Identification of the Amino Acid Residues Contributing to Monoclonal Antibody-Defined DQwl Epitopes Hellmuth Nordwig, William W. Kwok, and Judith P. Johnson
ABSTRACT: The reactivity of monoclonal antibodies (nAbs) RI, $1, and $5, shown previously to recognize polymorphic epltopes on HLA-DQ molecules, have been found to correlate with the presence of certain DQBI alleles, nAb S5 reacts with cells expressing DQB 1°0503, 0601, 0602, 0603, or 0604 alleles while RI and $1 react with all DQB1 alleles except "0201 and 0301. In the ABBREVIATIONS B-LCL B-lymphoblastoid cell line EBV Epstein-Barr virus
case of R1 and S1, sequence comparison of these chains suggests the involvement of residues 45-47 (GVY) in formation of the epitotms. This prediction has been confirmed by showing that a G ..o E mutation in posi-ion 45 of the DQBI*0~02 gene eliminates binding of both nabs. Huraa+ Immunology 31. 8 1 - 8 5 (1991J
IHWS nAb
International Histocompatibllity Workshop monoclonal antibody
INTRODUCTION HLA class II molecules are heterodimeric, highly polymorphic cell surface glycoproteins which control immune recognition through the presentation of foreign antigens to regulatory T cells [for a review, see ref. 1]. At least three different groups of these molecules can be distinguished: DR, DQ, and DP. The D Q molecules are polymorphic in both chains and are divided into the serological specificities DQw2, DQw4, DQw5, DQw6 (both D Q w l ) , DQw7, DQw8, and DQw9 (all DQw3). Sequence comparisons of D Q molecules indicate that these specificities are primarily determined by amino acid residues in the smaller molecular weight B chains [2]. A number of monoclonal antibodies (nAbs) have been produced against polymorphic epitopes on D Q molecules. These reagents are valuable as they enhance the understanding of major histocompatibility complex
Frorathe lnstitut fi~r lmmunologie, M+nchen, Germany (H.N.;J.P J.), and the Virginia Mason Research Center, Seattle, Washington Ogt.W.K.). Address reprint requests to Judith P. Johnson, lnstitut f+r lramunologie, Goethestrafle 31, D-8000 M~nchen 2, Germany. ReceivedSeptember 13, 1990; accepted November 16, 1990. Human Immunology31, 81-85 (1991) © AmericanSociety for Histocompatibilityand Immunogenetics,1991
polymorphyism and allow thc investigation of Iotasand allele-specific expression of HLA products. The reactivity patterns of many of these n A b s do not strictly correlate with serologically defined D Q specificities. However, residues that are critical for binding of these n a b s can often be predicted from a comparison of the amino acid sequences of reactive and unreacdve alleles [3]. Such predictions can then be tested by analyzing the reactivity of the antibody with transfectants expressing mutated HLA genes. In this study, we have applied this approach in an attempt to localize the epitopes of three such nAbs: R1, S1, and $5. R1 and $1 have previously been shown to be directed against polymorphic epitopes on all D Q w l molecules and on D Q molecules encoded on approximately half of the DR4 and DR8 haplotypes [4]. n A b $5 was shown to identify an epitope which divides the D Q w l specificity, as it is present on D Q molecules of the DRw13 Dw18 haplotype and most DR2 haplotypes [5]. Since the serological analysis in the Tenth International Histocompatibility Workshop (IHWS) resulted in the definition of new specificities and since polymerase chain reaction-based sequencing has revealed an 81 0198-8859/91/$3.50
82
H. Nordwig et ai.
positive cells. Thus they represent an "all but DQw2 and DQw7" binding pattern. An important feature of these mAbs is the split of the former DQw3 specificity which correlates with the new serologically defined subgroups: DQw8 and w9 are recognized by R1 and $1, whereas DQw7 is not. The same reaction pattern has been described for mAbs BT3.4 [8] and IIB3 [9]. These mAbs define, together with the complementary mAb TA10, an allelic series in HLA-DQ which is similar to the Bw4/6 system in HLA-B products [10]. The binding pattern of m A b $5 is unique. It reacts with all DQw6 cells and in addition with DQw5 molecules present on the DRw14 Dw9 haplotype. Therefore, $5 defines a subgroup of D O w l which is distinct from. the DQwS/w6 split.
