Increasing complexity of HLA-DR2 as detected by serology and oligonucleotide typing

Increasing Complexity of HLA-DR2 as Detected by Serology and Oligonucleodde Typing G. M. Th. Schreuder, P. M. van den Berg-Loonen, W. Verduyn, D. P. Geraets, J. ParlevHet, J. J. van Rood, and M. J. Giphart

ABSTRACT: Serological and uligonudeodde typing was performed on a number of HLA-DR2-posifive cells from different ethnic origin, including DR2 haplo~pes with various DQ associations. Exous 2 of DRBI and DRB5 of DR2-posldve individuals were locus-specific amplified and hybridized with a nm'aber of different oli8onucleotldes capable of discdm/nating between the v~'ious Dw2, Dwl2, Dw21, and Dw22 associated sequences. The linkage of DRB with DQAI and DQBI in these haplotypos w ~ analyzed. Among the DR2- positive cells we could define I0 different DR DQ haplotl~pes by serolngy and 13 by uligonucleotide typing. The DR2.ES specificity is a serological DRwI5 variant which could not be dlscdminated by oligonucleotide typing from a DRwl5 DQw5 haplotype. The DR2JA variant represents a

unique DRBI~1602 DRBS"0101 hapb~Ve. The DRI + 2s h~plocype conshm of a DRB1 DQ region from a Dwl :ad a DRB5 gem: from a Dw2 haplc~,,:pe. Its short DR2 serum [:~tem c ~ be exph/ned by the abse~e of a D R 2 DRB] gent product. DRBS~0101 sequences were found in assoc~ion with DRBl*I501, ~1502, "t602, and "010l alleles. Since the DRB5 gene/s capable of such different assodadom it is compcral~ ~ the DRB3 and DRB4 germs. This .may have implications for the definition of the broad DR2 specificity v~-hlchis predomitumfly encoded by the DRB5 gene produc~. New Dg2 haplotyt:ms iac[oded the following DQ combinations: DQw2-positive DQA1/BI®0301/0201 am] DQw6opOS~five DQAI/BI'0102/0601 and ®0102/0505 haplo~pes.

ABBREVIATIONS HTC homozysous typing cell HVR hypervarinble region mAb moaoclonal antibedy

PCR RFLP $SO

FI,~¢~ I m ~ g y

.32, 141-149 (1991)

polymeric ctmia reaction restriction fragmcm length polymorphi~m sequence-specific oli~nucleodde

INTRODUCTION Serolngically defined heterogeneity of HLA-DR2 was first described by R/chiardi et at. [1]. This resuked in the official recognition of D R w I 5 and D R w l 6 splks of D R 2 [2]. The DR2 subtypes are assoclgted with two serologicallS' distinct D Q w l subtypes; DQw5 and DQw6. la F ~ tb¢ Dc~rt~¢Rt of lmmNnabcn~toloSy and B l ~ n k , Unlacesir/Medical Cfnt~ Lelde~ (G.M.Tb.$.; w.V.;J.P.;J.J.uR.; M.J.GJ and TisJse Typin~ La~rat~ry (P.IH.¢dB.-L.; D.P.G.L Uni~nity Hospital lff~tricbt, The Netberlam~s. ~ l d ~ s ~prist ~ u ~ s to Dr. G.~LTb. Scbreuder, D ~ n r ~ t of I~a~tdegy a~d B l ~ s k , Bl~g l ~3-Q, Uni~,~ity Hospital, P.O. Box9600, 2300 RC L¢i~, T~¢ Netbeda~f. R~¢~i~ Fe~r~ar~ I, 1991; ~c~ptMJun¢ 17, 1991.

Humanlramuadog't32, 141-149(1991) © Ameri~ Societyfor ["[istocomtmfibilltVand IIamuao~eaeties,1991

most populations studied so far DRw15 is ~ o c i a r e d with DQw6 and D R w l 6 with DQwS. A r~re DRw16 DQw7 associ~on h~s been reposed for homozySous typing ce|! (HTC) ILFaM [3], which or'~giaates from Warao l n d / ~ s [4]. Other possible DR2 sob~'3Ves have been reported to occur in Orientals (D1L2.3) and in South Aft/can populatlons (DR2.4, DIL2LUM) [5, 6]. An apparent H L A - D R I + 2 s haplotype, segregating in a famib' causing D R triplets, al~a indi.c~ted involvemeat o f DR2 in further ckss It heterogeneity [7]. A number o f different DR2-related spedfiddes have been reported by cellular techniques [8], most of which could he class/fled as one of the four offici~y recognized DR2 D w specLfiddes. These four Dw specificities 141 0198.$859/91/$3.50

142

G.M. Th. Schreuder er al.

correlated with three different DR-DQ haplotypes: Dw2-DRwlS-DQw6, Dwl2-DRwlS-DQw6, Dw21-

