1997 90: 2398-2405
Miguel Angel de la Fuen e, Pila Pizcue a, Ma ga Nadal, Jaime Bosch and Pablo Engel
CD84 Leukocy e An igen Is a New Membe o he Ig Supe amily
h p://bloodjou nal.hema ologylib a y.o g/con en /90/6/2398. ull.h ml
Upda ed in o ma ion and se ices can be ound a :
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20036.
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Blood (p in ISSN 0006-4971, online ISSN 1528-0020), is published weekly by
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CD84 Leukocy e An igen Is a New Membe o he
Ig Supe amily
By Miguel Angel de la Fuen e, Pila Pizcue a, Ma ga Nadal, Jaime Bosch, and Pablo Engel
cDNA isola ed om a human B-cell line Raji lib a y was ana- (CD48 and HumLy9) ha e been mapped. CD84 monoclonal
lyzed and shown o encode he ull-leng h cDNA sequence an ibodies (MoAbs) we e shown o eac wi h cells ans-
o a no el cell-su ace glycop o ein, ini ially e med HLy9-
b
. ec ed wi h he cloned cDNA. These MoAbs we e u he
The p edic ed ma u e 307-amino acid p o ein was composed used o show ha CD84 is exp essed as a single chain cell-
o wo ex acellula Ig-like domains, a hyd ophobic ans- su ace glycop o ein o M 64,000 o 82,000, which was
memb ane egion, and an 83-amino acid cy oplasmic do- highly glycosyla ed. CD84 had a unique pa e n o exp es-
main. The ex acellula Ig-like domains p esen ed s uc u al sion, being ound p edominan ly on lymphocy es and mono-
and sequence homology wi h a g oup o membe s o he Ig cy es. Thus, he glycop o ein HLy9-
b
is ecognized by MoAbs
supe amily ha included CD2, CD48, CD58, and Ly9. No h- p e iously clus e ed as CD84 and ep esen s a newly iden i-
e n blo analysis showed ha he exp ession o HLy9-
b
was ied membe o he Ig supe amily ha may play a signi ican
p edominan ly es ic ed o hema opoie ic issues. Ch omo- ole in leukocy e ac i a ion.
some localiza ion s udies mapped he HLy9-
b
gene o ch o- q
1997 by The Ame ican Socie y o Hema ology.
mosome 1q24, whe e o he membe s o his Ig supe amily
T
Ly9, 2B4, and he ecen ly desc ibed signaling ecep o
SLAM.
9-11
HE INTERACTION be ween leukocy es and o he cells
egula e no mal immune unc ion. Along wi h he
polypep ides o ming he T-cell and B-cell ecep o com- CD84 is a clus e o di e en ia ion ha was es ablished
du ing he Fi h In e na ional Leukocy e Di e en ia ion An-plexes, a numbe o su ace molecules pa icipa e in B-cell
and T-cell ac i a ion. Some unc ion as cell-cell in e ac ion igen Wo kshop (Bos on, MA, 1994). Th ee MoAbs, 2G7,
152-1D5, and 153-4D9, de ined CD84 as a new su ace an i-molecules o as signal- ansducing elemen s, and some can
se e bo h unc ions.
1
A signi ican numbe o hese mole- gen p edominan ly exp essed by ma u e B cells and mono-
cy es.
12
Mo e ecen ly, biochemical analysis shows ha cules a e membe s o he Ig supe amily, which a e in ol ed
in p ocesses such as adhesion, mig a ion, p oli e a ion, di - CD84 MoAbs p ecipi a e a single-chain glycop o ein o ap-
p oxima ely 72,000 o 86,000 M .
13
e en ia ion, and e ec o unc ions o leukocy es.
2
Ex acellula po ions o CD2, CD48, and CD58 leukocy e This a icle desc ibes he molecula cloning, cha ac e iza-
ion, ch omosomal localiza ion, and issue-speci ic exp es-an igens a e p edic ed o ha e simila molecula s uc u es,
and hey o m a dis inc subg oup wi hin he Ig supe amily, sion o a new gene o he Ig supe amily ha encodes o
he cell su ace leukocy e an igen CD84.named he CD2 amily.
3
These p o eins ha e simila pa e ns
o conse ed disul ide bonds and comp ise N- e minal V-se
domains, which lack a disul ide bond, and C- e minal C2 se
MATERIALS AND METHODS
Ig domains. These ansmemb ane su ace glycop o eins a e
Isola ion o cDNA clones. A cDNA lib a y om he human
in ol ed in adhesion in e ac ions be ween T lymphocy es
Bu ki lymphoma cell line Raji in
l
g 11 (Clon ech Labo a o ies,
and accesso y cells and gene a e in acellula ac i a ion sig-
Palo Al o, CA) was used o isola e cDNA clones by c oss-species
hyb idiza ion, wi h a mix u e o di e en polyme ase chain eac ion
nals.
4-6
In i o s udies ha e shown ha he adminis a ion
(PCR)-gene a ed p obes co esponding o sequences epo ed o
o CD2 and CD48 monoclonal an ibodies (MoAbs) can in-
mu ine su ace molecule Ly9
9
; he ollowing p ime s we e used o
hibi T-cell esponses and p olong allog a su i al.