even greater diversity, we have reexamined the reactivity of these mAbs and attempted to localize the amino acid residues which contribute to their binding. MATERIALS AND METHODS The generation of mAbs directed against polymorphic epitopes o HLA class II molecules has been described [6]. Briefly, F1 mice received a single injection of 106 B-lymphoblastoid cell line (B-LCL) and 3 days later spleen cells were fused with the myeloma P 3 x 6 3 Ag8.653. A single cell ELISA was performed with the supernatants on a selected panel of HLA-typed B-LCL to detect a polymorphic binding pattern. The reactivity of the mAbs was further investigated by indirect immunofluorescence on 67 Epstein-Barr virus (EBV)transformed B-LCL from the 10th IHWS representing 30 different HLA class II haplotypes. The DQw3 flchain mutants and transfectants have been described
Correlation of mAb reactivity with the sequencesof DQ3 alleles. Table 1 shows the alleles of D Q a and-g chains of the investigated cells in the new W H O nomenclature [11]. Sequences have been published for each haplotype represented in Table 1, albeit not for each cell tested in this study. It can be seen from the grouping of the reacdvities that the binding pattern of all three antibodies correlates with the presence of certain 3, but not a chains, For example, the allele D Q A 1"0201 is present in R1 + $1 + as well as R 1 - $ 1 - cells and DQAI*0102 in $5 + as well as $ 5 - cells. We have therefore compared the sequences of the respective /3 chains to identify
[7]. RESULTS A N D D I S C U S S I O N
Binding patterns of the antibodies. The reactivity of the mAbs R1, $1, and $5 with the haplotypes represented by the 10th IHWS B-LCL used in this study is presented in Table 1. R1 and $1 are identical in their reactivity and recognize DQw5-, w6-, w8-, w9-, and DQw4-
TABLE 1
Binding pattern of the mAbs described in this study with the haplotypes represented by B-LCL of the 10th IHWS
Serological typing
a allele DQA 1°
fl allele DQB 1"
DRwl3 DQw6 DRwI3 DQw6 DRwI5 DQw6 DRwl5 DQw6 DRw8 DQw6 DRwl4 DQw5 DR1 DQw5 DRw16 DQw5 DR4 DQw8 DR9 DQw9 DR7 DQw9 DRw18 DQw4 DRw8 DQw4 DR7 DQw2 DRw17 DQw2 DR4 DQw7 DRwll DQw7 DRwl6 DQw7 DRwl4 DQw7 DRw8 DQw7
0102 0103 0102 0103 0103 0101 0101 0102 0301 0301 0201 0401 0401 0201 0501 0301 0501 0501 0501 0601
0604 0603 0602 0601 0601 0503 0501 0502 0302 0303 0303 0402 0402 0201 0201 0301 0301 0301 0301 0301
Reactivity
No. of cells tested
Example of cell
RI
SI
$5
4 4 5 1 1 3 7 3 4 2
$LE CB6B, HHKB DO208915 E4181324 TAB089 TEM $A, MZ070782 KAS011 WTS1, YAR DKB
+ + + + + + + + + +
+ + + + + + + + + +
~+ + + + + 0 0 0 0
I I
DBB RSH
+ +
+ +
0 0
3 7 7 3 7 1 2 1
MADUILA LBF QBL, COX JHAF, DEU SWEIG RML AMALA LUY
+ 0 0 0 0 0 0 0
+ 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
P o l y m o r p h i c Epitopes o n D Q w l Molecules
83
TABLE
2
Polymorphic positions in DQ/8 S1, and S5
DQB1 °
03
09
13
14
26
28
30
37
38
45
46
4~
52
53
55
56
57
66
67
70
71
74
75
77
0503 0601 0602 0603 0604 0501 0502 0302 0303 0402 0201 0301
S P ->
Y L F ?
G A -?