DRwI6-DQw5, Dw22-DRwI6-DQw7. DR2-bearing lymphncytes express two D R molecules with slightlydifferentelectrophoredcmobility, DRa31 and D R y 3 5 [7, 9]. D R 2 heterogeneitywas also observed in restrictionfragment lengthpolymorphism (RFLP) [I0-12]. When D Q polymorphism istaken into account the number of DR2- relatedhaplotypesfurther increased[6, 13-16]. Sequencing of the four cellularD R 2 types revealed two differentD R B genes,both genes showing polymorphism among the subtypes.These genes are now indicated as D R B I and D R B 5 with four alleleseach [17]. There is high homology among the differentDR2D R B I and D R B 5 sequences. W e observed further serological heterogeneity among both D R 2 and D Q in haplotypesfrom different racialorigins,using selectedD R 2 and D Q alloantisera and monoclonal antibodies(mAbs). Such heterogeneity may reflect differences in epitopes encoded by any of the DRB1, DRB5, DQA1, and D Q B I alleles. The availability o f sequence- and allele-specific oligonudeotides based on second exon sequence polymorphism o f the respective genes allowed the assignment of the most likdy alleles to each locus. Polymerase chain reaction (PCR)-amplified D N A of individuals with wellestablished and new DR2-relared speeificities was used to study the different D R D Q haplotypes in these DR2 variants.

The results indicate that DRB5 alleles can combine with different, also non-DR2 DRB1 alleles, and that a large variety of D Q A 1 and D Q B I combinations can be observed together with DR2. It is also possible to speculate on the origin of such variant haplotypes by assuming recombinational events.

MATERIALS AND METHODS

$era, The sera used for DR2 and D Q w l subtyping are shown in Table 1. The serological typing for D R and D Q was performed in Maastricht using the two-color fluorescence technique [18], and in Leiden using the propidiute iodide method [19].

Cdh. A number of different DR2 haplotypes was detected in individuals from different ethnic origins (Table 2). Where possible, their family members were studied for haplotype segregation. Homozygous typit~g cells were tested in parallel. Further details are shown in Table 2. Lymphocytes were used for the mieroeytom xicity test and for D N A extraction.

Oligonudeotide typing. Oligonucleotide typing was performed as described previously [20, 21]. In short the following procedures were used. D N A (0.5 tzg) was amplified by PCR using DRB-, DQA-, and DQB-specific primers in a final volume of 100 tzl. Dot blots were

T A B L E 1 Antiseraused for the recognitionof D R 2 variants No. Name 1. MSD75 2. VR62074 3. DER 03 4. MSD2 5. MgD16 6. MSD30 7. EB53 8. MSD4 9. 9887 10. VR42475 lt. VR4329 12. MSD21 13. MgDI4 14. VR53313 15. EB33 16. MgD19 17. LY1327 18. MSD70 19. llB3(mAb)

10th IHW° 5053

1024 5052

Origin

Specificity,

Mamtricht Leiden Dupom, Brussels Maastrieht Maastricht Maastricht De Waal, Amsterdam Maastricht Mervart, Toronto Leiden

DRwI6 DRwI5 DRw15 DR2 DR2 DR2 DR2 DR2 DR2 DR2 DR2 DQwl DQwl DQwl DQwl DQw5 DQw5 + DR1 DQw5 DQw6 + DQw8 + DQw9

Leiden

Maastricht Maastricht Leiden

~088

• Tenth International Histocompatibility Workshop designation.

De Waal, Amsterdam Maastricht Betuel, Lyon Maostricht Leiden

Increasing Complexity of HLA-DR2

TABLE 2

t'g3

DIL2-positive cells used for class I1 serology a~d/or oligonucleodde typing HLA class I| t~ping"

Name

DR

HTC VYF Dw2 h~C SAS Dwl2 6 panel cells Ast Mort Bru Cai Dov

w15 wl5 w15 DR4, wlS DKwl2, w15 DR3, w15 wt5 w15 wl~i

Bec

Sto Hat

2.ES

DRwI2, w15 w16 w)6 wl6 2.JA 2.JA I + 2s DR3, t + 2 s Dgw[3, 1 + 2s

HTC AZH Dw2l

5 panel cells HTC REM Dw22 Ja¢ N8 Gee

Bez $ch

DQ

Echaic origin

w6 ~6 w6 w6, DQw7 wl, DQw7 wl, DQw2 w2 w7 w5 wt ~-6,DQ~7 w5 w5 w7 wl wl w5 wS, DQ~2 wS, DQ~6

hi. Eur. Cauc~ Or/ental hi. Eur. C a ~ c . N. EUL Can't. Oriental N. E~. C~uc. Surinam N. Eur. Cauc. Marokkan Indonesian indonesian ok' N+ Eur. C~uc. Wa~ao Indian {4} [ndotmslan Chinese N+ Eur. Cauc. N. Eur. C.auc. nk

" l ~ class H phenntyl~eis shown from panel ceE~where the DR2 haplotype touM ~ t b¢ ¢onfin,n.edby #amily typing. 6North Eutopeim Caucasoid, 'nk = origin not known.

prepared using 5 / x l o f each individual P C R product. Hybridizations w e r e p e r f o r m e d with 32P-labeled sequence-specific oligoaucleotides (SSOs). DRB-amplified product was checked by SSO typing for the presence o f D R I - B I - , D R 2 - B I - , and D R B 5 specific sequences. Such D N A was further amplified in