7,8
O he
gene a e he p obes: Ly9 #1 (559 bp): sense p ime , 5
*
-AAGAAG-
membe s o he g owing CD2 amily a e he su ace an igens
CTTCCAAGAATGAGTCAGCAGCAG-3
*
, and an isense p ime ,
5
*
-CATCGTATGTATTTAAATGGGTGTC-3
*
; Ly9 #2 (470 bp):
sense p ime , 5
*
-GACACCCATTTAAATACATACGATG-3
*
, and
F om he Fundacio
´Clı
B
nic pe a la Rece ca Biome
`dica, he Uni- an isense p ime , 5
*
-CAGTCTCTTGTAGACAAGCAAGG-3
*
; and
e si y o Ba celona, Ba celona, Spain; he Depa amen de Gene
` - Ly9 #3 (445 bp): sense p ime , 5
*
-CCTTGCTTGTCTACAAGA-
ica Molecula , he Ins i u de Rece ca Oncolo
`gica, Ba celona, GACTG-3
*
, and an isense p ime , 5
*
-CAGAAAATTGTCCAT-
Spain; and he Hepa ic Hemodynamic Labo a o y, Li e Uni , Hos- GGCCAGTTG-3
*
.
pi al Clı
B
nic o Ba celona, Ba celona, Spain. cDNA was ob ained by e e se ansc ip ion polyme ase chain
Submi ed Feb ua y 7, 1997; accep ed May 19, 1997. eac ion (RT-PCR) o o al RNA isola ed om mu ine hymus using
Suppo ed by G an s No. PB-1562, SAF96-0120, and SAF97-0136 a Fi s S and cDNA Syn hesis Ki o RT-PCR (A ian Myelo-
om he Comision In e mins e ial de Ciencia y Technologia and blas osis Vi us [AMV], Re e se T ansc ip ase; Boeh inge Mann-
G an No. PIRB-00076 o he Comissio
´In e depa amen al de Re- heim, Mannheim, Ge many) and he Taq polyme ase Expand High
ce ca Tecnologia 1995. M.A.d.l.F. is a ellow o he Fondo In es i- Fideli y (Boeh inge Mannheim) ollowing manu ac u e’s ins uc-
gaciones Sani a ias (97/5246). The GenBank accession numbe o ions. The PCR p oduc s we e pu i ied, subcloned in o pSP64 ec o
he cDNA clone is U82988. (P omega, Madison, WI), and andom p iming labeled using [
g
-
32
P]
dCTP wi h he Ready-To-Go DNA labeling ki (Pha macia Bio ech,Add ess ep in eques s o Pablo Engel, MD, Fundacio
´Clı
B
nic,
Villa oel 170, Ba celona 08036, Spain. Uppsala, Sweden). Fil e hyb idiza ion was pe o med in Ex-
p essHyb hyb idiza ion solu ion (Clon ech Labo a o ies) a 45
7
C o The publica ion cos s o his a icle we e de ayed in pa by page
cha ge paymen . This a icle mus he e o e be he eby ma ked 2 hou s, ollowed by low s ingency washing in 2
1
SSC-0.05%
sodium dodecyl sul a e (SDS) a oom empe a u e. Plaques ha ‘‘ad e isemen ’’ in acco dance wi h 18 U.S.C. sec ion 1734 solely o
indica e his ac . we e posi i e on duplica e il e s we e picked, and phage inse DNA
was isola ed. cDNA inse s we e emo ed by EcoRI diges ion and
q
1997 by The Ame ican Socie y o Hema ology.
0006-4971/97/9006-0031$3.00/0 subcloned in o pSP64 (P omega). Res ic ion maps we e gene a ed
2398
Blood,
Vol 90, No 6 (Sep embe 15), 1997: pp 2398-2405
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Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
CD84 LEUKOCYTE ANTIGEN 2399
and nucleo ide sequences we e de e mined by dideoxynucleo ide Monocy es we e isola ed by adhe ence o blood mononuclea cells
o plas ic o 60 minu es a 37
7
C. Cell lines we e cul u ed in comple echain e mina ion by using he T7 Sequencing Ki (Pha macia Bio-
ech).
14
A compu e sea ch o nucleo ide and p o ein sequences was medium (RPMI 1640 supplemen ed wi h 10% e al cal se um
[FCS], an ibio ics, and 10 mmol/L glu amine). Cul u es o all cellconduc ed using he Blas GeneSea ch (Na ional Cen e o Bio ech-
nology In o ma ion, Na ional Ins i u es o Heal h, Be hesda, MD). lines we e spli he day be o e analysis and we e in loga i hmic
g ow h.Sequence analysis was pe o med by using PC/Gene So wa e (In el-
liGene ics Inc, Moun ain View, CA). COS cells we e ans ec ed wi h he HLy9-
b
cDNA subcloned in o
he pMT2 exp ession ec o using he die hylaminoe hyl-dex anRNA blo analysis. A posi i ely cha ged nylon memb ane, o
which poly A
/
RNAs om 50 human issue samples had been me hod.
16
Cell su ace exp ession was examined a e 48 hou s by
immunohis ochemical analysis as desc ibed.
17
S able ans ec an simmobilized in sepa a ed do s (The Human RNA Mas e Blo ), was
pu chased om Clon ech Labo a o ies. Poly A
/
RNA samples on we e p oduced by co ans ec ing he mu ine p e–B-cell lines 300.19
by using 40
m
g o pMT2-HLy9-
b
and 2
m
g o pCI-neo ec o con-Mas e Blo ha e been no malized o he mRNA exp ession le els
o eigh di e en housekeeping genes allowing he de e mina ion o aining he neomycin esis ance ma ke (P omega). The 300.19 cells
we e ans ec ed by elec opo a ion (280 V, 950
m
F) wi h a Gene he ela i e exp ession le els o a a ge mRNA in di e en issues.
The nylon memb ane includes poly A
/
om he ollowing issues: Pulse II Appa a us (Bio Rad, He cules, CA). A e elec opo a ion
he cells we e pla ed in la -bo omed 96-well issue pla es by lim-whole b ain, amygdala, cauda e nucleus, ce ebellum, ce eb al co ex,
on al lobe, hippocampus, medulla oblonga a, occipi al lobe, pu a- i ing dilu ion, and selec ion o s able ans ec an s was pe o med
by using G418 (1 mg/mL; GIBCO/BRL).
