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provided by Elsevier - Publisher Connector Immunity, Vol. 15, 363–374, September, 2001, Copyright 2001 by Cell Press Genetic and Functional Relationships Review between MHC and NK Receptor

John Trowsdale1 the , notably at the central class III re- kbp. It 12ف Immunology Division gion, where there is over one per Pathology Department encodes the most polymorphic human known University of Cambridge to date, the class I and class II molecules. Of the ex- Tennis Court Road pressed loci in the MHC, roughly 40% are associated Cambridge CB2 1QP with the . Some of the main immune United Kingdom system genes are shown in Figure 2. They include the classical class I, HLA-A, -B, and -C, nonclassical HLA-E, -F, and -G, as well as “postmodern” MICA and HLA class I and NK receptors are encoded within MICB genes (MHC class I chain-related genes). There dense clusters of immune loci. The MHC, at 6p21.3, are also nonexpressed pseudogenes representing all and the complex containing the KIR loci, at 19q13.4, three groups of sequence. The products of classical polymorphic class I genes, HLA-A, B, and C, interact both feature variation in the number of genes, as well with T cell receptor (TCR) molecules as well as with the as sequence polymorphism. In addition to T cell recep- products of the killer immunoglobulin-like receptor (KIR) tors, several variable class I-related molecules interact genes expressed on natural killer cells and some T cells. with polymorphic NK receptors. Some of the lectin- The KIR interaction sites on class I molecules corre- related NK receptor genes, at 12p13.1, also have li- spond significantly with known dimorphisms in HLA-B gands belonging to the extended class I family. The (Bw4 and Bw6) (HLA-B loci carry “public” epitopes expanding clusters of class I-related sequences and which are dimorphisms of amino acids 77–83, dividing their receptors, some of which evolved recently, reveal HLA-B sequences into Bw4 and Bw6) and HLA-C (group further complexity in immune recognition of disease. 1 and group 2 [HLA-C loci are dimorphic for residues 77–80. In general, those with residues N77 and K80(Cw02, 04, 05, 06...)interact with KIR2DL1, while The MHC (major histocompatibility complex) and the those with residues S77 and N80(Cw01, 03, 07 . . .) two sets of genes for NK (natural killer) receptors, the interact with KIR2DL2/3]). HLA-E, on the other hand, NKC (NK complex of lectin-related genes on chromo- interacts with products belonging to the lectin-like set some 12p13.1) and the LRC (leukocyte receptor com- of NK receptors in the natural killer complex (NKC). Ex- plex of immunoglobulin [Ig]-related genes on chromo- pression of the molecule depends on the presence of some 19q13.4), are large, dense clusters of immune loci. leader sequences from classical class I molecules, These complexes have some similar features, such as which are needed for HLA-E to reach the cell surface, the high levels of polymorphism, which may be main- where it then interacts with NKG2/CD94 ligand (Braud tained by resistance to infection. Diverse interactions et al., 1998). In mice, the Qa1 molecule performs a similar are emerging between the some of the main products function to HLA-E, interacting with the evolutionarily of MHC and NK clusters, class I molecules and NK re- conserved CD94/NKG2 heterodimer, although the two ceptors, respectively (Figure 1). These sets of polymor- class I genes may not be direct orthologs. The function phic cell surface molecules provide alternative ways of of HLA-F is not known, but it is capable of binding to recognizing disease and influencing immune responses. ILT2 and ILT4 receptors of the Ig superfamily, related The innate immune system, to which NK cells belong, to and mapping alongside the KIR genes (Lepin et al., has been overshadowed by adaptive immunity, with its 2000). HLA-G is restricted to the placenta, where it is rearranging receptors. The emerging genetic and func- thought to help protect the fetus from rejection by the tional relationships between the MHC and NK genes immune system of the mother (King et al., 2000). and their products help to provide a more balanced view The MIC genes are distant relatives of MHC class I of immune recognition. The growing numbers of NK- molecules, expressed on intestinal mucosa and on other related receptors and their class I-like partners reveal cells in response to stress (Bahram et al., 1994). These sophisticated recognition systems in which the adaptive genes contain heat shock elements in their promoters. immune system is modulated in novel ways. These inter- They do not appear to function in presentation of patho- ␣␤ actions have implications for understanding susceptibil- gen-derived peptides to T cells with TCRs but are ␥␦ ity to infection as well as autoimmunity and cancer. broadly recognized by intraepithelial T cells with TCRs. They can also act as costimulatory molecules for CD8␣␤ T cells after induction of expression by viruses MHC