- Email: [email protected]
Events in the adaptation of natural killer cell receptors to MHC class I polymorphisms
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Lopez-Botet, M. (1996), Functional analysis of otl[~l integrin in human natural killer cells. Eur. J. Immunol., 26, 2023-2029. Rabinowich, H., Lin, W., Manciulea, M., Herberman, R.B. & Whiteside, T. (1995), Induction of protein tyrosine phosphorylation in human natural killer cells by triggering via t~41~l or cz5[~l integrins. Blood, 85, 1858-1864. Rabinowich, H., Manciulea, M., Herberman, R.B. & Whiteside, T. (1996), 131 integrin-mediated activation of focal adhesion kinase and its association with fyn and zap-70 in human NK cells. J. Immunol., 157, 3860-3868. Raulet, D.H. (1996), Recognition events that inhibit and activate natural killer cells. Curt. Opin. Immunol., 8, 372-377. Ravetch, J.V. (1994), Fc receptors, rubor redux. Cell, 78, 553-560. Reth, M. (1989), Antigen receptor tail clue. Nature (Lond.), 338, 383-384. Ryan, J.C., Nienli, E.C., Nakamura, M.C. & Seaman, W.E. (1995), NKR-P1A is a target-specific receptor that activates natural killer cell cytotoxicity. J. Exp. Med., 181, 1911-1915.
Schwartz, M.A., Schaller, M.D. & Ginsberg, M.H. (1995), Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol., 11,549--599. Trinchieri, G. (1989), Biology of natural killer cells. Adv. lmmunol., 1989, 4, 187-376. Trinchieri, G. & Valiante, N. (1993), Receptors for the Fc fragment of IgG on natural killer cells. Nat. lmmun., 12, 218-234. Trotta, R., Kanakaraj, P. & Perussia, B. (1996), FcyRdependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-ct and early activation genes. J. Exp. Med., 184, 10271035. Yagita, H., Nakata, M., Kawasaki, A., Shinkai, Y. & Okumura, K. (1992), Role of pertorin in lymphocyte-mediated cytolysis. Adv. lmmunol., 51, 215242. Yokoyama, W.M. & Seaman, W.E. (1993), The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells : The NK gene complex. Annu. Rev. Immunol., 11,613-635. Yokoyama, W.M. (1995), Natural killer cell receptors, Curr. Opin. lmmunol., 7, 110-120.
Events in the adaptation of natural killer cell receptors to MHC class I polymorphisms E Parham
Departments o f Structural Biology and Microbiology and Immunology, Sherman Fairchild Building, Stanford University, Stanford, CA 94305-5400 (USA)
It was experiments in transplantation which led to discovery of the major histocompatibility complex (MHC) and its highly p o l y m o r p h i c class I genes. Tissue transplantation has since been perf o r m e d on a wide range o f multicellular organisms and the phenomena o f rejection observed in both invertebrate and vertebrate species (Hildemann, 1977). In pursuing these observations with m o l e c u l a r analysis, class I g e n e s were a l m o s t always found in the vertebrates but never in the invertebrates. This situation could be viewed in
Received April 21, 1997.
two rather different ways. First is that invertebrates do not possess MHC class I genes and that tissue rejections in vertebrates and invertebrates are separately e v o l v e d p h e n o m e n a w h i c h have c o n v e r g e d to appearing similar. S e c o n d is that class I genes exist in invertebrates, but the right e x p e r i m e n t s h a v e y e t to be d o n e . This o l d e r debate bears revisiting in the context of the explanations offered for the divergent class I receptors o f rodent and primate natural killer (NK) cells (Gumperz and Parham, 1995).
NK CELL FUNCTIONS
That molecules resembling either lectins or immunoglobulins are both class I receptors of mmnmalian NK cells is indisputably the result of convergent evolution. The alternative that these two classes of molecule share a common ancestor that was itself a class I receptor of N K cells seems improbable at best. Thus, Ly-49, CD94/NKG2 and other lectin-like receptors derive from an ancestral lectin that was not an MHC class I receptor; likewise, the killer cell inhibitory receptors (KIRs) derive from an immunoglobulin superfamily molecule that was not a class I receptor. Assuming convergent evolution, the question shifts to when and where these two molecular families were co-opted into the world of NK cells. In the remainder of this article, I will review chosen observations that bear on these questions.