18
Twen y clones om hemen, subs an ia nig a, empo al lobe, halamus, sub halamic nucleus,
spinal co d, hea , ao a, skele al muscle, colon, bladde , u e us, ans ec ion we e examined by indi ec immuno luo escence sc een-
ing, and he clone wi h he highes le el o exp ession was used inp os a e, s omach, es is, o a y, panc eas, pi ui a y gland, ad enal
gland, hy oid gland, sali a y gland, mamma y gland, kidney, li e , his s udy and main ained in he con inuous p esence o he selec ing
d ug.small in es ine, spleen, hymus, pe iphe al leukocy es, lymph node,
bone ma ow, appendix, lung, achea, placen a, e al b ain, e al MoAbs. The CD84 MoAbs 2G7 (IgG1), 152-1D5 (IgG1), and
153-4D9 (IgG1); and he MoAbs o he Unknown Panel we e ob-hea , e al kidney, e al li e , e al spleen, e al hymus, and e al
lung. No he n blo analysis was pe o med wi h nylon memb anes ained om he Fi h In e na ional Wo kshop on Human Di e en ia-
ion An igens, Bos on MA, 1994.pu chased om Clon ech Labo a o ies (Mul iple Tissue No he n
blo ) o which 2
m
g poly A
/
RNA om di e en issues (spleen, Immuno luo escence analysis. Cells we e kep a 4
7
C and exam-
ined immedia ely a e isola ion. Indi ec immuno luo escence analy-lymph node, hymus, appendix, lung, and li e ) ha e been blo ed
a e unning on a dena u a ing o maldehyde/1.2% aga ose gel. Do sis o iable cells was pe o med a e washing he cells wice. The
cells we e hen incuba ed o 20 minu es on ice wi h each MoAb asblo and No he n blo memb anes we e hyb idized wi h a 1,040-bp
p obe ha con ained he comple e eading ame sequence o he asci es luid dilu ed o he op imal concen a ion o immunos aining.
Iso ype-ma ched mu ine MoAbs ha we e un eac i e wi h humanHLy9-
b
clone and a 2,000-bp human
b
-ac in cDNA as con ol p obe.
Hyb idiza ion was pe o med acco ding o he manu ac u e ’s in- leukocy es we e used as nega i e con ols. A e washing, he cells
we e ea ed o 20 minu es a 4
7
C wi h FITC-conjuga ed goa an i-s uc ions. Memb anes we e au o adiog aphed o 72 hou s (HLy9-
b
) o 4 hou s (
b
-ac in) a
0
80
7
C by using wo in ensi ying sc eens. mouse Ig an ibodies (Cal ag, Sou h San F ancisco, CA). Single-
and double-colo immuno luo escence analysis was pe o med onAu o adiog aphy was scanned wi h a Bio-P o ile densi ome e
(Vilbe Louma , Ma ne la Vallee
´, F ance) and he ela i e do in en- an FACSCalibu (Bec on Dickinson, Immnocy ome y Sys ems, San
Jose, CA). A leas 5,000 cells we e analyzed o each sample.si y was de e mined wi h Bio-1D Analysis So wa e (Vilbe Loum-
a ). The ela i e do in ensi y alue was calcula ed as he op ical Immunop ecipi a ion analysis. A o al o 40
1
10
6
cells we e
washed h ee imes in phospha e bu e ed saline (PBS), esuspendeddensi y (OD) o he do minus he OD o he backg ound.
Ch omosomal mapping. Fluo escence in si u hyb idiza ion in 800
m
L saline, and su ace labeled wi h 200
m
L o Bio in 1 mg/
mL (Sigma) o 20 minu es a 4
7
C. A e washing he cells once in(FISH), using a 1,040-bp cDNA p obe ha con ained he comple e
eading ame sequence o he HLy9-
b
and a 5.4-kb PCR-gene a ed RPMI media wi h 10% FCS and ou imes in PBS, he cells we e
lysed in 1 mL o bu e con aining 0.5% ( ol/ ol) NP40 and p o easep obe o genomic DNA o he HLy9-
b
gene (sense oligo: 5
*
-TCT-
GCTTGTTCTCATTCTGTCTTCA-3
*
; an isense oligo: 5
*
-CAG- inhibi o s as desc ibed.
19
Immunop ecipi a ions we e ca ied ou us-
ing 15
m
g o he CD84 MoAbs 2G7 and 152-1D5. Cell lysa es we eTAAGAGTTGGGCAGAGAAGAT-3
*
) we e pe o med as p e-
iously desc ibed.
15
B ie ly, 2
m
g o each p obe was labeled wi h p eclea ed h ee imes o 2 hou s using 50
m
L (50% ol/ ol) o
mu ine Ig-coa ed beads a 4
7
C. Cell lysa es we e p eclea ed againbio in 16-dUTP (Boeh inge Mannheim) by nick ansla ion. A o al
o 400 ng o labeled p obe was p ecipi a ed along wi h 1
m
g o Co - o e nigh . The p eclea ed lysa e was hen incuba ed wi h 50
m
Lo
CD84 MoAb-coa ed beads (50% ol/ ol) o mu ine Ig-coa ed beads1 DNA (GIBCO/BRL, Gai he sbe g, MD) and 1 mg o salmon spe m
(Sigma, S Louis, MO), and he pelle was esuspended in a hyb id- wi h cons an o a ion a 4
7
C o 18 hou s. Immunop ecipi a es we e
washed and analyzed by sodium dodecyl sul a e-polyac ylamide geliza ion mix. Ten mic oli e s o he hyb idiza ion mix was applied o
each slide. A e hea dena u a ion o he p obe and he ch omosomal elec opho esis (SDS-PAGE) as desc ibed.