F I G U R E 1 Amino acid sequences of ,0t domains of D R B I ' 0 1 0 1 (consensus) and DR2 DRBI and DKB5 Mleles. The Amino Acid sequences are indicated for which SSOs have linen used. The SSOs encoding the boxed Amino Acids (1 and 2) were also used fur DR2 DRBI and DRB5 specific amplification. Further detials are given in Table 3.

a g r o u p specific fashion (o allow the detection o f H e respective D R B I and D R B 5 alleles (Fig. I). T h i s set o f SSOs could not discriminate between the DRBS+02OI and +0202 alleles. T h e r e f o r e they are indicated as DRBS*02. A set o f L1 D Q A I and 14 D Q B I SSOs alJowed the identification o f all eight D Q A l and all 13 D Q B 1 sequences that are presently known [ 17]. T h e sequences o f the p r h a e r s and oligoaucleoddes used, their ~ s i t i o n ing in the c D N A sequences, ~ well ~ their ~dle[k specitic/ties are shown in T a b l e 3. Oligonucleotide v/ping makes use o f known sequence polymorphism. Positive o r negative hybridiza-

- N ................................................. NN

|Q ZO 30 40 S9 60 70 60 ~| ~PLN~LKFECHF~CT$RV~LL`RC]YHQEE~YR~D$D~C~YR~VTlLG~D~EY~RSQKDLL|Q]~A~vDTYCRq~YGvQ|$~T~u~ Dm+l*|++1 D~SlIISO+ D~BIIISO! ~Z+L+I+02

0+2 +.2 0+12 0121

0t22

. . . . . . . .

-.

-P+~ . . . . . . . . . .

.

.

.

.

.

.

.

.

.

F-D-YP . . . . . . . . . . . . . . .

.

r-D-YF

. . . .

f. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 DRSS#01|I DUSe01O2

DS2 012

0HS*OZOl

Oez) D~22

0RS5¢0202

D~I2

iiDiiiiiiiii i iiiiiiiiiiiiiiiiiiiiii-. iiiiiiiii iiii 2

144 TABLE 3 A. Primers Local identificadon

Listing o f primers and SSOs

Sequence 5' ~ 3'

Used as

89-369~ 89-332" 89-053" 89-368"

CCG GTC GAC TGT CCC AGC ACG TIC TGG CAG CCT AAG AGG GAGTG T I G CAG CAG GAT AAG TAT GA GAA TIC TCG CCG CTG CAC TGT GAA GC

Generic DRB upstream DR2 BI upstream DR2 B~ apstreima Generic DRB downstream

90-123 90-124

GTG CTG CAG GTG TAA ACT TGT ACC AG GAC GGA TCC GGT AGC AGC GGT G

DQA upstream DQA downstream

90-125 90-126

CCG GTC GAC TCC CCG CAG AGG ATU TCG TG TGC TCT AGA GGGCGA CGC TCA CCT C

DQB upstream DQB downstream

B. SSOs Local identification

nt~

Hybridizes with alleles

88-148' 88-251' 89-332 89-053 89-054' 89-123' 89-44V 90-004~

TGT GGC AGC TI'A AGT TUG AA GAG CAG AGG CGG GCC GCG GT TGG CAG CCT AAG AGG GAGTG "FFG CAG CAG GAT AAG TAT GA GAG GAG GAC TrG CGC TIC GA GGT TCC TGC ACA GAG GCA TC GAC TIC CTG GAA GAC AGGCG GAC ATC CTG GAG CAG GCG CG

110-129 292-511 112-131 109-128 190-209 161-180 283-302 283-302

DRBIe0101, 0102, 0103 DRBI'0101, 0102, 0404, 0405 DRBI*IS01, 1502, 1601, 1602 DRBS"0101, 0102, 02 DRBS*OI01 DRBS"0102, 02 DRBI'I601, tl01, 1104, 0801, 0802 DRBI'ISOl, 1502, DRBS"02

89-058'

CTA CGG GGC TGT GGA GAG CT

333-352

90-113c 89-307" 89-308'

AAG GAC CTC CTG GAA GAC AG TAC GGG GTI GTG GAG AGC TT TAC GGG GTI GGT GAG AGC "IT

280-299 334-353 334-353

89-071 89-070 89-069 90-005 89-296 89-068 89-066 89-065 89-274 89-275 90-127

T I C AC-C AAA T I T GGA GGT TT ATG GAG ATG AGG AGT TCT AC ACC TGG AGA AGA AGG AGA CT GAG AGG AAG GAG ACT GCC TG TGG AGA TGA GCA GTI CTA CG TCT GGC CAG TUC ACC CAT GA CTG TIC CAC AGA CTT AGA "IT TGT TCC GCA GAT TTA GAA GA GTU CTC AGA CAA TIT AGA TT CTT GAA CAG TCT GAT TAA AC AT(= GCT GTG ACA AAA CAC AA ACA CAA CTA CGA GGT GGC GT