L M M ? M
G Y L L L
T --" --
H Y Y
Y D --
V
G
V
Y
P
Q
R
P
D
E D
V l
G R
R
S E E E E
V L L L L
R T T T T
M M M M M
L L
---
P P --
--
V V S A
A T T T T
D
l
T T D
L L
--
R R E
E E
L
T T T
L
L
--
/x
D
l
R
K
A
L
P
R
T
E
L
T
--
F
-A
L y
Y Y Y S y
S m
chains grouped according to their binding pattern with mAbs R1,
A A A
---I --
L L L
A A A E
A
F
L
E
React~ity DQB1 °
84
85
86
87
89
90
116
125
140
i67
182
185
197
203
220
221
224
R1
S1
$5
0503 o6ol 0602 0603 0604 0501 0502 0302 0303 0402 0201 0301
E
V
A
Y
G
I
?
>
>
?
>
>
?
>
>
-->
?
+
+
+
v
~
A
R
s
÷
s
v
R
Q
Q
+
+
+
? ?
H ? ?
-? >
N ? ?
! ? ?
? ?
? ?
? T ? T
? -? --
> N ? N
-? > -> i ? I
? -> --
. > I ? I 1
? H ? H H
? H ? H H
? > R ? -> ---
T
H
N
I
H
H
--
+ + + + + + + + 0 0
+ + + + + + + + 0 0
+ + + 0 0 0 0 0 0 0
F F ?
?
?
?
?
? G
Q Q Q Q
L L L L
E E E E
L L L L
T T T T
Q
L
E
L
T
T T T T
? -~ 1 ? -> ---
? > S ? A ? A A
T
--
A
The sequences are collected in tel 3 and polymorphic positions of the second domain in ref. 12. • ' ~ " indicates identity to the uppermost sequence.
p o l y m o r p h i c r e s i d u e s w h i c h m i g h t b e c r i t i c a l f o r ~pitope formation, T a b l e 2 lists t h e p o l y m o r p h i c r e s i d u e s o f all p u b l i s b e d D Q 13 c h a i n s [ 3 , 1 2 ] g r o u p e d a c c o r d i n g t o t h e i r antibody binding. In the case of $5, residue 125 (elyc i n e ) i n t h e m e m b r a n e p r o x i m a l d o m a i n is u n i q u e t o t h e c h a i n s p r e s e n t o n t h e r e c o g n i z e d c e l l s as f a r as t h e y h a v e b e e n s e q u e n c e d , i.e., D Q B I * 0 6 0 1 a n d 0 6 0 2 ( t h e r e is n o s e q u e n c e i n f o r m a t i o n f o r t h e alleles D Q B I * 0 5 0 3 ,
TABLE
3
First domain sequences of the transfected proteins 10
DQBI'0302 DQB l °0302ml 3 DQBl*0302m26 DQBI°0302m45 DQBl*0302m57 DQBI*0301 From tel 7.
0603, and 0604). Residue 125 may therefore be inv o l v e d in S 5 b i n d i n g . In the case of R1 and S1, a comparison of the polym o r p h i c p o s i t i o n s o f r e a c t i v e a n d n o n r e a c t i v e alleles reveals a potential involvement of residues/845 and 46 (possibly also 47). Both chains which are not recognized b y t h e m A b s , i.e., D Q B I * 0 2 0 1 a n d 0 3 0 1 , h a v e a n o n conservative exchange in one of these positions whereas t h e G V Y ( 4 5 - 4 7 ) s e q u e n c e m o t i f is c o m m o n t o t h e 1 0
20
30
40
50
60
70
80
RDSPEDFVYQFKGMCYFTNGTERVRLVTRYIYNREEYARFDSDVGVYRAVTPLGPP~SQKEVLERTRAELDTVCRHNY A¥ .E ~
A
Y-
.E
.D , ,,D
84
H. Nordwig et al.
R1
$1
IVD12 a
b
i.
!
C ]
specific antibody, IVD12 [14]. As can be seen, an exchange in residue 45 totally eliminated binding of R1 and $1, although IVD12 still reacted. Exchanges in the other residues did not influence R1 or $1 binding. Therefore, at least in DQBl*0302-bearing haplotypes, position 45 is involved in the formation of the R1/S1 epitope. The prediction made from the correlation of reactivity and sequences has been confirmed experimentally in this case, demonstrating the value of the former method. On the HLA class II tertiary structure model proposed by Brown et al. [15], the R1/S1 epitope is located on loops pointing outwards from the molecule, and thus predicted to be easily accessible to antibodies.
