w ;, ""?"?"

^{thatof"' al}pha and beta subunftsof 0'\ ' •

, .

.,

ACllOVU DSENU l s

~wish to expr ess.y IIp,preCiation'to the .e. ber s of .y

s u.pe~,:)~?' ~o:~};V~~

^{Dr. ".H.}

~~s1\i1l .

^Dr.

~.

^YOllng l\Usband

/an'~~;~.:~J. _~rr;·"l.:.a/ll .especially gra,te.f1Jl to.Dr. Mars ha l l fo r

hfs·encour ageme nt. sup por t andsou ndadvice.~

Co ns i dera~1e

^{inter est and}

sup~~rf

ha's 'be en

sho~n '

^{by the}

.rm·m u n O l 0 gy . ~ ep ar tm ~·n t ~' p~rt1-c-UI.a r;lY

^.Dr\ V. Sk a nes a.nd

Dr. B. Lars en who ha ve been gener ous with their;lt 1me a~ d ~ cou nc11.' r a;so wi'Sh to thank O.

COd ner.""H ·:-I: U1~ert.

^and

Y. "as s 1.whoha ve pr ovided·t e chn1cal. ass1.sta·nc e ~t.J:..a·r.iou s times.

In ad.d-it ion to the encour agne nt.and'~ora l.supp or t

. .

,prOVided by IIi IIlTtbTnltl'ecr, I am also appr ecia tive of. his c~ns1de,.able-es sfstenee in the preparat i.on of this thesis. particularly thegraphtcs.

. >.

HedJ.c1.ne andHea lthand"el fare Canada.

\

rtnencteI support has been provided by the Faculty of

....

.".:

- V TABLEOF CONTENTS

.ABST RACT • • • • •~ ACKI40WLEOGEMENTS••

.i l .1v T~BLEOF CONTENTS.••• •'• • • ••• • • • •'•• •• • •v LIST OF TABLES• • • • • • •• • • •• • •• • •~ •• • •x·i

1.3~IB Fus ion procedure

1.4 Objectives

-- '

11 / j I ' ^/ 17,

20' ' 0

'OJ

'~," 'I

22 '3 24 xiJ1 x,vii"

- ... .v,

.I NTRODUCTION

1.3.2 Screening for HlA~specHicities 1.3.2A Complemel't dependentcytoxietty 1.3.2B Cell b{ndlng_a~Says

1.1.1 ClaSS')II molecules _• •'•• ',' 1.1.1A Structure at the prote in le ve l 1.1 Over v iew of tl;e ,Ma'j o r His toc:omp a tibi.lft y

Co~plex •

.:.;#• • • '• • • •

1.1.1BPo l ymor ph i s mat the ONAlevel I.LlC Se r ologi c a l defin it io.n 1.2 Monoclonal Antibodiesto Class II Ant Igens 1.3 Preparation of Monoclonal Antibodies.to HlA-.O

Region Molecules •• • • • .1.3.1."- le' e hnique

1.3.IA Immuni zat ion.

LIST OFFIGURES.J• • • LIST0f't'~BBREVIATI~NS CHAPT ER I

i··.

r - ,

...

,.

~'('.~');"'.':,.'·i'~~•"', ''~';" .';.\'~:'.I "1.: .,·.co:.'".,i-','..i::.;''..:

CHAPTER II HAT ERI ALS.AND METHOD S

"

2.1 Productionof-N.FlD.HI Monocl onal Antibody 2.1 .1 Immu n1 ~ a t1 o nprot.OCr!

•2.1 .2 Fusion procedure • • •

25 25 25 2...-t-;2'A Reagents• • •:• • • • • • • ,"• • • 2S

-;'2.1 . 28

~ Fus l on

^{partnJr - ; . 'Z1}

~.1.2 C ^{Spleen¢ellS'/,}

, 2 \\ ,20 '""." ; ' ' 1 " " " "

2~1.3 Select-tnh and eetntenenceof hybrids.

- \ / '" I'

-. 2.13A( Se l eCt'ion

.I . "-t.: .

2.1.3 8.T-ransfer'and:Cl o ni n g

"fol . 3C [lCpansion- .1 2.1.3 0

-

Freezing

,

. ... 2. 1.3E Thawing • • • 2.1.4 Purif ica tion of NF:lD.Hl

'2 . 1. 4A Ascit es flufdI ••

2.2 Screen ingAs sa y s . .•

J ...

2.2.1

Ra~io

^Immune

Assa~1 (.R~A)

2.2.2 Enzyme·l 1nked l"1m Uln o s or b e ~ • 2~2.~A Reagents u.se.d-t'ar both ElISA-GAF

:and CEI.l S,I•• • • ••

2.2.28 ELISAon'G A1fh edce ll s ' -' 2.2 ',2 e ELISA on 1.Lv.e..-cel1s (CElISA)

2r3 Target Cells 0 0 . 0'. 0\" • 0 • 0 • 0 0

2. 3 .1 ce'll lines 0 0' 0 0

10 • 0 • •-0.•' 0

· . ,j _{- '}

'$c, 3 , 2

"'"''''...- J '-".':"""

27 28

'29

29 29 3.

31 31 32 32 33 34, 34

34 '3' 38 38

38

39

\

2.6 An~ l y s 1s of CEL rS~Dat a 2.6.-1-- Spe cHJctty tes t ing •

44 44 45 46 46

-

,'4 6 46 ' l i

\ -

39 39

4' 4D

.--

41·

41 41 41 42

42 42 43

.'-:-:-

..

^{' -,}

' ! "

. ..

-,:y'

"

cyies.• • ',' • •• • • • • "

2.3.3 PHA..Stimulate d.rccelt s • • • • • Is ot ;p ing

,

and Quintff1c at1 0n

^.

of NHD.Hl^.

^..

2.4.1 Ouchte r lony a.na 1y s 1 s

,

2.5 .1 Iodina tio nof cell surface prot e in s • Determi nant

2:3.2A Separationof perip9.errl,b-lood mon o~~~arcel ls, : • • • •'•• • 2,.3.28 Mo n ~ d~pletion• •• •• • • 2.3.?C seper-et.ton of T- and 8-lymphQ.

2.5 .2 .Immuno p recip ltat ion, • •• • • • •• .2.5'.3 O-ne-Di mensional' E.1e c tr o ph o r e s f s

(SDS,P~E): , • • • , • • • '2.S . 3A R~agen ts • • •'• •,. 2.5 .3g Prep ar a t i on of gels 2.5.3C Ele c t r oph-or e s i s 2.5.3 0 Auto r adiogra phy 2.4.2 QuantHlcat10n us in g EL'1SA

2.4 .2A Reagents 2.4.28 Assay

2.5 Mole cu la r Weight De t e r mlna t l o n o'f NFlD. HI 2.4

2.6.2 Compar iso n of "FlO.M I A to NFlD.MlB 48

"

,CHAPTERII I RESULTS

3.1 PreliminaryTesting of"Uncloned Cultur e SO 3.2 setection of a Screen ing Assay S2

.~

0,

3.t.1 ElBA on GA-Fixed Cells gave non- s pecif i c r-es ults " , , , , ,

; ~

^{, • • • • • 52}

---~3.6.1 C MFLD.HIA t.itration onB-Ce lls• •

..

/

S'4"- 54 51

55 6.

'I 61 63 67

"

72

75

.- 78

78

83 84 3.4 Cloning

! (

3.4.1 SUbC17fng.• •,: • • • •• • ••, ••• 3.S Imlllurroglobu)in Isotyping aod Quantlfica.tion • sero'lol"cal Pattern on Perip h'eral Blood Lympho c yt e s" '.' • • • • •• •• •• • ; .

7

^{.6 : 1 POL from}

~.::n related

^{controls . ,}

3.~fA Qual~ty of the---d a t a_ ._ •• 3 ...18 MFLO.Hi reactivity with T- and

I B-lymp hocyt.es •• •

3.2.2 COlllparison·of ELISAo~ GA·fhed cells versus CEll SA·:;• • •/~ • •• •• •• 3.2.2A VariabilitY·withinrepl icates. 3.2.28

Non :s'p~~,1f iJ/b ;ndin~

^{•• • •}

.3.2 .2C Gl ut ar al d;tIYde ,~o d ifie s some D-regi onmo}e c u l es • • • • • '., ' 3.2.3' CELISA

select/~ r

^scr e e ni ng assay

3.2:s.A 'Oll't1mi) conditions fOll-,GELISA

/

, /

-:-

3.3 Preliminary Scree n in gof MFUY.Hl in CElISA

. < ; . .

",:.- -' --

0',

,.~' tx '..,!

