Different NK Cell Surface Phenotypes Defined by the DX9 Antibody Are Due to KIR3DL1 Polymorphism

This information is current as Clair M. Gardiner, Lisbeth A. Guethlein, Heather G. of September 24, 2021. Shilling, Marcelo Pando, William H. Carr, Raja Rajalingam, Carlos Vilches and Peter Parham J Immunol 2001; 166:2992-3001; ; doi: 10.4049/jimmunol.166.5.2992

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Different NK Cell Surface Phenotypes Defined by the DX9 Antibody Are Due to KIR3DL1 Gene Polymorphism1

Clair M. Gardiner, Lisbeth A. Guethlein, Heather G. Shilling, Marcelo Pando, William H. Carr, Raja Rajalingam, Carlos Vilches,2 and Peter Parham3

KIR3DL1 and KIR3DL2 are NK cell receptors for polymorphic HLA-B and -A determinants. The proportion of NK cells that bind anti-KIR3DL1-specific Ab DX9 and their level of binding vary between individuals. To determine whether these differences are due to KIR polymorphism, we assessed KIR3D gene diversity in unrelated individuals and families. Both KIR3DL1 and KIR3DL2 are highly polymorphic , with KIR3DS1 segregating like an allele of KIR3DL1.AKIR haplotype lacking KIR3DL1 and KIR3DS1 was defined. The two KIR3DL1 alleles of a heterozygous donor were expressed by different, but overlapping, subsets of NK cell clones. Sequence variation in KIR3DL1 and KIR3DL2 appear distinct; recombination is more evident in KIR3DL1, and point mutation is more evident in KIR3DL2. The KIR3DL1 genotype correlates well with levels of DX9 binding by NK cells, but Downloaded from not with the frequency of DX9-binding cells. Different KIR3DL1 alleles determine high, low, and no binding of DX9 Ab. Conse- quently, heterozygotes for high and low binding KIR3DL1 alleles have distinct subpopulations of NK cells that bind DX9 at high and low levels, giving characteristic bimodal distributions in flow cytometry. The Z27 Ab gave binding patterns similar to those of DX9. Four KIR3DL1 alleles producing high DX9 binding phenotypes were distinguished from four alleles producing low or no binding phenotypes by substitution at one or more of four positions in the encoded : 182 and 283 in the extracellular Ig-like domains, 320 in the transmembrane region, and 373 in the cytoplasmic tail. The Journal of Immunology, 2001, 166: 2992–3001. http://www.jimmunol.org/

atural killer cells are lymphocytes of innate immunity CD94:NKG2 and KIR families contain inhibitory and activating that secrete immunomodulatory cytokines and kill cells receptors that differ in the lengths of their cytoplasmic tails and in N compromised by viral infection or malignant transfor- the signaling motifs they contain (2). mation (reviewed in Ref. 1). These functions are controlled by NK Selective expression of receptors in the CD94:NKG2 and KIR cell surface receptors that recognize MHC class I on prospective families gives clonal diversity to a person’s NK cell population target cells (reviewed in Ref. 2). Two distinct types of molecule (13, 14). Variation in the number of KIR genes present in a hap- provide human NK cells with MHC class I function. The lotype produces further diversity at the level of human populations by guest on September 24, 2021 CD94:NKG2 receptors recognize complexes of HLA-E and pep- (15, 16). In comparison with the KIR gene family, a part of the tides derived from HLA-A, -B, -C, or -G leader sequences (3, 4); leukocyte receptor complex on 19 (17), the CD94 their extracellular domains resemble the carbohydrate recognition and NKG2 genes of the NK complex on chromosome 12 are rel- domains of C-type lectins (5). In contrast, the killer cell Ig-like atively conserved (18). 4 receptors (KIR) are members of the Ig superfamily (6), and some Of 13 expressed human KIR defined to date, three are of the of them directly recognize polymorphic HLA-A, -B, or -C deter- type with three extracellular domains: KIR3DL1, KIR3DL2, and minants (7). Certain KIR2D are specific for HLA-C epitopes de- KIR3DS1. Whereas KIR3DL1 has been associated with HLA-B fined by polymorphism at positions 77 and 80 of the HLA-C heavy specificity (9) and KIR3DL2 with HLA-A specificity (11, 12), chain (8), KIR3DL1 recognizes the Bw4 determinant defined by KIR3DS1 is of unknown specificity. The KIR3DL1 receptor for sequence motifs at positions 77–83 of the HLA-B heavy chain (9, HLA-Bw4 was first identified and characterized using the 10), and KIR3DL2 has affinity for HLA-A (11, 12). Both the KIR3DL1-specific mAb DX9 (19). The Ab was subsequently used in flow cytometry to compare cell surface expression of KIR3DL1 Departments of Structural Biology and Microbiology and Immunology, Stanford Uni- by NK cells and T cells in a sample population of some 200 in- versity School of Medicine, Stanford, CA 94305 dividuals (20). Within this population an extensive heterogeneity Received for publication September 27, 2000. Accepted for publication December was observed both in the frequency of the NK cells and T cells that 14, 2000. bound DX9 and in the level of their binding (20). Individual do- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance nors could be characterized by whether their cells bound a high, with 18 U.S.C. Section 1734 solely to indicate this fact. low, or no amount of DX9. Particularly intriguing was that some 1 This work was supported by National Institutes of Health Grants AI22039 and individuals had distinct subpopulations of cells, binding high and AI17892 (to P.P.). C.M.G. was the recipient of a Leukemia Research Foundation low amounts of DX9. For any individual, the pattern of DX9 bind- Fellowship. C.V. was supported by a fellowship from the Instituto de Salud Carlos III, ϩ Spain (FIS BAE 98/5105). ing was stable over time, and the levels of binding to DX9 NK ϩ 2 Current address: Servicio de Inmunologı´a, Hopital Universitario Clı´nica Puerta de cells and DX9 T cells were the same. Moreover, twin compari- Hierro, San Martı´n de Porres 4, 28035 Madrid, Spain. sons and family studies suggested that the differences in frequency 3 Address correspondence and reprint requests to Dr. Peter Parham, Departments of and levels of DX9 binding were genetically controlled, but not by Structural Biology and Microbiology and Immunology, Sherman Fairchild Building, the presence or the absence of genes encoding the receptor’s HLA- Stanford University School of Medicine, Stanford, CA 94305. E-mail address: [email protected] Bw4 ligands (20). Such differences in the pattern of DX9 binding 4 Abbreviations used in this paper: KIR, killer cell Ig-like receptors; MFI, mean could be due to variation in the structure of KIR3DL1 or in other fluorescent intensity. components that control the transcription of the gene or the cell