Genes such as CMV (Groh et al., 2001). MICA and MICB are both -Mbp of DNA on chromo 4ف The human MHC covers polymorphic, with an unusual distribution of a number of some 6p21.3 and contains over 220 identified loci (MHC variant amino acids in their extracellular ␣1, ␣2, and ␣3 Sequencing Consortium, 1999). It has been divided into domains. MIC molecules are neither associated with three regions: class II (centromeric), class III, and class ␤2 microglobulin nor bound peptide, and only a small I (telomeric) with extended class I and class II regions on remnant of a groove remains. Null alleles have been either side. This is one of the most gene-dense regions of recorded, including haplotypes with no functional MICA or MICB expression, and mice do not appear to have 1 Correspondence: [email protected] MIC genes. MIC proteins interact with a common recep- Immunity 364

Figure 1. Interactions of the Products of Some MHC Class I-Related Genes with Receptors Encoded in the NKC and the LRC Some of the main sets of genes in the NKC, MHC, and LRC complexes are shown at their approximate chromosomal locations. Known receptor:ligand interactions are depicted by dotted lines. tor, comprising NKG2D homodimers, associated with as BTN and BTL, related to the milk butyrophilin, the adaptor DAP10 (Wu et al., 1999). The NKG2D genes are located in the class II-III border as well as the ex- are located in the NKC on 12. A MICA tended class I region. The encoded proteins are distantly crystal structure has been obtained as well as a complex related to the expanding family of coreceptor molecules of MICA with NKG2D (Li et al., 2001). Recently, other exemplified by B7.1 and B7.2 that are revealing increas- sets of sequences weakly related to class I have been ing complexity in costimulatory patterns regulating T found to interact with NKG2D. The human ULBP gene cell functions (Coyle and Gutierrez-Ramos, 2001). products, encoded on chromosome 6q24 (Cosman et MHC Evolution Is Restless al., 2001), and mouse RAEs (mouse chromosome 10- A good idea is worth imitating. Dot-matrix analysis re- syntenic to human 6q) (Yokoyama, 2000), may also indi- veals that both class I and class II genes, while main- cate stress. These molecules resemble the membrane- taining the same basic structure, have undergone re- distal parts of HLA class I molecules, the ␣1 and ␣2 peated duplication and loss, maintaining a handful of domains, attached to the cell surface by GPI linkage favored genes in most species, by a continuous “birth- (Cosman et al., 2001). and-death” process (Shiina et al., 1999). As novel gene Like class I, class II genes can be divided into classical family members emerged, they have diverged, some (HLA-DP, -DQ, and -DR) and nonclassical (HLA-DM have survived, and others have been lost. Interspersed and -DO). All class II molecules are heterodimers, en- within these variable regions are several islands of coded by pairs of loci. In all cases except HLA-DO, the unique sequence (e.g., LMP/TAP and RING3) which did A and B chain genes are adjacent in the genome. HLA- not obviously originate by cis-duplication. The expan- DM and -DO do not function directly as presenting mole- sions and contractions of sequences have apparently cules, and they do not have peptide binding grooves. taken place against a background of framework loci At steady state, these molecules are expressed mainly that are invariant in number and conserved in different in MIIC vesicles, where they aid in peptide loading onto species (Amadou, 1999). The class I region has been classical class II. Another set of MHC-encoded genes subjected to the most interspecies duplication events, plays a major role in loading peptides onto class I, and haplotypes can differ markedly in the number of namely TAP1/TAP2, LMP2/LMP7, and TAPBP, encoding loci (Pichon et al., 1996). Certain species of mice are the TAPASIN molecule (Lehner and Trowsdale, 1998). estimated to contain hundreds of class I sequences, Other immune system loci in the complex include cell most of which have become pseudogenes (Delarbre et surface molecules, such as an activating receptor al., 1992). One mechanism proposed to explain the gen- NKp30(1C7) on NK cells, IgSF molecules such as MOG, eration of the reiterated duplications involves capture complement components C2, C4, and factor B, inflam- by retroelements such as retroviruses (Dawkins et al., matory mediators such as TNF, and three HSP70 loci. 1999), but whether the MHC contains special features The organization of these genes is similar in man and that enable frequent duplication is not known. mouse (Allcock et al., 2000; MHC Sequencing Consor- As well as being polygenic, class I and class II genes tium, 1999). Some families of Ig superfamily genes, such are polymorphic. Sequence variation is not homoge- Review 365

Figure 2. Some of the Main Immune Genes in the MHC Classical class II genes are purple and nonclassical are pink. Classical class I are dark blue, nonclassical are blue, and others are pale blue.