,
ACKNOWLEDGMENTS The EBV-transformed B-LCL from the Tenth International Histocompatibility Workshop were kindly provided by Dr. R. Wank, Institute for Immunology, Munich. This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 217.
co ?.
"6 i
.:
/~
==
e
~
ii
f
.
°
log fluorescence FIGURE 1 FACS analysis of the reactivity of R1, S1, and IVD12 with the following cell lines (compare Table 3): a, MAT (untransfected, DQw2 homozygous); b, MAT transfected with a vector containing no insert; c, DQBI'0302 transfectant; d, DQBl*0302m13 transfectant; e, DQBI*0302m26 transfectant; f, DQB1*0302m45 transfectant; g, DQBl*0302m57 transfectant.
recognized chains. The involvement of residue 45 in determination of the R1/S1/IIB3 versus the TA10 allelic specificity has been predicted previously [3, 13].
Investigation of transfectants expressing mutated DQw3 [J chains. To directly examine the role of this residue in R1/S1 binding, cells expressing the DQBI*0302 product with mutations in positions 13, 26, 45, and 57 (Table 3) were investigated. In Fig. 1, binding of the mAbs R1 and $1 to these cells is compared with a DQw3-
REFERENCES I. Kappes D, Strominger JL: Human class II major histocompatibility complex genes and proteins. Ann Rev Biochem 57:991, 1988. 2. Horn GT, Bugawan TL, Long CM, Erfich HA: Allefic sequence variation of the HLA-DQ loci: R~:lationship to serology and to insulin-dependem diabetes susceptibility. Proc Natl Acad Sci USA 85:6012, 1988. 3. Marsh SGE, Bodmer JG: HLA-DR and -DQ epitopes and monoclonal antibody specificity. Immunol Today 10:305, 1989. 4. Johnson JP, Wank R: Identification of two cis-encoded HLA-DQ molecules that carry distinct alloantigenic specificities. J Exp Med 160:1350, 1984. 5. Johnson JP, Wank R: Biochemical dissection of DRw6 related epltopes using monoclonal antibodies. Hum Immunol 16:69, 1986. 6. Johnson JP, Contag I, Wank R: The isolation and characterization of murine monoclonal antibodies directed m polymorphic epitopes on HLA antigens. Hybridoma 6:17, 1987. 7. Kwok WW, Lotshaw C, Milner ECB, Knitter-Jack N, Nepom GT: Mutational analysis of the HLA-DQ3.2 insufin-dependent diabetes susceptibility gene. Proc Natl Acac $ci USA 86:1027, 1989. 8. Corte G, Moretta A, Cosulich ME, Ramarli D, Bargellesi A: A monoclonal anti-DC1 antibody selectively inhibits the generatiop of effector T-cells mediating specific cytolyric activity. J Exp Meal 156:1539, 1982. 9. Koning F, Raghoebar J, Schreuder GMT, Schuurman R, Bruning H: A monoclonal antibody detecting an HLA-
Polymorphic Epitopes on DQwl Molecules
DQwl-related determinant. Tissue Antigens 26:100, 1985. 10. Schreuder GMT, Maeda H, Koning F, D'AmaroJ: TA10 and 2B3, two new alleles in the HLt-DQ :egion recognized by monoclonal antibodies. Hum Iwanunol 16:127, 1986. 11. Bodmer JG, Marsh SGE, Albert E: Nomenclature for factors of the HLA system, 1989. Immunol Today 11:3, 1990. 12. Hurley CK, Steiner N: DQ polymorphism: An analysis of DQw4c, and ~8 membrane proximal regions. Hum Immunol 27:100, 1990. 13. Kenter MJH, AnholtsJDH, Schreuder GMT, van Egger-
85
mond MCJA, Ghyselen GM, van Rood JJ, Giphart MJ: Unambiguous typing.for HLA-DQ TA10 and 2B3 specificities using specific oligonucleotide probes. Hum immuno124:65, 1989. 14. Giles RC, Nunez G, Hurley CK, Nunez-Roldan A, Winchester IL Stasmy P, Capra JD: Structu~al analysis of a human I-A homologue asing a monoclonal antibody that recognizes an MB3-1ike specificity. J Exp Med 157:1461, 1983. Brown JH, Jardetzky T, Saper MA, $amraoui B, Bjorkman PJ, Wiley IX:: A hypothetical model of the foreign binding site of class II histocompatibility molecules. Nature 332:845, 1988.