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 2993 surface expression of its protein product. To begin distinguishing KIR3DL1 and KIR3DL2 subtyping among these possibilities we have investigated the structure of Generic KIR3DL1, KIR3DS1, and KIR3DL2 typing of genomic DNA was KIR3DL1 in individuals whose NK cells exhibit different patterns performed as previously described, with primers based in conserved re- of DX9 binding. The approach we took also facilitated assessment gions specific to each KIR family (15). For KIR3DL1 and KIR3DL2 sub- and comparison of variation within the three human KIR3D typing, primers designed to discriminate allele-specific polymorphisms groups: KIR3DL1, KIR3DS1, and KIR3DL2. were paired with KIR3DL1 or KIR3DL2 locus-specific primers. All primers are listed in Table II. Fig. 1 shows the alleles amplified by each primer set and describes primer combinations and PCR conditions. Amplification of Materials and Methods genomic DNA was performed in 25 ␮l reactions using 100 ng of DNA, KIR nomenclature 0.625 U of AmpliTaq polymerase, 2.5 ␮lof10ϫ buffer, and 0.2 mM dNTPs. Primers were used at a concentration of 0.5 ␮M. In naming KIR3DL1 and KIR3DL2 alleles the recommended KIR nomen- clature (21) was extended using principles established in the naming of Flow cytometry HLA alleles (22) (Table I). Following the gene designation is an asterisk and then three digits used to distinguish alleles that differ by nonsynony- PBMC were stained with the following Abs: CD3 (Leu 4) PerCP, and mous substitutions. The aim is for the numbers to correspond to the order CD56 (Leu 19) FITC (Becton Dickinson, Mountain View, CA). The anti- in which alleles are reported. Thus, NKAT3 (GenBank accession no. KIR3DL1 Ab DX9 was PE-conjugated as previously described (19). PE- L41269) (23) is KIR3DL1*001, and NKB1 (accession no. U31416) (24) is conjugated Z27 (anti-KIRp70) was purchased from Beckman Coulter (Mi- KIR3DL1*002. Fourth and fifth digits are used to distinguish alleles that ami, FL). Analysis was performed on a FACScan flow cytometer (Becton differ only by synonymous substitution, and they need only be used in Dickinson) using the CellQuest software package. contexts where silent substitutions are of relevance. When the context is clear, the letters KIR can also be dropped from the allele designation; thus Results Downloaded from KIR3DL1*001 can be shortened to 3DL1*001. An allele and its protein KIR3DL1 and KIR3DL2 are highly polymorphic genes product (allotype) are given the same name, but are distinguished by the use of italics for the allele name. This method of nomenclature has appli- Peripheral blood NK cells from 19 donors were analyzed for bind- cability to all KIR genes. ing the anti-KIR3DL1 mAb, DX9, using flow cytometry. Within this donor panel were found the four reaction patterns previously Cloning and sequencing of KIR3D described (20): a unimodal subset of cells with high binding, a RNA was isolated from PBMC using RNAzol (Tel-Test, Friendswood, unimodal subset of cells with low binding, a combination of cells http://www.jimmunol.org/ TX) according to the manufacturer’s instructions. First-strand cDNA was with high and low binding (bimodal pattern), and no binding at all. synthesized using oligo(dT) (PE Applied Biosystems, Foster City, CA) and AMV reverse transcriptase (Promega, Madison, WI). KIR3D were ampli- These patterns are not unique to the DX9 Ab, as they were also fied using the 3IG5Ј and NKR primers as previously described (14). The obtained with the Z27 anti-KIR3DL1 Ab (Fig. 2). Cells with low 3DL1*004 leader fragment was amplified from cDNA of donor 2 using the DX9 binding had a mean fluorescent intensity (MFI) of 38–208, following primers: forward, 5Ј-CTGCACCGGCAGCACCATGT-3Ј; and whereas cells with the high DX9 binding phenotype had an MFI of reverse, 5ЈTGGGAATGTGGATTCC-3Ј. PCR products were gel-purified using the Qiaex II Gel Extraction Kit 421–739. Fourteen of the donors were selected to represent the (Qiagen, Chatsworth, CA) and were cloned into pCR2.1-TOPO using the four patterns of DX9 binding, and RNA was made from their TOPO-TA Cloning Kit (Invitrogen, Carlsbad, CA). Plasmids were isolated PBMC. cDNA clones encoding KIR3DL1 were obtained by RT- from individual bacterial clones using the Qiaprep Spin miniprep (Qia- PCR and sequenced. As well as KIR3DL1, this approach yielded by guest on September 24, 2021 gen) and were sequenced using dye terminators and automated sequencing clones encoding KIR3DS1 and KIR3DL2, thus permitting analysis on an ABI 373A instrument (PE Applied Biosystems). To obtain a con- sensus sequence for each allele, at least three clones were fully sequenced and comparison of polymorphism in all three groups of human on both strands. The GenBank accession numbers for the new sequences KIR3D (Fig. 3). are given in Table I. cDNA sequences corresponding to 10 different KIR3DL1 alleles Sequences were aligned, and pairwise comparisons were performed us- were defined; three of them were previously described, and seven ing the GCG Wisconsin Package (version 10.0, Genetics Computer Group, Madison, WI). Protein structure was predicted using the Swiss-Prot pack- were new (Table I). Seven KIR3DL2 alleles were defined, of which age (25) (http://www.expasy.ch/sprot/). four were novel. When combined with other previously described sequences, nine KIR3DL2 alleles were defined. Only one form of KIR3DS1 was found, corresponding to the previously described Table I. Alleles of KIR3DL1 and KIR3DL2 KIR3DS1v (26). The KIR3DL1 and KIR3DL2 alleles were num- bered using a system based upon that used for HLA alleles (22) Sequence GenBank Reference (Table I). Gene Allelea Name Accession No. No. From the cDNA sequences, methods were developed for sub- KIR3DL1 3DL1*00101 nkat3 L41269 23 typing genomic DNA for KIR3DL1 and KIR3DL2 alleles. These 3DL1*00102 nnkat-3 AF262968 were used first to confirm the assignments made from sequencing 3DL1*002 nkb1 U31416 24 and second to determine genotypes for the members of the donor 3DL1*003 3DL1v AF022049 27 3DL1*00401 w204 AF262969 panel for whom sequences had not been determined (Fig. 3). Ten 3DL1*00402 m322 AF262970 panel members were heterozygous for KIR3DL1 variants; 3DL1*005 3DL1v2 AF262971 nine panel members expressed just one KIR3DL1 variant. In the 3DL1*006 NJN55 AF262972 latter group were the four individuals who had KIR3DS1. From 3DL1*007 r3k10 AF262973 3DL1*008 r3k2 AF262974 these data KIR3DS1 appears to segregate as though it were an KIR3DL2 3DL2*001 nkat4 L41270 23 allele of the KIR3DL1 locus (16). Fourteen of the panel members 3DL2*002 AMC5 X94374 11 were heterozygous at the KIR3DL2 locus. Three panel members 3DL2*003 nkat4A L76665 12 (donors 8, 9, and 10) appeared homozygous at both KIR3DL1 and 3DL2*004 cl-17.1C X93595 11 KIR3DL2 loci. Assignments of homozygosity should not be con- 3DL2*005 nkat4B L76666 12 3DL2*006 w3DL2b AF262966 sidered definitive because of the possibility that certain alleles 3DL2*007 b3DL2a AF262965 failed to be detected by the methods we used. Apparent from the 3DL2*008 r3k17 AF262967 number of alleles and the extent of heterozygosity in the donor 3DL2*009 rrk100 AF263617 panel studied is that both KIR3DL1 and KIR3DL2 are highly poly- a New alleles isolated in this study are in bold type. morphic genes. 2994 HIGHLY POLYMORPHIC KIR3DL GENES