-Mbp MHC region. In the non- after separation from the jawless fish. The adaptive im 4ف neous throughout the coding sequences, variation increases dramatically by mune system probably came to prominence only in spe- 5- to 50-fold flanking the most polymorphic gene loci cies that arose subsequent to the development of jaws. (Horton et al., 1998), which may be explained by hitchhik- Since genome duplication is a feasible precursor to de- ing along with the expressed, polymorphic class I and velopment of a novel network of interacting molecules, class II sequences. The most variable loci are HLA-DPB, it has been speculated that tetraploidisation of the early DQ, and DRB in class II and HLA-B and C in class I. vertebrate genome might have been one of the factors Variation is mostly localized within exons 2 and 3 for enabling the emergence of an adaptive immune system. class I molecules and exon 2 for class II. There is a There is no direct evidence for this, but the recent dis- predominance of nonsynonymous (i.e., protein chang- covery of the first living tetraploid mammal, the red vis- ing) substitutions in regions influencing the peptide cacha rat (Gallardo et al., 1999), lends some credence binding site, consistent with selection. This pattern of to the idea, as does the evidence of multiple rounds of variation is different than that in most other protein- polyploidisation in Xenopus. Certain MHC genes (TUBB, coding genes, where allelic variation tends to occur TNXB, PBX2, NOTCH4, RXRB, and RPS18) are syntenic more in noncoding regions. Over their entire length, in invertebrate genomes, such as D. melanogaster and class I/II alleles differ at 2%–7% of their sites, an order C. elegans, so the origin of at least part of the MHC of magnitude greater than the average proportion of predates the emergence of the adaptive immune sys- differences at other human nuclear genes, which is gen- tem. Two bony fish have been studied in detail at the -The generation of the unique pattern of genomic level, namely zebrafish and pufferfish. In tele .%0.1ف erally polymorphism in MHC molecules is the result of point ost fish, class I and class II genes are not linked, but mutations as well as, perhaps more importantly, either classical class I loci are closely associated with the conventional or gene conversion-mediated recombina- immune proteasome components LMP2 and LMP7 as tion (Little and Parham, 1999). In addition to sequence well as TAP transporter loci. In some sharks, there is variation in the peptide binding domain, DRB and C4 strong evidence for linkage between class I and class 500Myr association was lostفgenes are variable in number and position in different II, suggesting that the Ͼ haplotypes (Figure 3). at a later stage in the teleosts. A survey of a variety of The discovery of regions paralogous to the MHC at more closely related species is consistent with periodic 6p21.3 on such as 1, 9, and 19 has been upheaval of the MHC (Trowsdale, 1995). In some cases, proposed to result from ancient chromosomal duplica- such as humans and mice, there is no obvious orthology tions (see Flajnik and Kasahara, 2001 [this issue of Im- between class I loci. Apparently, rederivation of the rep- munity]). This has been disputed based on phylogenetic ertoire of loci took place subsequent to separation be- analysis, but it is clear that some related genes, such tween the ancestors of the two species, some 80 million as the NOTCH and RING3 families, are found on all four years ago (Parham, 1994). chromosomes. The list of genes found in two, three, or The chicken MHC provides evidence of linkage of key four of the locations already exceeds thirty. The se- MHC genes, including class I and II, TAP transporters, quence comparisons are consistent with duplications and C4 genes, in a tight cluster described as a “minimal taking place in a common ancestor of jawed vertebrates, essential MHC” (Kaufman et al., 1999). In this species, Immunity 366

Figure 3. Variation for Presence/Absence of Genes in Both the MHC and KIR Clusters A number of different arrangements for HLA- DRB genes are shown in the top panel and for KIR loci underneath. The positions of some of the KIR loci remain to be firmly established, and the arrangements are hypothetical. Note how some framework or anchor loci (red) are found on all haplotypes and others are highly variable. For the KIRs, the common “A” hap- lotype is shown on the top line. A useful web- site for KIR is: http://www.ncbi.nlm.nih.gov/ PROW/guide/679664748_g.htm

classical, polymorphic class I and class II genes are nations have been offered for association of autoim- found alongside TAP and TAPBP as well as C4. Many mune conditions with antigen-presenting molecules, but of the other genes associated with mammalian MHCs the precise mechanism remains obscure. Since there are deleted or moved. Interestingly, LMP genes are ab- are so many common class I and class II alleles in the sent. This may relate to the specificity of the peptide human population, there is effectively no wild-type. “Dis- binding site in chicken class I molecules, which, unusu- ease” alleles are part of the normal, unaffected popula- ally, accept peptides with negatively charged COOH tion, consistent with the notion that autoimmune condi- termini. The DO gene pair is absent, but DM equivalents tions are influenced by widespread contributory factors, are found. Two C-type lectin genes were identified in including non-MHC genes and environmental effects. the chicken MHC which may be related to natural killer No single class I or class II allele alone is necessary or receptor loci. sufficient to cause disease. In one of the most dramatic The recent draft sequence of the human genome con- models, HLA-B27 and ankylosing spondylitis, 95% of firms the finding that the MHC is one of the most gene- patients express B27, but only 3% of Caucasians with dense regions. It also coincides with a zone of extremely the allele will develop the condition (Edwards et al., high expression, a so-called RIDGE (for region of in- 2000). The association between narcolepsy and HLA- creased gene expression) (Caron et al., 2001). It occu- DQB1*0602 (A systematic nomenclature has been de- pies a region on external chromatin loops in interphase vised to accommodate the growing number of new al- nuclei, which may be related to transcriptional activity leles, with the unique locus designation followed by an (Volpi et al., 2000). As it is so well documented, the MHC asterisk and a four-digit allele identifier. So for example, is a useful model for other genome studies. Recombina- HLA-DRB1*0201 refers to the DRB1 locus, allele 2. The tion hotspots have been identified in the MHC, for exam- 01 refers to a subdivision, a minor variation of the second ple, in the class II region between HLA-DNA and RING3, allele. A further optional number can be added for synon- DQB3-DQB1, and TAP1 and TAP2 (Carrington, 1999). ymous nucleotide changes and noncoding allelic varia- Crossovers have been located in these regions to within tion. This nomenclature system is in force for all MHC a few hundred bp, and comparison with equivalent hot class I and II loci as well as other MHC genes, such as spots in rodents has identified recombinogenic chi-like TAP and LMP [see http://www.anthonynolan.com/HIG/ sequences in some of these locations, the significance index.html]) is another example where the sequence is of which remains to be determined (Jeffreys et al., 2000). present in nearly all patients and is a useful diagnostic The MHC and Disease Monitoring criterion, but the frequency of the allele in the normal, The MHC is associated with more diseases than any unaffected population approaches 25%. other region of the human genome and is linked to most, Complications arise in analysis of the relationship be- if not all, autoimmune conditions (Parham, 1999). Other tween MHC markers and infectious diseases (Meyer and nonimmune disease phenotypes have also been linked Thomson, 2001). There is no simple cause and effect with the region, from common cancers to enigmatic relationship between disease and alleles of class I and disorders such as narcolepsy. The highly polymorphic class II. Although infection is touted to be responsible class I and class II loci are the major determinants of for maintaining the profound polymorphism, there are MHC-associated disease, but the strong linkage dis- few good examples of HLA alleles conferring protection equilibrium across the complex makes it difficult to rule from infection. HLA-B*5301 is associated with resis- out a contribution from other linked genes, such as those tance to malaria, but only in specific populations. There in the class III or extended class I regions. Various expla- is evidence that evolution of the widespread, genetically Review 367

Figure 4. Interactions between MHC Class I Molecules and Inhibitory Receptors Both 2- and 3-domain Ig superfamily and lec- tin receptors are shown along with chromo- somal assignments. ITIM motifs are repre- sented as boxes. The Ig domains in the KIR family bend back into an elbow shape (Fan et al., 2001). ILT2/4 have been shown to be capable of binding the ␣3 domain of various class I molecules, such as HLA-A, B, F, and G.