8' 86 88 88 93

9' 9 '

'i-,"..."',~.'"•

3.10 One Dimensi onal Gel'Electrophore s is.

4.2.1 NFLO.HI:and OMspec1ficHy: 126 4.2.2 Association of NFLO.HI. wHh- ORw53 _',' 127 4.2.3 As~ociat1onof NFlO .Hl with 00)3c~l l~ 12' 4.2,'4 Segregation·ofNFlD.Hl Infamilies .,13 0 4.2.5 ReactionswHh"0!l4 ne g4,tiv e cells 131

'3 . 6. 10 NFlO.Hi.r~act ivit ywith PHA·

blasts'•• 3.6.2 FAMIl y'OATt.. • • • ••• • •

; ~3.6.2A Quality'of the data • ..."3.6.28 Interpreta tionof Data 3. 7· Ser o ng rc al Reactivit y,o ~ B-:Cell lines

3.7. } 'ltttettons • • • : • • • • • •"

(-"3.7.lA NFLO.Hl

tt

trettcns • •• • 3,7:_1~ Rela,t1vereactivityof .N(LO.Hl to.

HEI·I a • • • • •• • •• •• .104 3.7.2 Speclf1ci(ytesti,ng once.l.l,line s 104'

, \ '-

3.B Statisti cal AnalY~J~~:OfNFLO.HI • ; • •• .. 113 3.9, Comparison 0.(HFLO.H l ~withNFLO .HIB ._. 116 II '

, I '

I'

I' I

CrAPTERIV.~ : 01 SCUSSION

! i

',4.1

~ G l ~ t ar ~ l d e h,Y d e - f ixa t f o n.,

^{af Cells lddu ces}

Artifacts in EL1.5-A f",or .re stlns Honoclqnal' '~~t f bO~ ii!' " • • • • • • • •• ~. .-:-121

•• 2 Assigning Spe c i.f-.icity to HFLO.Hl 124

~,"'

4.3 Future plans for NFLO.HI ...133'

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LISf.OF REFE RENCES• .13'1

\.

APPENDIX A. ' . • • • • ~ • • • • • • • • •:-....:.-.:,,_<,/ , •.16'

D

','

,\

._.::,c;, ..~.';'._.' .,..•

~

•.. .

xl LISfOFTABlES

,

...~-v-

,:,";..--- I

~

^.•

~ ,Tlbh

^{1;HLA- Oregio n} serol ogi ca ll y de fin e d sp e cfft ctt hs in i.:~

- .

l1nt l g~ ~tsequ t1 1b,,' ull • • •• • . • •7

,

T.~'.e..2. Ruction s exp r e ss ee as Adj . 00 of viable cel ls ."and g1utarll~eh"Ydeff xed,"cell s (GA.F) 1n ELISA usin gt'lilO

l'IIonoe1~nl l anttb~:1 es. • ~'. : . •~'.' ^•

,

^{• • , . , . 56}

.Lebl e 3"..HLA

•

phenoty pes andCE~ISA resu l ~s of Clt'-cells . 65

~.

lIbJe 5,.CEllSA'results.as AdJ . 00.on sup e'rnat ant (SN) tro. selected cl ones (e) tes ted"a g ainst var fous ce11••71

-,

70'-- -.

-~

..,. ..•

66.

. .

^{" , '; ' '}

. .

hble 4. Resu lt s ofclo ni ng byf.tIl'1t 1ng dllut 1on\ •

Tlb~e 6. CEUSAresult s asA-dj~ ODon su p ern a t a n t fr o~~

N~lD."l Subclones (Sc)tes'ted againstvarious cells", 73

f.b1e7A.HLAPhuotypes PBl donor s

~

• • • • • • • • 79

c:z .

Table 7B. Res'u-lts IS AdJ. OD of "CAB

~ellc'ted

^wt'th

'.sepa rate·CI~;'::~· ·

PBL\" CELISA , •• •

" '~~'

. ,

~:.

. . ..

^,

{:'

. .

;.;:~..•.. .

'.

. ' . ^{, .}

.

..

^;

Table 8. St ..,:.t i c a l ana lysis of titrations .95

Table g. tatistical analyses of NFlO.HI ti t rat ions t three groups of ~ell lines in CElISA•.The s'are given as Adj .00 >Jfalues •• • • •• • • 103

,.'

Ta bl e.l o-A. NHO.Ml·Reactivity with Group 1 Cells (DR4 po;jtive ) " • •• • : • • • • " • • • •• ••• ••~^{• 105-}

.~

Ta b l e.10 B. _NFlO.~l ae^acttvtty _w~th Group Z cells (DR4 negative, O'RS orORw6positive) ••-. • ..• • • : 106

Ta bl e 10C. NHO.H l reactivitywfth.yroup3ce l l s (OR4 'ne ga t iv e.DRS neyathe'ii!,d DRw6 negative) ' • • • • •107

Tab le II. CElISA results from testing 42 B-lC l against

HFlO.HIand N~I-,te • • • 109

Tabl e 12. Correlatio nofMI with HlA· D Ant19_ens • 115.

Ta b le 13. Comparison ofpeacttons of NFl D.H1 A and NFl O.M lB·

wlth var i ou s cell lines in CElISA • • •• •'• • • • 117.

xif1 LJSTOFFlCURES

Fi gure 1. Hap of the major hts t ocolllpa t 1b l1 ft y cOlll plex on chr olllos ome6 showi ngth eHLA and complement

genes• • •• • • •• • •3

'F1 gui"e'2 . A s cheeettc 'r ep re s ent a t i o n of th e arra nge ment of nons that.enco de the domains found,fn the po'1ypep- ttd ! s.truct ur'e

o ~.

^{thealph'a ud betac.hains'of}

Clll~S

^II

..o1ecu1es

Figur e 3•.~chUlatic,lIlap';?--~ th e HLA-Oregion genes, adapt ed'

from Bodmer (1984)• . • . .12

\0:-,

",··,·~. i

F1gur e 4;Pre lim in ar y sc re eningo(".NFtO .Ml us i ng RU. Super- --na t a n t fr o1ll the .as ter cultu rl!.;.-wa.~ te s t e d against the.

hllun1z!ngcell (NB)anda panel o"~e ll lfnes. 51

... ..

^','

Fi gur e5.,R~acttonsof tr re levant~~yel ollla pr~tefnS tn Ell SA Jsin g 6A· fhed cells (GM~i~lh ••- •• •• '.';: 53

Figu r e 6. No_.n-spe c ific binding of we tr-relevent myelolll a- protetns-to'(;"3190cells tn'~ElISA. • •• • • 58

Figure 1. Bind.ing.of two Mo n o. or phi c DR"CAB to GH3 190 ce ll sIS.els ur edinELISA'. . • • •-. •• • • 59

. . ,..

/

Figure8. Effect of dtluting the conJuglt e on .en uring the rea~!..ton of AT..AB.DRin CELiS" •• • 12

Fi g u re 9. Effect of ce l l c ene e ntr'ette n on.lIeas u rin g the reactio.n of NEI- I i and cuttu r-e med ium withGM 3190

cells fn C£lISA. • •• • • • • 64

Fig u r e 10. ae aetten s of NFL D~ Hl ob t a ine d by scr ee nin g

pe r c en t age of the.:.AtAa,;,OR

• • • • • • t • • • • • •'0 . 74

expr e ssed cantro1..• • s ubc l en es

the sup erna ta nt f,.olft~the•._{~I? e r} cult ure 1n CELISA Ig.1nst a panel of ell an.dB-lel . '0 • • • • •-• •', 68

" " " i i , , ,' ' " . . " '" t hr ee ... .... eants ""

">' " " t

Fi gur e 12.00 as a func tion of IgGlco nce nt ,.. tfonmeasuredfn Ell SA• • ••~ • '.• •• • • • •• : -. 76

Figure13. CElISA results obtained for tltra t i ons of NFlD.Kt

'9.1n5t GM 3190 •• • 17

Fi gu r e14. Effictency of se para ti ngPSl into T· and B~ly mpho·

ty t e sas measur.edin CEl lSA and reactionof,MFLO.Hl wit h.

the separat edcel-ls , • • • • •• ....•• • • • •.", • • $2

Figure 15.'Reac tion of sp e etttc Ind non~ s pectfic ucHts

dUlls •• • • • • • • 85

,',

F1gure 1.6. Effec:t of PHA-sti mulit10n of T-cel ls on their reac:t 1o nsw1th'HCAB."• • • • • • • • • • • 81

F,1gure l1A-11D. ae e cttons of HCAB with c:e ll s fromfu11y lIelllber

.