Table II. KIR genotyping primers

Name Sequence (5Ј-3Ј) Exon Priming Sitea

KIR3DL1 sequence-specific primers 3DL1-202A (forward) TAC AAA GAA GAC AGA ATC CAC A 3 202 3DL1-223T (forward) TCC CAT CTT CCA TGG CAG AT 3 223 3DL1-1026C (forward) CAG ACA CCT GCA TGT TCT C 7 1026 3DL1-607T (reverse) GAG CTG ACA ACT GAT AGG A 4 607

KIR3DL1 locus-specific primers 3DL1-607C (reverse) GGA GCT GAC AAC TGA TAG GG 4 607 3DL1-560T (reverse) TAG GTC CCT GCA AGG GCA A 4 560 3DL1-1265C (reverse) GTA CAA GAT GGT ATC TGT AG 9 1265 3DL1-155G (forward) TCT TCG GTG TCA CTA TCG 3 155

KIR3DL2 sequence-specific primers 3DL2-322G (forward) TCA CTG GGT GGT CGG 3 322 3DL2-322A (reverse) GGG GTT GCT GGG TGT 3 322 3DL2-337G (forward) CAC CCA GCA ACC CCG 3 337 3DL2-337C (forward) ACC CAG CAA CCC CC 3 337 3DL2-474G (forward) CTT CTT TCT GCA CAG AGA G 4 474 3DL2-474T (forward) CTT CTT TCT GCA CAG AGA T 4 474 Downloaded from 3DL2-497A (forward) TGA GGA CCC CTC ACA 4 497 3DL2-497G (forward) TGA GGA CCC CTC ACG 4 497 3DL2-755T (reverse) CCC TGG ACA GAT GGT AGA 5 755 3DL2-755C (reverse) CCT GGA CAG ATG GTA GG 5 755 3DL2-1190C (reverse) GAT CCA ACT GTG CGT ACG 9 1190 3DL2-1190T (reverse) GAT CCA ACT GTG CGT ACA 9 1190

3DL2-122A (forward) CAG CAC TGT GGT GCC TCA 3 122 http://www.jimmunol.org/ 3DL2-122G (forward) CAG CAC TGT GGT GCC TCG 3 122 3DL2-394A (reverse) TCC TGA TTT CAG CAG GGT 4 394 3DL2-394C (reverse) TCC TGA TTT CAG CAG GGG 4 394

KIR3DL2 locus-specific primers 3DL2-393G (reverse) TCC TGA TTT CAG CAG GGG C 3 393 3DL2-67A (forward) GGG CCT GGC CAC TCA 2 67 3DL2-756C (reverse) TTC CCT GGA CAG ATG GTA G 5 756 3DL2-563T (forward) CGG TCC CTT GAT GCC TGT 4 563 3DL2-1019A (forward) TAT CTG CAG ACA CCT GCA 7 1019 by guest on September 24, 2021 a Priming sites correspond to the 3Ј end of each primer; nucleotide position is relative to the start codon.