stable Epstein-Barr virus is constrained by HLA class I. If subunits were encoded on different chromosomes, An epitope of nuclear antigen 4 of the virus dominates pairing of the heterodimer could be compromised (Ger- the class I response and is presented by HLA-A11. The main et al., 1985). Another example of this is provided epitope is conserved in Caucasian and African popula- by rat TAP alleles, which can differ by up to 30 amino tions, where A11 is relatively infrequent. Strains of the acids. Different allelic products can result in transport of virus from New Guinea, where HLA-A11 is highly preva- peptides with either positive or neutral COOHϪ termini, lent, carry a mutation in the epitope that precludes its which are only suitable for particular alleles of class I. binding to the class I molecule. Maintenance of so many Class I and TAP alleles on single haplotypes are nicely MHC alleles may be through heterozygous advantage matched to each other, ensuring transport of peptide or frequency-dependent selection (Parham and Ohta, for binding to the appropriate groove. This notion of 1996). Either model is consistent with multiple extant haplotypes with sets of polymorphic loci in counterpoint alleles. Heterozygous advantage of HLA class I in rela- may apply to the string of other immune genes in the tion to length of time to AIDS after HIV seropositivity MHC. Immunomodulatory genes, such as TNF, linked has been documented (Carrington et al., 1999). With a to particular HLA alleles may ensure an optimum level chronic infection such as HIV, where the virus sequence of immune response even with vastly different class I is unstable, it is reasonable to propose that heterozy- and II molecules (Gruen and Weissman, 1997). In the gotes have an increased chance of possessing an HLA class II region, there is evidence of a recombination hot allotype that can present an appropriate pathogen- spot between the TAP loci, resulting in linkage equilib- derived peptide for T cell recognition. In another study, rium. Perversely, DP and DQ/DR genes, either side of a single allele, HLA-B*5701, was found to have a protec- the TAP loci, are in linkage disequilibrium. This suggests tive effect on virus replication (Migueles et al., 2000). that selection favoring association of the class II loci Decades of MHC/disease studies have uncovered many counters the breakup of associations caused by recom- weak but significant associations. Haemochromatosis bination in the intervening region (Meyer and Thomson, is one of the few major diseases strongly identified by 2001). MHC variation, but it mapped some distance away, at the HFE locus. Paradoxically, HFE turned out to be re- NK Receptor Genes lated to class I, but it does not present to an immune The known roles of natural killer (NK) cells include modu- receptor. The extensive MHC linkage disequilibrium, lation of the immune system by the elaboration of cyto- which confounded identification of HFE for many years, kines as well as killing of infected cells. Detection of is also thought to be indicative of selection. targets by NK cells is mediated by two major families MHC Teamwork of receptor molecules belonging to the immunoglobulin The clustering of immune genes in the MHC may be superfamily (IgSF) and to the C-type lectin superfamily, simply fortuitous, although there could be advantages respectively. The ligands for many, but not all, of these in coinheritance of multiple polymorphic loci. Linkage receptors are MHC class I molecules. As summarized could help to coordinate gene expression and exchange in Figures 4 and 5, the receptors are either activating of sequences by nonhomologous recombination mech- or inhibitory. They quite often occur as pairs, comprising anisms such as gene conversion. Another putative ad- one of each type, although they are not necessarily im- vantage to linkage of polymorphic genes is cis-inheri- mediately adjacent in the linkage group. Effector func- tance of functionally interacting components. DQA and tions are controlled by sequences in the transmembrane DQB subunits, for example, are both highly polymorphic. and cytoplasmic tails (Vely and Vivier, 1997). Inhibitory Immunity 368

variety of different tissues, particularly in the haemo- poietic lineages. The products of some of these genes, such as the Ig-like transcripts (ILTs or leukocyte Ig-like receptors, LIR or CD85; the official HUGO nomenclature is LILR, see http://www.gene.ucl.ac.uk/nomenclature), are expressed on an extended range of cell types (Borges et al., 1997; Colonna et al., 1999). The NKC on chromosome 12p13 encodes over 15 type II transmembrane C-type lectin-like proteins. As well as NK cells, some NKC genes are found on a wider range of haemopoietic cell types. Multiple duplications must have given rise to this extensive cluster of related loci, which extends over Ͼ1.5 Mbp. There is evidence for recent duplication, as some of the genes, such as the NKG2 loci, are over 90% identical. There are obvious NKC orthologs in different species, such as CD69 and CD94, as well as species-specific loci (Barten et al., 2001). For example, three NKRP1 genes have been iden- tified in mouse and two in rat, whereas humans probably have only one functional gene. Mice have the NKR-P1A ortholog as well as the related NKR-P1C gene in addition to an ITIM-containing inhibitory molecule, NKR-P1B. The Ly49 family is subject to marked interspecies differ- ences. The exact number of murine Ly49 genes is not known, but there could be in the range of 12–15 genes, whereas there is only one pseudogene