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DP epitope mapping by using T-cell clones
DP Epitope Mapping by Using T-Cell Clones Arlette Urlacher, Anne Dormoy, and Marie Marthe Tongio
A B S T R A C T : To determine whether a correlation exists between the genomic HLA class II DP DNA polymorphism and cell surface expression and to detect the DP epitopes responsible for alloreactivity, anti-DP T-cell clones were generated against new PLT blank RFLP DPa and DPb-defined specificities. The clones were tested on the 10th IHWS B-LCLs and on local panel cells. Oligotyping of the tested cells made it possible to (a) correlate the DPa specificity with the DPBI*0402 specificity and
(b) split DPb into DPBI*1001 and DPB1*1401. By comparing DNA sequences of the second exon to panel reactivity, the epitopes responsible for DPB 1" 1001 and 1401 were defined and attributed to/~-chain residues contributing to peptide selection inside the HLA groove. However, DNA sequences could not explain anti-DPa allospecificity, indicating that another structure not yet definable may be involved. Human Immunology 35, 100-108 (1992)
ABBREVIATIONS
B-LCL RFLP IHWS
B-lymphoblastoid cell line restriction fragment length polymorphism International Histocompatibility Workshop
mAb MLC PBMC
monoclonal antibody mixed lymphocyte culture peripheral blood mononuclear cell
INTRODUCTION HLA class II DP antigens were first detected by Mawas et al. in 1978 [1] in secondary mixed lymphocyte cultures. The polymorphism of these antigens seemed to be limited, as only six specificities were known at the time of the 10th International Histocompatibility Workshop (IHWS). The development of molecular biology techniques, such as the restriction fragment length polymorphism (RFLP) analysis [2] and, more recently, sequencing and oligotyping, has shown that DP polymorphism is larger than predicted. Besides the classic D P w l to DPw6 specificities, RFLP analysis has detected new DP blank "specificities" called locally DPa, DPa', DPb, and DPc [3], and sequencing of the second exon of the DPB1 gene has revealed 19 DP alleles [ 4 7] and subsequently 36 alleles [26]. Typing of a large local panel and the 10th IHWS Blymphoblastoid cell lines (B-LCLs) by RFLP and oligoFrom the Histocompatibility Laboratory, Regional Center for Blood Transfusion, Strasbourg, France. Address reprint requests to Dr. A. Urlacher, Laboratoire d'Histocompatibilit~, Centre Regional de Transfusion Sanguine, I0 Rue Spielmann, F-67085 Strasbourg Cedex, France. Received September 2, 1991; acceptedAugust 10, 1992. I00 0198-8859/92/$5.00
typing has shown that (a) the RFLP DPa and DPa' specificities carry the DPBI*0402 sequence [3, 8], (b) RFLP DPc specificity is closely correlated with the DPB1*1701 sequence [9], and (c) the RFLP DPb specificity is heterogeneous, as will be seen further on. Although the polymorphism of the DP alleles has now been well defined by molecular biology techniques, little is known about its expression at the cellular level and even less is known about its function. In vitro, DP polymorphism is suspected of generating stimulation in mixed lymphocyte cultures (MLCs) [10, 11] and, in vivo, mismatches for DP antigens have been described as being responsible for graft-versus-host disease reactions [12]. However, these results need further confirmation. Some DP antigens have also been described as restriction antigens [ 13], but the role of polymorphism in the restriction mechanism also needs further study. The basis of alloreactivity and HLA restriction has been widely studied for class II DR and DQ antigens by analyzing panel reactivity of in vitro obtained T-cell clones raised in well-defined cell combinations [ 14, 15]. In this study, we used the same approach to determine whether the genomic DP D N A polymorphism was cotHuman Immunology 35, 100-108 (1992) © American Society for Histocompatibility and lmmunogenetics, 1992
DP Epitope Mapping
related with cell surface expression and to detect the DP epitopes responsible for generating alloreactivity. We chose closely related cell combinations in which stimulating and responding cells had identical class II DR and D Q specificities, but different DP specificities. The stimulating cells were homozygous for one of the new DP blank RFLP specificities defined above. In the first cell combination, the stimulating cells carried the RFLP DPa specificity (sequence DPBI*0402) and the responding cells carried the RFLP 2.1/4.1 specificities, (sequence DPB1*0201/0401). In the second combination, the stimulating cell was homozygous for RFLPDPb, and since at the time the study began DPb sequences were not known, the responding cells were chosen for their identical DR and DQ antigens tested by serologic typing. MATERIALS AND METHODS
101
and IVA, with two subclusters in IA and IIA called IA 1, IA2, and IIA1 and IIA2. For DPB, four main clusters were described, IB, IIB, IIIB, and IVB, with two subclusters in IIIB and IVB called IIIB1, IIIB2, IVB1, and IVB2 [3, 19]. Detailed patterns of DP2.1, 4.1, a, a', b, and c specificities are given in Fig. 1.
Oligotyping. Oligotyping was performed with 25 DPB and 4 DPA oligoprobes according to the 11th IHWS protocol after amplification of the second exon of the DPBA 1 and B 1 genes with specific primers given by the Workshop organizers (data to be published). MLC MLC was performed using the protocol technique of the 8th IHWS [20]. The data were analyzed using SRR (stabilized relative response) calculation.
HLA T y p e of the Cell Combinations Used to Raise Anti-DPa and Anti-DPb Clones
Generation and Maintenance of Alloreactive T-Cell Clones
DP specificities are given using the RFLP nomenclature and the corresponding DPB1 sequence extrapolated from the oligotyping is in parentheses.
Responding peripheral blood mononuclear cells (PBMCs) were primed in an MLC against the 25-Gyirradiated stimulating cells for 10 days. After a second priming that lasted for 6 days, blasts were separated from the nonreactive cells on a Percoll gradient and plated in limiting dilution (1/3 cell per well) with 1 x 105 25°Gy-irradiated pooled PBMCs as feeder cells. Six weeks after cloning, the grown cells were frozen in 5 × 106 aliquots and stored at -196°C until further testing.
Cell Combinations
Anti-DPa clones (DPBl*0402). Stimulating cell: SCHU.. A (local homozygous cell line included in the 10th IHWS studies = B-LCL no. 9 0 1 3 : A 3 B7 Cw7 DR2 DQ1 DPa (DPBI*0402). Responding cell: KLO.. G (local panel donor): A3 B7 Cw7 DR2 DQ1 DP2.1 (DPBI*0201)/A3 B56 Cw7 DR2 DQ1 DP4.1 (DPBI*0401). Anti-DPb RFLP clones. Stimulating cell: MEY.. Be: A3 B18 B55 Cw7 DR11 DR52 DQ7 DPb, DPb. Responding cell: WEI.. An: A2 A26 B18 B44 Cw7 DR11 DR52 DQ7 DP4.3/DPa (DPB1*0401/0402).
B-Cell Lines PBMCs and B-LCLs were used as stimulators in the proliferative tests of the clones. B-LCLs were either those provided by the 10th IHWS studies and/or locally transformed PBMCs from panel families. Proliferation Assays
HLA typing of the local cells was carried out using the classic microlymphocytotoxicity technique for HLA class I typing [16], and the Dynabeads technique was used for D R and D Q typing [17]. Sera from the 10th IHWS were used.
Proliferation assays were performed following the 10th IHWS protocol [21]. Briefly, 10,000 cloned T cells were cultured with 2.5 × 104 irradiated B-LCLs (100 Gy) or I × 105 irradiated PBMCs (25 Gy). The proliferative response was quantitated by a 18-hour pulse with [3H]thymidine (~H-TdR, 1/.~Ci/well). Results were expressed as the median counts per minute (cpm).
HLA-DP T y p i n g
Blocking Assays
RFLP. RFLP DP analysis was performed according to the 10th IHWS protocol with Msp I, Bst EII, Bgl II and Bam HI enzymes [18]. Hybridization was performed with ~2P-labeled DPA and DPB probes. RFLP fragments were subdivided into categories called main clusters and each main cluster included subclusters. For DPA, four main clusters were described, IA, IIA, IIIA,
The following monoclonal antibodies (mAbs) were used: W6/32 (anti-class-I monomorphic), L243 (antiDR~ [22]), Tti 22 (anti-DQ monomorphic + DR2 + DR7 [22]), TAL 14.1 (anti-DR/~ [22]), ILR1 (antiD P 2 + 3 + 4 b + D R 5 [23]), and B7.21 (anti-DP monomorphic [24]). For the inhibition studies of the clones, the 10th
Class I and Class II D R and D Q Serologic T y p i n g
102
A. Urlacher et al.
I RFLP
DP
2.1
DP
4.1
DP
a
DP
a'
DP
b
DP
c
DPA IA llA
1/41
IA2
IIA1
DPB III A IVA
1142
IIIB IIIB1
IVB
IB
liB
IIIB2 IVB1 IVB2
3am HI 8.67 neg 900 pos
FIGURE 1 RFLP patterns of DP subdivisions.
The subchisters are characterized by the presence of the following specific fragments IA 1
BstEII 25.40
Bglll 3.58
IA2
Bst Eli 21.50
Bglll 3.41
IIA1
Bst Eli 10.00
Barn HI 9.00
IIA2
Bst Eli 9.70
Bam HI 8.67
IliA
Bgl I17.24
IVA
Bgl II 5.29
IIIB 1
Bglll 18.(X)
Mspl 4.98
IIIB2
Bglll
Mspl 3.11
IVB1
Bglll 15.05
MspI 2.17
IVB2
Bglll 15.05
Mspl 1.07
IB
Bst Ell 4.40
liB
Bst Ell 4.00
18.00
Mspl 4.87
white boxes : fragment not present black boxes : fragment present hatched boxes : see notes on the right of the figure.
IHWS protocol for blocking assays was used [21]. The stimulating cells (original primer cells) were incubated with 50-/.d aliquots of antibodies for 1/2 hour at room temperature prior to adding the clones. Six wells were plated as unblocked controls, receiving 50 /zl of medium alone in place of antibody. The mean proliferation observed in these controls was considered as 100%. Tests were performed in triplicate. The percentage of proliferation was calculated for each antibody dilution: % proliferation = (mean cpm test/mean cpm unblocked control) × 100. RESULTS A n t i - D P a Clones Thirty clones showing proliferation with the original primer cells were grown. Tested on a restricted PBMC
panel, 10 of them showed the same specific reactivity. Clone 29, which grew quickly and displayed high proliferation values, was chosen for further analysis.