s• •• • • • • • • • • •'• • •• • • •". • • • gO Fig ~r e lBA. rfteettens of

\

~C"B aga ins t DR4 posi t iveB-L Cl in CELISA• • •• • • •.•".• • •• •'• • • • •'. • •• • 91

Fi 9u r e 18B.T1trat1on s of HCABag ai ns t DR~1.2 positive and DRw6 PQs1the B-LCL 1n CE~ISA• • • • , • • • • • 98

Fi9ure18C. Tttrationsof HC"B a9a1nst DRS pos1tive B-Lel in CELlSA • • • • .. • • ••• •• • • • • • ~ • • • • • 99

Fig ure lB~.Tttrationsof HCAS agltn s-t-DR2 pos itiv e S-LCt in CELISA • •• • • • • • • • ••• •.••

"

Figure 18E.,Tttratio ns of HCABagainst

,

^,

and DR"J.wB'posithe 8- LCL'in'CEllSA• •

100 // DRJ . 1 pos 1 t'iv e

,

/

, ,'• • ' / 101

Figu r~ IBF.·T1t r a t1. ons of HCABagainst OR1·p os tt iv e and

".DR7pos ft h e S"-LCl in.CEl,ISA. . . .. . 102

Figure 19••Freque ncy distribu ti o n plot of the Adj .aD value s for42B-LC Ltest ed a9a lnst MEI- Ja and

MFLD•JIll •• 112

fl9 u~e 2D. Frequency di stri butio n plo t of t~e , re la t ive reactivityof 'NFl D.,M l tested against 42B·LCL.·. . 114

figure·21. SOS- PAG E analysis of the

I l11m'unopr\c1Pi ~ated

moleculesfrOIll 125 1l~belled GH190S·cells. •• 120

/

I

"A

CDC

••CE_LISA- CLD Cl.L CM dH20 ElA Ell SA

Ust of Abbrevhttons

Bl0ck i"9buffer

B~lymphob~astoldcell tine s aovtne serum albumi n Complementl:Iependent cytoxicity C~lIulilirenzyme~1tnkedimrnuno's o r bent ass.ay Cloning by limit,ingdilution

Chrorii,c lymphat f~.leul:emia Clo~in9medium

Dist111ed water Enzyme 1I11~~rl o as s ay

EnzymeMltnked immunosorbentassay

xvt t

FBS Fetal bovineserum FH .Ficol1-Hypaque GA Glut.araldehyde GA·F Glut-a;"aldehYde~f ixed G~H Goat anti-mouse

HAT Hypox.anthan'lne-ee! nopterin~tI)ymfdine HH Hybri doma medium

HRP Horse radishperoxidase·.

HT ~ypoxantha.n1ne-thymi dt ne leL '/Lym~hob1astotd'ce l l lines LN2 Liquid nitrogen

"CAB Monoclonal antibOdy

.

~;

'

..

Protein A

Peri p he r a l bloodmon·OnU~lea.r Cel\/

Pe rtpher-e I bloodlymphocyte s \) Phosphatebuffered sa l I ne

Phosp-hatebuffered saline cont ai ning0.05'Twe e n - 20 Poly-l-Lys inent,

Po l yviny1 chloride PA

PBM PBl PBS_ PBS-T

> Pl l

PVC RAM RT

Rabbit anti-mouse Roomtemperature ^\,

(

Standard devtet.ton. SAS Saturatedamllloniumsu l pha t e

SDS'-~AGE ^Sodiumdodecyl su Iph atesp ulyacj-yLam.ide gel elec t r o- onc resls

T-lCl T-lymphoblasto id ce l l line s Stati stical

.

,'.~ ~

..

CHAPTERJ IITRODUCTIOJl

The work.de scr i bed in this thes i sdeal smain l ywith the ch~ r a c te r 1z a t«p n of a mon oclon a l antf bod y (NFlD.Mt) whi ch was produced in this laboratory , and the s el ection and applfca~'o n of.an approPr:' iat.e as sa y todete ct'andcharac t er- tae its specifici t y. PrelJm1na'ry'sc ree ni n gof supe rnatant __

~J r om th ~ '

hybr1doma

--c'u 1t u r~

had indicat ed that"'t\e ,a nt i body was re cogn1Z1ng a B-cell polymorphism. Sp e c if i cit y te st Ing

'U'S 1'~~

^{antfbod y.from'the}

clon~d

hybr1d oma su gge·s ts th'at NFlD:M~ ^{is d1rect'edto a dete'rm1nant} o~HLA-DR,4·molec ul e s.

In th1~'c ha pte'r' some of the relevant l1ter'a t ure on 'new dev~lopments in the HLAf1el..<f; p.ar~_c.~larlY the role played byeonoctcnet antibod 1es fn 1ncreas.1n'g,our kno..wledge of th1s

.c omp l.ex system, w1·ll·be reviewed . Emphasis,wfl'i be. placed on

HLA·D regfon molecules' ,.pe r ttcu l e r ly DR4and the clo s e ly essccte e ed ao t ecutes', DRw53 and OQw3. In add1t1 on l'Ae t ho ds used.e e identify and study the sp e c if ic ity of mono clona l ilI.nt1bod ies to~HLAstru ctu,!" es w_{i 1 l l} be revi ewe d.

) .

~.

~_.

flie HLA molecul e s form a c oepte x sy st emof cell surface glycoprote1ns that are encoded'by multiple gene~ in

. ..•

",;':

'. '

. .

,',"

\

the ma jo r histocompati bility comp lex (MHC) o!! the sho r t of ch romosome6

tfl...~

^{(figure I). The re}

"':'a~e

^{threema in}

~et s

of clo s el y rela ted gene swhi ch are co':'do mln a n t ly expressed and.t he pro duc ts of these'g~e n e s play import a nt role s In the immune respo nse .

Theclass

.

I genes encod eHlA- A, Cand^. B specificiti es^.of which the re are at lea s t 23 . B, and 47-:-a"llel i c varia nts,

. I '. I .

r eec ecttvejy• Each mol e cul e 1s composed o-f two chai ns.The hen yf/h alo, alpha (Hr · 43, 000 )

carrl ~; t~e

^all oa n tf genlc detnr minant s In its

mo~ t

^{extn;na l'}

dom~l.n ( al ;ha

^{3).It Is}

.

a~cho red

^{In the} membr a ne and-

non-co~alen ),y assoc ~ated

^{wIth a}

ligh t'·ch ain, be.t ag mf cr oglobfn (M.r.. 12,000) which Is not membra ne -bo u nd but is reqUire d'for expres s i onof Hl A-AC1 -- sp~ciHciti es. Beta 2 microg l ob-In i,s encoded en chromos ome15 -wtrere ~s.the heavy cha i n is enco de d intheMHConch ro mos ome

6.

In addition ther e are,some poorl i def ln~d clas s I mo l ecu le s tha t are st r uc t ur a lly similar to HlA·ACBand'are

. ~

-

'homologous'to the muri ne QaO-\,mo~ecul es. c~ ass I mol ecules are expressed on all nucteetee ce lls and on ptetetets •They aremainly in volve d tn'antJg e n pre s enta tion tocyt otoxi c

Y-cel1s'. • (

The HlA-D reg lo n,qr cl ass II genes.ar eco n si de r a bly more, comple x tha n the cl ass I ge ne s. There are ef 'l~~s t,t h r e e:

< •

sUb- r e gi ons (OPtOQ and DR) each withmu l t i pl,e genes, ~..any-of whi chare..k.nown to code for functional prot e ins.The gene

, -:

\

.','

s.

/"

- '

MAJOR HIS "j"0CO MPATIBILIT Y COMPLEX

CHROMOS OME 6

-

^OlO

, [ DP

CLAS SII. DO - DR

CLASS

II;[ }}B

21-0H C4A 21- 0 H

ClASS_~:[

^:

OAlT l

Ftgu re.J: KIp of the ."J orhlstocOlIp,a t1-btl1ty COII~l!~

chrOIlOSOlle 6 shovi n g th e Hl A.n~.eomp.' ellen t gen es; GlO.

gly c ol yas e : 2.1-0H,21- hydroxylne.

f '-

tha nthe cl ass I mo .l ec ul e s and muc h of our pre se nt kno wledge ha s C,o metront subr e gi on- sp eci f i c monoclo n a l'",n t1bod 1es and from bi oc hemi c alch a~terlut1onan d DNA-t e chno l o9)"(r- ev l ew- ed by Giles.and capr,@ 85; Mo ller 'e t al. 1985;.Boh e r et a1. 1984a; Korman et a1. 1985 ; Trowsdale et a1. 1985 ). The class II molecu les are import an t in anU gen p~fese nt atto~to 'rche lper and indu e,er ; :I IS and arlft'alnlY expr e s se d B-l ympho cyt es and other- an~n·pr e se nting cells such as dendr1 t l c macropha ges. Alth o u'gh they'are no t pres e nt on re s tf.ng T-e e.l 1s, expr-e s s tcn can~t~tge n . mitG'llen , and1- I nt e rf ero .n

st i m ula~to,:, .