in man, LY49L. There is little sequence available for the mouse NKC, but once this has been obtained it will be interesting to 2ف) account for the size difference of the human NKC MBp). This difference could 4ف) Figure 5. Interactions between MHC Class I-Related Molecules and MBp) and the mouse Activating Receptors be mainly due to expansion of the Ly49 gene family A range of activating receptors are shown along with their chromo- between the NK2GA and Ly49b loci (Barten et al., 2001). somal positions and class I ligands, where known. TM adapters are Southern blots showed that different inbred mouse shown next to each receptor molecule, with ITAM motifs repre- sented as discs. KIR2DL4 has been proposed to be an activating strains exhibit a high degree of variation as well as differ- receptor, but this has not yet been confirmed. ent numbers of Ly49 genes. These data, together with the limited sequence analysis, are consistent with haplo- typic variation in arrangement of Ly49 genes (Brown et molecules function via the well-documented immunore- al., 2000). ceptor tyrosine-based inhibitory motifs (ITIMs) (Long, Comparison of chimpanzee and human NKG2 genes 1999). Activating receptors have truncated cytoplasmic reveals recent evolutionary processes. The different domains lacking ITIMs. They associate with immunore- NKG2 loci, with the exception of NKG2D, are more simi- ceptor tyrosine-based activation motif (ITAM)-bearing lar within, than between, different species, such as hu- adaptor molecules (Isakov, 1998), such as the DAP12 mans and mice (Vance et al., 1999). This is consistent molecule. Association between the transmembrane re- with a rederivation of different loci in individual species gions of receptor and adaptor neutralizes oppositely by duplication of a single locus. Continuous incarnation charged residues, such as an aspartate in DAP12 and of genes is characteristic of other key immune loci (Nei a positively charged residue on the TM region of the et al., 1997), including MHC class I sequences, which receptor (Campbell and Colonna, 1999). are also nonorthologous between humans and rodents There Are Two Major Clusters of Genes for NK (Parham, 1994). Receptors, the NKC and the LRC A locus, Cmv-1, mediating resistance to mouse CMV Genes for inhibitory receptors are clustered on two chro- mapped to the Ly49 region. B6 mice but not BALB/c mosomes (Figure 6). The C-type lectin genes, such as are resistant to the virus. Resistance to ectromelia virus Mbp region of maps to a similar region which may not be identical 2ف CD94 and NKG2, are grouped in a human chromosome 12, the natural killer complex, or to Cmv-1 (Depatie et al., 1997). The two phenotypes, NKC (Brown et al., 2000). The Ig superfamily genes, on resistance to ectromelia virus-induced necrosis (Rmp1) the other hand, are clustered in the leukocyte receptor and acute splenic replication of mouse CMV (Cmv-1), complex (LRC) on human chromosome 19q13.4. Acti- are thought to be both mediated by NK cells. Recent vating versions of the receptors belonging to the same work points to the Cmv-1 locus as being allelic to Ly49h molecular superfamilies are interleaved with their inhibi- (Klra8) (Lee et al., 2001) and to the direct involvement tory counterparts (Moretta et al., 2000). Additional genes of an NK receptor in resistance to viral infection (Brown encoding activating or inhibitory receptors on NK cells et al., 2001). The proposed mechanism of action of the have been identified (Parham, 1997), and most of these encoded receptor is interesting. MCMV produces an also fit into the two extended superfamilies. Sequences MHC class I-like protein, gp144, that binds to inhibitory with varying degrees of relatedness are present on a receptors on NK cells, blocking activation, which would Review 369

Figure 6. Receptor Genes in the Human NKC and LRC Regions Only the lectin-related (NKC) and Ig superfamily (LRC) encoding molecules are shown. The maps are not to scale. be a consequence of downregulation of host class I by Other related molecules encoded centromeric of the the virus. It is suggested that mouse NK cells counter LRC include the sialic-acid binding Ig-like lectins (Sig- this strategy by producing a stimulatory receptor, en- lecs [Alternative names for Siglecs include sialoadhesin, coded by Klra8, which binds ligands specifically ex- CD169, CD22, CD33, MAG, CD170, and p75/AIRM-1 pressed by MCMV-infected cells. This example of a re- (Crocker and Varki, 2001)]), of which there are ten genes ceptor/ligand arms race may help to explain the so far (Crocker and Varki, 2001), and the CD66-related proliferation of other activating NK-related receptors, molecules. The Siglecs are expressed on a range of for which, in most cases, ligands remain to be found. haemopoietic cells other than CD4ϩ T cells. They most As expected from duplicated genes, the structures likely also form part of the innate immune system re- of the NKC-encoded C-type lectin-like loci are highly sponsible for sensing infection. The CD66 gene family related. Up to two exons encode the 3ЈUTR and cyto- includes the group of molecules commonly known as plasmic tail, and a single exon covers the transmem- CEA, for carcinoembryonic antigen. There are approxi- brane region. In each case, three separate exons encode mately 29 CD66 genes, 18 of which are expressed (Ham- the carbohydrate recognition domain (CRD). The only martsrom, 1999). Some 7 CEA genes encode mem- major variation is the separate exon for a stalk region brane-spanning cell surface molecules, with 1–4 Ig between the