the hla-a,b,c genotype of the class i negative cell line daudi reveals novel hla-a and -b alleles

Tissue Antigens 1995: 45: 177-187 Printed in Denmark . All rights reserved

Copyriglit 6 Mitnksgaard 1995

T I S S U E A N T I G E N S ISSN 0001-2815

The HLA-A,B,C genotype of the class I negative cell h e Daudi reveals novel HLA-A and -B alleles

M. J. Browning, J. A. Madrigal, I? Krausa, H. Kowalski, C. E. M. Allsopp, A-M. Little, S. Turner, E. J. Adams, K. L. Arnett, W. E Bodmer, J. G. Bodmer, l? Parham. The HLA-A,B,C genotype of the class I negative cell line Daudi reveals novel HLA-A and -B alleles. Tissue Antigens 1995: 45: 177-187. 0 Munksgaard, 1995

Abstract: Daudi, a lymphoblastoid B cell line derived from an African Burkitt lymphoma does not express HLA-A,B,C antigens at the cell surface. Although HLA-A,B,C heavy chains are made normally they do not assemble into functional molecules because P2-microglobulin is absent. Previous serological analysis of somatic cell hybrids indicated that the HLA haplotypes of Daudi encoded HLA-A1, AlO(A26), B17, and B16(38) antigens. Here we describe the application of molecular methods: ARMS- PCR, cDNA cloning and sequencing, immunoprecipitation and gel electrophoresis, to define the class I genotype of the Daudi cell line which is HLA-A*0102, A*6601, B*5801, B*5802, Cw*0302 and Cw*0602. With the exception of the B38 antigen, which is not a product of the alleles defined, the genotype is consistent with the Serological description. Two previously undiscovered alleles emerged from this analysis: A*0102 and B*5802. The A*0102 allele differs from A*0101 by 5 nucleotide substitutions within exon 2 where it has a motif shared with A*30 alleles; the B*5802 allele differs from B*5801 by 3 substitutions in exon 3 where it has a motif shared with B*14 alleles. Subtyping HLA-A1 alleles showed A*0102 was well represented amongst individuals typed serologically as A1 in an African population but was absent from Caucasoids. B*5802 has been found in a second individual. Thus the novel A and B alleles are not specific to the Daudi tumor. Overall, this analysis of a single East African cell illustrates the power of molecular methods to define new class I HLA alleles in non-Caucasoid populations.

Cytotoxic T cell (CTL) recognition of antigens pre- sented by class I HLA molecules can lead to elimination of virus infected or malignant cells (1). I n response to this defense mechanism viruses evolve ways to interfere with the function of class I H L A molecules, which often lead to reduced levels of class I expression at the cell surface (2). Anal- ogously, the absence of HLA-A or B molecules on certain tumors suggests that selection by the CTL response has favored the growth of such variant cells (3).

An extensively studied example of a class I negative variant is the Daudi cell line which is defective

M. J. Browning', J. A. Madrigal2', P. Krausa', H. Kowalski2, C. E. M. Allsopp3, A-M. Little2, S. Turner4, E. J. Adam2, K. 1. Arnett2, W. F. Bodmer', J. G. Bodmer' and P. Parham2 'Cancer Immunology Laboratory, ICRF, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom, *Departments of Structural Biology and Microbiology & Immunology, Stanford University, Stanford, California, USA, 3Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, 4Anthony Nolan Research Center, Royal Free Hospital, London, United Kingdom *Present address: Anthony Nolan Research Center, Royal Free Hospital, London, United Kingdom

Key words: class I - Daudi - ARMS-PCR - DNA sequencing

Received 4 November; accepted for publication 9 November 1994

in the synthesis of P2-microglobulin ( P2-m), the monomorphic light chain of class I molecules (43). Chromosome analysis of Daudi cells has shown that one allele of P2-m is absent due to a deletion in the long a r m region q14-q21 (ref. 6) of chromosome 15 where the Pz-m locus has been mapped (7-10). The second P2-m allele has a point mutation in the translation start codon (11) such that normal levels of m R N A are transcribed but not translated (12,13).

The Dnudi cell line was derived by culturing tumor tissue biopsied from a n East African patient suffering with Burkitt Lymphoma. It is not known

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if the primary tumor cells were class I negative or if the loss of expression was a consequence of in vitro culture. However, in either case it is possible that loss of class I expression was the result of selection by CTL.