Panelreactivity. Clone 29 was tested on 75 reference BLCLs from the 10th IHWS. Only RFLP DPa-typed cells were recognized (Table 1). Four cell lines, 9010, 9016, 9021, and 9037, which were RFLP DPa' typed were not recognized by clone 29 ([DPa' differs from DPa in one Bam HI fragment, 9.00 kb instead of 8.67 kb] [Fig. 1]). However, their sequence was published as DPB 1"0402 [5, 8]. Three other clones showed the same reactivity pattern as clone 29, but stimulated weaker proliferation. Local family studies showed a strict correlation between the presence of the RFLP DPa specificity and recognition by clone 29 (Table 2). This was confirmed by haplotypic segregation. The DPa specificity had an allelic incidence of about 15 % on unrelated donors, but we did not find the DPa' allele in any of the individuals tested.
Blocking tests. Blocking tests showed that the monomorphic B7/21 mAb was able to inhibit strongly the proliferation of clone 29 (data not shown), confirming that this clone recognized a determinant on the DP molecule.
MLC. If widespread DPa specificity can be accurately discriminated by T-cell clones, it should also be responsible for MLC stimulation. This was not the case when stimulating and responding cells were included in a classic MLC (Table 3). The observed RR values were 3 in one way versus 4 in the other. Anti-DPb Clones
Panel reactivity. Anti-RFLP DPb clones were obtained from two secondary mixed lymphocyte cultures using
DP Epitope Mapping
TABLE 1
103
Reactivity of anti-DPa clone 29 on the 10th IHWS cell lines
LCL identification
DPA 1 sequence a
DPB 1 sequence*
RFLP typing
Reactivity of clone no. 29
9013 9042 9045 9054 9071 9072 9091 9092
01 01 01 b 01 01 b 01 b 01 01
0402 0402 0402/02012 0402 0402/0301 0402 0402 0402
a/a a/a a/2.1 a/a a/3.1 a/a a/a a/a
91.9 ND ND 89.7 74.3 95.0 71.3 20.7 c
9064 9010 9016 9021 9037
01 ~ 030 lb 01 b 0201/0301 b 01 b
0402 0402 0402 0402/0101 0402
a'/a'
90.6
a'/a' a'/a'
6.7
a'/1.2 a'/a'
14.8 11.7 7.8
9002 9004 9005 9014 9025
01 b 01 01 01 b 01
0401 0401 0401 0401 0401
4.1/4.1 4.1/4.1 4.1/4.1 4.1/4.1 4.1/4.1
4.7 14.7 9.8 6.0 5.7
9036 9039 9068 9029
01 01 b 01 b 01 b
02012 02012 02012 02012
2.1/2.1 2.1/2.1 2.1/2.1 2.1/2.1
5.9 7.8 6.1 4.7
This table shows the reactivity pattern of clone 29 when tested on 75 reference B-LCLs from the 10th IHWS. Only results obtained with cell lines typed as DPBI*02012, 0401, or 0402 are reported. The other cell lines tested gave negative results. Data are given in cpm × 103. Each value is the median value of triplicate tests. The cell lines reported on this table were tested at least twice with clone 29. The DPA1 and DPBI genotypes given in this table have been taken from Kimera et al. [8]. bThese cell lines were also studied using PCR-RFLP by AI-Daccak et al. [27], who found the subtype DPAI*0301 for these cases. cThe reactivity of clone 29 with cell line 9092 was considered as positive despite the weak proliferation because this line weakly stimulated a pool of responding cells (28.0 cpm × 103).
TABLE 2
Family identification ANT ANT ANT ANT ANT ANT ANT TIP TIP TIP TIP TIP TIP
All (father) B~t (mother) Ant Eve Adr Pie Syl
Mau (father) Nel (mother) Hug Fr6 Jac Jea
WEI WEI WEI WEI WEI WEI
Th6 (father) Lin (mother) Eri And Ann Gab
RFLP typing
cpm × 103
DP4.1/DPb 4.1/a b/4.1 b/a b/a b/4.1 b/a
5.6 20.5 0.6 12.8 12.8 1.9 18.2
a/3
12.2
4.1/4.1
O. 1
3/4.1 a/4.1 a/4.1 a/4.1
0.5 15.8 19.4 13.9
4.3/4.3 3/a 4.3/3 4.3/a 4.3/a 4.3/a
0.4 17.9 0.4 11.4 11.3 13.8
This table shows the reactivity pattern of clone 29 when tested on local families. Only data on families carrying the RFLP DPa specificity are shown and given in cpm × 103. Each value is the median value of triplicate tests. Each cell was tested at least twice with clone 29.
tions with the RFLP DPc DPBl*1701 cells (group 4). Two clones (K and L) recognized the RFLP DPb DPB1*1401 cells and all of the DPBI*0301 panel cells (group 3). One clone (T) reacted with groups 3 and 4 combined.
Blocking tests. Block tests were performed with one clone representing each group (Fig. 2). N o inhibition
TABLE 3 the same stimulating responding cell combination as described in Materials and Methods. From the first priming, two pairs of clones, clones I and J and clones R and S, were obtained. From the second priming, 16 clones with specific panel reactivity were obtained. Data are summarized in Table 4. All of the clones were tested on a local DP RFLP and oligotyped panel because very few 10th IHWS B-LCLs carried the DPb specificity. In fact, oligotyping showed that the RFLP-DPb homozygous stimulating cell MEY disclosed the DPB1*1001/1401 sequences. Ten clones (A-J) recognized the DPb cells oligotyped as DPB1*1401 without any extra reaction (group 1). One clone (M) recognized the RFLP DPb DPBI*1001 specificity without any extra reaction (group 2). Six clones (N-S) recognized the RFLP DPb DPBI*1001 cells and showed strong specific extra reac-
Reactivity of anti-DPa clone 29 on local families
MLC data obtained with cell combinations used for generating anti-DPa and -DPb T-cell clones Stimulating cells
Responding cells
1
2
3
4
1.
S C H U And DPa/DPa (0402/0402)
1.965 ° 0b
3.227 4
65.684 146
52.312 112
2.
KLO GSr DP2.1/4.1 (0401/0201)
3.040 3
1.615 0
70.710 125
62.071 108
3.
MEY Ber D P b / D P b (1001/1401)
27.577 95
29.513 88
3.032 0
14.401 32
4.
WEI And DP4.3/DPa(0401,0402)
38.506 69
51.887 79
35.626 48
1.244 0
Data are given in cpm × 103~ and in SRRb. The MLC is considered as negative when SRR < 10.
104
A. Urlacher et al.
TABLE 4
Panel reactivity of anti-RFLP DPb clones CLONEJ0ENT~nOATION
Cell pd k~Y GJF LAG NE~r~ R'-;I4 BRA
RFIP bib
NO b/2 1 b/a hip
b/4 1
<3C#~
hi.;
[:~)1J GIJI
b/4 I bit 1
OIJGO A 140111001 I318 401/0201 28 8 401/0201 25 401/0301 i~J 401/1101 17 5 401/0401 NO h[) t~3
F{r[ +
Is/4 1
001/0401 001/0101 NO
ANC F{-~C l'{J M[3#
c/4 1 c/2 1 i~3 c/I 1
701/0401 701/0201 701/0601 701/0101
GQ~7 Z]E HEL {{()~~
14} el1 2 el2 1 b/2 1 b/2 1 3 113 1
301/0301 301/0101 30110201 301/1301 ~01/0201 !, 0 1 / 0 3 0 1
~t'A I{'~ I I'l'
[)l{J DfqA RII
SLA SMI VIL FP,E tf]~
MAR "ilP 9040 LFD CHI I4EF
(~/4
3 1 /4 1 3 1/1 2 hi) 3 I/4 1 Kl') } l/a 3 1/2 1 3 l/a 3 1/1 1 :J 1/3 1 3 2 3 1/4 3 3, 1/4 1 3 1/4 1
101/04b
B
417 117 41 3 ND 129 ND ND
C
105 86 118 ND 129 ND ND
D
319 179 189 ND 89 ND ND
E
116 126 125 ND 101 ND ND
F
G
254 187 15 149 183 155 10 6
456 433 366 349 436 469 23 2
H
361 381 308 342 411 41 4 16 9
519 7 328 175 112 i~) 287 46 1
J 324
K 44 3
t~)
48 1
iq3 11 2 t',O 154 11 7
339 31 385 459 338
419
35 9
L 28 8 27 1 188 16B 227 25.1 23 3
246
9 1
O 152
P 21 6
Q 39 1
34 4 303 292
11 8
20 9
7, 5 15 5
22 9 14 4
176 20 3 20 1 113
16 1 74 201 87
16 6 17 9 174 12 7
M
N
188
338
158 15 1 143
R 59 8
S 48 4
T 27 5 164 131 127 137 181 131
25 4
56 7
35 7 32 4
34 6 41 6
42 156 14
19 5 283 154
9 1 NO 98 8 4
MO 47 1 KO t~D
h/3 42 8 i~) ND
36 4 27 9 334 172 30 4
26 3
1
0301/0401 0301/0101 0:{01/0101 0301/0401 0301/0401 0301/04(/2 0301/0201 0301/0402 0301/0101 0301/0301 NO 2001/0401 2001/0401 2001/0401
58 51 4 71 1 39 I 88 5 44 8 51 9 80 6 66 2 52 1
65 53 71 36 80 30 37 70 47 31
70123
572
12 05
1 9 6 1 6 3
1 6 4 4
11 19
k13 NO NZ) t49 t,,D N3 KID hD
N3 N3
Panel reactivity of RFLP anti-DPb (DPBI*1001 and DPBl*1401) clones. All T-cell clones were investigated with all of the LCLs. Blank spaces indicate negative results. Results are median values from triplicate tests given in cpm x 103. ND, not determined. Four types of reactivities were observed: group 1, clones A-J recognize specifically DPB1*1401 cells; group 2, clone M is specific for the DPBI*1001 specificity; group 4, clones N - S corecognize DPBI*1001 and DPB1*1701 cells; and group 3, clones K and L corecognize DPB1*1401 and DPBI*0301 cells.
was noted with W 6 / 3 2 (anti-class-I) L243 (anti-DRa) or with T~i 22 (anti-DQ + DR2 + DR7). ILR1 ( a n t i - D P 2 + 3 + 4 b + D R 5 ) and TAL 14.1 (antiDR/~) strongly inhibited all the tested clones, but surprisingly, B7/21, the well-known anti-DP monomorphic antibody, inhibited only the specific antiDPB1*1401 (group 1) and D P B I * 1 0 0 1 (group 2). DISCUSSION The present study had two aims: (a) to determine whether the D P polymorphism discovered at the D N A level was expressed at the cell surface and (b) to determine, as previously done for D R and D Q antigens, the structural model for T-cell recognition of HLA class II DP-associated alloepitopes. Two RFLP D P specificities were chosen for this purpose. The first one, DPa, was present on some of the 10th IHWS homozygous B-LCLs. Further analysis of
the DP sequence of these cells showed that they carried the D P B I * 0 4 0 2 sequence. Because one of these DPa 10th IHWS B-LCLs came from our lab (no. 9013), we decided to use it as a stimulating cell. We chose a DR, D Q identical cell as a responding cell for which DP differed as little as possible from the D P B I * 0 4 0 2 sequence. A heterozygous cell was found that carried the DPB 1"0201 and DPB 1"0401 sequences, sequences on which only one and three amino acids in the first domain differed, respectively, from D P B I * 0 4 0 2 (Fig. 3). PLT tests showed that even if the reactivity pattern of the anti-DPa clone (clone no. 29) correlates with DPa, it is not an exact fit with the D P B I * 0 4 0 2 sequence typing pattern. In fact, with the exception of cell line 9064, cells RFLP types as DPa', which also carry the D P B I * 0 4 0 2 sequence, were not recognized by T-cell clone 29. If the DPB1 chain of DPa and DPa' specificities is DPB 1"0402 (as shown by a sequence analysis of several DP cell lines of the 10th IHWS [25]), then
DP Epitope Mapping
105
CLONE If GROUP 1}
CLONE N~GROUP 4}
iiiii~iiii!iiiil
i i i!i i i~i~!iil !{iiiiiiiiiii~i!i
[[:
)? ~;LONE M(GROUP 27
I
CLONE K (GROUP 3}
Pos.contr.