19
The samples we e un
in he p esence o 5% 2-me cap oe hanol ( educing condi ions). M sp eads, slides we e incuba ed o e nigh a 37
7
C. A e he pos hy-
b idiza ion washes he slides we e incuba ed wi h a idin- luo escein was de e mined using p es ained s anda d molecula weigh ma ke s
(GIBCO/BRL). The p o eins we e ans e ed by elec oblo ing wi hiso hiocyana e (FITC; Vec o Labo a o ies, Bu igame, CA). Slides
we e moun ed wi h 40
m
L o an i ade solu ion (Vec o Labo a o ies) glycine ans e bu e (96 mmol/L glycine, 12 mmol/L T is-base in
10% me hanol, pH 8.3) o poly inylidene di luo ide (PVDF) mem-con aining 150 ng/mL o 4
*
,6
*
-diamidino-2-phenylindole. Slides
we e s udied unde an Olympus AH-3 luo escence mic oscope b anes (Immobilon; Millipo e, Bos on, MA). A e 2 hou s o incu-
ba ion a oom empe a u e wi h blocking solu ion (10% non a milk(Olympus Op ical Co, L d, Tokyo, Japan) and images we e analyzed
wi h he Cy o ision Sys em (Applied Imaging In e na ional LTD, in PBS), a idin pe oxidase (100 ng/mL; Sigma) was added o 1
hou . The memb anes we e washed wi h PBS wi h 0.1% o Tween-San a Cla a, CA).
Cells. Blood was ob ained by p o ocols app o ed by he E hics 20, and he blo was de eloped using Ame sham’s Enhance Chemo-
luminiscence eagen (Ame sham In e na ional, Li le Chal on , UK).Commi ee o he Hospi al Clinic o Ba celona. Mononuclea cells
we e isola ed by Ficoll-Hypaque densi y g adien cen i uga ion. Fo N-deglycosysa ion, immunop ecipi a es we e diges ed o e nigh
AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood
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DE LA FUENTE ET AL2400
Fig 1. Nucleo ide sequence and p edic ed amino acid sequence o HLy9-
b
. Po en ial N-linked glycosyla ion si es a e unde lined. Hyd ophobic
s e ches ha would se e as a signal pep ide egion and a ansmemb ane egion a e double unde lined. * indica es he e mina ion codon.
a 37
7
C wi h 0.4 U o Pep ide-N-glycosidase F (PNGase F; Genzyme
The 1,040-bp nucleo ide sequence o clone HLy9-
b
#1 is
Diagnos ics, Camb idge, MA) in bu e p epa ed acco ding o he
shown in Fig 1. The open eading ame encodes a 328-
manu ac u e’s ins uc ions.
amino acid p o ein, o which he i s 21 amino acids com-
p ise a ypical hyd ophobic signal pep ide ha is clea ed
RESULTS
om he imma u e p o ein. The emaining 307-amino acid
Isola ion and cha ac e iza ion o HLy9-
b
cDNA clones p o ein has a calcula ed molecula mass o 34,435 M be o e
and deduced amino acid sequence. Low s ingency sc een- he addi ion o suga s. The e a e ou po en ial N-linked
ing o a human Bu ki lymphoma B-cell line Raji
l
g 11 glycosyla ion a achmen si es in he ma u e p o ein consis-
lib a y using mu ine Ly9 cDNAs as p obes yielded se e al en wi h he obse ed ela i e molecula weigh on SDS-
posi i e clones. Fou independen cDNA clones, named PAGE o 64,000 o 82,000 M . The sequence con ains 11
HLy9-
b
#1 (
Ç
2.4 kb), HLy9-
b
#2 (
Ç
1.6 kb), HLy9-
b
#3 se ine and 18 h eonine esidues, which could be O-glycosyl-
(
Ç
1.3 kb), and HLy9-
b
#4 (
Ç
1.4 kb), we e isola ed, and a ed. The e is a single 25-amino acid in e nal hyd ophobic
hey encoded a cDNA o he han ha desc ibed o HumLy9, segmen ha ep esen s he ansmemb ane egion and di-
he human homologue o mu ine Ly9 su ace molecule.
20
ides he p o ein in a 189-amino acid p edic ed ex e nal
A e diges ion o hese clones wi h EcoRI wo bands we e segmen and an 83-amino acid in acy oplasmic egion.
obse ed. All hese clones had an o e lapping band o ap- Analysis o he sequence sugges s ha he ex acellula po -
p oxima ely 1,100 bp ha showed a single open eading ion o HLy9-
b
is composed o wo Ig-like domains. The
ame o 987 bp. The o he EcoRI bands co esponded o NH
2
- e minal domain consis ed o a V-like domain, which
lacks he usually conse ed disul ide bonds be ween he
b
cDNA encoding 3
*
un ansla ed sequences.
AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood
Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
CD84 LEUKOCYTE ANTIGEN 2401
allowing us o de e mine he ela i e exp ession le els o
HLy9-
b
in di e en issues and du ing de elopmen . HLy9-
b
exp ession appea ed o be lymphoid issue speci ic: he
highes mRNA le el was de ec ed in lymph node, spleen,
and pe iphe al leukocy es (Fig 2A and B). Al hough a lowe
le els, hymus and e al spleen also exp essed signi ican
le els o mRNA (Fig 2B), whe eas i was e y low in bone
ma ow. Signi ican le els o mRNA o HLy9-
b
we e de-
ec ed in kidney and, o a lesse ex en , in lung (Fig 2A, C,
and da a no shown). These wo we e he only nonlymphoid
issues in which de ec able le els o mRNA we e obse ed.