Lectin r e c e p t o r s c a m e first It is now a matter of fact that humans and mice both have lectin-like molecules that serve as M H C class I receptors for N K cells. In mice they comprise the Ly-49 family of molecules ( Y o k o y a m a and Seaman, 1993), while in humans they are the C D 9 4 / N K G 2 molecules (Houchins e t al., 1991 ; L r p e z - B o t e t et al., 1997). In contrast, class I KIRs are m e m b e r s of the immunoglobulin superfamily that have only been found in the primates (Wagtmann et al., 1995). A parsimonious interpretation of these observations is that the application of lect i n - l i k e m o l e c u l e s to the j o b o f b i n d i n g M H C class I by N K cells predates that of the immunoglobulin-like m o l e c u l e s ( G u m p e r z and Parham, 1995). This evolutionary scheme is consistent with the H L A class I specificity of the CD94/NKG2 molecule. Although investigators disagree on the precise details as to which allotypes do and do not engage this receptor, what is clear is that it has a broad specificity for HLA-A, B and C allotypes (Lrpez-Botet et al., 1997). HLA-A, B and C are divergent loci which, unlike their m o u s e c o u n t e r p a r t s , rarely exchange information through the agency of gene conversion (Parham et al., 1995). With this history, the fact that CD94/NKG2 engages allotypes of all three loci argues for the C D 9 4 / N K G 2 interaction having predated the divergence of HLA-A, -B and -C from a common ancestral class I molecule. This divergence would have started on the lineage leading to m o d e m humans, apes and old world monkeys, after separation of the new world m o n k e y s . The MHC class I genes of modern species of new world monkey do not resemble HLA-A, -B, or -C genes and it therefore seems likely that this lineage of primates never had such genes (Watkins, 1995). The H L A class I specificities of the receptors of human NK cells have similarities in kind to the epi-
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tope specificities of antibodies specific for polymorphic class I determinants (Parham, 1985). There are essentially two categories of complementmTy determinant: one that is due to new substitutions and is defined positively by being on a subset of allotypes, and a second that is due to the ancestral structure, can cross between loci, and is present on many allotypes. The specificity of the KIRs fits into the first category, while that of C D 9 4 / N K G 2 fits into the second. C D 9 4 / N K G 2 appears to target a structure that might once have been a m o n o m o r p h i c feature o f ancestral primate class I. That CD94/NKG2 are lectin-like subunits suggests some sort of involvement with the single, invariant carbohydrate of H L A class I heavy chains which is attached at asparagine 86. Residues 84-89 are invariant in H L A - A , El and C molecules (Parham e t al., 1995) and the oligosaccharide structure itself has little structural heterogeneity or variation with protein allotype (Barber et al., 1996; Parham, 1996). This patch of conserved protein and carbohydrate, which encompa,;ses the carboxyl-terminus of the al helix and the following loop to which the carbohydrate is attached, could p r o v i d e the c o m m o n basis for the C D 9 4 / N K G 2 binding site. Allotypic specificity would then be the result o f effects due to protein p o l y m o r p h i s m in neighbouring regions of the antigen recognition site where variation is rife. I would argue that the: principal effects of these p o l y m o r p h i s m s is to reduce affinity of CD98/NKG2 for particular allotypes or subsets of allotypes. During the last 50 million years or so, the A, B and C loci as we know them have formed and their complicated polymorphisms elaborated. DmSing this time the CD94/NKG2 interaction, which hzLd been established much earlier, has not adapted to all the nuances. The long-term effect of this progression is that the proportion of class I allotypes that can regulate lectin receptors has decreased. In that situation, there could be benefit to be gained from the evolution of additional new receptors that interact with allotypes no longer working well with the lectins. The KIRs of the immunoglobulin superfami]y have properties that fit this bill.