~
w6/J2
I
I
L 243
~
B7.21
TU 22
~
ILR 1
TAL 14,1
FIGURE 2 Blocking tests of anti-RFLP DPb clones with anti-HLA monoclonal antibodies. One hundred percent represents the cpm value obtained by stimulation of the clone in RPMI medium clone. The other bars represent the cpm value when an anti-HLA mAb is added, compared with 100%.
clone 29, which carries the DPB1*0201/0401 sequences, should not recognize the single polymorphic positions 36, 5 5 - 5 6 , and 69. From these data, we conclude that individual amino acids do not confer the DPa specificity, but they do play a crucial role in determining the alloepitope. Punctual variations may change the conformation of the molecule, and in this case, the DPa epitope could be a conformational epitope. One possible hypothesis is that the polymorphism of the DP~ chain is implied. To date, eight DPA1 gene sequences encoded by the second DPA exon have been described [26]. T h e D P A I * 0 1 allele itself can be subdivided into D P A I * 0 1 0 1 , D P A I * 0 1 0 2 , and D P A I * 0 1 0 3
according to sequence differences in the transmembrane region. In their work on polymerase chain reaction (PCR)-RFLP, AI-Daccak et al. [27] were able to show that all of the DPa and DPa' 10th IHWS B-LCLs they studied carried the D P A I * 0 1 0 3 allele. Kimura et al. [8] found that two of these cells (nos. 9010 and 9021) carried the DPAI*0301 allele (Table 1). Whatever the results, neither of these studies suggests that the polymorphism o f the DPo~ chain is implicated. RFLP typing is known to be difficult and inaccurate in that it analyzes very large D N A fragments that are linked with the second exon coding for the DP specificity. Nevertheless our data show that this technique, contrary to the oligotyping, clearly discriminates DPa from DPa' as do the anti-DPa clones, with the exception of cell line 9064. DPa' differs from DPa in a Bam H I fragment (8.67 kb for DPa and 9.00 kb for DPa') that has been located on the DPA2 gene by Simmons and Ehrlich [25]. But if one refers to the DPA1 cartography, it could be located on the DPA1 gene, as the lengths of the above-mentioned fragments correspond to distances found between Barn H I restriction sites located on the DPA1 gene. The hypothesis that the different reactivity of clone 29 with RFLP DPa and with the DPa' cells is due to these antigens acting as restriction elements for class I and class II molecules [28, 29] was ruled out, as DPa + DPa' cells did not share the same HLA class I or class II molecules. The fact that some clones do not recognize all of the cells that carry an identical DPB1 nucleotide sequence in the second exon has also been described by De Koster et al. [30]. One of the last steps in our study was to examine the importance of the DPa antigen in the MLC. N o stimulation was observed in this test when the same combination in which the clones were obtained was used as stimulators versus responders. But weak stimulation was observed for the DPb specificity. This p h e n o m e n o n has already been observed by other authors using other responder-stimulator cell combinations [ 3 1 - 3 3 ] . Whether the combination raises suppressor cells has yet to be proven. Contrary to DPa, the RFLP-defined DPb specificity did not have a high incidence on the 10th IHWS BLCLs and thus no precise sequence could be attributed to this specificity on the basis of these cells. The problem was solved with our local panel where the incidence of RFLP-DPb was high and where a RFLP-DPb homozygous cell variety (MEY) could be used as a stimulating cell. Oligotyping showed that DPb includes several DPB1 alleles, among them the DPBI*1001 and D P B l * 1 4 0 1 sequences [9]. The stimulating cell MEY carried these two specificities. Because the panel reactivity of the different anti-DPb clones obtained was not as homogeneous as that which was observed for the
106
A. Urlacher et al.
10 0401
20
30
40
60
50
70
80
(NY)LFQ•R•E•YAFN•T•RFLERY•YNREEFA•FDSDV•EFRAVTELGRPAAEYWNSQKDILEEKRAVP•RM••HN•EL••••TL•(RR)
@4[!2
............................
V...................
DE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0201
............................
V..................
DE . . . . . . . . . . . .
E.....................
1001
VH-L
V. . . . . . . . . . . . . . . . . .
DE [ . . . . . . . . . . .
E ......
~ .......
DEAV---
. . . . . . . . . . . . . . . . . . . . . . . .
f 1701
VH-L
. . . . . . . . . . . . . . . . . . . . . . . .
V. . . . . . . . . . . . . . . . . .
bED ...........
E ......
[ .......
DEAV---
[JSf]l
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
V. . . . . . . . . . . . . . . . . .
DE . . . . . . . . . . . .
E ......
V .......
DEAV---
V. . . . . . . . . . . . . . . . . .
DED .......
v VV-L
14(?1
v,~ . . . . . . . . . . . . . . . . . . . . . . . .
L ..........
v VH-L
V .......
DEAV---
v,2 . . . . . . . . . . . . . . . . . . . . . . . .
V. . . . . . . . . . . . . . . . .
B
Amino acids the
bottom
C
Located of
DED ......
the
on
HLA
L ...........
D
Amino groove
anti-DPa clones, the data obtained with the totality of the grown T-cell clones are given in Table 4. In so far as 10 of the 20 clones analyzed disclosed strict antiDPB1*1401 specificity, it seems that this oligospecificity is immunodominant in the chosen cell combination. However, only one DPBI*1001 clone was obtained. A precursor frequency analysis would be necessary to confirm the immunodominance of DPB* 1401. Six clones recognizing the DPB 1" 1001 allele showed strong specific extra reactions with the RFLP-typed DPc cells oligotyped DPB1*1701. Strong similarities exist between the DPBI*1001 and B1"1701 alleles (Fig. 3). They differ only in two amino acids: D vs E in position 57 and V vs M in position 76. We hypothesize that the common epitope is a conformational epitope, which implies that the A, B, D, and F hypervariable regions are shared. The specific DPBI*1001 may be due to the amino acids in positions 57 and 76. According to the hypothetical model of class II antigens [34], both amino acids are located on the s-helix of the DP B molecule. The DPBI*0801 allele, which shows the same amino acids as DPBI*1001 in these positions but differs from it in the first hypervariable region, would have been useful in determining whether the specific alloepitope is created by the first hypervariable region A or by the amino acids in the A, C, and E regions. In the first hypothesis, the peptide presented would be of maximal importance. The first region is located on the B-pleated sheets in the groove of the molecule and probably plays a role in peptide binding. In the second hypothesis, allorecogni-
V. . . . . .
E
acids
the6(.helix
F
Located of
DEAV--
the
on HLA
FIGURE 3 DNA sequences of DP /3 chains [7]. (~¢) The amino acids involved in the specific alloepitopes of the described DP antigens. A, B, C, D, E, and F are the hypervariable regions of the HLA-DP second DPB1 exon.