In addition to the loss of expression of P2-m, in which it is unique among lymphoid cell lines (14,15), Daudi cells have other unusual properties making them a common target for investigation (16). They were initially found to accumulate IgM molecules at the cell surface without demonstrable expression in the cytoplasm ( 4 3 . In common with other Burkitt lymphoma cells Daudi has the 8:14 chromosomal translocation (17) which results in some immunoglobulin Vh genes becoming associated with chromosome 8 (ref. 18). Daudi cells are particularly sensitive to the growth inhibitory ef- fects of interferon (19,20). They contain many cop- ies of EBV DNA, mainly as episomal forms, that are weak producers of virus. These viral genomes have a large deletion compared to the wild type which may contribute to the unusual properties of Daudi cells (13).

There has been long-standing interest in the HLA-A,B,C alleles of Daudi cells. Serological analysis of somatic cell hybrids made between Daudi and P2-m expressing cell lines revealed expression at the cell surface of HLA-A,B antigens not found in the fusion partner and therefore of Daudi origin (21,22). Fellous et al. (21) and Arce- Gomez et al. (22) both identified the HLA-A10, B38 and B17 antigens; in addition Arce-Gomez et a1 found the A1 antigen and subtyped the A10 antigen as being A26. These studies suggested that Daudi synthesizes normal levels of HLA-A,B heavy chains, a result supported by the demonstration that transfection of Daudi cells with the mouse P2- m gene rescues expression of class I HLA heavy chains at the cell surface (23). Subsequently, Quillet et al. transfected the human P2-m gene into Daudi and serologically typed the HLA-A,B antigens of the transfectant as A10, A1 1 and B17 (ref. 24).

Serological identification of the class I HLA molecules of Daudi cell hybrids and transfectants is difficult, as evidenced by the differences in the results obtained by the three groups (21,22,24) and lack of assignments for HLA-C. A major factor contributing to the difficulty is that alloantisera used 'for HLA-A,B,C are defined on the basis of their cytotoxic reactions with peripheral blood lymphocytes, cells with different properties than Daudi cell derivatives. An additional complicating factor is that the HLA-A,B,C antigens of the East African population (Luo) from which Daudi cells were derived are poorly characterized (25). To re- solve the discrepancies in the serological analyses and define precisely the HLA-A.B,C alleles of

Daudi cells we have used a combination of molecular approaches.

Material and methods HLA-A,B,C typing

Serological HLA-A,B,C typing was performed on Ficoll-Hypaque separated PBL, fresh or cryopre- served, as described in either references 25, 26 or 27. DNA was extracted from either separated PBL or whole blood using standard methods (25,26). Samples from the Gambian population were the gift of Dr. AVS Hill, Oxford University.

PCR amplification primers

HLA class I sequence specific primers (SSP) were designed on the principle of the amplification refractory mutation system (ARMS) (28), such that the 3' residue of the primer coincided with a polymorphic residue in the HLA gene sequence. SSP were designed to have melting temperatures in the range 56-60°C (ref. 29), to ensure that the reactions would amplify under a standard set of conditions. Primer length varied from 16-20 bases. With the exception of two of the HLA-C reactions, the coding strand primers were specific for sites in exon 2 of the HLA heavy chain gene, and the non- coding primers were specific sites in exon 3. The specificity of each reaction was validated by testing the primer combination in PCR against target DNA from cells of known HLA specificity. Inter- nal control primers, specific for the P2-m gene or the adenomatous polyposis coli (APC) gene, were included in each PCR (30,32). The sequences of the majority of the SSP used have been published (29-33). Previously unpublished primer sequences are give in Table I.

PCR typing of class I alleles

The method for HLA class I typing by ARMS PCR has been described in detail elsewhere (30- 34). For HLA-A and -C, PCR were carried out in 5 0 ~ 1 volumes in 17 mM ammonium sulfate in a Hybaid thermal cycler (31). For HLA-B, the PCR were carried out in 1 3 ~ 1 volumes containing 40 mM KCI, 1.6 mM MgCL, 8 mM Tris-HC1, pH 8.8, 0.008% Triton X-100, 200 pM each dNTP, 5% DMSO, 1 pM each specific primer and 0.2 pM each control primer. The reactions were heated to 96°C prior to the addition of 0.75 units of Taq polymerase (Promega) and were run on a Gene- Amp PCR system 9600 (Perkin-Elmer Corp.) for a total of 30 cycles comprising: denaturation at 96°C for 30 s; annealing at 64°C for 50 s ( 5 cycles), 62°C ( 5 cycles), 60°C ( 10 cycles) and 55°C (10 cycles);