TM and the CRD, which several genes do domains. Another group of 11 genes encode secreted not have. Interestingly, the stalk is involved in the ligand molecules, belonging to the pregnancy-specific glyco- specificity of Ly49A and -C, as revealed by binding protein subgroup. Some CEA molecules possess homo- assays using chimeric molecules (Takei et al., 1997). So and heterotypic cell adhesion properties. Other Ig super- far, ligands have not been found for proteins without family genes map to the extended LRC, including the neck region. Some C-type lectins interact with car- FCGRT, encoding the class I-related neonatal FcRN, as bohydrate in a calcium-dependent manner, but carbo- well as CD79a and CD155. hydrate is not required for CD94/NKG2 interaction with Even further centromeric, at 19q13.1, are two of the HLA-E. genes for TM adaptor molecules. DAP12 complexes The LRC cluster of loci on chromosome 19q13.4 con- with activating receptors both within the LRC as well tains over 25 Ig superfamily genes, consistent with ex- as the NKC. DAP10/KAP10 associates with the NKC- tensive duplication (Figure 6) (Barten et al., 2001; Wilson encoded NKG2D molecule, the ligand for MICA/B, RAE, et al., 2000). Some loci in the LRC, such as NKp46, are H60, and ULBP (Cosman et al., 2001; Lanier et al., 1998). orthologous in humans and mice (Biassoni et al., 1999), The DAP12 and DAP10 genes are arranged tail to tail, bp. Unlike other adapters, DAP10 300ف so primordial genes must have been present before spe- separated by -million years contains a YINM motif in its cytoplasmic tail, the phos 80ف) ciation between man and rodents ago). Some of the other genes, such as the PIRs and phorylation of which can result in PI-3 kinase and Grb-2 the ILTs, are also likely to be orthologs (Kubagawa et association. al., 1999), but KIRs appear to be completely absent Another set of related genes, the SIRPS (signal regula- in rodents and may have only developed in primates tory protein), are encoded on chromosome 20. The CD47 (Khakoo et al., 2000). Chicken Ig receptors related to molecule (also IAP, for integrin-associated protein) acts PIRs and Fc receptors have been identified, consistent as a self-marker on red blood cells, which are cleared with a common origin (Dennis et al., 2000). The collagen from the bloodstream of mice if they are CD47-ve. CD47 receptor gene, platelet glycoprotein receptor VI (GPVI), on normal red blood cells prevented this elimination by maps to the telomeric end of the LRC. This gene is binding to the inhibitory receptor SIRP␣ (Oldenborg et related to the other loci in the LRC and, like ILT, the al., 2000). protein product associates with Fc common ␥ chain The arrangement of the signal sequence on two exons (FcR␥) for signal transduction. is characteristic of the KIRs as well as the related Fc Immunity 370

receptors. Exon-intron organization is generally con- between the Fc␣R and GPVI loci is closer to the genome served in the Ig receptor superfamily. KIR loci with only average of 3–5, consistent with the relative age of the loci two Ig domains seem to have been derived from genes compared to the KIR sequences. Minisatellite-related with three domains by exon skipping. In some cases, sequences are present in KIR loci. Each minisatellite the surplus exons have deleterious mutations. These contains multiple copies of imperfect repeats of a 19–20 mutations may help to facilitate elimination of the exon, bp sequence. Each KIR locus contains an invariant num- since nonsense or missense mutations have been dem- ber of repeat units, from 23 in the 2DL5 gene to 63 in onstrated to result in skipping of an exon in the BRCA1 2DS4. No allelic variation has been found in these re- locus (Liu et al., 2001). Inhibitory and activating versions peats so far. G ϩ C-rich minisatellites are associated of KIR loci are highly similar in sequence, and small with recombination and/or variation (Jeffreys et al., alterations in an inhibitory receptor are sufficient to cre- 2000), and the KIR minisatellites may possibly affect ate a charged residue upstream of a stop codon, making the stochastic regulation of expression in different NK an activating version capable of recruitment of DAP12 clones (Uhrberg et al., 1997). The KIR2DL4 gene is and loss of ITIM motifs. flanked by unique sequence, and, as opposed to the ف KIR genes are arranged head to tail 2 kbp apart in other KIR loci, the gene is expressed in most NK cells. a continuous reiterated sequence with just one unique Asimov devised three famous rules of robotics. Ac- section 14 kbp upstream of the KIR2DL4 locus (Wilson cordingly, in the “missing self hypothesis” (Karre, 1997), et al., 2000). In line with other data, this arrangement is a similar code of conduct could be devised for NK also consistent with recent duplication of KIR genes. clones: Like MHC class I and class II sequences, NK receptor 1. Express at least one inhibitory receptor for a self KIR loci are highly polymorphic (Uhrberg et al., 1997; class I molecule. Valiante et al., 1997). As shown in Figure 3, another 2. Do not change which receptors you express. feature of the MHC, particularly the class II genes, is variation for the presence or absence of loci, such as 3. Inhibition overrides activation. in the DRB and C4 clusters. And like these loci, KIR loci These rules do not preclude NK clones expressing are variable both in sequence as well as for presence/ receptors for ligands they do not have, which does arise. absence on different haplotypes (Figure 3). Some KIR It follows that KIR genes are each only