\ . The cl as s til g~nes code'for the comp lem e n t ccacc nents, e2,Fact or 8. C4A andC4 B,all of whi c he-r-ep~l y morp h 1 cwith theC4 com,ponents ha ving the mo st allelic vertents, Therol e produc t s have been dlfffcult to deflne sHo lo glca l1y.

pl ay ed by these ccep onents fn the hnmu ne res pon s e wlt h llles pec t to B-cel1 regula t Ion and diseas e- s uscep t lb l11t y is an area ot curr e nt 1nves t1g~~jo n.

1.1,1 Ch ss II_01 ecules

The ear ly-Vo rk on the tdenttttcettc n and cher e ctertz-, ettcn of the HLA-D regionspectttet ttes has been revi ew~d by sever a l au th or s Inc l ud 1 ng Bodmer (1971) and WInchester and

"

Kunkel (1979). Tae df s c ove ry.by-aatn et et- (196 4 ) that allogen i c cells from unrelated fndi vid uals andfromdizygotic

..

.'

~: ^{' (..}

/

twfn s,bu,i not trom.onol ygot f,c twf ns, sti mul a t ed eachot~' in'a .;he d leukocyte cultur e (MlC) sugges t ed an asso c iati o n, with trans.p la ntat i onan tl gens.The workof Bach andHfrs ~horn (196 4 ) and Bach and AJl os (1967) st r o nglysu gges tedth a t these cel l .uia rl y - deffne dantijle n~ were control led bya h1stoc ompat- fb1 T1 tylo cu s . It was not until·someyears later that the gene s det e r~1 n l n g thes e respons es were sh o wn to belinklld'to HlA (yan Leeuwen etel, 1971;"t unt-s and.Amos1973; van Roo d' et a1. 1·976)and that there were.ser olog1 c"al eq ufve lenrs of ,-

.- ". I

~IUlarl Y~def1ned ant igens'(H l A_-~) , '

. A modiflcation , of the HlCte c hni q-u-e us1ng homo zygou s typfpg cells (HTC) fromkn o wn HlA-O 1dentlcaldon o r s has s.tnc e be e n used todefine 19y~rian t s {sr o ss e-vttee etol'- al , 19B4). These verte nts (HlA.Ow') gene r a lly correlat ewfth

recogn ize d

.

at the 1971tnter nettcnat Hlsto-

.

~atlb l1 ty·Wo r k"shop . However, Ow varte ntsdo not always_

\~r :e l U .with the ORspeC1f1cl,t

y ;

part1cularly .1n't h ecase , of Dw4 and DR4,'Some of the discordan ce has r+ecent1y be e n ' expl ained byt.he pr es'.ence of'd1 f ferent SUbtypes wfth 1n a DR~pec~f1city, The contr f.butlonof ot.~__D.r egf onproducts such aslOQ toHLC·d'ef1ned OwSpec1ficftfet-- ~Xill requlres Clarif1catfonand will be d1scussed tnCha p t er IV.

~

The DP (prev10usly named SB) subregion

wh1Ch'encodes \~

least six etlette ,yar i a ntswas first de scrtb e d by Shaw.et;

C- el , (19BD) using secondaryst1mul a ti onof the HlC,Id e nt 1f i -

. K

^{•:.~, ,•.;,:.. •.J>-','. '.',,' .'.''•}

6 • cat ion of OPspe cificities has not beent eas tbtewitheenv ee-

t1o n!1enttsere since 'such anti se r a.arerare. Honoclonalt. an t ibodies spectrtc for DFr"molec ule s hue recentl ybe en rep o rted (Nadle r et,et,19 81; Wat s ~ net al.. 1983; Hurley e.t al. 1984 ).

In ad dltfon'toDR.,therear e other sero l~g1cal 1y -d ef1nl!d.

spectttct ttes inclUding OQ pr eviousl y ca ll ed H.B, DCand OS (Du q Ul!sno ye t a l . 1 979 ;:T,OSi etal : 1978 ; Goye r-t et 151.198 2 )'

I· . ., .

and_{lou s 1y c}the s upe r t_{a lled}yp_HTte DR_anjJ

j

_{BR (}^{Pe C1f1Cl}_~ark^til15_et ^DR52._{al. 198 0;}^{an~ ORw5 3,}Ta n fg ak f^{p;'e v-}'·ei^.·^..

al. 1983) . TheDQspe d 1c1 t1es'sh o w

allel 1~ asso~ iat'on

^with

~h~

^{DRspe c ifici ties ( abl e 1)}

wh 1~h

^{has made fdent 1'f1cat 1 0n',}

dif ficul t ~'nc e theal loantf sera Jlsedfor this purpo se of t e n

.

. .

con t aIn a ml»tu re at" :a nt 1bod1es . De spit ernt

s

'd r a wback of al lo antlbo dles fo ur teen alld thr ee..s.ero I 0 9 1~ a l varte nt s we" e ee scr tb e d , for DR"andDQ respe ct lvely duri ng the H1in.th In te r na tion al

l.,

^H1st oc om pat1b ll1ty Wor k.sh,o p (Bodme,r et al.

'1984.).

1·\) . ' .. •

r

~.

. ^..

1.1. 1A. Stru'tture at the proteln Je~el

The structure of the c.lass

. ...

I I molecu les (Figu r e 2) has -c. bqen reve a le d by one andtwo dim e ~sl.o~ a l etece repbore sts o,f.

femuncjre cfptr et ee mole cule s'as well as by aminoe ctd s eq uen ctng and 1I0 re recen tl y by'nucle otid e sequencing (revie we d fnSh a cke lfo r det a1. 1982: Gf l es and Capra'U8S).

. ".

DR. ~Q. and DP e ec b.c cn ststs of h/f!.terod1mer,s that are

. . .

^{~:., ;}

-

,;;.

- .

}

.::"'"

-/

^{.,--.:: ":r••.. ::•}

~"'":

.

Table l. HLA-O reg' on serol og1ca11y-de f 1nedspec tttc-tttes In

linkage dhequ11 tbr iull.'.

. ...

DR

D' SPL ITS D' ASSOC DO

)

HD HD- .1

LONG HD- .1

SHORT ND\ .1

.10 NO ~D .1

'~:.--

••

^{.13 .52 .1}

w14. .52 .1

.8 .52

5_ .11 .52 .3

.12 .5~ .3

~~2

^{.- '}

HD

. z

.HD .53

. z

".

j '. 1 .53 ._.w3

/

'. 2 .53 .3

4:3 .53 .3

"

.9 HIlo ~S3 .3

. ,

;.~;

... ".

^{".~., :}~,I ~

GENE ARRANGEMENTAND STRUCTU REOFCLASS IIMOLEC UL ES

Figure 2. A schematic rep r e s e n t a ti on of the arrangement of e x ons that encode the domains found in the polypeptide structure of the alp ha and beta chains of class II molecules (DR shown here). UT, u nt r e nslated; 55, signal sequence; TM, transmembrane;

en,

cyt ople smtc

vrn , c

z and {31 , {32 , external domains of the alpha and beta chains re s pe c t i v el y . (adapted from Stites et al. 19 8 4 )

... formed bytwo non-cove lentt y li~ ~ed glyc osylate dchains that

\rave rse the pl asma me mbran e {str-cn tnae ret al,19 15; Walsh and Crumpton 1971~ Sprl nge r ~t a1.1971 ; Owe n et el, 19 8 1 : Kv is t et a l • 1982 ). Eac hsub u nit has a li9ht chain, 'beta (l'l and a heavy enat n, alpha (a), with Hr rangin g fro m27, 0 00 - 29 , 000 and 31 , 000-3 4,000 respec tive l y: (Figu r e 2). InadditiO n the re'is an invariantcha in , called ga mma, Hr..31,00 0(Jone s et a l , 1979) . A1thoug~ its. function has'not been el uci da ted , it appears to be·i mpor tant In transf err in g the'a l p ha- b e ta dlm~r ^{t r-oe} the.end~p1asm ic ^{reticul umand} ins e rti n g it into the'~ 1asma me'mbran,e (KvlSt e't.'a l. 1:g8.2 ): OUl"f'ng tra-nsport some of the gammachains becom e in s e rte d i!!,.II re ver s ed ori e nt ati on with theca r bo xy l terminu sonthe plasma memb r a ne sid e and the amino ter-atnu s on the cyt opla s mic ~id e (Clae-

