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
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