223-242 161-180 185-204 190-209 162-181 136-155 220-242 221-240 220-242 288-307 268-287 305-324

90.128 90-130 90-131 90-132 90-133 90-134 90-135 90-136 90-137 90-139 90-293 90-290 90"291

GCT GGGGCT GCC TGC CGC CG TGG GGC GGC TTG ACG CCG AG GC~ GGC CTG TIG CCG AGT AC GGC GGC CTA GCG CCG AGT AC AGG GGC GGC CTG ACG CCG AG AGG GGC GGC CTG ATG CCG AG GCT GGGGCC GCC TGC CGC CG TGG GGC CGC CTG ACG CCG AG CGC GTG CGT TAT GTG ACC AG GAG CC.C GTG CGG GGT GTG AC GGG ACC GAG CTC GTG CGG GG TGC GTC 'ITG TGA CCA GAT AC TGC GTC TUG TAA CCA GAC AC

251-270 253-272 256-275 256-275 253-272 253-272 251-270 253-272 162-181 159-178 152-171 166-185 166-185

DRBI°0102, 01201, 1202, DRBS°02 DRBI*1602 DRBI°IS01 DRBI'1502, 1601. 1602, DRBSe0101, 0102 DQAI'0101, 0102, 0103 DQAI'010I DQAI'0103 DQAI*0101, 0102 DQAI*0102. 0103. 0501 DQAI*0103, 0201, 0601 DQAI'0201 DQAI'0301 DQAI°0401, 0501, 0601 DQA1)0501 DQAI'0401, 0601 DQBI°0501. 0502, 0503, 0601, 0602, 0603 DQBI'0201 DQBI'040t, 0402 DQBI*0501, 0604 DQBI*0502 DQBI'0503, 0601 DQBI'0602, 0603 DQBI*0302 DQBI°0301, 0303 DQBI°O601, 0301 DQBI°0501, 0502, 0503, 0402 DQBI'0401 DQBI'0302. 0303. 0602 DQBI'0603, 0604

89-440

Sequence 5' ~ 3'

• Combin~ion of primers 89-368and 89-369were used for amplificationof most exons 2 of HLA.DRBgenes;primers 89-532and 89-368for selectiveelon 2 zatpUficationof HLA-DR2BI genes;and primers 89-053and 89-368for selectiveamplificationof exons 2 of HI.A-DR2B5 genes. bat = marked position of the eDNAsequence ia class11alleles. , These S8Os~e used for hybridizationafter DRB1genericamplifcatloaand Dg2-atssociatedBI- ~ d BS-specificamplification.

Increas/ng Complexity of HLA-DR2

TABLE 4

145

Seanlogical patterns o f DR2 variants Antisoraa DR2 Pattern

DRwI6

DQwt ll

Cells

DR

DQ

1

2 5

w6 w6 wl w2a w7~ w5 wl w5

-,

Dov Bec Sin 5 I m + AZH

wt5 wt5 w15 w15 w15 wl5 2.ES w16

++ ++ ++ ++ ++ ++

REM

w16

w7 #

Jac, Ng Gee, Bez, Sch

2JA 1 + 2s

wl w5~

6p¢6 + VYF + Ast SAS + Hat Moo, Bru Cat

-

-

+

DQ~5

DQw6

DRwI5 45678901

+++++++÷ +++++~++ +++÷++++ +..-++++ ++++++++ ++++++++ + ¢ +---+÷++ -+++++÷+. _.+++++++ . . . . ++++++ . . . . ++++++

ltll 2545

ill 678

++.+ ++++ ++++ . . . . . . . . +~++ ++++ ++++ . . . . . ++++ ++++

--. . . . . . . . . . +++ --. + + . . --+++

1

9 + +

. .

-

• $¢ruatnumbers referto Table2. 610¢= ~nel cells. ' - = nega6v¢serum rcaedons; + = positiveserum re.dons. HI.A-DQw2,-DQw7.and -DRI serumreactionsare not shown. • . = not tested.

tion with a certain oligonucleotide does not prove the presence or absence of the complete allele per se. However, for the sake of simplicity we have adopted the present nomenclature assuming the presence of the complete sequence. RESULTS During routine HLA-DR and - D Q typing a number of DR2-selared patterns were observed in individuals from different ethnic origins. These individuals and, if possible, their family members were further studied by serology (Table 4). When only the DR2-related sera were considered the regular D R w l 5 pattern, which is the most common in Caucasoids, could be clearly distinguished from the D g w l 6 pattern. Among the cells reactive with the D K w l 5 set'a, some were not reactive with several broad DK2 sera (Table 4). This pattern segregated in the faln/lies of two of the panel members studied (data not shown), and was called DR2.ES. Another DR2 pattern, locally called DR2.JA, was nonreactive with D R w l 5 and some broad DR2 sera- A simihr DRwl6-1ike pattern was also observed on cells that c a ~ r , / D R I + 2 s triplets. With D R I + 2 s lmplotypes the DR.2 and D R I antisera are both reactive [7]. In total five different DR2 patterns were distinguished when

Further polymorphism was observed when HLA-DQ typing was taken into account. Some DRwI5 hapiotypes appeared to be DQwl-positive but DQw6-negatire. In one family clear DQwS- reactions were seen, but in other DQwl-positive ceRs neither DQwS- nor DQw6-specific sera were reactive. Cells with such a D Q pattern are indicated as D Q w L In one family D g w l 5 segregated with DQw2, and in another with DQwT. i-|TC REM is known to be Dw22 D R w l 6 DQw7 [3]. N o further cells with this DR D Q pattern ~ r e detected. By combining D R and D Q serology we could distinguish 10 different patterns (Table 4).