~son-we1sh

^{et a l, 1984 }-.}

,

Llke 'mo s t tra ns memb r a n e glyc o,e.ro t e in s the ca r b ox y l ter mi n i of the alpha and bet a'cha in s arc lo ca ted on the , cy to p l a s mic std e of th e e eser-e ne with' the hyd ro pHobic se gment s tr~v er s fn9 itandthe aminotermin i on the external side of the membrane (Kaufman and Str omfnger 1979; Korman et, et ,1982) .The DR alpha cha in is compos ed o,f 229amin o aci~ s (IS..in te r n a l l y , 23 ~ra n s m ~m b r a n e , and 191 externall y ), ,Whereas the beta cha i n-has'237 residu'c s (16 fnt e r n a l ly , 22 t;ansmclllbrane •.and 1'99 externally) (Kratz in et a I , 1981 ; Yang et et ,,1982 ; Kaufman and Stro'llIinger 198~) .'80 t hhave,domain~

1 He structures woft h the beta ch'at,nhavingtwo imm u n o ~

St ro mi "g el" 198.2).

,10 globu li n - lHe intr a c hain dtsu lph id e bond s whe rea s the alpha eh'ai n has only one immu no glo bulin- l1ke domain (Ka u f ma n and' Mo!'ecular stu dies using se~q ue nces der,l ve d from clo ne d cONA and f(om "gen omicDNAhave sho wn tha t ea ch of the domai ns

J

seenin the pr ot e in st r uc t ure is encoded by II sepa-rft e ge ne segmen t (F i g u re 2).Ho st:ct the pol ymorph i sm re ~t d e s on the themos t ex t ern al'domain ( 1.)of theOR beta chai ns while the alpha chai nis inv a ria nt (Char ro n and McDevitt 19 79:Shackel-

l '. -

fordet a1.,19 82).Howev -erDQalpha'c ha i ns aswel l"a s OQbet a chains have beenshown.-~o be polymorphic(de Kr-etser-et el, 1983: Co rt e et at, 1981 ;Hurl.ey et al.t9 8 4~.

The DR bet a ch ai ns have one aspa ra gi ne- li nke d ollgo~

s a ccha rtde at posit lon·19whil e th~ alphaehetn has two es car e atneettne ee amino acids at positi on s 78·and 11 6 (St ro mi nge r·1 980 ;

Shack elfor~

^{and St}roillinge r 1982 ). Bio c he m- ical st ud ies using tun icamy~in to inhibit the tcr-a etton of asparagine-linked:s u gar s or en~oglycos idas es toremove suga rs h eve sho wn th a t the poly morph ism·1s res tric t e d to the polype pt 1de s wit hthecar bo hyd r a te s cont r ib utl,ng ver y )ittl e to the het erogen e it y (Sh ac k e l f o r d et al, 1983). n ee ene rep ort s by Nepom,'eta1. (1983 ) however sugges t ~hat the Dw vart e'nts Dw4 and Dwl4 differ on1 Y,.1n ~he post~tran s1at ional modifi cation of the mol ecule s due to ol t go s ac c h4f' 'kIe dlffer ·'.

enee s, but thi s has·not been confirmed.

"

t~·. \

:':,....^"

I.. .

'>

. 'i

1.1.18.~o1 7.orp hi u•• t the

au

le nl

There Ire ~t lea s t thre e sub r egions in the Ht A·D regi a n wtt ha.tn1l11 uIIof six alpha gene s udeigh t betagenes tde nt t f te d so far. Most of the genes for eac h 5ubr e gton wer e lIap p ed usin g a cOllb{n at,l o n of eDNAand ge n Olllc clo nin g (r e vt e we d byr:orll u et al. 1985; Trowsd aleet a1. 1985). Ihe order of the s~b r e g1o n s (Figure 3) 15 Ilo s tl y esta blishe d alth ough the wh o l e region has not yet be e n map ped with ove r l a ppi n'g COS'mids (revfe wed.'by Trowsda l e et el ,1985l:The

·map.( ada pt ed and mod1f f ed fr om Bodme r eeet ,1984a) 15b~sed' on studies involving molecular data .;" well as on re comb in- ations'wtt h in famn n s (Shaw et a1. 198D).a~d HLA-deletlon lIutant"S" (Kav a t has et et, 198 1 ) .

In each of theDP andOQsub r egio ns ther e are twopa1rs

. I ' .

of beta and al.ph. aene s,but.onl y onep. 1r for ~ach subregi o n '15 def1n1telyknowlI"'.to be expre ssed. It 1s tho ught"tha t

palr of the DP gen es (fh al ) 15n.otexp r esse de tnce both the genes cont •.in frallesh tft ..ut~ tt ons . Re cent ly tt hal.bee.n_

.shownthat both~he

131

ge ne andtheat ge ne are polY lllorph 1c.

Inc r e as e d reco mbina tion betwee n OP and DQ.,possibly eue toa hot spottelol1lert oto DP. fs said to eaplatnthe lack of linkage d1sequ~l1br1ulllbetwee nDP and the othe r class II mol e cules .

One pair of the'OQgenes is high l y polymo rphic whereas ther e 15 l1mtted.·po l y mor ph 1 s m of the other pa1,r wh 1ch is

~"."

12

HLA-D REGION

Figure 3. Schematic se p of the HlA-D region genes. adapted from Bodmer (1984). The pI acement of the DO beta gene between OP and OQis based on the work of Tonnelle et al. (198 5) but it may lie between OQ-DR and the complement genes. *putative recombination hot spots.

13 sometime s calle'dOX..(Tro wsda l e e.t a1. 1985). Rece nt wor k at th e protein lev e I su g ges ts that SOAlehomo z yg ou sce l ls do expr es s two differen tOQ molecule s (Gl1es and Capra1985).

80th DQa andf3 a.re

4 1~hl Y

polym orphi c and sho w stron g lfnkage di seq uilfbrfumwHh ea c~ ot h e r and w1.th ttle DRbeta genes . This

w as

the rat fon ale'th;tBodme r et at, (1984 a) used -',"for placing'the m togeth er and to the r1 ght of two.puta tiVI!,

. • 7

recomb1Q"lItfonhot spo ts,'The o rjter twoDQ"gene" (OXa and ''''OXP )areles s polymorphic tha n t'he oth,er se t of OQg~nl! s.· .-

~,"thO U 9 h t~ey,are 1'n,l i n ka ge d1sequ111br1u m.wHhel ch other.

they are not 1n linkag e.d 1sequ fl 1 b r.1 umwith eithe r the DQ

I ' -, .. '

genes'or the,OR"bet'a'aenes,Aga i n this'e x p latns whyth e y,wer e placed ne x,t'toeac h,o t he r,a!"d betwe en the two hotspo ts.

The DR subregion ha s been placed tel ce e rtc to DQ by ana l ogy with-the I~A and I. E'~s'ubregio ns of the mous e . In

add1t,i~n

^{t.her e fs some eV.i.dence}

~f

^{a ""} cros sov erthat puts OQ

c~omeric

^{to O..R'(r e ported inGile san}d Cap r a 1985 ) .The OR subregio"nconsis ts of o~ly, o~e,a l ph,a gene b~t the number of bet-a"g e n e s may be'as high as fou r depending on the haplotype . One of the beta gene s·apP"ls to be a ps eudo- -ge ne (Trowsda1eet a1. '·1985; Moller et at. 1985 ). Unl.1ke the aJpha genes of'the DQ su_bre9ion. the OR e l pbe is not pol ymor- phi c.butth e,functional beta genes He highly polymorphic with the exceptio n of'the ORseco ~d beta gene whfch enc odes the.DR supertypicspe ett tetttes, ORw52~nd ORw53 .

',~'

" ... .

.. ... .

14 In additionthere may be othersub,.e9ion~. An alpha gene originally ca ll e d 01. then renamed DO (lnc ko et /11. 1985), appears to be the same as or allelic to the DZ alpha gene (Tr ows da l e et /11. 1985) and has be.!" mapped to"t heO-reg ion . Recently. a eDNA clone containing a beta gene, called DO was is o l a t ed frolll...the mRNA.qf a hemizygous cell line (Tonnelle et et,'1985).Using mapping techniques. they'have shown that It

is most'likel'y situated either between.the DP genes and the

DQ:-OR' genes.or betWllen the'DQ- DR genes e nd the campl eeent genes.