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HLA-A,B,C alleles of Daudi

TABLE 1. Oligonucleotide Primer Sequences

Primer Sequence Coding

Specificity

AL#16 AL#21 AL#22

AL#32

AL#34 AL#37 AL#38

AL#42

Non-coding AL#P AL#S AL#X AL#Z AL#AB AL#AM AL#AT AL#AU AL#AW

G A C C A G G A G A C A C G G A A T A A C T A C A A C C A G A G C G A G G A C A C T C C A T G A G G T A T T T C T T

C G A G T G G A C C T G G G G A C

C T C A C A G A C J G A C C G A G C C C T C G T C C C C A G G C T C T A G C C C C G C T T C A T C GC C

G A C G C C G C G A G C C A G A A

G C C T C C C A C T T G C G C T G C G C C T C C C A C T T G C G C T T G C C C G T C C A C G C A C C G G G T A T C T G C G G A G C C C G C A C G T C G C A GC C A T A C A T T A C G T C G T A G G C G T C C T G C C C T G C T C C GC C G C A T G G A G T G C T T G G T G G T C T G A G C T T G G C C C C T G G T A C C C G T

A1 A36 A1 A36 A1 0 (not A’6602) A1 1 A80 [Cw6,7]

A1 A36 A2 A3 A10 (not A25) A l l A28 A19 (not A32) [some

A1 A36 A26 A43 A29 A80 A2

A1 A36 A’0201-4/7/9/11/12 A3 A32 A74 A80 [Cwl]

HLA-B; HLA-E,G]

A1 A36 A3 A9 A10 A l l A19(nOt A30) [Cw3, HLA-F,G]

A1 A36

A’2301 A19 (A10) [HLA-B,C,F] A1 A36 A3 A24 A l l A80 [HLA-GI A1 [A25 A26 A43 A‘6601 A1 11 A1 A23 A*2402 A80 A1 A36 A3 A’3402 A1 1 A30 A80 A1 A36 A3 A’3402 A l l A29 A’6801 A80 A1 A36 A1 A36 A2 A l l A28 HLA-A IOCUS

Internal Control Primers B2M#5 B2M#6 T A A C T A T C T T G G G C T G T G A p2-microglobulin non-coding

A G A T T C A G G T T T A C T C A C G pp-microglobulin coding

Specificities given in [brackets] represent potential cross reactivities

extension at 72°C for 50 s, with a final 5 min. extension at 72°C (ref. 32). The difference in these cycling conditions was due simply to different opti- mal conditions for ARMS PCR on the thermal cyclers used. Following PCR, lop1 of the reaction was run on a 2% agarose gel, and the result of each reaction was read by the presence or absence of an appropriate sized PCR product.

PCR gene mapping

By using a fixed allele or group specific ARMS- designed SSP in either exon 2 or exon 3 to set a context of defined HLA specificity, in combination with a panel of PCR primers specific for alterna- tive motifs at polymorphic sites in the other exon, we were able to construct PCR gene maps of the polymorphic sites of exons 2 and 3 of the HLA- A1 and A10 alleles of Daudi, and to extrapolate DNA sequence information from these maps. In- dividual PCR were carried out using primers as listed in Table 1, under the conditions described above for genotyping HLA class I by ARMS PCR. The presence or absence of an amplified PCR product for each primer combination was determined by agarose gel electrophoresis.

lsoelectric focusing of HLA-A,B,C heavy chain

HLA class I heavy chains were immunoprecip- itated from radiolabelled Daudi lysates as described (39, using the following antibodies: “ABR2”, a rabbit antiserum (5pl) which reacts with the cytoplasmic tail of HLA-B and some HLA-A molecules (36), the IgG2a monoclonal antibodies: LA45 (5pl of a 1/10 dilution of ascites), which reacts with free class I heavy chains pos- sessing arginine at residue 62 and asparagine at residue 63 in the class I heavy chain (37,38) and 41/28 (5pg) which reacts with both P2-m associated and free class I heavy chains (39), which pos- sess the residues proline 193, glycine 207 and glutamate 253 (40).