tion could be attributed to the MHC molecule alone. Unfortunately, the only DPBI*0801 sequenced cell line (PIAZ) is no longer available. Clones K and L recognized all of the DPb B1"1401 cells and showed specific extra reactions with DPBl*0301-typed cells. The DPBI*2001 cells, whose sequence is identical to that of DPBI*0301 except in region E, were not recognized. Published DPB 1" 1401 and 0301 sequences differ only in one amino acid in the first hypervariable region in position 9. According to the model proposed by Brown et al. [34], this amino acid is located on the ~3-pleated sheets in the bottom of the groove of the HLA class II molecule, and therefore plays a crucial role in peptide selection. The common epitope recognized by clones K and L must be a confornational epitope confered by specific amino acids in the B, C, D, E, and F hypervariable regions. The valine residue in position 76 is also important because DPBI*2001 cells that do not carry this amino acid are not recognized by the clones. De Koster et al. [30] recently described T-cell clones obtained with DPw3 stimulating cells. They found anti-DPw3 + DPw14 clones, as we did when using DPw14 as the stimulating antigen. Figure 3 shows the putative location of the different DPa and DPb allospecificities drawn on the amino acid sequence of the DPB 1 chains. The allocated specific Tcell alloepitopes are distributed on the bottom of the groove and on the 0e-helixes. These results raise the question of the basis of alloreactivity. Blocking test data were rather surprising: only the
DP Epitope Mapping
specific anti-DPBl*1001 and DPB1*1401 were inhibited by the well-known monomorphic anti-DP B7/21 antibody whereas ILR1 blocked the reactivity of all of the clones. ILR1 mAb has been described as reacting with the DP2, DP3, and DP4b (equivalent to DPa) molecules, all of which carry amino acids (aa) V in position 36, D in position 55, and E in position 56 [23]. It has also been pointed out that mAb ILR1 recognizes the DR5 specificity, whose DR11 subdivision also carries the abovementioned aa in positions 38, 57, and 58, respectively. Because DPb has a two-amino acid deletion occurring on DP/3 chain after aa 23, the DP~ 36, 55, and 56 correspond to the D R 3 38, 57, and 58 positions. DR11 is the only D R specificity that carries E in position 58. After carrying out mutagenesis studies, Yu et al. [23] concluded that E 58 for DR11 and E 56 for DPa contribute to the formation of the epitope recognized by the ILR1 mAb. If this is true, the other D P specificities that carry E 56 (DPIB1*0601, DPBI*0801, D P B I * 0 9 0 1 , D P B I * 1 0 0 1 , D P B l * 1 4 0 1 , DPB1*1601, DPB1*1701, DPB1*1801) should also react with mAb ILR1. This was confirmed for the specificities studied in this work, because mAb ILR1 inhibited the proliferation of anti-DPB 1" 1001, DPB 1" 1401 and -DPB 1" 1001 + DPB 1" 1701 clones. We excluded the possibility that the blocking activity could be due to the anti-DR11 antibody because not only was the DR11, D P B I * 1 0 0 1 / 1401-stimulating cell MEY blocked, but the stimulating cells G U E (DR7 DPB1*1401/0201) and G U I (DR1/ DR3 DPB1*0101/1001) were blocked as well. This suggests that amino acid E in position 56 plays a crucial role in creating a common epitope for T-cell clones and monoclonal antibodies in the DP/3 chain. The absence of blocking reactivity o f clones N and K by mAb B7/21 remains unexplained as this antibody is known to recognize the DP/~ chain, even in interisotypic ~ and/3 chain assembly. Unfortunately, the precise epitope of this mAb is not yet known. Inhibition by the anti-DR/3 TAL 14.1 may be due to shared epitopes with DR/3 and RFLP-DPb molecules. However, this needs further confirmation as previous studies have shown that cell lines which were transfected with various DP/~ chains did not fix this mAb [22]. CONCLUSION HLA class I! D P antigens can now be studied extensively by molecular biology analysis, and correlations with cellular data make it possible to study the functional importance o f these little-known molecules. Although RFLP analysis should be avoided for HLA-DP typing, as about 7% discrepancies have been found be-
107
tween RFLP typing and PLT and oligotyping [9], this technique may be useful for detecting some specificities which cannot be screened by the sequencing of the second exon or by oligotyping (e.g., DPa'). Correlation of oligotyping and T-cell clone analysis reveals the important functional structures. Our data confirm those recently published by De Koster et al. [30]. These authors, however, determined shared T-cell epitopes on DP molecules and our study determines specific alloepitopes of RFLP DPa and DPb antigens. Our analysis shows that, for clinical purposes such as bone marrow grafting, D P specificity matching by molecular biology techniques alone may not be sufficient in all cases. T-cell epitope mapping may turn out to be a better solution for this problem in the future. ACKNOWLEDGMENTS
The authors are grateful to Mrs. A. Mathern, A. Rutz, and A. Schell for technical work and to Mrs. C. Saalbach for preparing the manuscript. This work was supported by the INSERM (Institut de la Sant~ de la Recherche M~dicale) no. 893014 and the ARC (Association pour la Recherche sur le Cancer) no. 6185. REFERENCES 1. Mawas C, Charmot D, Sivy M, Mercier P, North ML, Hauptmann G: A weak human MLR locus mapping at the right of a crossing over between HLA-D, Bf and GLO. J Immunogenet 5:383, 1978. 2. Hyldig-Nielsen J, Morling N, Odum N, Ryder LP, Platz P, Jakobsen B, Svejgaard A: Restriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes. Proc Natl Acad Sci USA 84:1644, 1987. 3. Mitsuishi Y, Urlacher A, Mayer S, Tongio MM: DP RFLP studies of the 72 core cell lines selected for the Southern Blot Protocol. In Dupont B (ed): Immunobiology of HLA I. New York, Springer-Verlag, 1989. 4. Bugawan TL, Horn GT, Long CM, Mickelson E, Hansen JA, Ferrara GB, Angelini G, Erlich HA: Analysis of HLA-DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DPa and DPb loci. J Immunol 141:4024, 1988. 5. Angelini G, Bugawan TL, Delfino L, Erlich HA, Ferrara GB: HLA-DP typing by DNA amplification and hybridization with specific oligonucleotides. Hum Immunol 26:169, 1989. 6. Bugawan TL, Angelini G, Larrick J, Auricchio S, Ferrara GB, Erlich HA: A combination of a particular HLA DP/3 allele and on HLA-DQ heterodimer confers susceptibility to coeliac disease. Nature 339:470, 1989. 7. Bugawan TL, Begovich AB, Erlich HA: Rapid HLA-DPB typing using enzymatically amplified DNA and nonradioactive sequence-specific oligonucleotide probes. Immunogenetics 32:231, 1990.
108
8. Kimura A, Dong RP, Harada H, Sasazuki T: DNA typing of HLA class II genes in B-lymphoblastoid cell lines homozygous for HLA. Tissue Antigens 40:5, 1992. 9. Dormoy A, Urlacher A, Tongio MM: Complexity of the HLA-DP region: RFLP analysis versus PLT typing and oligotyping. Hum Immunol 34:39, 1992. 10. Cesbron A, Moreau P, Milpied N, Muller JY, HarrousseauJL, Bignon JD: Influence of HLA-DP mismatches on MLR I: responses in unrelated HLA-A, B, DR, DQ, Dw identical pairs in allogeneic bone marrow transplantation. Bone Marrow Transplant 6:337, 1990. 11. Olerup O, M611er E, Persson U: HLA-DP incompatibilities induce proliferation in primary mixed lymphocyte culture in HLA-A, B, C, DR and DQ identical individuals. Transplant Proc 22:141, 1990. 12. Odum N, Platz P, Jakobsen BK, Munck-Petersen C, Jacobsen N, M611er J, Ryder LP, Lamm L, Svejgaard A: HLA-DP and acute graft versus host disease. In Dupont B (ed): Immunobiology of HLA II. New York, SpringerVerlag, 1989. 13. Eckels DD, Lake P, Lamb Jr, Johnson AH, Shaw S, Woody JN, Hartzmann RJ: SB restricted presentation of influenza and herpes virus antigen to human T lymphocyte clones. Nature 301:716, 1983. 14. Termijtelen AM: T cell allorecognition of HLA class II. Hum Immunol 28:1,1990. 15. Reinsmoen NL, Bach FH: Structural model for T cell recognition of HLA class II associated alloepitopes. Hum Immunol 27:51, 1990. 16. Terasaki PI, Mac Clelland JD: Microdroplet assay to human cytotoxins. Nature 204:998, 1964. 17. Vartdal F, Gaudernack G, Funderud S, Bratlie A, Lea T, Ugelstad J, Thorsby E: HLA class I and II typing using cells positively selected from blood by immunomagnetic isolation: a fast and reliable technique. Tissue Antigens 28:301, 1986. 18. Marcadet A, Connell PO, Cohen D: Standardized Southern blot workshop technique. In Dupont B (ed): Immunobiology of HLA I. New York, Springer-Verlag, 1989. 19. Mitsuishi Y, Dormoy A, Urlacher A, Tongio MM: Routine HLA-DP typing by RFLP analysis. Nouv Rev Fr Hematol 31:387, 1989. 20. Dupont B, Braon DW, Yunis ES, Carpenter CB: HLA-D by cellular typing. In Terasaki PI (ed): Histocompatibility Testing. Los Angeles, UCLA, 1980. 21. Flomenberg N, Hansen JA, Klein JS, Dupont B: T-cell recognition of HLA class II molecules: background, goals, and experimental design. In Dupont B (ed): Immunobiology of HLA, vol I. New York, Springer-Verlag, 1989. 22. Altmann DM, Heyes JM, Ikeda H, Sadler AM, Wilkinson D, Madrigal JA, Bodmer JG, Trowsdale J: Fine mapping of HLA class II monoclonal antibody specificities using transfected L celIs. Immunogenetics 32:51, 1990.
A. Urlacher et al.
23. Yu WY, Watts R, Karr RW: Identification of amino acids in HLA-DPw4b/3 and DR5/31 chains that are involved in antibody binding epitopes using site directed mutagenesis and DNA mediated gene transfer. Hum Immunol 27:122, 1990. 24. Robbins PA, Evans EL, Ding AH, Warner NL, Brodsky FM: Monoclonal antibodies that distinguish between class II antigens (HLA-DP, DQ, and DR) in 14 haplotypes. Hum Immunol 18:301, 1987. 25. Simmons MJ, Ehrlich HA: RFLP: sequence interrelations at the DPA and DPB loci. In Dupont B (ed): Immunobiology of HLA, vol I. New York, Springer-Verlag, 1989. 26. Bodmer JB, Marsh SGE, Albert ED, Bodmer WF, Dupont B, Erlich HA, Mach B, Mayr WR, Parham P, Sasazuki T, Schreuder GMTh, Strominger JL, Svejgaard A, Terasaki PI: Nomenclature for factors of the HLA system, 1991. Tissue Antigens 39:161, 1992. 27. AI-Daccak A, Wang FQ, Theophille D, Lethielleux P, Colombani J, Loiseau P: Gene polymorphism of HLADPB1 and DPA1 loci in Caucasoid population: frequencies and DPB1-DPA1 associations. Hum Immunol 31:277, 1991. 28. Essaket S, Fabron J, De Preval C, Thomsen M: Corecognition of HLA-A1 and HLA-DPw3 by the human CD4+ alloreactive T lymphocyte clone. J Exp Med 172:387, 1990. 29. De Koster HS, Anderson DC, Termijtelen A: T cell sensitized to synthetic HLA-DR3 peptide give evidence of continuous presentation of denatured HLA-DR3 molecules by HLA-DP. J Exp Med 169:1191, 1989. 30. De Koster HS, Kenter MJH, d'Amaro J, Luiten RM, Schroeisers WEM, Giphart MJ, Termijtelen A: Positive correlation between oligonucleotide typing and T cell recognition of HLA-DP molecules. Immunogenetics 34:12, 1991. 31. A1-Daccak R, Loiseau P, Miramont P, Rabian C, Raffoux C, Colombani J: Evaluation of HLA-class II identity between unrelated individuals by serological typing. DNARFLP method and mixed lymphocyte reaction. Hum Immunol 29:189, 1990. 32. Termijtelen A, Erlich HA, Braun LA, Verduyn W, Drabbels JJM, Schroeijers WEM, van Rood JJ, De Koster HS, Giphart MJ: Oligonucleotide typing is a perfect tool to identify antigens stimulatory in the mixed lymphocyte culture. Hum Immunol 31:241, 1991. 33. Salazar M, Yunis I, Alosco SM, Chopek M, Yunis EJ: HLA-DPB1 allele mismatches between unrelated HLAA, B, C, DR (generic) DQAl-identical unrelated individuals with unreactive MLC. Tissue Antigens 39:203, 1992. 34. Brown SH, Jardetsky T, Saper MA, Samraoui B, Bjorkman P, Wiley DC: A hypothetical model of the foreign antigen binding site of class II histocompatibility molecules. Nature 332:845, 1988.