High, low, and no binding of DX9 to NK cells are properties of with this assignment was that donor 15, who also had NK cells different KIR3DL1/3DS1 alleles with a unimodal low binding phenotype, had the 3DL1*005 allele. Within the donor panel, levels of DX9 binding to NK cells were Donor 15 also had the 3DL1*007 allele, which could have con- correlated with KIR3DL1/3DS1 genotype (Fig. 3). NK cells from tributed either a low binding phenotype or, alternatively, a no bind- donor 1 did not bind DX9, showing that both the 3DL1*00401 and ing phenotype. 3DS1 genes of this donor gave a null phenotype. This assignment for 3DL1*00401 was confirmed by the data from donor 2, who Clonal expression of the two KIR3DL1 alleles in a KIR3DL1 only had 3DL1*00401 and whose NK cells did not bind DX9. heterozygote NK cells from donors 3, 4, and 5 bound DX9 at high levels with A panel of 44 NK cell clones was established from donor 7, who unimodal distribution and had 3DS1 in combination with either is heterozygous for alleles KIR3DL1*002 and KIR3DL1*004, the 3DL1*001 or 3DL1*003.As3DS1 gives a null phenotype, both first of which is associated with high DX9 binding, and the second the 3DL1*001 and 3DL1*003 genes must independently produce a with no DX9 binding. Each NK cell clone was assessed for its high binding, unimodal phenotype. Similarly, NK cells from do- expression of the two KIR3DL1 alleles by RT-PCR typing and for nors 6 and 7 had unimodal high binding phenotypes produced by binding the DX9 mAb (Table III). Seventeen clones bound the genotypes consisting of a 3DL1*004 allele in combination with DX9 Ab, and they were the same 17 clones that typed for the either a 3DL1*001 or a 3DL1*002 allele. As 3DL1*004 gives a KIR3DL1*002 allele. Seven of the 17 clones also typed for ex- null phenotype, 3DL1*001 was responsible for the unimodal high pression of KIR3DL1*004. Four additional clones typed for binding phenotype of donor 6, and likewise, 3DL1*002 was re- KIR3DL1*004 expression in the absence of KIR3DL1*002 expres- sponsible for the high binding phenotype of donor 7. Donors 8–13 sion, and these clones did not bind the DX9 Ab. The remaining 23 all had unimodal high binding phenotypes and were typed as hav- NK clones expressed neither KIR3DL1 allele. This result demon- ing either one or both of the high binding alleles, 3DL1*001 and strates that the two KIR3DL1 alleles are differentially expressed in 3DL1*002. the NK cells of donor 7 and are thus independently expressed in a Donor 14 had NK cells that bound DX9 at low levels with clonal fashion. Consequently, these data also provide a direct dem- unimodal distribution. As 3DL1*005 was the only gene of the onstration that the different cell surface phenotypes detected by the 3DL1/3DS1 group found in this donor, these data indicated that DX9 Ab are due to expression of different alleles. Although the this allele gives a unimodal low binding phenotype. Consistent two KIR3DL1 alleles can be expressed independently of each The Journal of Immunology 2995

FIGURE 1. Primer combinations, PCR conditions, and expected results for KIR3DL1 and KIR3DL2 genomic subtyping. The first two columns list the forward (F) and reverse (R) primers used in the subtyping of KIR3DL1 (A) and KIR3DL2 (B). Alleles that are am- plified by each primer set are indicated by black boxes. Approximate product sizes are shown to the right. The am- plification conditions are listed in the table as H, N, or L, reflecting the an- nealing temperature used. For reactions using condition N, initial denaturation was 95°C for 5 min; five cycles of 97°C for 20 s, 62°C for 45 s, and 72°C for 90 s; followed by 26–30 cycles of 95°C for 20 s, 60°C for 45 s, and 72°C for 90 s; and finally a 7-min extension at 72°C. For condition H, annealing Downloaded from temperatures were 68°C during the ini- tial five cycles and 64°C for the re- maining cycles. For condition L, the temperatures were 60 and 58°C. All other parameters of the amplification remained constant. http://www.jimmunol.org/

other, the observed frequency of NK cell clones expressing both had 3DL1*001, a common allele that produces a high DX9 binding KIR3DL1 alleles (16.0%) is greater than that expected from the phenotype, in combination with 3DL1*006, an allele found only in product of the individual frequencies of expression from the two this member of the panel. Thus, expression of 3DL1*006 is prob- alleles (9.6%). It is therefore possible that initiation of transcrip- ably responsible for the subset of donor 19’s NK cells that bind tion at one KIR3DL1 allele in a cell increases the probability of DX9 at a low level. transcription at the second allele. In the clones that expressed both This analysis shows that different KIR3DL1 alleles and their by guest on September 24, 2021 KIR3DL1 alleles, the mean level of DX9 binding was the same as combinations are responsible for the variety of cell surface phe- that for clones expressing only KIR3DL1*002. Thus, the level of notypes detected with the KIR3DL1-specific mAb DX9. We find expression of one allele was not affected by expression of the that alleles 3DL1*001, 002, 003, and 008 determine high binding, other. alleles 3DL1*005, 006, and 007; low binding and the 3DL1*004 alleles determine no binding. Bimodal patterns of DX9 binding to NK cells are the property of certain combinations of KIR3DL1 alleles NK cells from both donors 16 and 17 had bimodal patterns of DX9 Inheritance of patterns of DX9 binding in families binding and were heterozygotes with one KIR3DL1 allele that To test further the correlations between KIR3DL1 genotype and gives a high binding phenotype (3DL1*002) and a second allele cell surface phenotype made from analysis of the donor panel, we that gives a low binding phenotype (3DL1*005; Fig. 3). In these re-examined families in which the DX9 binding patterns had been two individuals, the bimodal pattern of DX9 binding is due to the previously described but could not be explained (20). When family clonal expression of these two alleles. Thus, we can infer that the members were typed for KIR3DL1 alleles, a precise correlation NK cells binding DX9 at low level were those expressing only between genotype and DX9 binding pattern was observed. Data 3DL1*005, whereas those binding DX9 at high level comprised cells expressing only 3DL1*002 plus the smaller subpopulation of from two representative families are shown in Fig. 4. In both fam- cells expressing both 3DL1*002 and 3DL1*005. ilies it can be seen that children whose NK cells gave a bimodal Two other members of the panel, donors 18 and 19, also had NK pattern of DX9 binding have inherited a high binding KIR3DL1 cells with a bimodal pattern of DX9 binding. These data also cor- allele from one parent and a low binding KIR3DL1 allele from the related with heterozygosity for KIR3DL1 alleles determining low other parent. Members of family A have either unimodal high or and high DX9 binding. Donor 18 has the 3DL1*007 allele, which bimodal DX9 binding patterns that arise from segregation of the from the analysis of donor 15 is known to confer either a low high binding KIR3DL1*001 and 3DL1*002 alleles and the low binding phenotype or a no binding phenotype (see above). Thus, in binding 3DL1*005 allele. In family F the segregation of KIR3DL1 combination, the data from donors 15 and 18 show that 3DL1*007 alleles that determine high (3DL1*002), low (3DL1*005), and no confers a low binding phenotype and is responsible for the NK (3DL1*004, 3DS1) binding of the DX9 Ab results in siblings with cells in donor 18 that bind DX9 at low levels. Consequently, ex- NK cells exhibiting three different phenotypic patterns of DX9 pression of 3DL1*008, donor 18’s other KIR3DL1 allele, was re- binding: no binding, unimodal low binding, and bimodal binding. sponsible for producing the subset of NK cells with the high DX9 These data clearly show how the pattern of DX9 binding by human binding phenotype. Among the panel members, donor 18 was the NK cells is determined by the combination of KIR3DL1/3DS1 al- only individual to have the 3DL1*008 allele. In contrast, donor 19 leles a person inherits. 2996 HIGHLY POLYMORPHIC KIR3DL GENES

that the B3 haplotype is also distinguished by a novel recombinant form of the KIR2DL2 gene (KIR2DL2v1 GenBank accession no. AF285433) (28).