One dimensional isoelectric focusing (IEF) was performed as described (33). Two dimensional electrophoresis, with electrofocusing in tube gels for the first dimension and size separation in an SDS slab gel for the second dimension was performed by a modification of the method described by O’Farrell (41), using Hoefer Scientific 2D gel electrophoresis apparatus. 1.5-mm diameter tube gels were prepared using the same recipe as for 1D IEE After completion of electrofocusing (15 hours), the gels were removed from their tubes and

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HLA-A

A*01, A*10

HLA-B

B*17 (B*58)

HLA-C

Cw*03, Cw*OG

Figure 1. Low resolution ARMS PCR typings of Daudi HLA-A,B,C genes. PCR primer combinations and reaction specificities were as follows: HLA-A: size markers (lane 1); negative control (lane 2); AL#16/AL#AT, A1/36 (lane 3); #37/#AW, A2 (lane 4); #7/#D, A3 (lane 5 ) ; #8/#H, A9 (lane 6); #7/#C, A10 (lane 7); #6/#I, A1 1 (lane 8); #6/#H, A28 (lane 9); #7/#F, A19 (not A30) (lane 10); #12/#G, A30 (lane 11); blank (lane 12); #30/31/#H, A2,9,28 (lane 13); #30/3l/#L, A1,3,10,11,19 (lane 14); internal control primers P2-m#1/P2-m#2 (ref. 31, 33). HLA-B: size markers (lanes 1,2); B#Sa2/B#Fl, B27/73 (lane 3); #4a.2/#D3, B*40/47 (lane 4); #la/#Glb, B41 (lane 5); #la/#Glc, B*4001 (lane 6); #6a/#H, €313 (lane 7; #6a/#F3, B44 (lane 8); #Sb/#F3, B12 (lane 9); #la/#C3, B45/21 (lane 10); #4b/#C3, B22 (lane 11); #14/#E2, B16 (lane 12); #14/#1, B14 (lane 13); #Sc/#Glc, B7 (lane 14); #8/#GIb, B8 (lane 1.5); AS#l/#Dl, B18 (lane 16); #4c/AS#A, B37 (lane 17); #5g/#A3, B35/53 (lane 18); #4c/#C4, B5 (lane 19); #G183/#F2, B17 (lane 20); #5e.2/#K, B58 (*5801) (lane 21); #5b/#C2, B*1501-8 (not *1502), *1512, *1514, '4802, *4003 (lane 22); #4d/#F2, B*1501-7 (not *1503), *I51 1-17,46, 57 (lane 23); #S'Al/#Glb, B42 (lane 24); #Sb/#Glc, B'4001, *4801 (lane 25); #5a.l/#F2, B*1509,1510,1518 (lane 26); size markers (lanes 27,28); internal control primers PIC#l/PIC#A (ref. 32). HLA-C; size markers (lane I) ; negative control (lane 2); #136/#35, Cw*Ol (lane 3); #27/#145, Cw*O2/Cw41-42 (lane 4); #31/135, Cw*03, (lane 5); #27/#143, Cw*04 (lane 6); #27/#42, Cw*OS (lane 7); #27/#127, Cw*06/CLlOv (lane 81: #130/#41, Cw*07 (lane 9); #31/42, Cw*08 (lane 10); internal control primers Pr-m#S/P2-m#6 (ref. 34).

incubated in 0.0625M Tris HC1, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol for 30 minutes. The tube gels were then layered on top of a 10% SDS-polyacrylamide gel (0.75 mm thick), prepared with a 1-cm stacking gel, by placing the tube gel inside the cathode (top) buffer tank along the opening which exposes the slab gel to the cathode buffer tank. Pre-stained molecular weight size markers (Bio-Rad, low range) were mixed with 0.125M Tris HCl, pH 6.8, 1% agarose, 0.1% SDS and applied to the end of the tube gel. Electro- phoresis was performed at 110V until size markers were seen to enter the resolving gel, after which the voltage was increased to 170V Electrophoresis was terminated after complete separation of the molecular weight markers was seen. Gels were fixed in 15% methanol, 7% acetic acid for 20 minutes and then treated with Amplify (Amersham Interna- tional) for 20 minutes. Gels were dried under vac- uum and exposed to X-ray film.