ABSTRACT: The reactivity of monoclonal antibodies (nAbs) RI, $1, and $5, shown previously to recognize polymorphic epltopes on HLA-DQ molecules, have been found to correlate with the presence of certain DQBI alleles, nAb S5 reacts with cells expressing DQB 1°0503, 0601, 0602, 0603, or 0604 alleles while RI and $1 react with all DQB1 alleles except "0201 and 0301. In the ABBREVIATIONS B-LCL B-lymphoblastoid cell line EBV Epstein-Barr virus
case of R1 and S1, sequence comparison of these chains suggests the involvement of residues 45-47 (GVY) in formation of the epitotms. This prediction has been confirmed by showing that a G ..o E mutation in posi-ion 45 of the DQBI*0~02 gene eliminates binding of both nabs. Huraa+ Immunology 31. 8 1 - 8 5 (1991J
IHWS nAb
International Histocompatibllity Workshop monoclonal antibody
INTRODUCTION HLA class II molecules are heterodimeric, highly polymorphic cell surface glycoproteins which control immune recognition through the presentation of foreign antigens to regulatory T cells [for a review, see ref. 1]. At least three different groups of these molecules can be distinguished: DR, DQ, and DP. The D Q molecules are polymorphic in both chains and are divided into the serological specificities DQw2, DQw4, DQw5, DQw6 (both D Q w l ) , DQw7, DQw8, and DQw9 (all DQw3). Sequence comparisons of D Q molecules indicate that these specificities are primarily determined by amino acid residues in the smaller molecular weight B chains [2]. A number of monoclonal antibodies (nAbs) have been produced against polymorphic epitopes on D Q molecules. These reagents are valuable as they enhance the understanding of major histocompatibility complex
Frorathe lnstitut fi~r lmmunologie, M+nchen, Germany (H.N.;J.P J.), and the Virginia Mason Research Center, Seattle, Washington Ogt.W.K.). Address reprint requests to Judith P. Johnson, lnstitut f+r lramunologie, Goethestrafle 31, D-8000 M~nchen 2, Germany. ReceivedSeptember 13, 1990; accepted November 16, 1990. Human Immunology31, 81-85 (1991) © AmericanSociety for Histocompatibilityand Immunogenetics,1991
polymorphyism and allow thc investigation of Iotasand allele-specific expression of HLA products. The reactivity patterns of many of these n A b s do not strictly correlate with serologically defined D Q specificities. However, residues that are critical for binding of these n a b s can often be predicted from a comparison of the amino acid sequences of reactive and unreacdve alleles [3]. Such predictions can then be tested by analyzing the reactivity of the antibody with transfectants expressing mutated HLA genes. In this study, we have applied this approach in an attempt to localize the epitopes of three such nAbs: R1, S1, and $5. R1 and $1 have previously been shown to be directed against polymorphic epitopes on all D Q w l molecules and on D Q molecules encoded on approximately half of the DR4 and DR8 haplotypes [4]. n A b $5 was shown to identify an epitope which divides the D Q w l specificity, as it is present on D Q molecules of the DRw13 Dw18 haplotype and most DR2 haplotypes [5]. Since the serological analysis in the Tenth International Histocompatibility Workshop (IHWS) resulted in the definition of new specificities and since polymerase chain reaction-based sequencing has revealed an 81 0198-8859/91/$3.50
82
H. Nordwig et ai.
positive cells. Thus they represent an "all but DQw2 and DQw7" binding pattern. An important feature of these mAbs is the split of the former DQw3 specificity which correlates with the new serologically defined subgroups: DQw8 and w9 are recognized by R1 and $1, whereas DQw7 is not. The same reaction pattern has been described for mAbs BT3.4 [8] and IIB3 [9]. These mAbs define, together with the complementary mAb TA10, an allelic series in HLA-DQ which is similar to the Bw4/6 system in HLA-B products [10]. The binding pattern of m A b $5 is unique. It reacts with all DQw6 cells and in addition with DQw5 molecules present on the DRw14 Dw9 haplotype. Therefore, $5 defines a subgroup of D O w l which is distinct from. the DQwS/w6 split.