No he n blo analysis om di e en lymphoid issues and
lung showed a band o 4.4 kb (Fig 2C). No smalle RNA
species could be de ec ed, e en a e a p olonged exposu e
o he memb anes, sugges ing ha he HLy9-
b
mRNA has
a long 3
*
-un ansla ed egion.
HLy9-
b
is loca ed a ch omosome 1q24. To de e mine
he ch omosomal localiza ion o he HLy9-
b
gene FISH was
pe o med. By using hecDNA p obe, pHLy9-
b
, a consis en
bu no conclusi e hyb idiza ion signal on ch omosome 1
(band q24) was obse ed. To loca e he gene mo e p ecisely,
he FISH me hod was pe o med using a 5.4-kb genomic
agmen o he HLy9-
b
gene, ob ained by PCR. A o al o
85% o he me aphases showed signal on 1q24, con i ming
he localiza ion ob ained wi h he cDNA p obe (Fig 3).
Iden i ica ion o HLy9-
b
as CD84. To check whe he
p eexis ing MoAbs we e able o ecognize he gene p oduc
o HLy9-
b
cDNA, he Unknown Panel o MoAbs o he
Fi h In e na ional Wo kshop on Di e en ia ion An igen was
sc eened o MoAbs eac i e wi h pMT2-HLy9-
b
– ans-
ec ed cells. Th ee MoAbs, 2G7, 152-1D5, and 153-4D9,
eac ed wi h COS cells ans ec ed wi h he HLy9-
b
cDNA,
bu did no eac wi h cells ans ec ed wi h he exp ession
ec o alone o wi h an un ela ed cDNA (da a no shown).
Fig 2. mRNA analysis o HLy9-
b
. (A) Do blo analysis o poly(A)
"
These h ee MoAbs showed an iden ical and unique pa e n
RNA om di e en issues using labeled HLy9-
b
and
b
-ac in cDNA
as p obes. Au o adiog aphy was scanned wi h a Bio-P o ile densi-
o exp ession and had been clus e ed as CDw84 du ing he
ome e (Vilbe Louma , Ma ne la Vallee
´, F ance). (B) Quan i ica ion
Fi h In e na ional Wo kshop.
12
These MoAbs also speci i-
o mRNA in di e en lymphoid issues. Do ela i e in ensi y as mea-
cally s ained a mu ine p e–B-cell line (300.19) s ably ans-
su ed wi h Bio-1D Analysis So wa e (Vilbe Louma ). (C) No he n
ec ed wi h he 1,040 bp pHLy9-
b
cDNA (Fig 4). Exp ession
blo analysis o HLy9-
b
.
o CD84 on he ans ec ed cells was de e mined on he basis
o an i-CD84 MoAb eac i i y, measu ed by immuno luo-
escence s aining. The CD84 MoAbs did no eac wi h un-
shee s. The second domain had he s uc u al ea u es o an ans ec ed pa en cells no wi h cells ans ec ed wi h ec o
Ig- unca ed C2 se domain wi h wo pu a i e disul ide alone. In all cases he eac i i y pa e n o he MoAb was
bonds. A p o ein sequence homology sea ch showed ha iden ical. These esul s show ha CD84 MoAbs iden i y he
his p o ein sha ed signi ican sequence iden i y wi h a g oup p o ein encoded by he HLy9-
b
cDNA.
o membe s o he Ig supe amily, including Ly9, CD48, CD84 is highly glycosyla ed. The CD84 MoAbs we e
CD58, and CD2. These da a indica e ha he HLy9-
b
gene coupled o beads and used o immunop ecipi a e he CD84
encodes a p o ein ha belongs o he Ig supe amily. p o ein om de e gen -solubilized ex ac s o bio inyla ed
The p edic ed cy oplasmic domain o he ma u e p o ein cell lines. Op imum esul s we e ob ained using he Raji cell
con ains i e y osines (Ty ). In e es ingly, ou o hese Ty line because he le el o CD84 exp ession was highe han
(Ty 241, Ty 258, Ty 278, and Ty 303) con ain mo i s in o he cell lines (Fig 5). The CD84 an ibodies speci ically
simila o hose p e iously desc ibed as po en ial SH2 do- immunop ecipi a ed p o eins ha mig a ed as a b oad single
main binding mo i s. These a e sequence mo i s o kinase band in he egion om
Ç
64,000 o 82,000 M in SDS-
molecules such as s c-kinase ype y osine kinases.
21
The e- polyac ylamide gels (Fig 5).
o e, sequences in he cy oplasmic ail sugges in ol emen To de e mine he molecula mass o he p o ein in he
o his molecule in signaling unc ions. absence o N-linked suga s, su ace-labeled CD84 p o ein
De ec ion o HLy9-
b
mRNA in hema opoie ic issues. was diges ed wi h endoglycosydase F and analyzed by SDS-
mRNA exp ession was examined by do blo analysis using
poly A
/
RNA isola ed om 50 di e en human issues, PAGE. A e enzyme diges ion, a loss o he
Ç
64,000 o
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DE LA FUENTE ET AL2402
Fig 3. Ch omosomal localiza ion o HLy9-
b
gene. Me aphase showing FISH wi h he HLy9-
b
5.4-kb genomic p obe. Double spo s o hyb idiza-
ion signal on ch omosome 1 is indica ed by an a ow. The HLy9-
b
gene was assigned o 1q24.
Fig 4. CD84 MoAbs eac i e wi h he p o ein
p oduc o he pHLy9-
b
cDNA. 300.19 cells ans-
ec ed wi h HLy9-
b
cDNA we e examined by using
he CD84 MoAbs 2G7, 152-1D5, and 153-4D9 in indi-
ec immuno luo escence assays wi h low cy ome-
y analysis. Fluo escence his og ams gene a ed us-
ing 300.19 ans ec ed cells a e shown as solid lines.