Cloning and sequencing HLA-A,B,C cDNA

The methods used were those of Ennis et al. (42) as modified by Zemmour et al. (43) and Dornena et al. (44). An additional modification made in experiments to isolate B*5802 cDNA was the addition of 5% DMSO during PCR amplification. The new sequences defined here, A*0102 and B*5802, have been deposited in the Gene Bank under accession numbers U07161 and L33923 respectively.

Results and discussion

Independently, two approaches were taken to define the HLA-A.B and C alleles of the Daudi cell line. The first used pairs of oligonucleotide primers based upon polymorphic regions of known HLA- A,B and C sequences to generate specific amplification products which were analyzed by gel electrophoresis. This method is termed ARMS (Am- plification refractory mutation system) PCR (28). The second approach was to clone and sequence HLA-A,B and C cDNA from mRNA isolated from Daudi cells.

Low resolution ARMS-PCR typing of Daudi DNA indicated the presence of genes associated with the serological antigens HLA-A1 ,A10; B17(B58); Cw3, Cw6 (Figure 1). Subtyping of the A10 showed it was either an A*34 or A*66 allele, and subtyping of the Cw3 gene was consistent with Cw*0302. Further analysis showed that the allele encoding the A1 antigen was distinct from A*0101 and appeared to be a novel subtype of A*01 containing an element in exon 2 shared with A*30 alleles. Exons 2 and 3 of the HLA-A alleles were analyzed in greater detail by constructing PCR genes maps of each exon for the A*01 and A10 genes (Figure 2).

In the construction of these maps, PCR amplifications were performed using one primer to define either the A1 or A10 allele paired with a series of other primers each defining particular sequence motifs. Only when sequences corresponding to

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B*5801 that encodes the B58 antigen; Cw*0602 that encodes the Cw6 antigen, C*0302 that encodes the Cw3 antigen and the novel subtype of Al, termed A*0102. This last allele differs from A*0101 by 5 nucleotide substitutions in codons 9- 24 of exon 2 where A*0102 shares a motif with A*30 alleles (Figure 3). The sequence data and that of the PCR map are entirely consistent.

These five alleles can account for the A 1 ,A 1 O,A 1 1 and B 17 serological reactions observed for somatic cell hybrids and Pz-m transfectants of Daudi cells (21-24). The A1 and A1 1 antigens are cross-reactive serologically and it is reasonable that the novel A*0102 subtype could type as either A1 or A1 1 depending upon the alloantisera employed. Similarly, the A66 and A26 antigens are cross-reactive and there remain unre- solved ambiguities in distinguishing serologically the products of the A*2601 and A*6601 alleles. Neither ARMS PCR nor cloning and sequencing, however, identified a gene consistent with the serological definition of B38. To exclude the presence of a B*38 variant differing from B*3801 at one of the PCR primer polymorphic sites, several additional primer combinations were tested which should have identified the presence of a B*38 variant. gene in Daudi. No PCR product consistent with the presence of any B*38 gene was obtained (data not shown).

From the initial analysis it appeared that Daudi was homozygous for the B*5801 allele. To test this hypothesis we performed isoelectric focusing analysis of class I HLA heavy chains immuno- precipitated from Daudi cells with antibodies specific for epitopes of the free heavy chains. A band in addition to those corresponding to the A*6601, A*0102, B*5801, Cw*0302 and C*0602 heavy chains was observed, which had an isoelectric point between those of B*5801 and Cw*0602 (Fig- ure 4). Two dimensional gel electrophoresis using isoelectric focusing in the first dimension and SDS- PAGE in the second dimension showed this band to have a molecular weight corresponding to that of a complete class I heavy chain (Figure 5). It was therefore unlikely to be either an irrelevant con- taminant or a proteolytic degradation product of one of the class I heavy chains already defined. These analyses indicated that the additional band represented the product of a second HLA-B allele distinct from HLA-B*580 1.

Four antibodies, all having polymorphic specificity, were used to immunoprecipitate the heavy chains of Daudi cells and their patterns of precip- itation are shown in Table 11. Particularly informa- tive were the monoclonal antibodies HClO and LA45 which recognize epitopes centered on arginine at position 62 in the al helix. The only HLA-

181

HLA-A*Ol

Exon 2

Daudi

Exon 3

Daudi

Vavy Vavy

Figure 2. PCR gene maps of exons 2 and 3 of the HLA-A*O1 genes of Daudi and A*0101 cell line Vavy. Exon 2 gene maps were constructed using fixed primer AL#AT in exon 3 and, in order (with reaction specificity), exon 2 primers AL#22 (AU 36/*3001), #5 (A*3001/*3002), #I2 (A30), #38 (A1/36), #7