Patterns of variation in KIR3DL1 and KIR3DL2 are distinct Comparable numbers of KIR3DL1 and KIR3DL2 alleles were de- fined in this study. However, by several criteria there are striking differences in the polymorphism of the two genes. In nucleotide sequence KIR3DL1 alleles are more divergent from each other than are KIR3DL2 alleles, as measured either by simple pairwise comparisons (Fig. 6, A and B) or by the numbers of polymorphic nucleotide positions: 36 for KIR3DL1 and 15 for KIR3DL2. Con- sequently, the average genetic distance between KIR3DL1 alleles (1.1% difference in nucleotide sequence) is Ͼ5-fold greater than for KIR3DL2 alleles (0.2% difference). These differences between the genes would have been even greater if KIR3DS1 had been included in the KIR3DL1 analysis. Although KIR3DL2 has less nucleotide diversity than KIR3DL1,

a comparable difference is not seen in pairwise comparisons of the Downloaded from amino acid sequences. Almost all nucleotide substitutions in KIR3DL2 alleles are nonsynonymous, whereas KIR3DL1 alleles differ by a mixture of synonymous and nonsynonymous substitu- tions (Fig. 6, C and D). A further difference is that the KIR3DL1 alleles have a greater proportion of shared nucleotide substitutions

(74%) than do KIR3DL2 alleles (23%). Thus, there is greater ev- http://www.jimmunol.org/ idence for recombination in the generation of KIR3DL1 diversity, a mechanism that is also likely to have produced KIR3DS1.In contrast, point mutation is more apparent in the pattern of KIR3DL2 polymorphism. More than 80% of the substitutions in the extracellular domains of KIR3DL1 are in loops of the Ig-like domains, whereas for KIR3DL2 Ͼ80% of the substitutions are in strands of the ␤-pleated sheets (Fig. 7). These striking differences FIGURE 2. The DX9 and Z27 Abs give four similar patterns of binding all point to the combined forces of mutation, selection, and drift to human peripheral blood NK cells. The panels on the left show binding having operated in very different ways upon the human KIR3DL1 by guest on September 24, 2021 of NK cells to PE-conjugated DX9 Ab, the panels on the right show bind- and KIR3DL2 genes. ing to PE-conjugated Z27 Ab. Each of the four pairs of panels shows analysis of cells from a different donor whose numbers correspond to those Discussion given in Fig. 3. Going from top to bottom, a unimodal high binding pattern, a bimodal binding pattern, a unimodal low binding pattern, and a no bind- KIR3D genes encode inhibitory receptors that engage HLA-A ing pattern are shown. Near each peak are values for the percentage of NK and -B ligands, products of the most polymorphic HLA class I cells in the peak and their MFI. genes. Our study demonstrates that the KIR3DL1 and 3DL2 genes are also highly polymorphic, and for KIR3DL1 the polymorphism has been directly correlated with differences in cell surface phe- notype as detected with the KIR3DL1-specific mAb, DX9 (19, 20). Definition of a KIR haplotype lacking KIR3DL1 and KIR3DS1 This analysis also shows for the first time that in heterozygotes the in a family two alleles of a KIR gene (KIR3DL1) are expressed differentially Analysis of the African-American family C was particularly in- in clonal fashion, giving rise to four subpopulations of NK cells: formative and revealed a KIR haplotype that has neither the positive for one allele, positive for the second allele, positive for KIR3DL1 nor the KIR3DS1 gene (Fig. 5). Two types of KIR hap- both alleles, and negative for both alleles. lotype have been distinguished from the correlation of Southern From sequence analysis of just 14 donors, eight different inhib- blotting patterns with KIR genotype (15): A haplotypes are char- itory KIR3DL1 allotypes were defined. In addition, the noninhibi- acterized by having KIR2DS4 as the only gene encoding a short- tory KIR3DS1 is particularly homologous to KIR3DL1 in the ex- tailed KIR with the D1 ϩ D2 configuration of Ig-like domains, and tracellular domains, and in this donor panel its gene distributed as B haplotypes are characterized by KIR2DL5 (27), additional short- though it were an allele of KIR3DL1. The eight KIR3DL1 allo- tailed KIR, and a large 24-kb HindIII fragment. Five KIR haplo- types subdivide into three groups according to how NK or T cells types were shown to segregate in family C, two of which are A expressing them bind the DX9 Ab: with high level binding, low haplotypes (A1 and A2) and three of which are B haplotypes (B1, level binding, or no binding. These different levels appear to de- B2, and B3). Each haplotype could be distinguished by the pend solely upon the KIR3DL1 allele expressed, not upon other KIR3DL1 or KIR3DS1 genes it does or does not carry. The A1, A2, genetic or environmental factors including HLA class I type (20). and B2 haplotypes carry the 3DL1*004, 3DL1*002, and In heterozygotes who have a high and a low binding KIR3DL1 3DL1*001 alleles, respectively. In contrast, haplotype B1 has allele, their independent and clonal expression produces a charac- KIR3DS1, and haplotype B3 has neither a KIR3DL1 gene nor teristic bimodal pattern of DX9 binding in flow cytometry. In these KIR3DS1. Further supporting this conclusion were sequence anal- patterns the small proportion of KIR3DL1-expressing cells that yses of KIR3DL cDNA from all available family members and of express both alleles probably group with the cells expressing just all KIR cDNA from family member C5. The latter study showed the high binding allele, a conclusion supported by the analysis of The Journal of Immunology 2997 Downloaded from FIGURE 3. DX9-binding NK cell phenotype and KIR3D genotype of 19 healthy human donors. The figure shows a compilation and summary of the results obtained. The KIR3DL1 and KIR3DL2 alleles assigned to each donor were based upon the combination of cDNA sequence analysis and typing of genomic DNA. Alleles sequenced are those shown in boxes shaded gray. The allele nomenclature and its rationale are explained in Materials and Methods; correspondence with previous designations is given in Table I. Alleles that differ only in the fifth digit (the pairs 3DL1*00101/3DL1*00102 and 3DL1*00401/3DL1*00402) have nucleotide sequences differing by single synonymous substitutions, and they thus encode identical . http://www.jimmunol.org/ NK cell clones. The strength of our conclusion is the ability to that the D1 and D2 domains were essential for expression of the predict the cell surface phenotype of family members based solely DX9 epitope. In further support of this model is the observation upon their KIR genotype. that DX9-binding chimpanzee KIR3DL has the same residues at Through high resolution analysis of an informative family we positions 182 and 283 as high binding KIR3DL1 allotypes (31). Ј have been able to demonstrate the presence and segregation of a Previously it was shown that Fab and F(ab )2 of the DX9 Ab KIR haplotype that lacks both KIR3DL1 and KIR3DS1. Thus, null dissociated with similar kinetics from high and low binding NK haplotypes also represent an element in the population diversity of cells from the same donor, and that similar titration curves were KIR3DL1. We