even greater diversity, we have reexamined the reactivity of these mAbs and attempted to localize the amino acid residues which contribute to their binding. MATERIALS AND METHODS The generation of mAbs directed against polymorphic epitopes o HLA class II molecules has been described [6]. Briefly, F1 mice received a single injection of 106 B-lymphoblastoid cell line (B-LCL) and 3 days later spleen cells were fused with the myeloma P 3 x 6 3 Ag8.653. A single cell ELISA was performed with the supernatants on a selected panel of HLA-typed B-LCL to detect a polymorphic binding pattern. The reactivity of the mAbs was further investigated by indirect immunofluorescence on 67 Epstein-Barr virus (EBV)transformed B-LCL from the 10th IHWS representing 30 different HLA class II haplotypes. The DQw3 flchain mutants and transfectants have been described
Correlation of mAb reactivity with the sequencesof DQ3 alleles. Table 1 shows the alleles of D Q a and-g chains of the investigated cells in the new W H O nomenclature [11]. Sequences have been published for each haplotype represented in Table 1, albeit not for each cell tested in this study. It can be seen from the grouping of the reacdvities that the binding pattern of all three antibodies correlates with the presence of certain 3, but not a chains, For example, the allele D Q A 1"0201 is present in R1 + $1 + as well as R 1 - $ 1 - cells and DQAI*0102 in $5 + as well as $ 5 - cells. We have therefore compared the sequences of the respective /3 chains to identify
[7]. RESULTS A N D D I S C U S S I O N
Binding patterns of the antibodies. The reactivity of the mAbs R1, $1, and $5 with the haplotypes represented by the 10th IHWS B-LCL used in this study is presented in Table 1. R1 and $1 are identical in their reactivity and recognize DQw5-, w6-, w8-, w9-, and DQw4-
TABLE 1
Binding pattern of the mAbs described in this study with the haplotypes represented by B-LCL of the 10th IHWS
Serological typing
a allele DQA 1°
fl allele DQB 1"
DRwl3 DQw6 DRwI3 DQw6 DRwI5 DQw6 DRwl5 DQw6 DRw8 DQw6 DRwl4 DQw5 DR1 DQw5 DRw16 DQw5 DR4 DQw8 DR9 DQw9 DR7 DQw9 DRw18 DQw4 DRw8 DQw4 DR7 DQw2 DRw17 DQw2 DR4 DQw7 DRwll DQw7 DRwl6 DQw7 DRwl4 DQw7 DRw8 DQw7
0102 0103 0102 0103 0103 0101 0101 0102 0301 0301 0201 0401 0401 0201 0501 0301 0501 0501 0501 0601
0604 0603 0602 0601 0601 0503 0501 0502 0302 0303 0303 0402 0402 0201 0201 0301 0301 0301 0301 0301
Reactivity
No. of cells tested
Example of cell
RI
SI
$5
4 4 5 1 1 3 7 3 4 2
$LE CB6B, HHKB DO208915 E4181324 TAB089 TEM $A, MZ070782 KAS011 WTS1, YAR DKB
+ + + + + + + + + +
+ + + + + + + + + +
~+ + + + + 0 0 0 0
I I
DBB RSH
+ +
+ +
0 0
3 7 7 3 7 1 2 1
MADUILA LBF QBL, COX JHAF, DEU SWEIG RML AMALA LUY
+ 0 0 0 0 0 0 0
+ 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
P o l y m o r p h i c Epitopes o n D Q w l Molecules
83
TABLE
2
Polymorphic positions in DQ/8 S1, and S5
DQB1 °
03
09
13
14
26
28
30
37
38
45
46
4~
52
53
55
56
57
66
67
70
71
74
75
77
0503 0601 0602 0603 0604 0501 0502 0302 0303 0402 0201 0301
S P ->
Y L F ?
G A -?