(A*3001), #34 (A1/36), #32 (not Bw4), #21 (AU36). Exon 3 gene maps were constructed using fixed primer AL#16 in exon 2, and, in order (with reaction specificity), AL#AB (A1/36/30), #O (A30), #AM (A1/36), #AU (A1/36), #S (A1/36), #P (A30), #I (A1/36), #K (A*3001), #T (A*3002/*3003), #A (A1/36), #Q (A30), #X (A1 not 36), #Z (A1 not 36). The A*01 gene of Daudi differed from the A*0101 gene of cell line Vavy in its reactivity with primers #22, #5, #I2 and #38 which define polymorphic sites in the 5’ region of exon 2.

(HLA-A), #42 (A1/36), #I0 (A*3001/*3002), #I6 (A1/36), #6

both primers are present in the same allele will amplification products of appropriate length be formed. Thus by scoring the amplifications a map of sequence elements present in a particular allele can be built up. In Figure 3 the results of this type of analysis for the A1 alleles of Daudi and the Vavy cell line are compared. The A1 allele of Vavy gives an amplification pattern consistent with the A*0101 sequence whereas that from Daudi was consistent with a novel A*01 subtype. Primers derived from the 5’ part of exon 2 gave distinct patterns of amplification for the A1 alleles of Daudi and Vavy whereas primers derived from the 3’ part of exon 2 and all of exon 3 gave identical patterns. The PCR gene map of the A10 gene was consistent with the expected pattern of reactivity of A’6601 (not shown).

Consistent with these results were those obtained from initial experiments to clone and sequence HLA-A,B and C cDNA from Daudi mRNA. Clones encoding five alleles were obtained: A*6601, a member of the AIO family that is cross-reactive serologically with A36 (ref. 45);

Browning et al.

A.0101 A'0102 A'3601 A.3001

.... A.0302 ..... A.1101 A.1102

601 700 GAGACGCTGC AGCGCACGGA CCCCCCCAAG ACACATATGA CCCACCACCC CATCTCTGAC CATGAGGCCA CCCTGAGGTG CEGC€CCTG GGCTICTACC A'0101

A.1102

A.0101 A.0102 A93601 A.3001 A'3002 A.3003 , A'0301 A.0302 A'1101 A'1102

800 CCrnCAGAA

895 TCACCcniAG ATGGG

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TABLE 2. Antibody

Allele LA45 HClO w 2 a ABR2 ~~~

- ++ + Cw’0302 - + +++ ++t ~40102 -

A’6601 +++ +++ + ++ 8‘5801 - - ~ * 5 a o 2 - Cw’0602 -

+++ +++ - +++ +++

- ++ ++

B heavy chains not to react with these antibodies are those of the B17 group which includes the products of the B*5701, B*5702 and B*5801 alleles (36). The “additional” band failed to react with HClO and LA45 though reacting strongly with the monoclonal antibody 41/28 and the rabbit antiserum ABR2 directed against the cytoplasmic tail. This resulted suggested that the second HLA-B allele of Daudi was, like B*5801, a member of the B17 family.

Further experiments to clone HLA-B from Dau- di cDNA were performed with the modification that 5% DMSO was added to the PCR amplifications. We reasoned that the second HLA-B allele may have failed to amplify due to the formation of secondary structures and that addition of DMSO would reduce this possibility. Two classes of HLA- B cDNA clones were then obtained: the first corre- sponded to B*5801 and the second to a closely related allele designated B*5802. The B*5802 allele differs from B*5801 by 3 nucleotide substitutions (Figure 6) which change the amino acids at posi- tions 94, 95 and 97 in the az domain (Table 111). The isoelectric point predicted from the amino acid sequence of B*5802 corresponds precisely to that of the “unknown” band in the isoelectric focusing gels.

Previously a single clone having the B*5802 sequence was isolated from cDNA derived from the Toto cell line (HLA-A28,30: B13,51: Cw6). This clone represented a third HLA-B allele to be obtained from that cell line and further analysis suggested it was the result of contamination with an unknown second cell line which may well have been Daudi. In a compilation of amino acid sequences the B”5802 sequence was previously listed as B? (46).