PCR digo typing.

D N A was amplified from: (I) a number of HTCs including the four Dw types, (2) a number o f local panel cells, (3) most o f the above described DR2 variants, and (4) three different DR). +2s-positive individuals. The results are summarized in Table 5. Group-specific amplification was performed to distinguish DR1- BI- DR2-BI-, and DRBS-specific sequences by oligonucleodde typing. DRB S$O typing sevea[ed seven different haplotypes. Dw2 H T C s and most of the DgwlS-positive panel cells show~l the DRBI~IS01 together with the DRBS'0101 sequence h-respective of ~heir D Q type. A DwX2 H T C SA$ showed the D RBI * I S0 2 together with the DRBS®0102 sequence. T h e ~urm combination was found for cells Bec and Sto, but panel donor H a t

1~6

G.M. Th. Schreuder et~.

TABLE 5

Assignment of HLA-DRI31, -DRB5, -DQA1, and -DQB1 alleles based on oligonucleotide typing of a panel of DR2 variants

Cells HTC + 6pc° Ast Mort + Bru Cai Dov SAS Bec Sro H~r AZH + 5pc

Dw

DR

DQ

DQBI

DQAI

DRBI

DRB5

w2 --; ---w12 ---w21

w6 w6 wl w2 w7 w6 w5 wl w6 w5 w7 wl

0602 0603 0502 0201 0301 0601 0501 0501 0601 0502 0301 0502

0102 0102 0102 030l 050l 0103 0101 0101

1501 1501 1501 1501' 1501 1502 1502

0102

0102 0501 0102

1502 1601 1602 1602

010l 0101 0101 0101 0101 0102 0102 0102 0101 02 02 0101

w5

0501

0101

0101

0101

gEM

w22

Jac + Ng

--

wl5 w15 w15 w15 w15 w15 wl5 2.ES wl5 w16 w16 2.JA

Gee + Bez + Sch

--

1 + 2s

1502

• pc = panelcells. Dwtypenot known. "TheDRBI'lS01of thiscellappearedto h~vea recentlypublishedAmericanblackvariant[281.afterhybridization with a locally synthesizedSSO whichis specificfor thisvariantDR2.The variantis provisionallydesignatedas DRBI'lS0~.

displayed a DRBI*1502 with DR.BS*0101. In individuals with the DR2.JA variant the DRB 1" 1602 allele was detected with DRBS*0101. Generic amplification of DNA from D R I + 2 s individuals revealed the presence of DR1-BI*0101 and DR2-BY*0101 sequences by SSO typing (Table 5). However, no DR2-B l-specific sequences could be detected. Neither was any PCR. product obtained after DR2-B1 specific amplification. The DRBS~0101 sequence was found in association with four different DRBI genes (Table 5). DQA1 and DQBI SSO typing revealed eight different DQ haplotypes among the DNA of DR2-positive individuals (Table 5). Apart from the well-known combinations we observed DQAI/B1 haplotypes for DQw6 with "0102/0601 and "0102/0603, a DQw2 haplotype with "0301/0201, and a DQw5 haplotype "0101/0501 which is usually seen with Owl. Combining serology and SSO typing we found 13 different haplotypes, four of which could not be distinguished by serology and two not distinguished by SSO typing.

DISCUSSION The combination of DR. and DQ serology with DR.B and D Q SSO typing revealed extensive polymurphism among DR2 haplotypes. Based on DR serology we could distinguish five different serum natterns, all of

which showed clear segregation in families (data not shown). After allele-specific hybridization for DR2 DKB1 and ORB5 sequences, all sequences could be classified as one of the known alleles. Such an assignment of alleles is of course arbitrary, since none of these genes or their products have as yet been sequenced completely. Three new DRB 1 DRB5 combination,-, occurred, involving the DRBS*0101 allele. This allele was found in combination-with the ORB1 allele "1502 in Hat:, with ~1602 in DR2.JA, and with "0101 in D R I + 2 s (Table 5). Sequencing needs to be performed to prove that this ORB5*0101 allele as defined by SSO typing is indeed identical in all haplotypes. No DR2 DRB1 gene sequences were detected after group-specific hybridization of D N A of the DR1 +2s haplotype. This confirms previous protein and RFLP analyses [7, 22]. SSO typing did not detect any differences between the Dgw15 alleles of cell Bec as compared to the DR2.ES variant of Sto. Sequencing of the respective DRB1 and DRB5 alleles may prove further heterogeneity and explain the serologically detected variability. Preliminary experiments prove that these DR2 subtypes are different from DR2LUM [6] (data not shown). It is tempting to speculate on the location of different DR2 epitopes that can be recognized by the respective antisera. Previous absorption experiments with transfected mouse L cells did show that broad DR.2 alloantlsera (Table 4, nos. 10 and 11), reactive with all DR2 suhtvr~_ rr,.cog'niTe thf,. nrocluet of the