A B S T R A C T : To determine whether a correlation exists between the genomic HLA class II DP DNA polymorphism and cell surface expression and to detect the DP epitopes responsible for alloreactivity, anti-DP T-cell clones were generated against new PLT blank RFLP DPa and DPb-defined specificities. The clones were tested on the 10th IHWS B-LCLs and on local panel cells. Oligotyping of the tested cells made it possible to (a) correlate the DPa specificity with the DPBI*0402 specificity and
(b) split DPb into DPBI*1001 and DPB1*1401. By comparing DNA sequences of the second exon to panel reactivity, the epitopes responsible for DPB 1" 1001 and 1401 were defined and attributed to/~-chain residues contributing to peptide selection inside the HLA groove. However, DNA sequences could not explain anti-DPa allospecificity, indicating that another structure not yet definable may be involved. Human Immunology 35, 100-108 (1992)
ABBREVIATIONS
B-LCL RFLP IHWS
B-lymphoblastoid cell line restriction fragment length polymorphism International Histocompatibility Workshop
mAb MLC PBMC
monoclonal antibody mixed lymphocyte culture peripheral blood mononuclear cell
INTRODUCTION HLA class II DP antigens were first detected by Mawas et al. in 1978 [1] in secondary mixed lymphocyte cultures. The polymorphism of these antigens seemed to be limited, as only six specificities were known at the time of the 10th International Histocompatibility Workshop (IHWS). The development of molecular biology techniques, such as the restriction fragment length polymorphism (RFLP) analysis [2] and, more recently, sequencing and oligotyping, has shown that DP polymorphism is larger than predicted. Besides the classic D P w l to DPw6 specificities, RFLP analysis has detected new DP blank "specificities" called locally DPa, DPa', DPb, and DPc [3], and sequencing of the second exon of the DPB1 gene has revealed 19 DP alleles [ 4 7] and subsequently 36 alleles [26]. Typing of a large local panel and the 10th IHWS Blymphoblastoid cell lines (B-LCLs) by RFLP and oligoFrom the Histocompatibility Laboratory, Regional Center for Blood Transfusion, Strasbourg, France. Address reprint requests to Dr. A. Urlacher, Laboratoire d'Histocompatibilit~, Centre Regional de Transfusion Sanguine, I0 Rue Spielmann, F-67085 Strasbourg Cedex, France. Received September 2, 1991; acceptedAugust 10, 1992. I00 0198-8859/92/$5.00
typing has shown that (a) the RFLP DPa and DPa' specificities carry the DPBI*0402 sequence [3, 8], (b) RFLP DPc specificity is closely correlated with the DPB1*1701 sequence [9], and (c) the RFLP DPb specificity is heterogeneous, as will be seen further on. Although the polymorphism of the DP alleles has now been well defined by molecular biology techniques, little is known about its expression at the cellular level and even less is known about its function. In vitro, DP polymorphism is suspected of generating stimulation in mixed lymphocyte cultures (MLCs) [10, 11] and, in vivo, mismatches for DP antigens have been described as being responsible for graft-versus-host disease reactions [12]. However, these results need further confirmation. Some DP antigens have also been described as restriction antigens [ 13], but the role of polymorphism in the restriction mechanism also needs further study. The basis of alloreactivity and HLA restriction has been widely studied for class II DR and DQ antigens by analyzing panel reactivity of in vitro obtained T-cell clones raised in well-defined cell combinations [ 14, 15]. In this study, we used the same approach to determine whether the genomic DP D N A polymorphism was cotHuman Immunology 35, 100-108 (1992) © American Society for Histocompatibility and lmmunogenetics, 1992
DP Epitope Mapping
related with cell surface expression and to detect the DP epitopes responsible for generating alloreactivity. We chose closely related cell combinations in which stimulating and responding cells had identical class II DR and D Q specificities, but different DP specificities. The stimulating cells were homozygous for one of the new DP blank RFLP specificities defined above. In the first cell combination, the stimulating cells carried the RFLP DPa specificity (sequence DPBI*0402) and the responding cells carried the RFLP 2.1/4.1 specificities, (sequence DPB1*0201/0401). In the second combination, the stimulating cell was homozygous for RFLPDPb, and since at the time the study began DPb sequences were not known, the responding cells were chosen for their identical DR and DQ antigens tested by serologic typing. MATERIALS AND METHODS
101
and IVA, with two subclusters in IA and IIA called IA 1, IA2, and IIA1 and IIA2. For DPB, four main clusters were described, IB, IIB, IIIB, and IVB, with two subclusters in IIIB and IVB called IIIB1, IIIB2, IVB1, and IVB2 [3, 19]. Detailed patterns of DP2.1, 4.1, a, a', b, and c specificities are given in Fig. 1.
Oligotyping. Oligotyping was performed with 25 DPB and 4 DPA oligoprobes according to the 11th IHWS protocol after amplification of the second exon of the DPBA 1 and B 1 genes with specific primers given by the Workshop organizers (data to be published). MLC MLC was performed using the protocol technique of the 8th IHWS [20]. The data were analyzed using SRR (stabilized relative response) calculation.
HLA T y p e of the Cell Combinations Used to Raise Anti-DPa and Anti-DPb Clones
Generation and Maintenance of Alloreactive T-Cell Clones
DP specificities are given using the RFLP nomenclature and the corresponding DPB1 sequence extrapolated from the oligotyping is in parentheses.
Responding peripheral blood mononuclear cells (PBMCs) were primed in an MLC against the 25-Gyirradiated stimulating cells for 10 days. After a second priming that lasted for 6 days, blasts were separated from the nonreactive cells on a Percoll gradient and plated in limiting dilution (1/3 cell per well) with 1 x 105 25°Gy-irradiated pooled PBMCs as feeder cells. Six weeks after cloning, the grown cells were frozen in 5 × 106 aliquots and stored at -196°C until further testing.
Cell Combinations
Anti-DPa clones (DPBl*0402). Stimulating cell: SCHU.. A (local homozygous cell line included in the 10th IHWS studies = B-LCL no. 9 0 1 3 : A 3 B7 Cw7 DR2 DQ1 DPa (DPBI*0402). Responding cell: KLO.. G (local panel donor): A3 B7 Cw7 DR2 DQ1 DP2.1 (DPBI*0201)/A3 B56 Cw7 DR2 DQ1 DP4.1 (DPBI*0401). Anti-DPb RFLP clones. Stimulating cell: MEY.. Be: A3 B18 B55 Cw7 DR11 DR52 DQ7 DPb, DPb. Responding cell: WEI.. An: A2 A26 B18 B44 Cw7 DR11 DR52 DQ7 DP4.3/DPa (DPB1*0401/0402).
B-Cell Lines PBMCs and B-LCLs were used as stimulators in the proliferative tests of the clones. B-LCLs were either those provided by the 10th IHWS studies and/or locally transformed PBMCs from panel families. Proliferation Assays
HLA typing of the local cells was carried out using the classic microlymphocytotoxicity technique for HLA class I typing [16], and the Dynabeads technique was used for D R and D Q typing [17]. Sera from the 10th IHWS were used.
Proliferation assays were performed following the 10th IHWS protocol [21]. Briefly, 10,000 cloned T cells were cultured with 2.5 × 104 irradiated B-LCLs (100 Gy) or I × 105 irradiated PBMCs (25 Gy). The proliferative response was quantitated by a 18-hour pulse with [3H]thymidine (~H-TdR, 1/.~Ci/well). Results were expressed as the median counts per minute (cpm).
HLA-DP T y p i n g
Blocking Assays
RFLP. RFLP DP analysis was performed according to the 10th IHWS protocol with Msp I, Bst EII, Bgl II and Bam HI enzymes [18]. Hybridization was performed with ~2P-labeled DPA and DPB probes. RFLP fragments were subdivided into categories called main clusters and each main cluster included subclusters. For DPA, four main clusters were described, IA, IIA, IIIA,
The following monoclonal antibodies (mAbs) were used: W6/32 (anti-class-I monomorphic), L243 (antiDR~ [22]), Tti 22 (anti-DQ monomorphic + DR2 + DR7 [22]), TAL 14.1 (anti-DR/~ [22]), ILR1 (antiD P 2 + 3 + 4 b + D R 5 [23]), and B7.21 (anti-DP monomorphic [24]). For the inhibition studies of the clones, the 10th
Class I and Class II D R and D Q Serologic T y p i n g
102
A. Urlacher et al.
I RFLP
DP
2.1
DP
4.1
DP
a
DP
a'
DP
b
DP
c
DPA IA llA
1/41
IA2
IIA1
DPB III A IVA
1142
IIIB IIIB1
IVB
IB
liB
IIIB2 IVB1 IVB2
3am HI 8.67 neg 900 pos
FIGURE 1 RFLP patterns of DP subdivisions.
The subchisters are characterized by the presence of the following specific fragments IA 1
BstEII 25.40
Bglll 3.58
IA2
Bst Eli 21.50
Bglll 3.41
IIA1
Bst Eli 10.00
Barn HI 9.00
IIA2
Bst Eli 9.70
Bam HI 8.67
IliA
Bgl I17.24
IVA
Bgl II 5.29
IIIB 1
Bglll 18.(X)
Mspl 4.98
IIIB2
Bglll
Mspl 3.11
IVB1
Bglll 15.05
MspI 2.17
IVB2
Bglll 15.05
Mspl 1.07
IB
Bst Ell 4.40
liB
Bst Ell 4.00
18.00
Mspl 4.87
white boxes : fragment not present black boxes : fragment present hatched boxes : see notes on the right of the figure.
IHWS protocol for blocking assays was used [21]. The stimulating cells (original primer cells) were incubated with 50-/.d aliquots of antibodies for 1/2 hour at room temperature prior to adding the clones. Six wells were plated as unblocked controls, receiving 50 /zl of medium alone in place of antibody. The mean proliferation observed in these controls was considered as 100%. Tests were performed in triplicate. The percentage of proliferation was calculated for each antibody dilution: % proliferation = (mean cpm test/mean cpm unblocked control) × 100. RESULTS A n t i - D P a Clones Thirty clones showing proliferation with the original primer cells were grown. Tested on a restricted PBMC
panel, 10 of them showed the same specific reactivity. Clone 29, which grew quickly and displayed high proliferation values, was chosen for further analysis.