L M M ? M
G Y L L L
T --" --
H Y Y
Y D --
V
G
V
Y
P
Q
R
P
D
E D
V l
G R
R
S E E E E
V L L L L
R T T T T
M M M M M
L L
---
P P --
--
V V S A
A T T T T
D
l
T T D
L L
--
R R E
E E
L
T T T
L
L
--
/x
D
l
R
K
A
L
P
R
T
E
L
T
--
F
-A
L y
Y Y Y S y
S m
chains grouped according to their binding pattern with mAbs R1,
A A A
---I --
L L L
A A A E
A
F
L
E
React~ity DQB1 °
84
85
86
87
89
90
116
125
140
i67
182
185
197
203
220
221
224
R1
S1
$5
0503 o6ol 0602 0603 0604 0501 0502 0302 0303 0402 0201 0301
E
V
A
Y
G
I
?
>
>
?
>
>
?
>
>
-->
?
+
+
+
v
~
A
R
s
÷
s
v
R
Q
Q
+
+
+
? ?
H ? ?
-? >
N ? ?
! ? ?
? ?
? ?
? T ? T
? -? --
> N ? N
-? > -> i ? I
? -> --
. > I ? I 1
? H ? H H
? H ? H H
? > R ? -> ---
T
H
N
I
H
H
--
+ + + + + + + + 0 0
+ + + + + + + + 0 0
+ + + 0 0 0 0 0 0 0
F F ?
?
?
?
?
? G
Q Q Q Q
L L L L
E E E E
L L L L
T T T T
Q
L
E
L
T
T T T T
? -~ 1 ? -> ---
? > S ? A ? A A
T
--
A
The sequences are collected in tel 3 and polymorphic positions of the second domain in ref. 12. • ' ~ " indicates identity to the uppermost sequence.
p o l y m o r p h i c r e s i d u e s w h i c h m i g h t b e c r i t i c a l f o r ~pitope formation, T a b l e 2 lists t h e p o l y m o r p h i c r e s i d u e s o f all p u b l i s b e d D Q 13 c h a i n s [ 3 , 1 2 ] g r o u p e d a c c o r d i n g t o t h e i r antibody binding. In the case of $5, residue 125 (elyc i n e ) i n t h e m e m b r a n e p r o x i m a l d o m a i n is u n i q u e t o t h e c h a i n s p r e s e n t o n t h e r e c o g n i z e d c e l l s as f a r as t h e y h a v e b e e n s e q u e n c e d , i.e., D Q B I * 0 6 0 1 a n d 0 6 0 2 ( t h e r e is n o s e q u e n c e i n f o r m a t i o n f o r t h e alleles D Q B I * 0 5 0 3 ,
TABLE
3
First domain sequences of the transfected proteins 10
DQBI'0302 DQB l °0302ml 3 DQBl*0302m26 DQBI°0302m45 DQBl*0302m57 DQBI*0301 From tel 7.
0603, and 0604). Residue 125 may therefore be inv o l v e d in S 5 b i n d i n g . In the case of R1 and S1, a comparison of the polym o r p h i c p o s i t i o n s o f r e a c t i v e a n d n o n r e a c t i v e alleles reveals a potential involvement of residues/845 and 46 (possibly also 47). Both chains which are not recognized b y t h e m A b s , i.e., D Q B I * 0 2 0 1 a n d 0 3 0 1 , h a v e a n o n conservative exchange in one of these positions whereas t h e G V Y ( 4 5 - 4 7 ) s e q u e n c e m o t i f is c o m m o n t o t h e 1 0
20
30
40
50
60
70
80
RDSPEDFVYQFKGMCYFTNGTERVRLVTRYIYNREEYARFDSDVGVYRAVTPLGPP~SQKEVLERTRAELDTVCRHNY A¥ .E ~
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84
H. Nordwig et al.
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specific antibody, IVD12 [14]. As can be seen, an exchange in residue 45 totally eliminated binding of R1 and $1, although IVD12 still reacted. Exchanges in the other residues did not influence R1 or $1 binding. Therefore, at least in DQBl*0302-bearing haplotypes, position 45 is involved in the formation of the R1/S1 epitope. The prediction made from the correlation of reactivity and sequences has been confirmed experimentally in this case, demonstrating the value of the former method. On the HLA class II tertiary structure model proposed by Brown et al. [15], the R1/S1 epitope is located on loops pointing outwards from the molecule, and thus predicted to be easily accessible to antibodies.
,
ACKNOWLEDGMENTS The EBV-transformed B-LCL from the Tenth International Histocompatibility Workshop were kindly provided by Dr. R. Wank, Institute for Immunology, Munich. This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 217.