These results show that the HLA-A,B,C genotype of the Daudi cell line is HLA-A*0102,

F@rre 3. Comparison of the nucleotide sequence of A*0102. with those of other alleles in the AI/A3/A1 I ftimily. Dashes indicate identity with A*0107, dots indicate not determined.

Figure 4. One dimensional isoelectric focusing gel of class I HLA heavy chains irnmunoprecipitated from extracts of biosynthetically radiolabelled Daudi cells using different antibodies. Lane 1: ABR2. Lane 2: LA45 Lane 3: 41/28.

Figure 5. Two dimensional gel of HLA-A,B heavy chains precipitated from extracts of biosynthetically radiolabelled Daudi cells using a rabbit antiserum (ABR2) raised against a synthetic peptide corresponding to part of the cytoplasmic tail of HLA- B heavy chains. This antiserium precipitates the HLA-A and B heavy chains from Daudi but not the HLA-C heavy chains. “Spots” corresponding to the different HLA-A,B heavy chains are indicated by arrows.

A*6601; B*5801,B*5802,Cw*0302,Cw*0602. Two novel alleles, A*0102 and B*5802, were found in this analysis. One possibility was that these alleles are specific to the tumor cell line and associated with malignancy. To test this hypothesis individuals from a caucasoid population and a West African population typed serologically for A1 were PCR typed for the A*0101 and A”0102 subtypes and the results confirmed by limited PCR gene maps. None of 36 unrelated caucasoids typed for A*0103 whereas 9 of 22 unrelated Gambians were positive for this allele (Figure 7). Moreover, five of

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GGCCC'EACC B'5801 Be5802 B'5701 B.5702 B'1516 B'1517 B'3501 B+1401 B'1402

B'5801 B'5802 B'5701 B'5702 B'1516 B'1517 B'3501 B'1401 B'1402

B'5801 B'5802 B'5701 B'5702 B'1516 B'1517 B'3501 B'l.401 B'1402

B'5801 B'5802 B'5701 B'5702 B'1516 B'1517 8'3501 8'1401 B'1402

B'5801 B'5802 B'5701 8'5702 8'1516 B'1517 8'3501 8'1401 8'1402

101 CCGCCATGTC

-9-------- -57--------

TCGCGCTCCG CTACTACAAC CAGAGCGAGG CCGGGICTCA CATCATCCAG AGGATGTATG GCTGCGACCT

CCCGTGTGGC 8'5801 8'5802 8'5701 Bt5702 B'1516 B'1517 8'3501 8'1401 B'1402

601 700 GAGACGCTGC AGCGCGCGGA CCCCCCAAAG ACACACG'TGA CCCACCACCC CGTCTCI13AC CA'EAGGCCA CCC'EAGGTG CTGGGCCcrC: GGCTITTACC B'5801

B'5802 B'5701 8'5702 8'1516 8'1517 B'3501 B'1401 B'1402

8'5801 B'5802 B'5701 B'5702 B'1516 8'1517 B'3501 Bt1401 B'1402

TGAGGGGCTG B'5801 8'5802 8'5701 8'5702 8'1516 B'1517 B'3501 8'1401 B'1402


TABLE 3.

position position+ Gambian A*Ol's

Nucleotide 98 121 123 126 144 Aminoacid 9 17

A*0101 T C C G C F R A'0102 C A T A s s

Codon 9 17 17 18 24 1 3 5 7 9 Daudi

Nucleotide 353 355 361 Amino acid 94 95 97 2 4 6 8 1 0

B*5801 T A A 8'5802 C C T

Codon+ 94 95 97

I I R T L W Caucasian A*Ol's

+ numbering starts with the amino-terminus of mature heavy chains

the nine A*0102 positive individuals also expressed the HLA-B49 antigen, suggesting a haplotype association between A*0102 and B49. In contrast none of the samples typed for the A*0101 allele expressed the B49 antigen. No associations were found between A*0102 and HLA-C specificities as typed by serology. These results demonstrate that A*0102 is a normal allele of African populations and not the result of a tumor associated mutation.

From the nucleotide sequences, a PCR panel for distinguishing HLA-B 17 subtypes (including B*5801 and *5802) was designed (Figure 8). In screening a panel of DNA samples from cell lines previously defined as B17, one cell line typed as B*5802 (RCE 56, a cell line deposited in the BSHI rare cell exchange) in addition to Daudi. This cell line was derived from an Afro-Caribbean individual, and had been characterized previously as

(Cw*17). This finding shows that the B*5802 allele is a normal allele of human populations and was not formed as a consequence of tumorigenesis in Daudi. Preliminary evidence suggests that, in some instances, the B*5802 allele may have been incor- rectly assigned previously by serology as B57, based on its association with Cw6 (M Bunce, per- sonal communication).