Panelreactivity. Clone 29 was tested on 75 reference BLCLs from the 10th IHWS. Only RFLP DPa-typed cells were recognized (Table 1). Four cell lines, 9010, 9016, 9021, and 9037, which were RFLP DPa' typed were not recognized by clone 29 ([DPa' differs from DPa in one Bam HI fragment, 9.00 kb instead of 8.67 kb] [Fig. 1]). However, their sequence was published as DPB 1"0402 [5, 8]. Three other clones showed the same reactivity pattern as clone 29, but stimulated weaker proliferation. Local family studies showed a strict correlation between the presence of the RFLP DPa specificity and recognition by clone 29 (Table 2). This was confirmed by haplotypic segregation. The DPa specificity had an allelic incidence of about 15 % on unrelated donors, but we did not find the DPa' allele in any of the individuals tested.
Blocking tests. Blocking tests showed that the monomorphic B7/21 mAb was able to inhibit strongly the proliferation of clone 29 (data not shown), confirming that this clone recognized a determinant on the DP molecule.
MLC. If widespread DPa specificity can be accurately discriminated by T-cell clones, it should also be responsible for MLC stimulation. This was not the case when stimulating and responding cells were included in a classic MLC (Table 3). The observed RR values were 3 in one way versus 4 in the other. Anti-DPb Clones
Panel reactivity. Anti-RFLP DPb clones were obtained from two secondary mixed lymphocyte cultures using
DP Epitope Mapping
TABLE 1
103
Reactivity of anti-DPa clone 29 on the 10th IHWS cell lines
LCL identification
DPA 1 sequence a
DPB 1 sequence*
RFLP typing
Reactivity of clone no. 29
9013 9042 9045 9054 9071 9072 9091 9092
01 01 01 b 01 01 b 01 b 01 01
0402 0402 0402/02012 0402 0402/0301 0402 0402 0402
a/a a/a a/2.1 a/a a/3.1 a/a a/a a/a
91.9 ND ND 89.7 74.3 95.0 71.3 20.7 c
9064 9010 9016 9021 9037
01 ~ 030 lb 01 b 0201/0301 b 01 b
0402 0402 0402 0402/0101 0402
a'/a'
90.6
a'/a' a'/a'
6.7
a'/1.2 a'/a'
14.8 11.7 7.8
9002 9004 9005 9014 9025
01 b 01 01 01 b 01
0401 0401 0401 0401 0401
4.1/4.1 4.1/4.1 4.1/4.1 4.1/4.1 4.1/4.1
4.7 14.7 9.8 6.0 5.7
9036 9039 9068 9029
01 01 b 01 b 01 b
02012 02012 02012 02012
2.1/2.1 2.1/2.1 2.1/2.1 2.1/2.1
5.9 7.8 6.1 4.7
This table shows the reactivity pattern of clone 29 when tested on 75 reference B-LCLs from the 10th IHWS. Only results obtained with cell lines typed as DPBI*02012, 0401, or 0402 are reported. The other cell lines tested gave negative results. Data are given in cpm × 103. Each value is the median value of triplicate tests. The cell lines reported on this table were tested at least twice with clone 29. The DPA1 and DPBI genotypes given in this table have been taken from Kimera et al. [8]. bThese cell lines were also studied using PCR-RFLP by AI-Daccak et al. [27], who found the subtype DPAI*0301 for these cases. cThe reactivity of clone 29 with cell line 9092 was considered as positive despite the weak proliferation because this line weakly stimulated a pool of responding cells (28.0 cpm × 103).
TABLE 2
Family identification ANT ANT ANT ANT ANT ANT ANT TIP TIP TIP TIP TIP TIP
All (father) B~t (mother) Ant Eve Adr Pie Syl
Mau (father) Nel (mother) Hug Fr6 Jac Jea
WEI WEI WEI WEI WEI WEI
Th6 (father) Lin (mother) Eri And Ann Gab
RFLP typing
cpm × 103
DP4.1/DPb 4.1/a b/4.1 b/a b/a b/4.1 b/a
5.6 20.5 0.6 12.8 12.8 1.9 18.2
a/3
12.2
4.1/4.1
O. 1
3/4.1 a/4.1 a/4.1 a/4.1
0.5 15.8 19.4 13.9
4.3/4.3 3/a 4.3/3 4.3/a 4.3/a 4.3/a
0.4 17.9 0.4 11.4 11.3 13.8
This table shows the reactivity pattern of clone 29 when tested on local families. Only data on families carrying the RFLP DPa specificity are shown and given in cpm × 103. Each value is the median value of triplicate tests. Each cell was tested at least twice with clone 29.
tions with the RFLP DPc DPBl*1701 cells (group 4). Two clones (K and L) recognized the RFLP DPb DPB1*1401 cells and all of the DPBI*0301 panel cells (group 3). One clone (T) reacted with groups 3 and 4 combined.
Blocking tests. Block tests were performed with one clone representing each group (Fig. 2). N o inhibition
TABLE 3 the same stimulating responding cell combination as described in Materials and Methods. From the first priming, two pairs of clones, clones I and J and clones R and S, were obtained. From the second priming, 16 clones with specific panel reactivity were obtained. Data are summarized in Table 4. All of the clones were tested on a local DP RFLP and oligotyped panel because very few 10th IHWS B-LCLs carried the DPb specificity. In fact, oligotyping showed that the RFLP-DPb homozygous stimulating cell MEY disclosed the DPB1*1001/1401 sequences. Ten clones (A-J) recognized the DPb cells oligotyped as DPB1*1401 without any extra reaction (group 1). One clone (M) recognized the RFLP DPb DPBI*1001 specificity without any extra reaction (group 2). Six clones (N-S) recognized the RFLP DPb DPBI*1001 cells and showed strong specific extra reac-
Reactivity of anti-DPa clone 29 on local families
MLC data obtained with cell combinations used for generating anti-DPa and -DPb T-cell clones Stimulating cells
Responding cells
1
2
3
4
1.
S C H U And DPa/DPa (0402/0402)
1.965 ° 0b
3.227 4
65.684 146
52.312 112
2.
KLO GSr DP2.1/4.1 (0401/0201)
3.040 3
1.615 0
70.710 125
62.071 108
3.
MEY Ber D P b / D P b (1001/1401)
27.577 95
29.513 88
3.032 0
14.401 32
4.
WEI And DP4.3/DPa(0401,0402)
38.506 69
51.887 79
35.626 48
1.244 0
Data are given in cpm × 103~ and in SRRb. The MLC is considered as negative when SRR < 10.
104
A. Urlacher et al.
TABLE 4
Panel reactivity of anti-RFLP DPb clones CLONEJ0ENT~nOATION
Cell pd k~Y GJF LAG NE~r~ R'-;I4 BRA
RFIP bib
NO b/2 1 b/a hip
b/4 1
<3C#~
hi.;
[:~)1J GIJI
b/4 I bit 1
OIJGO A 140111001 I318 401/0201 28 8 401/0201 25 401/0301 i~J 401/1101 17 5 401/0401 NO h[) t~3
F{r[ +
Is/4 1
001/0401 001/0101 NO
ANC F{-~C l'{J M[3#
c/4 1 c/2 1 i~3 c/I 1
701/0401 701/0201 701/0601 701/0101
GQ~7 Z]E HEL {{()~~
14} el1 2 el2 1 b/2 1 b/2 1 3 113 1
301/0301 301/0101 30110201 301/1301 ~01/0201 !, 0 1 / 0 3 0 1
~t'A I{'~ I I'l'
[)l{J DfqA RII
SLA SMI VIL FP,E tf]~
MAR "ilP 9040 LFD CHI I4EF
(~/4
3 1 /4 1 3 1/1 2 hi) 3 I/4 1 Kl') } l/a 3 1/2 1 3 l/a 3 1/1 1 :J 1/3 1 3 2 3 1/4 3 3, 1/4 1 3 1/4 1
101/04b
B
417 117 41 3 ND 129 ND ND
C
105 86 118 ND 129 ND ND
D
319 179 189 ND 89 ND ND
E
116 126 125 ND 101 ND ND
F
G
254 187 15 149 183 155 10 6
456 433 366 349 436 469 23 2
H
361 381 308 342 411 41 4 16 9
519 7 328 175 112 i~) 287 46 1
J 324
K 44 3
t~)
48 1
iq3 11 2 t',O 154 11 7
339 31 385 459 338
419
35 9
L 28 8 27 1 188 16B 227 25.1 23 3
246
9 1
O 152
P 21 6
Q 39 1
34 4 303 292
11 8
20 9
7, 5 15 5
22 9 14 4
176 20 3 20 1 113
16 1 74 201 87
16 6 17 9 174 12 7
M
N
188
338
158 15 1 143
R 59 8
S 48 4
T 27 5 164 131 127 137 181 131
25 4
56 7
35 7 32 4
34 6 41 6
42 156 14
19 5 283 154
9 1 NO 98 8 4
MO 47 1 KO t~D
h/3 42 8 i~) ND
36 4 27 9 334 172 30 4
26 3
1
0301/0401 0301/0101 0:{01/0101 0301/0401 0301/0401 0301/04(/2 0301/0201 0301/0402 0301/0101 0301/0301 NO 2001/0401 2001/0401 2001/0401
58 51 4 71 1 39 I 88 5 44 8 51 9 80 6 66 2 52 1
65 53 71 36 80 30 37 70 47 31
70123
572
12 05
1 9 6 1 6 3
1 6 4 4
11 19
k13 NO NZ) t49 t,,D N3 KID hD
N3 N3
Panel reactivity of RFLP anti-DPb (DPBI*1001 and DPBl*1401) clones. All T-cell clones were investigated with all of the LCLs. Blank spaces indicate negative results. Results are median values from triplicate tests given in cpm x 103. ND, not determined. Four types of reactivities were observed: group 1, clones A-J recognize specifically DPB1*1401 cells; group 2, clone M is specific for the DPBI*1001 specificity; group 4, clones N - S corecognize DPBI*1001 and DPB1*1701 cells; and group 3, clones K and L corecognize DPB1*1401 and DPBI*0301 cells.
was noted with W 6 / 3 2 (anti-class-I) L243 (anti-DRa) or with T~i 22 (anti-DQ + DR2 + DR7). ILR1 ( a n t i - D P 2 + 3 + 4 b + D R 5 ) and TAL 14.1 (antiDR/~) strongly inhibited all the tested clones, but surprisingly, B7/21, the well-known anti-DP monomorphic antibody, inhibited only the specific antiDPB1*1401 (group 1) and D P B I * 1 0 0 1 (group 2). DISCUSSION The present study had two aims: (a) to determine whether the D P polymorphism discovered at the D N A level was expressed at the cell surface and (b) to determine, as previously done for D R and D Q antigens, the structural model for T-cell recognition of HLA class II DP-associated alloepitopes. Two RFLP D P specificities were chosen for this purpose. The first one, DPa, was present on some of the 10th IHWS homozygous B-LCLs. Further analysis of
the DP sequence of these cells showed that they carried the D P B I * 0 4 0 2 sequence. Because one of these DPa 10th IHWS B-LCLs came from our lab (no. 9013), we decided to use it as a stimulating cell. We chose a DR, D Q identical cell as a responding cell for which DP differed as little as possible from the D P B I * 0 4 0 2 sequence. A heterozygous cell was found that carried the DPB 1"0201 and DPB 1"0401 sequences, sequences on which only one and three amino acids in the first domain differed, respectively, from D P B I * 0 4 0 2 (Fig. 3). PLT tests showed that even if the reactivity pattern of the anti-DPa clone (clone no. 29) correlates with DPa, it is not an exact fit with the D P B I * 0 4 0 2 sequence typing pattern. In fact, with the exception of cell line 9064, cells RFLP types as DPa', which also carry the D P B I * 0 4 0 2 sequence, were not recognized by T-cell clone 29. If the DPB1 chain of DPa and DPa' specificities is DPB 1"0402 (as shown by a sequence analysis of several DP cell lines of the 10th IHWS [25]), then
DP Epitope Mapping
105
CLONE If GROUP 1}
CLONE N~GROUP 4}
iiiii~iiii!iiiil
i i i!i i i~i~!iil !{iiiiiiiiiii~i!i
[[:
)? ~;LONE M(GROUP 27
I
CLONE K (GROUP 3}
Pos.contr.