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log fluorescence FIGURE 1 FACS analysis of the reactivity of R1, S1, and IVD12 with the following cell lines (compare Table 3): a, MAT (untransfected, DQw2 homozygous); b, MAT transfected with a vector containing no insert; c, DQBI'0302 transfectant; d, DQBl*0302m13 transfectant; e, DQBI*0302m26 transfectant; f, DQB1*0302m45 transfectant; g, DQBl*0302m57 transfectant.
recognized chains. The involvement of residue 45 in determination of the R1/S1/IIB3 versus the TA10 allelic specificity has been predicted previously [3, 13].
Investigation of transfectants expressing mutated DQw3 [J chains. To directly examine the role of this residue in R1/S1 binding, cells expressing the DQBI*0302 product with mutations in positions 13, 26, 45, and 57 (Table 3) were investigated. In Fig. 1, binding of the mAbs R1 and $1 to these cells is compared with a DQw3-
REFERENCES I. Kappes D, Strominger JL: Human class II major histocompatibility complex genes and proteins. Ann Rev Biochem 57:991, 1988. 2. Horn GT, Bugawan TL, Long CM, Erfich HA: Allefic sequence variation of the HLA-DQ loci: R~:lationship to serology and to insulin-dependem diabetes susceptibility. Proc Natl Acad Sci USA 85:6012, 1988. 3. Marsh SGE, Bodmer JG: HLA-DR and -DQ epitopes and monoclonal antibody specificity. Immunol Today 10:305, 1989. 4. Johnson JP, Wank R: Identification of two cis-encoded HLA-DQ molecules that carry distinct alloantigenic specificities. J Exp Med 160:1350, 1984. 5. Johnson JP, Wank R: Biochemical dissection of DRw6 related epltopes using monoclonal antibodies. Hum Immunol 16:69, 1986. 6. Johnson JP, Contag I, Wank R: The isolation and characterization of murine monoclonal antibodies directed m polymorphic epitopes on HLA antigens. Hybridoma 6:17, 1987. 7. Kwok WW, Lotshaw C, Milner ECB, Knitter-Jack N, Nepom GT: Mutational analysis of the HLA-DQ3.2 insufin-dependent diabetes susceptibility gene. Proc Natl Acac $ci USA 86:1027, 1989. 8. Corte G, Moretta A, Cosulich ME, Ramarli D, Bargellesi A: A monoclonal anti-DC1 antibody selectively inhibits the generatiop of effector T-cells mediating specific cytolyric activity. J Exp Meal 156:1539, 1982. 9. Koning F, Raghoebar J, Schreuder GMT, Schuurman R, Bruning H: A monoclonal antibody detecting an HLA-
Polymorphic Epitopes on DQwl Molecules
DQwl-related determinant. Tissue Antigens 26:100, 1985. 10. Schreuder GMT, Maeda H, Koning F, D'AmaroJ: TA10 and 2B3, two new alleles in the HLt-DQ :egion recognized by monoclonal antibodies. Hum Iwanunol 16:127, 1986. 11. Bodmer JG, Marsh SGE, Albert E: Nomenclature for factors of the HLA system, 1989. Immunol Today 11:3, 1990. 12. Hurley CK, Steiner N: DQ polymorphism: An analysis of DQw4c, and ~8 membrane proximal regions. Hum Immunol 27:100, 1990. 13. Kenter MJH, AnholtsJDH, Schreuder GMT, van Egger-
85
mond MCJA, Ghyselen GM, van Rood JJ, Giphart MJ: Unambiguous typing.for HLA-DQ TA10 and 2B3 specificities using specific oligonucleotide probes. Hum immuno124:65, 1989. 14. Giles RC, Nunez G, Hurley CK, Nunez-Roldan A, Winchester IL Stasmy P, Capra JD: Structu~al analysis of a human I-A homologue asing a monoclonal antibody that recognizes an MB3-1ike specificity. J Exp Med 157:1461, 1983. Brown JH, Jardetzky T, Saper MA, $amraoui B, Bjorkman PJ, Wiley IX:: A hypothetical model of the foreign binding site of class II histocompatibility molecules. Nature 332:845, 1988.