Increasing Complexity of HLA-DR2

DRB5*0101 but not o f the D R B I ' I S 0 1 allele [23]. Thus the broad DR2 epitope appeared to be absent on D R B I ' I S 0 1 products. D R w l 5-specific allusera (Table 4, no. 2) reacted with the product of transfecred DRBI*IS01. Such seca neither react with transfecred nor with normal DRBS*0101 (DR2.JA and D R I + 2 s negative), but do re cognize the DRB 1* 1502 products. Thus D g w 15 specific epitopes are probably only present on D R B I ' 1 5 0 1 and "1502, but absent On DRB5 producgs. The Dgwl6-specific antiserum MSD75 (Table 4, no. I) seems to be specific for determinants on the DRB 1* 1601 gene product. Until now DRw 16 was characterized by negative serum reactions only. The MSD75 alloserum is the first DRw16-specific serum described so far and proves further heterogeneity among DRWI6 and related DR2 short specificities. The DRBS*0101 allele is found in association with four different D R B I genes. High homology among the DRB5 alleles and their strong associations with certain D R B I alleles suggests a comparison with DRB3 especially but also with DRB4 genes. The presence o f a DR2 epitope on the DRB5 product changes DR2 from a private D R to a public specificity comparable to DRw52 and DRw53. The D R w l 5 is a true D R specificity for which several specific antisera are available. A variety of D Q A D Q B associations was observed on these haplotypes. Not all D Q w l haplotypes could be clearly typed as either DQw5 or DQw6. D Q w l-positive Implotypes with the D Q B I ' 0 6 alleles always reacted with mAb IIB3 [24] (Table 4, no. 19) but DQBI*05 alleles often failed to react with the DQwS-speciilc sera, particularly when testing Oriental haplotypes. Apart from affinity problems with the alloaarisera this might also be due to hitherto unknown D Q B I sequence differences which are, of course not recognized by SSO typing. This is strengthened by the SSO typing identity of haplotypes Bec and Stu, which differ both for D R and D Q serology. Earlier studies [ 13, 16] describing D Q A 1/B l varisot combinations do mention the DQA1/BI'0102/0601 seen in the D Q w l haplotype of cell Hat, although not in combination with the D R B I ' 1 5 0 2 . The D Q w l positive D Q A I/B 1~0102/0603 haplotype of cell Ast seems to be different from earlier described variants. A DQw2-posidve haplotype was found in the family Cal. SSO typing revealed a DQAI*0301 in association with DQBI*0201. This combination has been described to occur in American blacks with DR7 and DR9 DQw2 haplotypes [25], and again demonstrates that the DQB1 and not the DQA1 allele is responsible for the serological DQw2 typing. The DQw7 haplotypes of the DRwlS-positive cell Dov anneamd identical to that of the Dw22 H T C REM.

t47

In a recent study reported by Mehra et al. [261, additional DIL2 D Q w l hapk~v~.pes were detected in Asian Indians using SSO typing. These hapbtypas were in part identical to the ones recently described by Sing~ e~ al. [16], although DRB5 potymorphism was ant included in the latter study. Together with the new DR2 D Q haplotypes reported here, these studies demonsrrare extensive polymorphism among non-Caucasoid populations even for the DR2 specificity that h ~ always been considered as we|l defined. The DR2 heterogeneit3" should also be kept in mind for the analyses of the effect of D R w~tching in organ and tissue transplantation, especially where non-Caucasoid populanous are concerned. Can anything be said about the origin mad geaere~/on of these variant hapbtypes? Xn Fig. 2 some of them are shown together with their presumably "parental" haplotypes. Some of these haptotypes were detected in normal Dutch Cauca.~ids. The first one, ce]l Ast, has a D Q B 1"0603 which is generally found in DRwl 3-Dwl8 haplotypes and could thus represent a recombination between Dw2 and D w l 8 sequences. T'ne .~cond one concerns the D R I + 2 s haplotyl~. This haplo~tpe contains predominantly D w l genes to which a DRB5 gene has been added. Haplotype Bec could represent a recombination between the D Q region o f a D w I and die DR region o f a D w l 2 haplotype. The seemingly identical Sto haplotype must be closely feinted although its serological pheaotTt~e indicates that both D R and D Q sequences should be different. The Dw21-associated D Q A I " 0 | 0 2 D Q B I ' 0 5 0 2 combination can occur with several ocher DR2 haplotypes. The same is true for the Dw5- associated DQw7 combination DQAI*0501 DQBI*0301. However, DR4 haplotypes have also been soggested to function as ancestor for the DQw7 o f cell REM [27t. For the nonCaucasoid haplotypes the ar~umpdon of such recombinational events remains speculative, k m a y not be true that the best defined haplotypes are "parental" to the rare and new ones. Caucasoid haplotypes are the best defined and sequenced but them is evidence that the Caucasoid population m/ght have evolved through a bottleneck and because of that lost a lot of its variation compared to, for example, African popul~ions (Olerup et al. in preparation). Therefore, the "rare" haplotypes might in fact represent p a r e a ~ ones that stood at the origin of our presently so well-defined haplotypes. Maybe future family segregation studies will reveal the actual recombinational events by which the b a p l o ~ s can be geaecated. In conclusion, we have shown that in a panel of DR2positive cells as m~ny as 13 different D R D Q haplo~pes could be distinguished when combining oligonudeotide typing with serology. 'The F~R1 +2~ han[otvtm can h~, considered aL~a new