The two novel alleles defined in Daudi, A*0102 and B*5802, both differ from familiar alleles (A*0101 and B*5801 respectively) by clusters of nucleotide substitutions found in other alleles (Table 111). Furthermore, the differences between the pairs of A*01 and B*58 subtypes involve residues of the peptide binding site (47). This is a

HLA-A33, A34; B42, B58 (Bw4/6); C W ~ , C W ~

Figure 6 . Comparison of the nucleotide sequence of B*jSOZ with B*5801, with other iilleles in the B17 family (B*5801 and B*5702), with alleles (B*1516 and B*1517) encoding the serologically cross-reactive B63 antigens, with B*350 I which is structurally related and with B* 14 alleles which are potentinl donors for the sequence differing between B*5802 and B*jSOI ..

1 3 5 7 9 11 13 Daudi 2 4 6 8 10 12 14

Figure 7. Identification of A*0102 in 10 Gambian and 14 Cau- casian individuals typed as HLA-A1 by serology. For each individual the PCR correspond to A*01/*36 (primers AL#16/#AT) and A*0102 (primers AL#I2/#AT). In each panel, Daudi is shown as a positive control for both reactions. A*0102 was identified in 5 of the Gambian individuals illustrated, but was not detected in any of the Caucasians.

characteristic pattern for class I HLA alleles and one which suggests that conversion or double recombination between alleles has been the mechanism responsible for the difference. A*0102 differs from A*OI01 by a motif shared with A*30 alleles and could therefore have been formed by conversion or double recombination between A*0101 and an A*30 allele (48). Similarly B*5802 differs from B*5801 by a motif shared with B14 alleles and might therefore have been formed by conversion or double recombination between B*5801 and a B*14 allele. That Daudi expresses neither an A30 or a B14 allele further argues against these alleles having been formed cle nova in the tumor cells from which the Daudi cell line was made.

Elimination of class I HLA molecules at the cell surface through loss of P2-m expression is not unique to the Daudi cell line. The human colon cancer cell line HCT has a deletion in one copy of the Pz-m gene and a point mutation in the other that together produces the same phenotype as seen in Daudi (49). Similarly, a deletion in the P2-m gene of the human melanoma cell line SK-MEL- 33 is correlated with absence of class I molecules at the cell surface (50). Loss of P2-m represents perhaps the most effective way for tumors to pre- vent antigen presentation by class I molecules and attack by CTL. With two mutational events a dip- loid cell completely suppresses class .I mediated presentation of tumor antigens whereas individual mutation in a heavy chain gene affects only the expression of one out of six HLA-A,B,C alleles. Thus mutation in P2-m may be a particularly effec-

185

Browning et al.

€3 *57 B*5801 B*5802 B*5801/*5802

DBB wT49 RCE56 Daudi

Figure 8. Subtyping HLA-B17 by ARMS PCR. Cell lines illustrated are DBB (B*57), WT49 (B*5801), RCE 56 (B*5802) and Daudi (B*5801/*5802). For each cell line the reaction specificities were B 17 (primers B#5e.2/#F2, reaction lane 1); B*57 (#5e.2/#K2, lane 2); B*58 (#5e.U#A3); B*5801 (#Se.U#K, lane 4); and B*5802 (#5e.2/#1, lane 5).

tive way for tumors to escape the host’s CTL response.

Acknowledgments

We thank Dr. AVS Hill, Oxford University, for DNA from Gambian individuals, and Dr. J. Mark- wich, Charing Cross Hospital, London, for allowing us to present data from the cell line (RCE 56). This research was supported by National In- stitutes of Health grants A122039 and A124258 to Peter Parham. JAM was supported by a postdoc- toral fellowship from the Leukemia Society of America. HW was supported by the FWF Erwin- Schroedinger-Fellowship, Vienna.

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1941 -1 948.

Address: P. Parham Departments of Structural Biology and

Stanford University Stanford, CA 94305 USA

Microbiology & Immunology

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the hla-a,b,c genotype of the class i negative cell line daudi reveals novel hla-a and -b alleles

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