S aining o he 300.19 un ans ec ed cells a e shown
as do ed lines. A MoAb agains CD22 (HB22.7) was
un eac i e wi h bo h ans ec ed and un ans ec ed
cell lines. Fluo escence in ensi y is shown on a h ee-
decade log scale.
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CD84 LEUKOCYTE ANTIGEN 2403
Fig 5. Immunop ecipi a ion o cell su ace CD84
p o ein. De e gen lysa es o su ace-labeled Raji cell
line, 300.19 cells ans ec ed wi h HLy9-
b
, and 300.19
un ans ec ed cells we e immunop ecipi a ed wi h
he 2G7 plus 152-1D5. The immunop ecipi a ed ma-
e ials we e ea ed wi h o wi hou N-glycosidase
(PNGase F) and analyzed unde educing condi-
ions on a 10% SDS-polyac ylamide gel. Molecula
weigh s (kD) we e de e mined by he mig a ion o a
known p o ein s anda d.
82,000 M band and he appea ance o a majo componen p o ein wassimila in bo h cDNA- ans ec ed cells (
Ç
59,000
o 77,000; Fig 5) and Raji cells (
Ç
64,000 o 82,000; Fig 5).a
Ç
45,000 and a ain e band o
Ç
36,000 M was obse ed
(Fig 5). Compa ible wi h his obse a ion CD84 has ou Despi e his di e ence in elec opho e ic mobili y be ween
he immunop ecipi a es om he wo cells, he bands ob-po en ial N-linked glycosyla ion si es (NXT/S) in he ex a-
cellula domain. se ed a e ea men wi h N-glycanase we e he same o
bo h cell ypes (Fig 5). These esul s show ha CD84 unde -Immunop ecipi a ion wi h CD84 MoAbs o he 300.19
cell s ably ans ec ed wi h 1,040-bp pHLy9-
b
cDNA clone goes ex ensi e pos - ansla ional p ocessing as i was ex-
p essed as a single-chain molecule, ye he de e mined M showed a b oad band simila o he p o ein ha was immuno-
p ecipi a ed om he Raji cells, excep ha he M was was mo e han wice he p edic ed size o he co e p o ein.
In ac , di e en ial use o hese N-linked si es may explain
Ç
57,000 o 77,000 (Fig 5). A e ea men o he immuno-
p ecipi a ed ma e ial wi h endoglycosydase F, a d ama ic he he e ogenei y in size o he CD84 p o ein.
educ ion in he molecula mass was also obse ed. A majo Compa ison o he CD84 amino acid sequences wi h o he
band o 45,000 and a ligh e one o 36,000 M iden ical o p e iously iden i ied p o eins showed a s iking s uc u al
hose ound in immunop ecipi a es o he Raji cells line could homology wi h he ex acellula Ig-like domains o a sub-
be de ec ed. These esul s showed ha he di e ences in g oup o he Ig supe amily su ace ecep o s ha includes
molecula mass be ween he p o ein in Raji cells and in he CD48, CD58, Ly9, and CD2, named he CD2 amily. CD84
ans ec ed cells we e because o di e ences in he deg ee is mos simila o mu ine and human Ly9, which a e p e-
o glycosyla ion. dic ed o encode a p o ein wi h ou Ig-like domains in he
ex acellula egion.
9,20
In con as o Ly9, which has ou Ig-
DISCUSSION
like domains, CD84 like o he membe s o he CD2 amily is
composed o only wo Ig-like domains. Compa ison o Ig-
In his s udy, we ha e isola ed cDNA clones encoding o like domains in human Ly9 (HumLy9) and CD84 showed
he leukocy e an igen CD84. The s uc u al ea u es o he ha he i s domain o CD84 had 34.6% amino acid se-
CD84 p o ein, p edic ed om nucleo ide sequences de i ed quence iden i y wi h domain 1 and 30.3% wi h domain 3 o
om he cDNA clone HLy9-
b
(Fig 1), showed ha i en- HumLy9; and he second domain o CD84 had 38.9% iden-
codes a ansmemb ane ype-I glycop o ein con aining wo i y wi h domain 2 and 33.3% wi h domain 4.
Ig-like domains in he ex acellula egion: a hyd ophobic Figu e 6 shows sequence homology be ween CD84,
memb ane spanning domain, and an 83-amino acid cy o- HumLy9, and human CD48. Al hough he sequences o he
plasmic egion (Fig 1). I is likely ha he en i e coding domains sha e ela i ely li le iden i y (
Ç
25% o 39%), he
egion o CD84 was iden i ied as ans ec ion o cell lines co e s uc u es o he Ig-like domains a e ema kably simila .
wi h he pHLy9-
b
cDNA-gene a ed cell-su ace exp ession
o he p o ein (Fig 4) and he M o he immunop ecipi a ed Domain 1 o CD84 lacks a disul ide bond bu is o he wise
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DE LA FUENTE ET AL2404
Fig 6. Ig-like domains o HLy9-
b
. HLy9-
b
Ig-like domains a e compa ed wi h HumLy9 and human CD48. Solid lines abo e he sequence
p edic ed
b
-shee s. Conse ed esidues appea in s ippled boxes. Gaps a e indica ed wi h b oken line o allow op imal alignmen o he
p o eins. (A) Fi s Ig-Like domain; (B) Second Ig-like domain. * indica es conse ed amino acids o he Ig supe amily.
simila o V-like domains ound in o he p o eins o he CD2 ch omosome 1. Human CD2 and CD58 genes a e loca ed
on ch omosome 1p13, whe eas he CD48 and HumLy9 genes amily.