148

G.M. Th. Schreuder et al.

Haplotype

Dw

P(arentat):

Dw2

OR

DQ

DQB1 DQA1 DRBI DRB5

w15

w6

0602 0102:1501

w15

w6

0600

010~:: 1501

0!0!

P:

Dw18 w13

w6

0~0

0103 1301

---

P:

Dw2

w6

0602

0102 1501 0101

~!:

0101~ 0 1 0 !

Ast:

Gee,Bez,Sch P:

w15

1+2s w5 Dwl

Be(:: Sto:

1

w5

W15

W5

0101 ~'L

10101

0q01 ....

010!

15021:,0102

OIO1

150~ : 0!02

2.ES

wl

P:

Dw12 w15

w6

0601

P:

05020102

0103 ~1,502 0102

Dw21 w16

w5

Mon,Bru

w15

wl

Jaq,Ng

2.JA

wl

~

0102 1602 0101 0501

P:

Dw5

0102

1601 0201 1501 0101

w11

w7

~

Dov:

w15

w7

0~.,I '0501

1501 0101

REM

Dw22 w16

w7

0301

1 6 0 2 0202

0601

D R specificity which combines a DQA/B and DRB1 region from the DR1-Dwl haplotype with the DRB5 gene of a DR2 haplotype. DRB5 alleles can associate with more than one DRB1 allele placing the DRB5 gene at a level comparable to DRB3 and DRB4. DR2associated D Q A and D Q B polymorphism is much increased in non-Caucasoid populations. Several of the variant haplotypes could be explained by the assumption of recombinational events between any of the D Q and D R loci.

1101

. . . .

FIGURE 2 Proposed recombinational events for the generation of variant DR2 haplotypes. Shaded areas represent those parts of the parental haplotypes thought to be involved in the recombination.

Dutch National Reference Center for Histocompatibili W Testing. REFERENCES 1. Richiardi P, Belvedere M, Borelll l, De Marehi M, Curtoni E8: Splits of HLA-DRw2 into subtypie specificities closely correlated to ~ o HLA-D products. Immuungenetics 7:57, 1978. 2. WHO-HLA Nomenclature Committee: Nomenclature for factors of the H L A system, 1987. Vox Sans 55:119,

AC [¢dqOWLEIX~MENTS

The authors wish to thank Jos Drabbels, Harma Karsens, and Ans Niterink for theirexcellent technical assistanceand Drs. H. Bcmel, E. Dupont, H. Mervart, and L. de W0al for their

generous serum gifts. The cooperation of the panel donors and their family members is gratefully acknowledged. This work was supported in part by the Dutch Foundation of Medical and Health Research (Medigon), the J.A. Cohen Institute for Radiopatholngy and Radioproteetion, and by the

1988. 3. Schreuder GMT, Doxiadis l, Parlevliet J, Grosse-Wllde H: HLA-DR, DQ, LB, and T A I O specificities of Ninth Workshop homozygous typing cells. In Albert ED, Banr MP, Mayr WR (eds): Histocompadbility Testing 1984. Berlin, Spfinger-Verla8 1984, p 243. 4. Layfisse Z, 8imoney N, Park MS, Teras&i PI: HLA.D and DRw determinants in an American indigenous isolate. Transplant Pro(: 11:1788, 19795. Barchelor JR, Curtoni S, Mervart H, Noreen H, Davey NJ, Moses JH, Amoroso A: Antigen socieW" no, 22 re-