estimate this type of haplotype may be present in given by the two types of Ab fragment in binding to both high and human populations at frequencies of about 7% (data not shown), low binding cells (20). These data favor a model in which the cell by guest on September 24, 2021 suggesting that individuals lacking KIR3DL1 and KIR3DS1 alto- surface KIR3DL1 molecules on the high and low binding NK cells gether could be present at frequencies of up to 0.5%. In this regard bind the Ab with similar affinity, but the number of epitopes ac- analysis of an Australian panel of 147 donors revealed three who cessible to Ab differ on the two types of cell. Knowing now that typed for neither KIR3DL1 nor KIR3DS1 (29). Such individuals the cells express different KIR3DL1 alleles suggests that the could lack the genes or, alternatively, have novel KIR3DL1 alleles 3DL1*005 allele, conferring a low binding phenotype, gives rise that did not anneal to the typing primers. either to fewer protein molecules on the cell surface than the high Comparison of KIR3DL1 allotypes identifies four positions binding 3DL1*002 allele or to molecules for which a substantial within the amino acid sequence (182, 283, 320, and 373) where proportion do not bind the DX9 Ab. That 3DL1*005 is only dis- polymorphic substitutions present in more than one allotype cor- tinguished from the high binding 3DL1*001 allele at positions 182 relate with DX9 binding phenotype. Thus, all four alleles and 283 in the extracellular domains, argues against these substi- (3DL1*001, 002, 003, and 008) associated with high levels of DX9 tutions acting to reduce the affinity of binding to DX9. binding encode proline 182, tryptophan 283, isoleucine 320, and The remaining two positions of substitution (320 and 373) that glutamate 373. By contrast, the four alleles (3DL1*004, 005, 006, distinguish low and no binding KIR3DL1 allotypes from high and 007) associated with the low or no binding DX9 phenotypes binding ones are found in the transmembrane region (position 320) have one or more of the following substitutions: serine 182, and the cytoplasmic domain (position 373). Their importance is leucine 283, valine 320, and glutamine 373 (Fig. 7A). The highlighted by the low binding allele 3DL1*007 for which these 3DL1*004 allele that gives no binding has all four substitutions, are the only substitutions that distinguish it from the group of high whereas the three alleles that give low binding have either one or two of them. In addition, substitutions that are unique to individual allotypes might also contribute to the DX9 binding phenotype. For Table III. Expression of KIR3DL1 alleles by NK clones from donor 7 example, the unique cysteine residue at position 277 of 3DL1*006 might form disulfide-bonded homo- or heterodimers in which the NK Cell Clonesa ϭ DX9 epitope becomes obscured. KIR (n 44) Two of the shared positions of substitution are in the extracel- ␤-actin 3DL1*002 3DL1*004 DX9 Binding No. Frequency (%) lular Ig-like domains and could directly affect the structure of the ϩϪϪϪ23 52.3 epitope recognized by DX9. These are residue 182 in the D1 do- ϩϩϪϩ10 22.7 main and residue 283 in the D2 domain. A model in which the D1 ϩϪϩϪ4 9.1 and D2 domains form the site of the DX9 epitope is consistent with ϩϩϩϩ7 15.9 the results reported by Rojo et al. (30), who from analysis of a Clones were obtained from donor 7 (see Fig. 3) who is heterozygous for KIR3DL1 mutants lacking individual Ig-like domains concluded KIR3DL1*002 and KIR3DL1*004. 2998 HIGHLY POLYMORPHIC KIR3DL GENES Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. DX9-binding cell surface phenotype correlates with KIR3DL1/3DS1 genotype in families. Pedigrees for two families, A and F, FIGURE 5. Segregation of a KIR haplotype lacking KIR3DL1 and 3DS1 are shown. Family members are designated A1–A5 and F1–F6. Within the in a family. Five members of family C (designated C1–C5) were typed for box or circle is given the KIR3DL1/3DS1 genotype of the family member all known KIR genes. KIR3DL1 alleles were assigned by allele-specific and a superscript, hi, lo, or no, indicating the DX9 binding phenotype typing and nucleotide sequencing. Haplotypes were assigned and are des- associated with each allele (high binding, low binding, or no binding, re- ignated B or A depending on the presence of KIR2DL5 and whether 2DS4 spectively). The observed phenotype of DX9 binding to NK cells (high, is the only gene encoding a short-tailed KIR. The pedigree is shown in the low, bimodal, or no binding) is given under each box or circle. Beneath this upper half of the figure, and the genes constituting each haplotype are descriptor are given the percentage of NK cells binding DX9 and the level shown in the lower half. Haplotype B3 in family members C3 and C5 is of binding as the MFI. For bimodal distributions these parameters are given that lacking KIR3DL1 and 3DS1. In all family members analyzed the NK separately for each component of the distribution. cells were either negative for DX9 binding (shaded black) or gave a uni- modal distribution of cells with high binding (shaded gray). The percentage of DX9-binding NK cells for each family member is shown. Family mem- binding alleles. These substitutions cannot directly affect the struc- ber C5 was donor 2 in the panel described in Fig. 3. In the haplotypes the ture of the extracellular epitope recognized by the DX9 Ab, so KIR3DL1*004 and KIR3DS1 alleles that have a DX9-negative phenotype their effects must be indirect in nature. It is possible that they are shaded black. Other KIR genes are shaded gray. The order of genes is influence some aspect of the folding of the protein, its translocation based upon Wilson et al. (16). Haplotype B3 also contains KIR2DS1 but is to the cell surface or interactions there with other molecules. Al- not shown due to error. ternatively, nucleotide polymorphisms in regulatory, noncoding regions that are in linkage disequilibrium with the polymorphisms in codons 320 and 373 could reduce or eliminate transcription or trends can be seen in our data, such as 3DL1*001 being generally translation of the alleles conferring low or no binding phenotypes. expressed by a higher proportion of NK cells than 3DL1*002. Although polymorphism in the KIR3DL1 gene is sufficient to Polymorphism in the KIR3DL1 promoter, which need not be in explain differences in the level with which the DX9 Ab binds to complete linkage disequilibrium with the structural polymorphisms NK cells and T cells, it alone cannot account for differences in the defined here, could contribute to differences in the frequency of frequency of DX9-binding NK cells that distinguish individuals KIR3DL1-expressing cells. In this regard, polymorphisms in the within the population. Thus, donors 16 and 17 have the identical promoter of the KIR2DL5 gene may set a precedent (32). Also of combination of 3DL1 alleles, but donor 16 has twice as many DX9 likely importance are the identities of the other KIR genes in an binding NK cells as donor 17 (Fig. 3). On the other hand, possible individual’s genotype and the strengths of their promoters. The Journal of Immunology 2999