I m m u n o g i o b u l i n - l i k e receptors are new, like HLA-C Three KIR specificities for H L A class I epitopes have been defined and they resemble the fir,;t category of antibody-defined epitope to which I alluded above. They are defined positively by small numbers of substitutions found on a subset of allotypes. More importantly each of the three specificities is confined to the allotypes of a single locus - - two to HLA-C and one to HLA-B - - suggesting that these
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receptor specificities have evolved subsequent to the divergence of the HLA-A, -B and -(3 loci. This in itself is evidence for the K I R type of receptor specificity being newer than that of the lectins. The H L A class I locus most committed to interaction with KIRs is HLA-C. All H L A - C allotypes interact with a K I R and these l i g a n d - r e c e p t o r s divide into two g r o u p s b a s e d upon a m i n o acid substitutions at positions 77 and 80 of the c t t h e l i x ( C o l o n n a et al., 1993). T h u s , f o r H L A - C , the interaction with K I R is fixed: every human being possesses at least one H L A - C ligand that can bind to a KIR. Only one third o f H L A - B alleles are k n o w n to i n t e r a c t w i t h K I R s w h i c h t a r g e t sequence motifs at residues 77-83 of the a t helix that determine the Bw4 serological epitope (Celia et al., 1994; Gumperz et al., 1995). Within populations, B w 4 epitopes are present at c o m p a r a b l e frequencies. Italians, for e x a m p l e , h a v e a B w 4 gene frequency of about 35 %, such that 42 % of Italians do not possess a Bw4 ligand for the H L A B-specific KIR (Imanishi et al., 1992). The conclusion to be drawn f r o m these o b s e r v a t i o n s is t h a t H L A - C i n t e r a c t i o n s w i t h K I R are m o r e i m p o r t a n t than H L A - B i n t e r a c t i o n s with K I R , and/or the H L A - B interactions with KIR are more recently evolved than those of H L A - C and have yet to b e c o m e fixed. For HLA-A, -B and -C, a hierarchy emerges with regard to their i n v o l v e m e n t with KIR. H L A - C is fully c o m m i t t e d , H L A - B is e x p e r i m e n t i n g and H L A - A appears diffident, if not totally detached. The functional segregation of the three H L A class I loci with regard to K I R interaction reflects their structural relationships: H L A - B and C are much more similar to each other than either is to HLA-A. Combining this observation with the close proximity of the HLA-B and HLA-C genes, and their distance from HLA-A (Trowsdale, 1995), makes a good case for H L A - B and H L A - C having e v o l v e d by gene duplication from a common ancestor, long after that ancestor had diverged from HLA-A. When did the gene duplication that produced HLA-B and C occur? Information addressing this question has emerged from the study of M H C class I genes in n o n - h u m a n primate species (Watkins, 1995). R e c o g n i z a b l e A, B and C genes are only found in humans, apes and old world monkeys. Thus, the features that distinguish these loci have evolved since divergence of old and new world monkeys. A recognizable C gene has only been found in humans and apes. That no species of monkey has been found to express HLA-C shows that evolution of the characteristics that distinguish m o d e r n - d a y B and C occurred subsequent to the divergence of old world monkeys and apes, some 30-40 million years ago. Recognizable B-like genes are, however, present in old world monkeys, indicating that HLA-B is more
like the common ancestor, while HLA-C has derived novel characteristics. It therefore seems most likely that the m o d e m B and C genes derive from an ancestral B-like gene w h i c h duplicated. This d u p l i c a t i o n w o u l d h a v e given rise to two identical B-like genes, and with time these two genes diverged. Selection on one of the genes (that we now call B) would have tended to preserve ancestral characteristics, while the second gene (that we now call C) acquired new features that placed it under a different type of' selection. The duplication may have occurred prior to the diverg e n c e o f a p e s and old w o r l d m o n k e y s , but the acquisition of C-like character in one of the daughter g e n e s p r o b a b l y o c c u r r e d s u b s e q u e n t to that divergence. Within this scheme for the evolution of the HLAB and -C loci, I hypothesize that the selection which drove the divergence of C from B was that coming from interactions with NK cells and competing with the pre-existing pressures from cytotoxic T cells. With gene duplication, one locus could still fulfill the r e q u i r e m e n t s o f C T L while the other c o u l d change to accommodate N K cells. At the heart of this accommodation was the development of a functional interaction with the ancestral KIR molecule.