Charron et /11.'{l98S1 and Giles and Capra (1985) have

. .

reported that an additional source of polymorphism is possible with the expr e ssfon of hybrid OQeotecut es.on the cell surf a c e.~p p a re n t l y the alpha chain prodLftedtr om the DQ .ge ne of onech r omo s ome can tr ans as s ocle e e with the beta chain

produced tram the OQ beta gene of the other chromosome to produce a new DQ molecule.

1.1:1C SerologIcal definItIon

Molecules encoded by the HlA-D SUbreglon~ro~~en dif.flcuft to define serologically a·nd biochemically usi,fg standard~ypin9reagents spCh as alloant15era and heteroantl- sera.This 15 malnly due to the many polyclonal reactivities th~,t are pr,es~nt In an"'t'lvra. Even s o-eelled~onosP~ci~C <...- antisera often react w~th t~o or nor-e cto s el.y related

IS determinan t s that are pres e nt on differ ent eet e eutes, 111 addttton, most of the allo antisera have low affinlty con- st ant s~a('e

- \ - O f

^{t Her andare 1n l}l mite d sup pl y,

- ,Durin gthe 1984 Nt stoc ompattbfL'[tyWo rk.s ht>pa total of 10 DR spec1f ic ltf e,s (1-wIO) were conrtraeo.,~ y se ro logical typi ng mai n ly using ati-oentts er e,Ther e wa s no evide nce tha t ORI, 3• .7, w9 and wlOmolec ul es ca rrie dmor e than oneall e lic

til vari ant ; splLts of DR5[\till and w12) an.d gRw6 ("13 and w14) were recognize d on the b ests ~f react i on s with .antJ se r a and: monc:iclonal.a nti bodi es (Bet u eret , a l. 1984; Schre ude,r. et'

.

-

a l, 1984) . Alt ho ugh not gh en 'w ork sh opstatu s,DR2appear sto ..r;a·ve

,,,v)

least two ser-cloatcel va ria nts (Singa 1 et al. 198 5,~_

M~rvar t ^{et ej, 19B3 ). while DR4 appe ar s to ha ve thr e e} sero logi cal varian t s (W.41 Ifa msonet a1. 1984). DRw815 qul t e complicat ed and wfl1 11k.el y be split enen specifi c anti se r a Of"IROn Q c1~nal antibodie sb~c ome ava11abl e.

The DR4 specffi clty has prov ed esp ecf a l l y diff icul t to _cha r ac t e ri ze wlthstandard typ in g reag ent s and 1t'was found tocorrelate poorly.wlth'thece ll u la r ly - d'efi ne d Dw4spectrt-.

city. (Rein smo en and Bac h 1982).There are seeer-et'possi ble explanationsfor thi s including the pr;e;;nce of antfb od1es t-~'/

the productsof cl os e ly related g~nes.such ~s tho~e e~cod 1ng th e DRw53 and DQw3 spe c ific i t ies in efl c enttse r e which can eo'n f u s e the result s .As prev i ousl y mentfoned DR4 ha s tentat1~ely ^beensubd iv1 ded-tntc three-~erotypes ^on.th~ basis ""-.

'~.

.

.-:- .

.. ⁽

16 of reactio ns wlth three clust er s of alloan t 1s era anal yz ed In the la s t worksh o p. In add ition severa l 1nvesti gato rs usin g Hl C.and lmmun oc hemi Cli1)a na l yse s hav e sho wn that DR4 ts II s up ertyctc s pe ctt tctty which Inc lu d es severa l subty pes. ThJsu ~t y p e s ^{d eftne d by HlC (Re1nsmoe n and BaCh'19 B2 ;} Thomp s en .e t el, 19 8 3 ) corre l at e wel l with the'str uc t ur a l va r1 .atio ~.s.se en tn-tne DR beta ctre tnstha t wer e 1mmuno prec1 pa ftate d wlth mcnc c'f cneLsent tbc ctes and""ana lyze d by 2-0.ge ls (Gron~r^{et. et,} 1983; Nepom et si. 1983) .

Re ce nt_·1mmunoch emi ca.l analy s is ofOQ molec u le s deriv e d f;Qmhomoz yg ou s ce ll. lin e s showedtha t these are at least as' comple x as DRmo le c ules (Gl1e s an~ Ca pra 198 5) .Thereappear to be thr ee va r ian t s of alpha chains and ttve v erte nts o.t beta chains so that each OR ty p e ts a' s s o ciate d_wi-t h a dis t in c t DQ type. For example . although bot hOR4 and DRSare as soc i ate d with OQw3, the.OQ in each type is a diff eren t allel i c var1an t: There f or e .It seel.~.like ly't hat the sero log -. Ica.! ly- d ef1 ne dOQ spectt tetttes have severej.s ubtypes . Fu rth- . er more the.pre sen ce of shor t ant ib odies in 1I.11? a ntis e ra to'OQ alle li c var ia nts woul d be al~ ~~.t.~lmp o s s ib l e to d1,st1ng uis h from theclc s ety as s oc 1'at ed ORspec ific it i es. St ast ny et ~1.

(1984) neve-shownthatsomeof the alloarl'tibod 1e s to DRspec - ifJ c1ti e s '"als o.,conta in

anti bod~ es

^{t ;}

s;e~.lftc

^OQ

va~ts

^•

...

. ...

-:

."

1.2 1II0n'octo ..1Antibodies to Chss II Ant igens

..

froll Illyow~'experie n c e and jUdg1ngfrolllthe ab u n d anc e of repor t s tn the1He r-at ur e. thepro~uct ton of 1II'i!1ne mono- clona l antibodie s which recogniz e mon ollorph t c cell

su -;'f ac e" '

d.eterlllnants 15,rela:~h e..1 y eas y.Howev~r'1t Is mOrl! djf f t· cult to make 1II000c100.1 antibodies capable of'distingu is hlng the spe ettr c prO~f one ge n~or all e le,fram"-.that ~f a.

cleselyjreleted,g ene or allele•.Man y of the mon oclon al ant I bOdi es th,at-'have bee." described'are'sub region.spe c if ic

sU~h'

as NEt-antt·

i.

^{which reco.gntze s}

monom~ "p hlc

deter minant s .o nORmolecu les (H~ n s e n et-af~ 1'~80 }-"Other s fn addit i on

t o

rec og nlz i"g .11 the produc t s

,

of on e sub reg to"n

.

IU y also

.

recog n iz e an all e lic product of anpt h..e r su b r egton.FO,r exupl e CA.206, wh1S;h're co gni zes a 1I0nomo r phf c DR,determ~

1nant,'has been shown recent ly to re c0 9n1ze adee eretnent on DQeeteeute s of DR7 cells (Cha r r onet el , 19841.

As has been poin t e d out by Gil es and Capra (-19 85) . Nany'o f th e.or1 9 'nal Il~noclonal enttbe ete s were'poo rly descrt be d because the cOliplexHy of the D. r €9l on was no t' full yappr ecia t ed and lIlanyof t'he cel ls ~se dto cha r ac terize ", the spe et ttctete s were not alway s homozygo us. Never-tnel ess they were, and con t t~ u e t9 be tnval'uable for 1mmunoc helll1c a l anll y s h.·

Monoclo na l ant1bod 1es wer-etested,p r operl y for the f1rst

.•r:•. ••.•

".~,~

18 tfme inthe 198 4 Internat..t.ona l Hlsto eompatlb fl lty W,orkshop. Of 110 HLA·D'r eg1o n mo nocl o nal antibod ie s that we re submit t - ed, 92 we re accep t e d for fUrr te'stfn g (Bod me r et et . 1984b ). Inthe final analy se s , 54 of these we r e grouped lnt o

I .

twenty clus..te rs depe ndi ng on the produ ct recognlzed.The r e we re two cluster-s for OQwl, one ea c hfor,OQw3 , TA10 (subs e t of DQw3),ORw5 2 . OR4, and the rest 0.1the cIuster-s fnclud ed mo r e ihanone kno wnor unknown spe c1 flc1ty ,

The thi"rd list ing of the ecnocl c ne l antib od y'regis t ry

(Co ~ban,1

^{et a1.1984) Inclu ded all those} submit t e d to the _) wor ksh opas well' as p"re v1o us ent r ies (C~lo m"b a n1et at, 1982;;, _

Colomb a n1et al, 1983) and new ene r tes up to June, 198 4•.Qf the class II monoc lonal ant1 bodfes ,·3S (44 .9 1) were "dettned POly mOrPh fc : wh11 e: 10 (12.81) were

."'u nde,f 1 n~d P01 Y~~rPh i · -:I_'

12 (15.4%)wer~ ·defi ne d eon ce crp ntc " and 21 (26. 91 ) w re

"u ndeffned monom ~rPh 1 c · .On l y am1,~ or ity of the polylll o r~+c- / ent-tbpdtes r ec o qnteed'. the cl a ss ifa l e netqe n s de fi ned by al lo a n tfse r a andthe se were: one ellctl f~rOR1- 1 lk e, OR2-a ctf- vate d ce ll s. DR3+ monomorp~1c OQ. OR~, DRS, .DR1, OR7+wl S ", DRw8+wl S, ORw6 +wS. ORl+4 +10, DRl +4.I1ke . DR2+4+6, OR7+3+S+6 ;

t~"o e~ch

^{':o r} DR4+1+9 and ORwS3; three for ORw52.Th'e r-eeetn- fn9 monoclon al antibodies rec ognJ. zed polymorph1 sms deflne~;_

aS,e1the rOQw1, OQw3,or related spec if ic it i e s . When one cons 1,.rs,that ...1~ HlA·D reg'1on vir1ants ser olog l call y recognlze~d in, ,t he Ninth Inte rnat10nal Workshop

. '

.,/~,

--' ,

"

.