FIGURE 6. Distinctive polymorphism of the KIR3DL1 and KIR3DL2 genes. Pair- wise comparisons were made for the nu- cleotide and encoded amino acid se- quences of the KIR3DL1 and KIR3DL2 alleles. Frequency histograms of the num- ber of differences were plotted. On the left are shown histograms for the nucleo- tide substitutions in KIR3DL1 (A) and KIR3DL2 (B). On the right are shown the histograms for the amino acid differences in KIR3DL1 (C) and KIR3DL2 (D) allo- types. KIR3DS1 was not included in the analysis. Downloaded from

The results we present here, the genomic analysis of Wilson et al. though it were an allele of the same locus as KIR3DL1. From se- (16), and other population studies of KIR genotype (29, 33) paint a quence analysis of one KIR haplotype having the 3DS1 gene and consistent picture in which KIR3DL2 appears to be an invariant com- another having the 3DL1 gene Wilson et al. reached the identical ponent of human KIR haplotypes, whereas KIR3DL1 and KIR3DS1 conclusion; they found KIR3DL1 and KIR3DS1 to be closely related http://www.jimmunol.org/ are only present on some haplotypes. In the donor panel we studied in sequence and to occupy orthologous positions on the 3Ј side of the KIR3DS1 was represented by a single sequence that was distributed as invariant KIR2DL4 gene. Analysis of partial sequences for the by guest on September 24, 2021

FIGURE 7. Distinctive patterns of amino acid substitution in KIR3DL1/3DS1 and KIR3DL2 proteins. In the alignments of 3DL1/3DS1 (A) and 3DL2 (B), only the positions of difference are shown. Each allotype is compared with the consensus sequence and identities are indicated with a dash. n.d. at a position indicates that the residue was not determined, because the corresponding codon was not included within the region PCR-amplified and sequenced. Names of the novel sequences defined here are in bold. Substitutions from the consensus that are shared by two or more allotypes are shaded with either stipple or black. The residues shaded black are implicated in modulating the DX9 binding phenotype, which is given in the column on the right. The transmembrane cytoplasmic domains of KIR3DS1 diverge greatly from the corresponding region of KIR3DL1 and were not included in the alignment. D0, D1, and D2 refer to membrane distal, middle, and membrane proximal Ig domains. S, stem; Tm, transmembrane domain; Cyt, cytoplasmic domain. The predicted loop and strand elements of secondary structure are indicated by lp and sd, respectively, under the alignment. 3000 HIGHLY POLYMORPHIC KIR3DL GENES