expressed on a are present on all or most haplotypes. Between these certain fraction of the NK cell population, yet they share framework sequences the number of KIR genes varies. identity over their upstream promoter regions of over Gain/loss variation throughout evolution may take place 91%. The flanking, anchor loci KIR3DL3 and KIR2DL4 by nonreciprocal recombination. The reiteration of KIR have more divergent promoter sequences of 89% and loci in a head-to-tail arrangement may help to facilitate 69% identity, respectively. Few physiological transcripts this, resulting in multiple haplotypes with different gene of KIR3DL3 have been reported. KIR2DL4 is expressed arrangements. Most KIR loci also have multiple alleles. on 100% of NK cell clones. How is control over gene The inhibitory KIR3DS1 and activating KIR3DL1 se- expression exercised to satisfy rule 2? The high degree quences are apparently alleles at a single locus (Gardi- of suggests that KIR genes are ner et al., 2001; Uhrberg et al., 1997; Wilson et al., 2000). likely to be regulated by a stochastic mechanism, possi- There is no indication so far that regions flanking highly bly involving methylation. The high level of homology of polymorphic KIR loci also show polarized variation, as KIR promoters does not allow identification of transcrip- in MHC class I and class II genes (Beck and Trowsdale, tion factors specific for individual KIR expression. The 2000). Marked variation in KIR sequences is, as ex- finding of nonexpressed KIR2DL5 variants suggests that pected, found in those residues that impact class I struc- a mutation in a putative AML transcription factor site tures (Gardiner et al., 2001). Variation elsewhere in the may prevent these variants from being expressed genes may influence expression either through alter- (Vilches et al., 2000). The binding site for AML is con- ation in the transmembrane region or in the promoter served between all other KIR promoters including sequences. KIR2DL4. This family of transcription factors regulates The immunoglobulin-like transcript (ILT, LIR,orLILR) a variety of hematopoietic genes. They can act both as genes are in two clusters of 6–7 loci, each linked to a positive as well as negative regulators of transcription. single leukocyte-associated inhibitory receptor (LAIR) Divergent, Convergent, and Coevolution of NK locus, orientated in opposite directions. In contrast to Receptors and Ligands the KIRs, the ILT introns and intergenic regions are not Although some primate lineages may have functional highly homologous, and ILT loci are probably older than Ly49 genes, a single pseudogene is now left in man. the KIR genes. Comparison with murine PIRs suggests that these loci are orthologous in the two species (Blery Similarly, there is no identifiable trace of KIR genes in et al., 1998). Apart from the single exception of ILT6, mice. The logical conclusion, now generally accepted, ILT loci are present on all haplotypes. Some of the ILT is that mouse Ly49 and human KIR genes are functional sequences are highly variable, including ILT5 and ILT8 homologs, although they come from different structural (Colonna et al., 1997). families. The two sets of genes share some striking AluS sequences and no AluJs in each features. As well as recognizing MHC class I, they are 9ف There are KIR gene, so the AluS/J ratio is extremely high over the both expressed in a clonal fashion on different NK cells -and they are both polymorphic. They use similar mecha .(3ف KIR region (cf. the MHC above, where the ratio is This is also consistent with the recent derivation of KIR, nisms for signal transduction. The existence of genes restricted to primates (Rajalingam et al., 2001). The S/J from different superfamilies performing related func- ratio over ILT sequences as well as over the region tions in different species is an intriguing example of Review 371

inversions on could have relocated the CD155 and the class I-like FCGRT gene (encodes FcRN, which is responsible for transfer of IgG molecules across the placenta) to 19q13.2 (Du Pasquier, 2000). Inversions may account for scrambling MHC paralogous genes on chromosome 1 and for separation of the class I-related ULBP loci to 6q, near to T complex genes, which are MHC linked in mice. Another LRC gene, FcAR, is related to other C2-set Ig superfamily molecules (Dennis et al., 2000). All of the other Fc receptor loci are on human chromosome 1. One end of the NKC on chromosome 12 contains a small set of MHC-paralogous loci including a gene related to TAPBP (Du Pasquier, 2000). Much of the genomic clustering of NK-receptor- related genes such as the KIRs has resulted from re- peated cis-duplication. Duplication of IgSF genes has obviously been a major factor in shaping the immune system, colonizing multiple chromosomes. Homotypic or heterotypic associations of IgSF proteins are com- mon. Multimerization may have been a factor in the development of NK receptors, as it could lead to “zip- pering” of a mixture of inhibitory and activating recep- tors with MHC class I ligands when an NK cell interacts Figure 7. Divergent and Convergent Evolution of NK Receptors by with its target (Boyington et al., 2000). Cis-cis associa- Development of KIR Genes in Human and Ly49 in Mouse tion of inhibitory and activating receptors could be an The maps are not to scale. attractive evolutionary intermediate on the path to mod- ulating target recognition. The structures of both KIR convergent evolution (Figure 7) (Barten et al., 2001). The and Ly-49 reveal two different interfaces with class I