19 (Albert and Hayr 1985).a~d^{that numerous} laborat'or'1es have been engaged.

fn

the productfon of monoclonal ent tbcetes to HlA antlgens for almost a decade. i tIs.s u r p Hs in gthat there 15 5\111 a paucHy of HLA-D regfon eonccjcnet antibodles for routlne HLA-D typtng reagents. However , it'mu st be po inted

'o ut ,t ha t 1n addf ttc e to those discu ssed above, there are

other-reported llIortOclonal antibodfes to."polymOrph 1c determ~_ tnents , bu.t either they'a.re.:not cYtot'Olt1~·.(~.11,,~n t1 bOd 1 I! S_' .wer-e.tested by comp1ement.·d~pende~t c~·iox. 1~\t;^{',1.n...the-Le}st;

:O" .Sh~;)O~h

^{e v}

ebmde~cr;bedfl:te

^the

w,~; . • ,I~OP" "

⁾

A.lthoug~ ~t,1_s~1'$appo1ntlng·,t~at.mo.st bf th!!! monoclon al , ent tecei e s have'not p.r O'Yedus,ef~l.for 'u r o l o g i c a l'an.alys'1s ; ,th ey nave been invaluable. ·pa'r t i c u l arl y"1n.:1mmu·n o c h e m1c a l a n.al y s.e s , In "tn c re estn s our u nder-ste ndLn g of class JI 'nio 1e c u ies.

8eca~se

acnccu one l

antfb~d.i'S

^are

'ultra~peCifiC

,

.. - -

reagentS_l t'~ey are' often'c a pa ble o~ d1scriminat,1I\g betwee~ ,.,;

two'closely related determ1.nants,' For example.:1n.t h e:las~, WQ'r-kShop'. OR~l-3 (a ORw6',splH).c ou l d be dist1'ngu1.shed h'om ORw12 (a OR5s,pl1t) 'on thebasi$-'of their-r-eacttcns w,fth:fcur

mO/~ 'Clonal a~t:1bOdfe~ (S.c-hreUde·~ 'et· ~~'. i9~:4l ;C~~'ia:ldf' :~~

"~1' (1985) has de s c r tbed an interesting bro.adl~ rl!acting

nal antibOdy which'r e a c ts wHh

, on ,

'OR'W8 ,.~ nd~'.D Rw9'bu t can a arently ~1fferent1ate between,O~;wI3 ^an"d__o~wi4"'(Q,RW6 subtype's) and.betwe~n010110'a nd Ow4(~R4·~'i'b·1;YP.~s)o''He nc e~ven ' a

mo~o~'lona.l'

, ' .

an'ttbo~/W1th.

-'

a .broad:·~·p·~c

^{" ' ,}..

ifl (~t~

- ^.,

'c a.~'. ~,e'

".

~ -,

." _., t

_"(

'C I

'r

.1

20 useful ty p ing rel gent.

') 1.3 ' rep "r At ton of Monoe l0 nll Ant 1bod 1e s to HlA·D bgt.o"

Mole cules

--

Wit h Oe ad v ent 0) morioel~nal an t~~y tech.nO)o·g~ / ' (K.oh loer and Mils t ein 197 ~ ). s eve r e.l tnv esttaetcr s hl,we:

attempte d to produ c.e aoncctonat:ant 1.o d1e s.which re cogn ize spe et t tc det\rmt na n ts on HlA

: ~O:.l·eC ~ l ~ $::

^{S1nce ther e are'}

~ Ye ra l

^good

re ~1ew

^{eret el e s (Godt ng}

~~80 )

^{an d"textbook s}

(Ke nne tt et el, 1981 ; Godi ng 1983 >'whlc h de"S.crfbe 1n detatl

·the pr e pa rat 1o-nofmono c l o nal antl bodies.on ly.• brt el ove r - viewof the tecnn t q ue as_. i t perta·,_\n·~to thiswor kwil"be

:re-

vtevec ner-e , ElIlphas!s,wl l l beplac~^{onUUY,Sused-to}sc r.e.~n for and cber ectertee~on ocl onal ant1&od1'es to HLA ant igen s .

) .

1.3.1 Tl!!c~nt q ue ..1-

--.~

l.l.IA •• • un1utton

"':

.

'.

Whtl esome1nves t1g!ltors (Brodsk y et eL, 1919)have IISIt'd,

s,em.' .~ ~r if 1 ed

^HLA

ll\~O l ~icu les'>pr\p a red " f r ~~ ~ e l l

^lySat,U.,

ot bers have s1mpfy use Ifhol e'C~l 1s~O ^'mmu n1zl! mit e, usually:'

.

.;.

~ ,: :

Balb/ c (reueee et.a1. 191 9 ). Tbe',dOSe and th e number O,f'

1 mnru n i za t lo ~!

^{are vertaete bu t}

~e"era ll y .t.wo 1mmU'~; Zlt.t ons'· ·;

.' • :':10-'" . .:.

,

:' .

v., :'

...~

.

^{~' :}

^.

^';,"

"

/1

21 seve ra l w~ek s ap.,- t are give n fntr ' pe rHon ea l l y anda f,".l iMmun1 zat 1ong1ven1nt ra~e n o usly threedays prt o r to~fus fon . InMos t cases the allha l w1'11 res pond we ll to cell ular

I

Intf,ge n s..t'u t.the.re s~onseIs predomina tely spec1es-spe c1f t~. ' Sfnce ftndtn g a spe c

.

ts-tc.a-cett laa~ 1ngant1bodyto a p ertte u- hI' HLAan t tle n allong the nUllerp us ot her 1mllune a-ceHs In the

sple~ .

^.fs

te;'~ ' h'l t ca llY ..

^dlff'

r

^cult

"

^1I0St

,

^{1n v estlg ato r s}

" ,

"per f o r mseeettc-c ett tus tons w1thljut prevlo.,.se lecttcn,

, . I ,"': ' .

1.1.38. 'FUSfon.proced ure" I

Th e r e are several 8-azaguantne r e stse ant myeloma line s that are defici ent in theenzyme, hypounth 1ne "'guan 1ne-phos- pho ribos ylt ra ns

.

fe r ls e (HGP RT as e ) wh'tch lIake s'u 1tabl e-fusion 'part ner s: It 15peete r-ebIe to use one of the,n on-sec re t or s

suchIS SP2l0-Ag14 (Sllul'IIIn et a1.'1918).'

. • ·'1 . "

Fus10n t.chn1q uu ur-y from one labora tory to.~ano~he r wfth re's pe c t to the co.~ o s 1tfon and e enc e nt r-etten of the pol y ethylen e 9"lycol. th~ ratio of,sple e n ce11 s to mye lO'lna

"

--.,. .

cel ls . the duration of the t\ybrt.dt zat 1on. the cu ltu r eeee tue, supplellents. and'the use of a fe ed er layer. Hy brtd s are

.·s e le c t e d fn HAT

med1~ .

^{'wh1ch contaIns' t}hymtd1 ne a'ndhypOXt -

"thine IS the exogenous.source~'\of DNA nucleotide'precursors

for the salvagepathway (Llttlefield 19641. They ar e required because'the. ~pathway 15 blocked'by the aminopterIn, a foltc acid ana lo g , whichstop sth~HGPRTase de f1c1 e nt myeloma

.. . . .

^{' .,.'}

...- ^{.r» >}

~:

.

.