KIR3DL1 and KIR3DS1 genes in B cell lines derived from American Acknowledgments Indian populations is also consistent with them being alleles (C. We thank those who have donated blood to this project, and Drs. Stewart Vilches and M. Pando, unpublished observations), Cooper and Salim Khakoo for their help in obtaining blood samples. We Other results, however, indicate that the situation in some KIR thank Drs. Marco Colonna, Lewis Lanier, Eric Long, and Charles Lutz for haplotypes may be more complicated. In KIR genotyping an Irish their comments on the draft manuscript. panel, Crum et al. (33) identified one person, from 90 studied, who typed for two variants of KIR3DL1 as well as KIR3DS1. In ongo- References ing studies of patients undergoing bone marrow transplantation we 1. Biron, C. A., K. B. Nguyen, G. C. Pien, L. P. Cousens, and T. P. Salazar-Mather. 1999. Natural killer cells in antiviral defense: function and regulation by innate have also identified an individual with a similar genotype (H. Shil- cytokines. Annu. Rev. Immunol. 17:189. ling, unpublished observations). Thus, in these individuals one KIR 2. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359. haplotype must contain either KIR3DL1 and KIR3DS1 (the inter- 3. Braud, V. M., D. S. Allan, C. A. O’Callaghan, K. So¨derstro¨m, A. D’Andrea, G. S. Ogg, S. Lazetic, N. T. Young, J. I. Bell, J. H. Phillips, et al. 1998. HLA-E pretation proposed by Crum et al.) or, alternatively, two variants of binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391:795. KIR3DL1. Evidence for the latter type of haplotype is the report by 4. Brooks, A. G., F. Borrego, P. E. Posch, A. Patamawenu, C. J. Scorzelli, M. Ulbrecht, E. H. Weiss, and J. E. Coligan. 1999. Specific recognition of Vyas et al. (31), who found four different KIR3DL1 variants to be HLA-E, but not classical, HLA class I molecules by soluble CD94/NKG2A and expressed by one donor. The close juxtaposition of the KIR genes NK cells. J. Immunol. 162:305. and their separation by highly homologous segments are properties 5. Boyington, J. C., A. N. Riaz, A. Patamawenu, J. E. Coligan, A. G. Brooks, and P. D. Sun. 1999. Structure of CD94 reveals a novel C-type lectin fold: implica- that could favor unequal recombination and the production of hap- tions for the NK cell-associated CD94/NKG2 receptors. Immunity 10:75. lotypes with duplicated genes (16). In this regard a haplotype con- 6. Long, E. O., D. N. Burshtyn, W. P. Clark, M. Peruzzi, S. Rajagopalan, S. Rojo, N. Wagtmann, and C. C. Winter. 1997. Killer cell inhibitory receptors: diversity, Downloaded from taining two copies of KIR2DL5 has been recently described (32). specificity, and function. Immunol. Rev. 155:135. The low frequency of individuals with KIR3DS1 and two 7. Boyington, J. C., S. A. Motyka, P. Schuck, A. G. Brooks, and P. D. Sun. 2000. Crystal structure of an NK cell immunoglobulin-like receptor in complex with its KIR3DL1 variants suggests that haplotypes containing such dupli- class I MHC ligand. Nature 405:537. cations are relatively rare and probably represent recent derivatives 8. Colonna, M., G. Borsellino, M. Falco, G. B. Ferrara, and J. L. Strominger. 1993. of haplotypes carrying just one KIR3DL1/3DS1 gene. If that is the HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells. Proc. Natl. Acad. Sci. USA 90:12000. case the existence of haplotypes carrying two KIR3DL1/3DS1 9. Gumperz, J. E., V. Litwin, J. H. Phillips, L. L. Lanier, and P. Parham. 1995. The http://www.jimmunol.org/ should not be seen as a refutation of the model that KIR3DL1 and Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell KIR3DS1 have an allelic relationship, as has been proposed (33, clones that express NKB1, a putative HLA receptor. J. Exp. Med. 181:1133. 10. Cella, M., A. Longo, G. B. Ferrara, J. L. Strominger, and M. Colonna. 1994. 34), but as an exception to this general rule. NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA Deletion and duplication of the KIR3DL1 gene as well as for- alleles with isoleucine 80. J. Exp. Med. 180:1235. 11. Pende, D., R. Biassoni, C. Cantoni, S. Verdiani, M. Falco, C. di Donato, mation of the short-tailed form KIR3DS1 are all changes likely to L. Accame, C. Bottino, A. Moretta, and L. Moretta. 1996. The natural killer cell have resulted from unequal crossing over between KIR haplotypes. receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains Recombination between KIR3DL1 alleles is also implicated in the and is expressed as a 140-kD disulphide-linked dimer. J. Exp. Med. 184:505. generation of new KIR3DL1 alleles (26). In contrast to KIR3DL1, 12. Dohring, C., D. Scheidegger, J. Samaridis, M. Cella, and M. Colonna. 1996. A the KIR3DL2 gene appears relatively resistant to recombination. A human killer inhibitory receptor specific for HLA-A. J. Immunol. 156:3098. by guest on September 24, 2021 13. Moretta, A., C. Bottino, D. Pende, G. Tripodi, G. Tambussi, O. Viale, A. Orengo, single KIR3DL2 gene encoding a long-tailed KIR appears a stable M. Barbaresi, A. Merli, E. Ciccone, et al. 1990. Identification of four subsets of feature of human KIR haplotypes, and point substitutions are a human CD3ϪCD16ϩ natural killer (NK) cells by the expression of clonally dis- tributed functional surface molecules: correlation between subset assignment of more characteristic feature of KIR3DL2 polymorphism than is the NK clones and ability to mediate specific alloantigen recognition. J. Exp. Med. case for KIR3DL1. Indeed, the higher frequency of nonsynony- 172:1589. mous substitutions in KIR3DL2 could be due to diversifying se- 14. Valiante, N. M., M. Uhrberg, H. G. Shilling, K. Lienert-Weidenbach, K. L. Arnett, A. D’Andrea, J. H. Phillips, L. L. Lanier, and P. Parham. 1997. lection in a gene in which low recombination does not facilitate Functionally and structurally distinct NK cell receptor repertoires in the periph- hitchhiking of synonymous substitutions. Although both the eral blood of two human donors. Immunity 7:739. 15. Uhrberg, M., N. M. Valiante, B. P. Shum, H. G. 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Immunol. 28:3959. immune functions of these molecules. 18. Brown, M. G., A. A. Scalzo, K. Matsumoto, and W. M. Yokoyama. 1997. The Comparison of human and common chimpanzee KIR shows that natural killer gene complex: a genetic basis for understanding natural killer cell function and innate immunity. Immunol. Rev. 155:53. the lineage of KIR, which in humans comprises KIR3DL1 and 19. Litwin, V., J. Gumperz, P. Parham, J. H. Phillips, and L. L. Lanier. 1994. NKB1: KIR3DL2, is represented by a single gene (Pt-KIR3DL1/2) in com- a natural killer cell receptor involved in the recognition of polymorphic HLA-B molecules. J. Exp. Med. 180:537. mon chimpanzee (31). The structure of Pt-KIR3DL1/2 is a pas- 20. Gumperz, J. E., N. M. Valiante, P. Parham, L. L. Lanier, and D. Tyan. 1996. tiche of elements shared with either KIR3DL1 or KIR3DL2 in Heterogeneous phenotypes of expression of the NKB1 natural killer cell class I which the extracellular domains are more like KIR3DL1 and the receptor among individuals of different human histocompatibility leukocyte an- tigens types appear genetically regulated, but not linked to major histocompati- intracellular domains are more like KIR3DL2. Correlating with bility complex haplotype. J. Exp. Med. 183:1817. this structure, the Pt-KIR3DL1/2 receptor has an inhibitory spec- 21. Long, E. O., M. Colonna, and L. L. Lanier. 1996. Inhibitory MHC class I recep- tors on NK and T cells: a standard nomenclature. Immunol. Today 17:100. ificity that is different from those defined for the human KIR3DL 22. Bodmer, J. G., S. G. E. Marsh, E. D. Albert, W. F. Bodmer, R. E. Bontrop, but which includes both MHC-A and -B allotypes. This compar- D. Charron, B. Dupont, H. A. Erlich, R. Fauchet, B. Mach, et al. 1997. 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