~
w6/J2
I
I
L 243
~
B7.21
TU 22
~
ILR 1
TAL 14,1
FIGURE 2 Blocking tests of anti-RFLP DPb clones with anti-HLA monoclonal antibodies. One hundred percent represents the cpm value obtained by stimulation of the clone in RPMI medium clone. The other bars represent the cpm value when an anti-HLA mAb is added, compared with 100%.
clone 29, which carries the DPB1*0201/0401 sequences, should not recognize the single polymorphic positions 36, 5 5 - 5 6 , and 69. From these data, we conclude that individual amino acids do not confer the DPa specificity, but they do play a crucial role in determining the alloepitope. Punctual variations may change the conformation of the molecule, and in this case, the DPa epitope could be a conformational epitope. One possible hypothesis is that the polymorphism of the DP~ chain is implied. To date, eight DPA1 gene sequences encoded by the second DPA exon have been described [26]. T h e D P A I * 0 1 allele itself can be subdivided into D P A I * 0 1 0 1 , D P A I * 0 1 0 2 , and D P A I * 0 1 0 3
according to sequence differences in the transmembrane region. In their work on polymerase chain reaction (PCR)-RFLP, AI-Daccak et al. [27] were able to show that all of the DPa and DPa' 10th IHWS B-LCLs they studied carried the D P A I * 0 1 0 3 allele. Kimura et al. [8] found that two of these cells (nos. 9010 and 9021) carried the DPAI*0301 allele (Table 1). Whatever the results, neither of these studies suggests that the polymorphism o f the DPo~ chain is implicated. RFLP typing is known to be difficult and inaccurate in that it analyzes very large D N A fragments that are linked with the second exon coding for the DP specificity. Nevertheless our data show that this technique, contrary to the oligotyping, clearly discriminates DPa from DPa' as do the anti-DPa clones, with the exception of cell line 9064. DPa' differs from DPa in a Bam H I fragment (8.67 kb for DPa and 9.00 kb for DPa') that has been located on the DPA2 gene by Simmons and Ehrlich [25]. But if one refers to the DPA1 cartography, it could be located on the DPA1 gene, as the lengths of the above-mentioned fragments correspond to distances found between Barn H I restriction sites located on the DPA1 gene. The hypothesis that the different reactivity of clone 29 with RFLP DPa and with the DPa' cells is due to these antigens acting as restriction elements for class I and class II molecules [28, 29] was ruled out, as DPa + DPa' cells did not share the same HLA class I or class II molecules. The fact that some clones do not recognize all of the cells that carry an identical DPB1 nucleotide sequence in the second exon has also been described by De Koster et al. [30]. One of the last steps in our study was to examine the importance of the DPa antigen in the MLC. N o stimulation was observed in this test when the same combination in which the clones were obtained was used as stimulators versus responders. But weak stimulation was observed for the DPb specificity. This p h e n o m e n o n has already been observed by other authors using other responder-stimulator cell combinations [ 3 1 - 3 3 ] . Whether the combination raises suppressor cells has yet to be proven. Contrary to DPa, the RFLP-defined DPb specificity did not have a high incidence on the 10th IHWS BLCLs and thus no precise sequence could be attributed to this specificity on the basis of these cells. The problem was solved with our local panel where the incidence of RFLP-DPb was high and where a RFLP-DPb homozygous cell variety (MEY) could be used as a stimulating cell. Oligotyping showed that DPb includes several DPB1 alleles, among them the DPBI*1001 and D P B l * 1 4 0 1 sequences [9]. The stimulating cell MEY carried these two specificities. Because the panel reactivity of the different anti-DPb clones obtained was not as homogeneous as that which was observed for the
106
A. Urlacher et al.
10 0401
20
30
40
60
50
70
80
(NY)LFQ•R•E•YAFN•T•RFLERY•YNREEFA•FDSDV•EFRAVTELGRPAAEYWNSQKDILEEKRAVP•RM••HN•EL••••TL•(RR)
@4[!2
............................
V...................
DE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0201
............................
V..................
DE . . . . . . . . . . . .
E.....................
1001
VH-L
V. . . . . . . . . . . . . . . . . .
DE [ . . . . . . . . . . .
E ......
~ .......
DEAV---
. . . . . . . . . . . . . . . . . . . . . . . .
f 1701
VH-L
. . . . . . . . . . . . . . . . . . . . . . . .
V. . . . . . . . . . . . . . . . . .
bED ...........
E ......
[ .......
DEAV---
[JSf]l
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
V. . . . . . . . . . . . . . . . . .
DE . . . . . . . . . . . .
E ......
V .......
DEAV---
V. . . . . . . . . . . . . . . . . .
DED .......
v VV-L
14(?1
v,~ . . . . . . . . . . . . . . . . . . . . . . . .
L ..........
v VH-L
V .......
DEAV---
v,2 . . . . . . . . . . . . . . . . . . . . . . . .
V. . . . . . . . . . . . . . . . .
B
Amino acids the
bottom
C
Located of
DED ......
the
on
HLA
L ...........
D
Amino groove
anti-DPa clones, the data obtained with the totality of the grown T-cell clones are given in Table 4. In so far as 10 of the 20 clones analyzed disclosed strict antiDPB1*1401 specificity, it seems that this oligospecificity is immunodominant in the chosen cell combination. However, only one DPBI*1001 clone was obtained. A precursor frequency analysis would be necessary to confirm the immunodominance of DPB* 1401. Six clones recognizing the DPB 1" 1001 allele showed strong specific extra reactions with the RFLP-typed DPc cells oligotyped DPB1*1701. Strong similarities exist between the DPBI*1001 and B1"1701 alleles (Fig. 3). They differ only in two amino acids: D vs E in position 57 and V vs M in position 76. We hypothesize that the common epitope is a conformational epitope, which implies that the A, B, D, and F hypervariable regions are shared. The specific DPBI*1001 may be due to the amino acids in positions 57 and 76. According to the hypothetical model of class II antigens [34], both amino acids are located on the s-helix of the DP B molecule. The DPBI*0801 allele, which shows the same amino acids as DPBI*1001 in these positions but differs from it in the first hypervariable region, would have been useful in determining whether the specific alloepitope is created by the first hypervariable region A or by the amino acids in the A, C, and E regions. In the first hypothesis, the peptide presented would be of maximal importance. The first region is located on the B-pleated sheets in the groove of the molecule and probably plays a role in peptide binding. In the second hypothesis, allorecogni-
V. . . . . .
E
acids
the6(.helix
F
Located of
DEAV--
the
on HLA
FIGURE 3 DNA sequences of DP /3 chains [7]. (~¢) The amino acids involved in the specific alloepitopes of the described DP antigens. A, B, C, D, E, and F are the hypervariable regions of the HLA-DP second DPB1 exon.