molecules, resulting in cocrystals with extensive or- evidence is consistent with the gradual replacement of dered ligand-receptor oligomerization. If these struc- the functions of Ly49 and the loss of LY49 genes as tures are recapitulated at the immune synapse, they KIRs emerged, very recently, in some primates. On the could facilitate the formation of an extensive bridging other hand, KIR genes may have never developed in network. The cooperative effect of this arrangement, rodents, although the syntenic region can be identified. which has been proposed for coreceptors such as As shown in Figure 7, the two major groups of receptor CTLA4/CD28, would be to boost avidity. The precise genes, the LRC and NKC, are both identifiable as syn- way in which signals from both inhibitory and activating tenic clusters. The Fc␣R and gpVI loci effectively tag NK receptors are integrated is not fully understood, but one end of the LRC in man and mouse. Many of the ways could be imagined in which the network could be genes in both the LRC and NKC complexes have re- disturbed in this situation by mixtures of activating and mained in order in the diverse species even though the inhibitory receptors. The signaling components, which functional, polymorphic NK receptor subsets eventually are common to other systems, call for close integration polarized to different gene sets. of inhibitory and activating receptors. Why did KIR genes develop in only a few species, The range of expression patterns, ligands, and func- one of which considers itself the most advanced? The tions of Ig superfamily molecules along chromosome 19 simplest explanation is that the genes arose subsequent is extensive. So far, all the known ligands for KIRs are to, and to compensate for, deletion of Ly49. Another class I molecules. The variety of ligands for related mole- possibility is that KIRs perform novel functions, and their cules such as GPVI, CD66, and NKp46 indicate the ex- emergence initially provided a key selective advantage. treme versatility of this molecular family, which extends To explain the rapid evolution of KIR, it has been sug- to hematopoietic receptors such as human growth hor- gested that they interact with ligands different than the mone receptor, prolactin receptor, and erythropoietin known class I structures (Rajalingam et al., 2001). receptor (Fan et al., 1997). Cells can become activated Whether or not this intriguing idea turns out to true, NK when NKp46 binds via sialic acid to flu haemagglutinin, receptor evolution must be to some extent tied in with taking advantage of an essential structural component class I ligands, which, as pointed out above, are often of the virus (Mandelboim et al., 2001). The in vivo ligands unique to each species. for most of the ILTs remain to be found. Some viral It has been speculated that lectin genes, rearranging proteins, such as UL18 from CMV, bind ILTs, presumably IgSF genes, and their receptors all emerged from the disrupting host receptor/ligand interaction and poten- MHC. There are a few scraps of evidence of ancient tially modulating ILT functions in monocytes (Borges et genetic links, such as the putative NK receptor genes al., 1999). in the chicken MHC, which could be coevolving with The MHC and LRC both have highly conserved sub- linked class I alleles (Kaufman et al., 1999). The short sections that consist of multiply duplicated genes, such arm of human chromosome 19 at 19p13.1-13.3, where as the class I or class II genes in the MHC and the ILT two lectin-related DC-SIGN genes and the CD23 gene genes in the LRC. Both also feature a segment with are located, is paralogous with the MHC. Pericentric isotypic as well as allotypic diversity. In both complexes, Immunity 372

features of the DNA sequence are characteristic of terri- innate and adaptive immunity are truly integrated and tories with different gene content. In the MHC, GC rich- highly complex (Brown, 2001). They suggest novel initia- ness demarcates transition to different zones. In the tives for investigating immune regulation, tumor immu- LRC, the ILT and KIR regions may be distinguished by nosurveillance (Smyth et al., 2001), and autoimunity (Yen variation in Alu repeat content, in addition to intragenic et al., 2001) based on the existence of two interacting distance and the presence of KIR-specific minisatellites sets of highly polymorphic loci. (Wilson et al., 2000). The extreme variation in both re- gions is likely to be driven by resistance to infection, Acknowledgments consistent with extreme species differences in type and arrangement of MHC and LRC loci (Hill, 1999). The strik- Research in my laboratory is supported by the Wellcome Foundation ing feature of the variation in both regions is the mainte- and the Medical Research Council. I would like to thank all of my nance in the population of a large number of alleles, each laboratory colleagues who have contributed to the work summarized here. at a low frequency (Parham and Ohta, 1996). Variation in most other genes is usually manifested as a small num- References ber of alleles at high frequency along with a number of rare variants. The existence of two sets of polymorphic Allcock, R.J.N., Martin, A.M., and Price, P. (2000). The mouse as a surface molecules that interact with each other provides model for the effects of MHC genes on human disease. Immunol. interesting possibilities for cooperation. Since they are Today 21, 328–334. on separate chromosomes, some combinations of MHC Amadou, C. (1999). 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