A d a p t a t i o n of H L A class I to K I R Today, HLA-B is expressed at high levels on the cell surface, while HLA-C is expressed at low levels (McCutcheon et al., 1995). StructuraJ[ comparisons show that HLA-B retains ancestral qualities, whereas H L A - C has acquired new traits. If this is also true for the level of expression, then we can conclude that reduced expression is an adaptation to KIR interaction and the control of NK cells. This could stem from the relative independence of K1R interaction from the particular species o f peptide bound to a class I molecule. Whereas, for a T-cell receptor, the target is limited to the minute subset of class I molecules carrying a particular peptide, that is clearly not the case for KIR and thus functional r e c e p t o r - l i g a n d interaction can, in principle, be developed with lower levels of expression, and may indeed be desirable. The alternative KIR binding sites of' H L A - C allotypes are defined by having either serine 77 and asparagine 80 (group 1) or aspamgine 77 and lysine 80 (group 2). Experiments in mutation show that the group 1 specificity can be introduced into a group 2 molecule by replacement of asparagine for serine at residue 77 and that the group 2 specificity can be introduced into a group 1 molecule by substitution of lysine for asparagine at position 80 (Biassoni et al., 1995). Within the context of the H L A - C locus, the difference between the two specificities is trivial
NK CELL FUNCTIONS
in terms of evolutionary divergence. These do not s e e m to be alternative structures crafted by the steady accumulation of ever-refining change. One or two substitutions is all it took ! O f the four amino acids that define the group 1 and 2 H L A - C determinants, only lysine 70 of the group 2 motif is unique to the HLA-C locus, suggesting that group 2 specificity is the most recently derived. Consistent with this model is the fact that many HLA-B allotypes have the group 1 sequence motif, although, with one exception, they do not interact with KIR. The exceptional H L A - B allotype that interacts with KIR is HLA-B*4601. This allele is one of few that have been formed by a gene conversion between two HLA class I loci. The recipient gene in the conversion was HLA-B*1501, which has serine 77 and asparagine 80 but does not interact with group 1 KIR. The donor gene in the conversion was HLACw*0102, which has serine 77 and asparagine 80 and does interact with group 1 KIR. The effect of the gene conversion was to replace residues 66-76 in B * 1 5 0 1 w i t h the h o m o l o g o u s s e q u e n c e o f Cw*0102. This change involves a total of six amino acid substitutions, including the residues lysine 66, arginine 69 and valine 76 which are fixed in HLA-C and absent in HLA-B (Zemmour et al., 1992). The single gene conversion event that created B ' 4 6 0 1 f r o m B*1501 and C w * 0 1 0 2 appears, in terms of functional immunology, to have reversed the effect of millions of years of divergence of the HLA-B and C loci. Although still an allele of HLAB, the functional properties of B ' 4 6 0 1 are those of an HLA-C allele: it interacts with group-l-specific KIR, it stimulates cytotoxic T cells that cross-react with HLA-Cw*0102 not B*1501, and its pattern of glycosylation is characteristic of HLA-C (Barber et al., 1996).
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linked to C w l , 77.5% are linked to both Cw*0102 and A2, and 47.7% are linked to C w l , A2, DR9 and D Q 3 ( I m a n i s h i e t a l . , 1992). T h e s t r o n g a n d extended linkage disequilibrium, which is reiterated in Thais and other southeast Asian populations, suggests that recombination within this haplotype has been suppressed either by selection or by being physically frozen, perhaps as a consequence of the gene conversion.
Conclusion In this article, I have attempted to develop some order to the historical events that formed the set of H L A class I receptors of N K cells that now function in the h u m a n i m m u n e response. The lectins with their broad cross-locus specificity for H L A class I d e t e r m i n a n t s c a m e first, to be f o l l o w e d b y the i m m u n o g l o b u l i n - l i k e K I R m o l e c u l e s with their tighter intra-locus specificities. Both the HLA class I oligosaccharide, to which a lectin could bind, and the protein polymorphisms that determine KIR specificities, localize to the same area of the class I structure, providing further unity of target to these structurally different receptors. The development of KIR specificity and the gene duplication and divergence that produced distinctive H L A - B and C loci p r o b a b l y occurred c o n c o m i tantly, under selection imposed by NK cells. Gene duplication allowed the HLA-C locus to commit to N K cell regulation at the expense of its contribution to the T-cell response. This is manifest in hs lower cell surface expression which reflects the relative indifference of K I R receptors to specific peptides bound by class I.
These observations are particularly striking when one considers that the minimum difference between an H L A - B and an H L A - C allele is 55 nucleotide substitutions or 24 amino acid substitutions (Parham et al., 1995). The inference is that much of this considerable d i v e r g e n c e is of little or m i n o r consequence to the basic immunological functions.