.. ---

²²

ce ll s fr OIl growing.Only fuse d cell s which espres s th e HGPRTas e en'll'J1e trOll the splee n ce'l) pi rt ner wil l pr 01tf:

e ea ee,The unfused spleen cel ls are tncap a ble of con t 1nuou s grow th in vitro and·d l e na t ura11y. A fuston and sub se qu e nt cloning of'posltive hybrids can re su lt 1n hund reds of -ete eeeu teur es

.I

~tha trequir een eff

.

ic ient scree ni n glIe t hod.

1.3.2 SCl"eenl!'1g'forHLA-s pec if ic i t t es

1.3.2A Callph_n t.depend e nt.cytC?x1ctty

For app'rox 1mat e'ytwenty ,ears,HLA Srflo.9istshave'us e d the,coDlPlement~depe nde n tcyto ltf c H y (CDC)''a a, (Terasakt and Hc Cl~ l 1and 1964) to def in e the HLAant entc syst em and to charact eri ze al lo ant.isera whtc h ar"e us e d to"HLA. ty pe.and crossn~tch pa t i e nt s andtheir pros pec tiveorgan dono r s .AeJl.c-

r tions of lDany of the lIonoclon al anti bod 1es ar e'n ot det ec table

~y

^{COCoEit he r}

thet ~

^Fe.

r~~ ions

^do

~"ot

^{bin d}

the. ~con.Pl elle nf~

c eepcn e nt, Clq. ef f i c ie nt ly as 1s the cas e'for IgGI anti_.

'bOd~~or they bin d epit opes toofa~apa rt to be c'ros ~- l1n k ed' by Cl q"(jruc c c et a1. 1980) .Thi s cro ss l fn king fs requir e d for the tfl H f a t1 0n of the cl e sstcal comple llent cas c a d e . Su c h ~ technical·p{ OblellIt ~,r~ rar e ly enc o un te re d When,antisera with th c"rmanyPOlyclo\al s pectttettte s are used. In·o.rdernot to

. , ~ l

^{monoc lonal} antibodi es

ag a l n~

^{.r are spectttcttte s , ft}

1 J ,

~".

impo rt a nt ther ef or e.t e use.an al t ernat iv e lIe t ho d th. t is..

. _. ,

"

..

. '

,

•

23 sensitive, specific, and'~ep"~du ttbl e. It should, also be sImple and fast because successful fusi on and clontng can yIeld hundreds of cultures that require immediate scre enin g since

new

hybrids are unstable (Goding 1~80).

1.3\28 Cell binding assays -.

Many.of the standard immu no l o g ic a l assays in cl u ~ 1ng '{n d't r ect lmmu.nofl~roscence and irn m u n ~ p e r o x i d a se st ai-ni n g are- tedious to perform ~.n large numbers of .?mpl.es and have tfie disadvantage,of reader bia s. Solid(hase assay,s S~Ch ^as radio 1mmune assa ys (RIA) and e nzymecl!nked te s u n o scrbent assays (ELISA) are usually pr-ete rred, Most of the origin al RIA were performed on cells in suspension and specifi c . monoclo~ antibody that bound to the cellswas detected with· 125lodine ,;,lab ell ed

r~bbit

or goa-t-anti-mou,se

irlmunoglobu1in~.

Certain drawback s fnc l u dtn s the in h e r e nt -d a n ge r.of using 125 1. its

r!,la~1vely

^{short h attcttr e , and'th;}'"numerous centrifugation steps,required. 'l e dse v e r a l in v e s t i g ato rs to develop,the ELISA as'analternathe methd.

Although th~^{RIA as} described by Tsu et at, (1980) 'was' originally used to detect NFlO.M,I, i twas decided to evaluate an ELISA,t h a t used cells stuck t.o polY·~"lysinetreated wells of a.mt,crotttre plate followed by'fhatJlJn with glutaralde- hyde (Stocker and Heusser 1979; Kennett 1981). D'es.plte'the

~

. .

; obvious advantages of hav.ing cells §dhered to plastic. th'ls

4."

\";\;-,'~-,.:.>.. ^r.

....

^~..

met hod had to be abandon e d ee c eus e the sl ute rete eh yde selectlvely destroyed some'HlA-DR'ep"ftop"es and incre as e d n?n- s pe c ifi ebtndl nq, The metho d that was found to be the most reliable was thOe cell u l ar enzyme -lf nke d fmmu nosor be nt assay (CElI SA) performed e ssenttetty as descr~bed by Horrh et.at , (19 8 2 ) ~ In prfnctple 1t is sfmi1ar"~o the ELI SAb~t vfab le un t reated cells are us e d ins t ea d of glutarald·ehy·de.

fi xe ,\ cel ls'.Thi s Ileth od wa s"used for all'the scre en ln g an~' spec i"hC itytest1ngand 15de scrib e d

fn~

^{:d'eta1 l f"nChapte r II.}

"J..4' Obj e c th es

-u

ne of the"monoclo nal antibodles '(NFl D. Hl l pr od uce d 111

this la bo ra t or y appear e d to r'eect·w1bh a polymorllhic eet e r-ert- nailla0 n h~.'Ran B-ly mp h~Cytes .The ma-i nobject1ves of thls proj ect we r e:

~) to determfne the specHicfty of NFlD .iu us1 ng a cellu la r enzyme -11 nked immunoa s sa y (C'El IS A)

2) to st udy the expr-e s stcn of theHI epHope on dIfferent ce lls•

3) to do preli ml n ar.1\ mflec ula r ana l y s i s of the mol e c u le s fmm,:nop rec1P 1":ated

bY~l.)

^{. '}

'r.

,-'.

25 acAPTER II MATERIALS AJlD METHODS

2..1 Production of NFLO."lMonoe 1 ona 1 Antibody

2. 1.1 r ••lln1Ziti on protocol

. .

1:xl07B-lymphpcytes from"a chr on ic lymph ocyt i cleuke mi c patient wi th the'H'LAphenotype: AltAIr;,B1 5. Bw35; Cw3. Cw4;

OR4, were inje c ted intr a pe rit o ne a l 1y (I P ) int o a fema l e neonatal ncus e , le s s than 24 hoursold. This was ,followe dby fu rth e r Inj ect io ns at 1 week, 2 we ek s and 6 week s.Afinal immuniz a tio n was gi..-en at age 3 mon t hs and Ute }1J;ion ~as done 3 days1at~r.~

".

2 .1.2 Fusion procedure

2.1.2A Reagents r ,Hybrl doma'm.edium(HH)

50 011I 1 RPMI1640~ ~OWlab'or a to r ie s General , Mele""' . Vi,."g1n1a 22102USA)

100- 1lI1 hea t-tna'ct hat ed fetal

bovlnJ.f~er·um,

^{(FRS). (Flow}

Labo ratories )

"1.0 ml20 0 m" L-glutamtne (Floll Labo r atcr h s ) 5 ml 5x iO~3H 2~ me r c a-p t oet h a n o l (2a I 1 E ) . (Si gmaChe m'tca l

c .

·; -'.'''';

_ 26

,Co .

,

St Louis.Missouri 6]l2.8USA).

11. 100 x HAT solut10n

136IIghypounthfne (51 gila) ,

i .

76.g U1fnopterfn (S1gill) and 38~8 IIgthYIl1.d1ne (S( gllla ) wer e dis~1ved In 80 .1 dH20 with..,a fewdrops of,5" HaOH added to facl1ttate dis s ol vtng .'It was,the n lIade up to 100'1111, ster111z; d th rou-gh 0. 22",mfflt er (now Laborator t.e s ) andsto red,at -2 0⁰

e

1n·SIll!"eltquc t

s,

111: 100 xMT solut10n

Thi s Will S p r e pa r-eu In the same wa y as 100 Il HAT , but the ulnopt ~ r1nwas omitted.

...

tv. HAT Iled tu.. and HTmed 1ulll

One .1of either 100/X HAT or 100 xHT was added to 10 0

III1 HM to give a f1nal e e nee nt r attcn of 1 x 10.4 M hypounthtne , 4 Jt {0-7 Ha1ll1n opt e rfn....nd 1.6.x 10;5 H t'h;II~dfne.

P01yethylen ~ghc o l (PEG) , 501

• Twogill. PEGlIlw. 40 00(J.T.Ba ker Co., PhlJ,11PSbur'g",

N .J ~V

--wa.s

me 1te ~ bY

^{autoclavlng ."Two m1}

prewar~~um

free RPHI-1 6 40 ('SF-RPMI) was adde d to t~.e"_P£tr vhen tt "

had coo l e d toappr oxtmatel y50° C.

: ' .v..