2,3
The second domain also sha es homology wi h he
unca ed C2 domain ound in his g oup o membe s o he a e si ua ed on ch omosome 1q21-24.
20,22,23
These genes a e
igh ly linked wi h he ATP1A genes on ch omosome 1,Ig supe amily. Fou Cys esidues, which a e conse ed in
all membe s o he CD2 amily, we e ound in he pu a i e which ha e e ol ed by gene duplica ion.
24
By in e ence, i
is likely ha duplica ion o a p imo dial gene also ga e isesecond ex acellula domain o CD84 (Fig 6). These Cys a e
p obably in ol ed in he o ma ion o wo disul ide bonds. o he s uc u ally ela ed CD2, CD48,CD58, Ly9, and CD84
genes. I has been sugges ed ha Ly9 and mouse CD48 sha eOne pai o hese esidues a posi ions 134 and172 delinea es
an Ig-like domain c ea ing he cha ac e is ic Ig-like loop a common ances o ha duplica ed and di e ged, o gi e ise
o a p ecu so o he Ly9 and CD48 genes, wi h a seconds uc u e and his domain con ained many o he hallma k
amino acids ha de ine he C2 se o Ig-like domains.
2
duplica ion e en p oducing he ou -domain s uc u e ob-
se ed in Ly9.
9
The sequence simila i ies be ween CD84,The 83-amino acid cy oplasmic domain o CD84 con-
ained i e y osine esidues ha could se e as po en ial Ly9, and CD48, oge he wi h he mapping o hese genes
o he same ch omosomal egion, sugges s ha hese h eesi es o phospho yla ion. Fou o hese Ty we e con ained in
wo mo i s Ty -X-X-Leu/Ile and Ty -X-X-Val wi h a gene al genes sha e a common ances o .
CD84 exp ession appea ed p edomina ely o be lymphoid-sequence con ex ha can se e as po en ial si es o SH2
domain ecogni ion.
21
These ypes o mo i s ha e been issue es ic ed as shown by Do blo and No he n blo
analyses (Fig 2A, B, and C) and immunohis ological analysisshown o be si es o in e ac ion wi h mul iple cy osolic p o-
eins, including y osine kinases, and a e ound in he cy o- o di e en issues (da a no shown). The highes le els o
CD84 mRNA we e de ec ed in lymph node, spleen, andplasmic domain o di e en signal ansduc ion molecules,
indica ing ha CD84 may be in ol ed in signal ansduc ion. pe iphe al leukocy es (Fig 2A and B). These esul s a e con-
sis en wi h he obse a ion ha CD84 is exp essed by lym-The CD84 gene was mapped o ch omosome 1q24 (Fig
3), whe e o he membe s o his Ig supe amily (CD48 and phocy es and monocy es.
13,25
S aining o lymph node and
spleen shows ha CD84 is weakly exp essed by lymphocy esHumLy9) ha e also been mapped. The genes o he membe s
o his amily a e loca ed in wo pe icen ic loci on human bu s ongly in sca e ed cells in he in e ollicula zone,
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CD84 LEUKOCYTE ANTIGEN 2405
S oneman E, Benne M, Kuma V: Cloning and cha ac e iza ion o
which we e mos likely mac ophages.
13
CD84 exp ession
he 2B4 gene encoding a molecule associa ed wi h non-MHC- e-
may be an ea ly e en in T-lymphocy e de elopmen as hy-
s ic ed killing media ed by ac i a ed na u al kille cells and T cells.
mocy es showed low bu de ec able le els o mRNA (Fig 2)
J Immunol 151:5328, 1993
and cell su ace exp ession (da a no shown). In con as
11. Cocks BG, Chang CC, Ca ballido JM, Yssel H, de V ies JE,
e y low le els o mRNA we e de ec ed in bone ma ow,
A e sa G: A no el ecep o in ol ed in T-cell ac i a ion. Na u e
which is consis en wi h he obse a ion ha CD84 is no
376:260, 1995
p esen in p o-B o p e-B cells.
25,26
In only 2 ou o he 39
12. Tedde TF, Wagne N, Engel P: B-cell an igens sec ion epo ,
nonlymphoid issues analyzed, de ec able le els o mRNA
in Schlossman S (ed): Leucocy e Typing V. Ox o d, UK, Ox o d
we e obse ed. Signi ican le els o mRNA o CD84 could
Uni e si y P ess, 1995, p 483
13. de la Fuen e MA, Pizcue a P, Engel P: CDw84 wo kshop
be de ec ed in he kidney and, o a lesse ex en , in he lung
epo , in Kishimo o T (ed): Leucocy e Typing VI. New Yo k, NY,
(Fig 2 and da a no shown). The exp ession o CD84 by
Ga land Publishing Inc, 1997 (in p ess)
hese issues dese es u he in es iga ion.
14. Sange F, Nicklen S, Coulson AR: DNA sequencing wi h
F om he s uc u al in o ma ion ob ained in his s udy, i
chain- e mina ing inhibi o s. P oc Na l Acad Sci USA 74:5413, 1977
appea s ha CD84 is a newly desc ibed cell su ace molecule
15. Nadal M, Mila M, P i cha d M, Mu A, Pujals J, Blouin JL,
ha sha es homology and ch omosomal localiza ion wi h a
An ona akis SE, Balles a F, Es i ill X: YAC and cosmid mapping
g oup o Ig supe amily membe s. Al hough he unc ion o
o an unbalanced ch omosomal localiza ion causing pa ial isomy
CD84 is s ill unknown, i s s uc u al simila i y wi h o he
21 and Down synd ome. Hum Gene 98:460, 1996
membe s o he Ig supe amily, as well as he p esence o
16. Engel P, Nojima Y, Ro hs ein D, Zhou L-J, Wilson GL, Keh l
JH, Tedde TF: The same epi ope on CD22 o B lymphocy es medi-
ou po en ial SH2 domain binding mo i s ound in he cy o-
a es he adhesion o e y h ocy es, T and B lymphocy es, neu ophils
plasmic domain, sugges ha i may be in ol ed in cellula
and monocy es. J Immunol 150:4719, 1993
in e ac ions and signal ansduc ion.