tion could be attributed to the MHC molecule alone. Unfortunately, the only DPBI*0801 sequenced cell line (PIAZ) is no longer available. Clones K and L recognized all of the DPb B1"1401 cells and showed specific extra reactions with DPBl*0301-typed cells. The DPBI*2001 cells, whose sequence is identical to that of DPBI*0301 except in region E, were not recognized. Published DPB 1" 1401 and 0301 sequences differ only in one amino acid in the first hypervariable region in position 9. According to the model proposed by Brown et al. [34], this amino acid is located on the ~3-pleated sheets in the bottom of the groove of the HLA class II molecule, and therefore plays a crucial role in peptide selection. The common epitope recognized by clones K and L must be a confornational epitope confered by specific amino acids in the B, C, D, E, and F hypervariable regions. The valine residue in position 76 is also important because DPBI*2001 cells that do not carry this amino acid are not recognized by the clones. De Koster et al. [30] recently described T-cell clones obtained with DPw3 stimulating cells. They found anti-DPw3 + DPw14 clones, as we did when using DPw14 as the stimulating antigen. Figure 3 shows the putative location of the different DPa and DPb allospecificities drawn on the amino acid sequence of the DPB 1 chains. The allocated specific Tcell alloepitopes are distributed on the bottom of the groove and on the 0e-helixes. These results raise the question of the basis of alloreactivity. Blocking test data were rather surprising: only the
DP Epitope Mapping
specific anti-DPBl*1001 and DPB1*1401 were inhibited by the well-known monomorphic anti-DP B7/21 antibody whereas ILR1 blocked the reactivity of all of the clones. ILR1 mAb has been described as reacting with the DP2, DP3, and DP4b (equivalent to DPa) molecules, all of which carry amino acids (aa) V in position 36, D in position 55, and E in position 56 [23]. It has also been pointed out that mAb ILR1 recognizes the DR5 specificity, whose DR11 subdivision also carries the abovementioned aa in positions 38, 57, and 58, respectively. Because DPb has a two-amino acid deletion occurring on DP/3 chain after aa 23, the DP~ 36, 55, and 56 correspond to the D R 3 38, 57, and 58 positions. DR11 is the only D R specificity that carries E in position 58. After carrying out mutagenesis studies, Yu et al. [23] concluded that E 58 for DR11 and E 56 for DPa contribute to the formation of the epitope recognized by the ILR1 mAb. If this is true, the other D P specificities that carry E 56 (DPIB1*0601, DPBI*0801, D P B I * 0 9 0 1 , D P B I * 1 0 0 1 , D P B l * 1 4 0 1 , DPB1*1601, DPB1*1701, DPB1*1801) should also react with mAb ILR1. This was confirmed for the specificities studied in this work, because mAb ILR1 inhibited the proliferation of anti-DPB 1" 1001, DPB 1" 1401 and -DPB 1" 1001 + DPB 1" 1701 clones. We excluded the possibility that the blocking activity could be due to the anti-DR11 antibody because not only was the DR11, D P B I * 1 0 0 1 / 1401-stimulating cell MEY blocked, but the stimulating cells G U E (DR7 DPB1*1401/0201) and G U I (DR1/ DR3 DPB1*0101/1001) were blocked as well. This suggests that amino acid E in position 56 plays a crucial role in creating a common epitope for T-cell clones and monoclonal antibodies in the DP/3 chain. The absence of blocking reactivity o f clones N and K by mAb B7/21 remains unexplained as this antibody is known to recognize the DP/~ chain, even in interisotypic ~ and/3 chain assembly. Unfortunately, the precise epitope of this mAb is not yet known. Inhibition by the anti-DR/3 TAL 14.1 may be due to shared epitopes with DR/3 and RFLP-DPb molecules. However, this needs further confirmation as previous studies have shown that cell lines which were transfected with various DP/~ chains did not fix this mAb [22]. CONCLUSION HLA class I! D P antigens can now be studied extensively by molecular biology analysis, and correlations with cellular data make it possible to study the functional importance o f these little-known molecules. Although RFLP analysis should be avoided for HLA-DP typing, as about 7% discrepancies have been found be-
107
tween RFLP typing and PLT and oligotyping [9], this technique may be useful for detecting some specificities which cannot be screened by the sequencing of the second exon or by oligotyping (e.g., DPa'). Correlation of oligotyping and T-cell clone analysis reveals the important functional structures. Our data confirm those recently published by De Koster et al. [30]. These authors, however, determined shared T-cell epitopes on DP molecules and our study determines specific alloepitopes of RFLP DPa and DPb antigens. Our analysis shows that, for clinical purposes such as bone marrow grafting, D P specificity matching by molecular biology techniques alone may not be sufficient in all cases. T-cell epitope mapping may turn out to be a better solution for this problem in the future. ACKNOWLEDGMENTS
The authors are grateful to Mrs. A. Mathern, A. Rutz, and A. Schell for technical work and to Mrs. C. Saalbach for preparing the manuscript. This work was supported by the INSERM (Institut de la Sant~ de la Recherche M~dicale) no. 893014 and the ARC (Association pour la Recherche sur le Cancer) no. 6185. REFERENCES 1. Mawas C, Charmot D, Sivy M, Mercier P, North ML, Hauptmann G: A weak human MLR locus mapping at the right of a crossing over between HLA-D, Bf and GLO. J Immunogenet 5:383, 1978. 2. Hyldig-Nielsen J, Morling N, Odum N, Ryder LP, Platz P, Jakobsen B, Svejgaard A: Restriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes. Proc Natl Acad Sci USA 84:1644, 1987. 3. Mitsuishi Y, Urlacher A, Mayer S, Tongio MM: DP RFLP studies of the 72 core cell lines selected for the Southern Blot Protocol. In Dupont B (ed): Immunobiology of HLA I. New York, Springer-Verlag, 1989. 4. Bugawan TL, Horn GT, Long CM, Mickelson E, Hansen JA, Ferrara GB, Angelini G, Erlich HA: Analysis of HLA-DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DPa and DPb loci. J Immunol 141:4024, 1988. 5. Angelini G, Bugawan TL, Delfino L, Erlich HA, Ferrara GB: HLA-DP typing by DNA amplification and hybridization with specific oligonucleotides. Hum Immunol 26:169, 1989. 6. Bugawan TL, Angelini G, Larrick J, Auricchio S, Ferrara GB, Erlich HA: A combination of a particular HLA DP/3 allele and on HLA-DQ heterodimer confers susceptibility to coeliac disease. Nature 339:470, 1989. 7. Bugawan TL, Begovich AB, Erlich HA: Rapid HLA-DPB typing using enzymatically amplified DNA and nonradioactive sequence-specific oligonucleotide probes. Immunogenetics 32:231, 1990.
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8. Kimura A, Dong RP, Harada H, Sasazuki T: DNA typing of HLA class II genes in B-lymphoblastoid cell lines homozygous for HLA. Tissue Antigens 40:5, 1992. 9. Dormoy A, Urlacher A, Tongio MM: Complexity of the HLA-DP region: RFLP analysis versus PLT typing and oligotyping. Hum Immunol 34:39, 1992. 10. Cesbron A, Moreau P, Milpied N, Muller JY, HarrousseauJL, Bignon JD: Influence of HLA-DP mismatches on MLR I: responses in unrelated HLA-A, B, DR, DQ, Dw identical pairs in allogeneic bone marrow transplantation. Bone Marrow Transplant 6:337, 1990. 11. Olerup O, M611er E, Persson U: HLA-DP incompatibilities induce proliferation in primary mixed lymphocyte culture in HLA-A, B, C, DR and DQ identical individuals. Transplant Proc 22:141, 1990. 12. Odum N, Platz P, Jakobsen BK, Munck-Petersen C, Jacobsen N, M611er J, Ryder LP, Lamm L, Svejgaard A: HLA-DP and acute graft versus host disease. In Dupont B (ed): Immunobiology of HLA II. New York, SpringerVerlag, 1989. 13. Eckels DD, Lake P, Lamb Jr, Johnson AH, Shaw S, Woody JN, Hartzmann RJ: SB restricted presentation of influenza and herpes virus antigen to human T lymphocyte clones. Nature 301:716, 1983. 14. Termijtelen AM: T cell allorecognition of HLA class II. Hum Immunol 28:1,1990. 15. Reinsmoen NL, Bach FH: Structural model for T cell recognition of HLA class II associated alloepitopes. Hum Immunol 27:51, 1990. 16. Terasaki PI, Mac Clelland JD: Microdroplet assay to human cytotoxins. Nature 204:998, 1964. 17. Vartdal F, Gaudernack G, Funderud S, Bratlie A, Lea T, Ugelstad J, Thorsby E: HLA class I and II typing using cells positively selected from blood by immunomagnetic isolation: a fast and reliable technique. Tissue Antigens 28:301, 1986. 18. Marcadet A, Connell PO, Cohen D: Standardized Southern blot workshop technique. In Dupont B (ed): Immunobiology of HLA I. New York, Springer-Verlag, 1989. 19. Mitsuishi Y, Dormoy A, Urlacher A, Tongio MM: Routine HLA-DP typing by RFLP analysis. Nouv Rev Fr Hematol 31:387, 1989. 20. Dupont B, Braon DW, Yunis ES, Carpenter CB: HLA-D by cellular typing. In Terasaki PI (ed): Histocompatibility Testing. Los Angeles, UCLA, 1980. 21. Flomenberg N, Hansen JA, Klein JS, Dupont B: T-cell recognition of HLA class II molecules: background, goals, and experimental design. In Dupont B (ed): Immunobiology of HLA, vol I. New York, Springer-Verlag, 1989. 22. Altmann DM, Heyes JM, Ikeda H, Sadler AM, Wilkinson D, Madrigal JA, Bodmer JG, Trowsdale J: Fine mapping of HLA class II monoclonal antibody specificities using transfected L celIs. Immunogenetics 32:51, 1990.
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23. Yu WY, Watts R, Karr RW: Identification of amino acids in HLA-DPw4b/3 and DR5/31 chains that are involved in antibody binding epitopes using site directed mutagenesis and DNA mediated gene transfer. Hum Immunol 27:122, 1990. 24. Robbins PA, Evans EL, Ding AH, Warner NL, Brodsky FM: Monoclonal antibodies that distinguish between class II antigens (HLA-DP, DQ, and DR) in 14 haplotypes. Hum Immunol 18:301, 1987. 25. Simmons MJ, Ehrlich HA: RFLP: sequence interrelations at the DPA and DPB loci. In Dupont B (ed): Immunobiology of HLA, vol I. New York, Springer-Verlag, 1989. 26. Bodmer JB, Marsh SGE, Albert ED, Bodmer WF, Dupont B, Erlich HA, Mach B, Mayr WR, Parham P, Sasazuki T, Schreuder GMTh, Strominger JL, Svejgaard A, Terasaki PI: Nomenclature for factors of the HLA system, 1991. Tissue Antigens 39:161, 1992. 27. AI-Daccak A, Wang FQ, Theophille D, Lethielleux P, Colombani J, Loiseau P: Gene polymorphism of HLADPB1 and DPA1 loci in Caucasoid population: frequencies and DPB1-DPA1 associations. Hum Immunol 31:277, 1991. 28. Essaket S, Fabron J, De Preval C, Thomsen M: Corecognition of HLA-A1 and HLA-DPw3 by the human CD4+ alloreactive T lymphocyte clone. J Exp Med 172:387, 1990. 29. De Koster HS, Anderson DC, Termijtelen A: T cell sensitized to synthetic HLA-DR3 peptide give evidence of continuous presentation of denatured HLA-DR3 molecules by HLA-DP. J Exp Med 169:1191, 1989. 30. De Koster HS, Kenter MJH, d'Amaro J, Luiten RM, Schroeisers WEM, Giphart MJ, Termijtelen A: Positive correlation between oligonucleotide typing and T cell recognition of HLA-DP molecules. Immunogenetics 34:12, 1991. 31. A1-Daccak R, Loiseau P, Miramont P, Rabian C, Raffoux C, Colombani J: Evaluation of HLA-class II identity between unrelated individuals by serological typing. DNARFLP method and mixed lymphocyte reaction. Hum Immunol 29:189, 1990. 32. Termijtelen A, Erlich HA, Braun LA, Verduyn W, Drabbels JJM, Schroeijers WEM, van Rood JJ, De Koster HS, Giphart MJ: Oligonucleotide typing is a perfect tool to identify antigens stimulatory in the mixed lymphocyte culture. Hum Immunol 31:241, 1991. 33. Salazar M, Yunis I, Alosco SM, Chopek M, Yunis EJ: HLA-DPB1 allele mismatches between unrelated HLAA, B, C, DR (generic) DQAl-identical unrelated individuals with unreactive MLC. Tissue Antigens 39:203, 1992. 34. Brown SH, Jardetsky T, Saper MA, Samraoui B, Bjorkman P, Wiley DC: A hypothetical model of the foreign antigen binding site of class II histocompatibility molecules. Nature 332:845, 1988.