Increasing Complexity of HLA-DR2

149

port (DR2). In Dupont B (od): immunobiolegy of HLA, 18. Rood JJ van, van Leeuwen A, Pioem JS: Simulta~qeous sol 1. New York, Springer-Verlag, 1989, p 240. detection of two cell popuim~ns by tw-o-colottrllu~'es. cence and ~ppl/c~ion co r.her~ognidon of B cell dtmr6. Oodshnorn M, Du Toil ED, Martell RW: New HLAmimmts. Nature 262:799, t976. DR2 related specific|tiesdetected in South Africanblacks and individuals of mixed ancestry. Ttas~e Antigens 19. Naiimi A, D'Am~troJ, B.-ulfing.~/, van Leeuwen A, ,¢~m 35:211, 1990. Rood ).,I: A u ~ m d reading of p r o # d i m iodMe lymphocxrtotoxiciw ~-~:sfor HLA-DR, MB, MT typit~. Tis7. Bontrop RE, Schreuder GMT, Elfefink DG, Mikulski sue Antigens 24:302, I984. MMA, Geerse R, Giphart MJ: Molecular, serologic, and functional evidence for an apparent HLA.DR triplet. J 20. Kenter MJH, Anholt¢JDH, Sch~reudetCaM'Y,m ~ggerImmunol 137:211, 1986. mend MCJA, Ghyselen GM, ~ Rood JJ, Gipl'~i MJ: Urmmblguo,astyp/ng for HLA-DQTAI0 and 2B3 sped8. Reinsmoen NL, Layrisse Z, Berael H, Freidel AC, Bach tic|tinsusing specific o[/gonucleotide probes. Hum ImFH: A study of HLA-DR2 ~msociatedHLA-Dw/LDspecimunoI 24:65, 1989. fic|ties. Hum Immunol 11:105, 1984. 21. Vljsorberg K, Scbreoder GMT, Keater MJH, N~ipal 9- Yabe T, Reinsmoen NL, Wu S, Bach FH: Protein atmlysls AM, van Rood JJ, Giph,~t Mj: App]ic.ab~- Of HL~.ofHLA-DR2.3 haplowpe. Hum Immunol 19:127, 1987. DQB o|igonucleodd¢ typing for ~ e TAIO and 2B3 s~-c10 Singal DP, Butler I., LamonssgueL: Identification of two ificidns in romine H I ~ typlng. Tissue Antigens 35:t65, subtypes of HLA-DR2 by Southern blot analysis. Tissue 1990. Antigens 28:29, 1986. 22. Tiiaans MGJ, van F~germoad MCJA, Fei H, Scb~e~r 11. Tilanus MGJ, van Eggermond MCJA, van dee BijI M, GMT, Giph~t MJ: A fmni]ywith ~mep~,rent HLA-DR Morolli B, Schreuder GMT, de Vries RRP. Gipharr MJ: triplet. ~xp Ciln I m m u ~ 6:162, 1989. HLA class 11DNA analysisby RFLP reveals novel class II 23. Schreuder GMT, Marsh SGE, Heyns JM, Mnscs JH, polymnsphisms. Hum Immunol 18:265, 1987. Kratt~t P, Bcxlmer JG: Mkrocyt'~'x~xklty a.~e~y u d ~ 12. BidwellJ: DNA-RFLP analysis and genowping of HLA. mouse L cells transfected with huraan MHC d~ss 1i DR and DQ antigens, lmmunol Today 9:18, 1988. genes. Tissue Antigens 36:79, 1990. 13. Bidwelt LJ, Bidwell EA, Savage DA, Middleron D, CUl24. Schreuder GMT, Macd~ H, Koni~ F, D'Am~o J: TAI0 len C, Klouda Fr, Bradley BA: Identification of Dw2, and 2B3, two new alleles in dxe HLA-DQ region recogDwl2, and 'short" DR2 splits with sequential exon-spenixed by monodonal antibodies. Hum L,,nmunol I6:127, eilic DRg, DQ0, and DQ~, eDNA probes. In Dupont B 1986. (ed): iramunobiology of HLA, rot 2. New York, 25. Todd JA, Mi}ovi¢C, Fletcher J, Jenkins D, Bradweti AR, $pringer-Vetl~g 1989, p 198. Emrtmt~AH: Identification of susccpdbRity loci for insn14. Wu S, Lu D, Madden M, Liu C-P, MiyokawaN, Bach FH, lin-delmndent d/lbems meltitius by wans-rac/~[gene mapSaundessTL: Full length DOff eDNA sequences of HLAping. Nature 338:.587, 1989. DR2/DQwl suhwpes. Hum Immunol 27:305, I990. 26. Mehra NK, Verdulin W, Tmleja V, D~bbels J, Sigh 15. Howell WM, Evans PR, $~ge DA. Wilson PJ, Smith JL: SPN, Giph~x MJ: Analysis of HLA-DR2 polymorphis~ms HLA typing by DNA-RFLP: Identification of a novel by oligonucl¢otMehybridize|on in an Asian Ind~.n popuDR2.-associated RFLP in a Thai individual. Tissue Am/lation. Hum Immunol (in press). gens 35:189, 1990. 27. Liu C-P, Bach FH, Wu S: Molecular studies of a rcaxt 16. SingalDP, D'Souza M, Snod SK: Polymorph/smof HLADR2/LDgdDQw3 HLA class II haplotype, j lmmunol DR2, DQwl haplotyges in Asian Indians. Tissue Anti140:3631, 1988. gens 36:164, 199028. Demopuins iT, HodBe TW, Woomn V, Acron RT: A 17. Bodmer JG, Marsh SGE, Albert E: Nomenclature for novel DPd~I allele in DR2-pnsRiso Ameriean blacks. fgtors of the HLA system, 1989. lmmunol Today 11:3, Hum lmmunol 30:41, 1991. 1990.