17. Engel P, Wagne N, Mille A, Tedde TF: Iden i ica ion o he
ligand binding domains o CD22, a membe o he immunoglobulin
ACKNOWLEDGMENT
supe amily ha uniquely binds a sialic acid-dependen ligand. J Exp
Med 181:1581, 1995
We hank Vic o ia To a and Isabel Sa
´nchez o assis ance wi h 18. Chen A, Engel P, Tedde TF: S uc u al equi emen s egula e
hese expe imen s. endop o eoly ic elease o he L-selec in (CD62L) adhesion ecep o
om he cell su ace o leukocy es. J Exp Med 182:519, 1995
REFERENCES
19. Tedde TF, Schlossman SF: Phospho yla ion o he B1
1. Weiss A, Li man DR: Signal ansduc ion by lymphocy e an i- (CD20) cell su ace molecule exp essed by no mal and malignan
gen ecep o s. Cell 76:263, 1994 human B lymphocy es. J Biol Chem 263:10009, 1988
2. Williams AF, Ba clay AN: The immunoglobulin supe amily- 20. Sand in MS, Henning MM, Lo MF, Bake E, Su he land GR,
domains o cell su ace ecogni ion. Ann Re Immunol 88:381, McKenzie IFC: Isola ion and cha ac e iza ion o cDNA clones o
1988 Humly9: The human homologue o mouse Ly9. Immunogene ics
3. Da is SJ, an de Me we PA: The s uc u e and ligand in e ac- 43:13, 1996
ions o CD2: Implica ions o T-cell unc ion. Immunol Today 21. Songyang Z, Shoelson SE, McGlade J, Oli e P, Pawson T,
17:177, 1996 Bus elo XR, Ba bacid M, Sabe H, Hana usa H, Yi T, Ren R, Bal i-
4. Sp inge TA, Dus in ML, Kishimo o TK, Ma in SD: The lym- mo e D, Ra no sky S, Feldman RA, Can ley LC: Speci ic mo i s
phocy e unc ion associa ed LFA-1, CD2 and LFA-3 molecules: Cell ecognized by SH2 domains o Csk, 3BP2, ps/ es, GRB-2, HCP,
adhesion ecep o s o he immune sys em. Ann Re Immunol 5:223, Syk, and Va . Mol Cell Biol 14:2777, 1994
1987 22. Kingsmo e SF, Wa son ML, Moseley WS, Seldin, MF: Physi-
5. Bie e BE, Sleckman BP, Ra no sky SE, Bu ako SJ: The cal linkage o genes encoding he lymphocy e adhesion molecule
biologic oles o CD2, CD4 and CD8 in T cell ac i a ion. Ann Re CD2 and i s ligand LFA-3. Immunogene ics 30:123, 1989
Immunol 7:579, 1989 23. Kingmo e SF, Sou yal CA, Wa son ML, Pa el DD, Seldin
6. A ulanandam ARN, Moingeon P, Concino MF, Recny MA, MF: Physical and gene ic linkage o he genes encoding Ly-9 and
Ka o Y, Yagi a H, Koyasu S, Reinhe z EL: A soluble mul ime ic CD48 on mouse and human ch omosome 1. Immunogene ics 42:59,
ecombinan CD2 p o ein iden i ies CD48 as a low-a ini y ligand 1995
o human CD2: Di e gence o CD2 ligands du ing e olu ion o 24. Wong YW, Williams AF, Kingsmo e SF, Seldin MF: S uc-
humans and mice. J Exp Med 177:1439, 1993 u e, exp ession, and gene ic linkage o he mouse BCM1 (OX45 o
7. Gu
¨ckel B, Be ek C, Lu z M, Al e og P, Schi mache V, Blas 1) an igen. E idence o gene ic duplica ion gi en ise o he
Kyewski BA: An i-CD2 an ibodies induce T-cell un esponsi eness BCM1 egion on mouse ch omosome 1 and he CD2/LFA3 egion
in i o. J Exp Med 174:957, 1991 on mouse ch omosome 3. J Exp Med 171:2115, 1990
8. Qin L, Cha in KD, Lin J, Yagi a H, B ombe g JS: An i-CD2 25. Engel P, Smi h H, Tedde TF: Pheno ypic analysis wi h he
and an i-CD2 ligand (CD48) an ibodies syne gize o p olong allo- B-cell unknown panel mAb, in Schlossman S (ed): Leucocy e Typing
g a su i al. J Exp Med 179:341, 1994 V. Ox o d, UK, Ox o d Uni e si y P ess, 1995, p 607
9. Sand in MS, Gumley TP, Henning MM, Vaughan HA, Gomez 26. Rehmann JA, LeBien TW: Exp ession and unc ion o epi-
LJ, T apani JA, McKenzie IFC: Isola ion and cha ac e iza ion o opes ecognized by wo kshop B-cell unknown panel mAb du ing
cDNA clones o mouse Ly-9. J Immunol 149:1636, 1992 ea ly human B-cell de elopmen , in Schlossman S (ed): Leucocy e
Typing V. Ox o d, UK, Ox o d Uni e si y P ess, 1995, p 62610. Ma hew PA, Ga ni-Wagne BA, Land K, Takashima A,
AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood
Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om