As a consequence of the focus of HLA-C upon the simple ligands that bind to KIR, polymorphism at HLA-C is lower than at HLA-B. Interallelic conversion events have been less common in the evolution of HLA-C alleles and it is striking that there is only one example of a conversion targeted to residues 77-80 defining the KIR ligands. In contrast, the Bw4 motif recognized by HLA-B-specific KIR is the most common target for interallelic HLA-B conversion.
Although the product of a rare intergenic conversion event, B ' 4 6 0 1 itself is h a r d l y rare. H L A B ' 4 6 0 1 is predominantly localized to Asian populations, although the recent patterns of trade and human migration have carried it elsewhere. In Italians, of whom Marco Polo founded the China trade, HLA-B*4601 has a frequency of 0.1% compared to 0.0% in their rivals, the Spanish and Portuguese, and 15.1% in the Singapore Chinese. The linkage disequilibrium of HLA-B*4601 with alleles at other H L A loci is very striking. From the total number of B ' 4 6 0 1 alleles in the Singapore Chinese: 97..4% are
Both experiments of nature and mutagenesis in the laboratory show that the H L A class I epitopes recognized by K I R are rather simple entities that can be removed or replaced by single events that commonly contribute to H L A class I evolution. Similarly, it appears that the functional diversification of the HLA-B and C loci is simpler than structural differences would suggest. These properties are all consistent with the relative youth of the inte,ractions between H L A class I molecules and KIR, suggesting there may be considerable potential for their future improvement.
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References
Barber, L.D., Patel, T.P., Percival, L., Gumperz, J.E., Lanier, L.L., Phillips, J.H., Bigge, J.C., Wormald, M.R., Parakh, R.B. & Parham, P. (1996), Unusual uniformity of the N-linked oligosaccharides of HLAA, -B, and -C glycoproteins. J. Immunol., 156, 32753284. Biassoni, R., Falco, M., Cambiaggi, A., Costa, P., Verdiani, S., Pende, D., Conte, R., Di Donato, C., Parham, P. & Moretta, L. (1995), Amino acid substitutions can influence the natural killer (NK)-mediated recognition of HLA-C molecules. Role of serine-77 and lysine-80 in the target cell protection from lysis mediated by "Group 2" or "Group 1" NK clones. J. Exp. Med., 182, 605-609. Cella, M., Longo, A., Ferrara, G.B.,Strorninger, J.L. & Colonna, M. (1994), NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80. J. Exp. Med., 180(4), 1235-1242. Colonna, M., Borsellino, G., Falco, M., Ferrara, G.B. & Strominger, J.L. (1993), HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and N K 2 - s p e c i f i c natural killer cells. Proc. Natl. Acad. Sci. U S A , 90, 1200012004. Gumperz, J.E., Litwin, V., Phillips, J.H., l,anier, L.L. & Parham, P. (1995), The Bw4 public epitope of HLAB molecules confers reactivity with natural killer cell clones that express NKB 1, a putative HLA receptor. J. Exp. Med., 181, 1133-1144. Gumperz, J.E. & Parham, P. (1995), The enigma of the natural killer cell. Nature (Lond.), 378,, 245-248. Hildemann, W.H. (1977), Specific immunorecognition by histocompatibility markers: the original polymorphic system of immunoreactivity characteristic of all multicellular animals. Immunogenetics, 5, 193-202. Houchins, J.P., Yabe, T., McSherry, C. & Bach, F.H. (1991), DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells. J. Exp. Med., 173(4), 1017-1020. Imanishi, T., Akaza, T., Kimura, A., Tokunaga, K. & Gojobori, T. (1992), WI5.1: Allele and haplotype frequencies for HLA and complement loci in various
ethnic groups, in "HLA 1991, Proceedings of the Eleventh International Histocompatib:ility Workshop and Conference, Vol. 1" (K. Tsuji, M. Aizawa & T. Sasazuki) (pp. 1065-1220). Oxford University Press, Oxford. L 6 p e z - B o t e t , M., P 6 r e z - V i l l a r , J.J., C.arretero, M., Rodrfguez, A., Melero, I., Bell6n, T., Llano, M. & Navarro, F. (1997), Structure and function of the CD94 C-type lectin receptor complex involved in recognition of H L A class I molecules. Immunol. Rev., 155, 165-174. McCutcheon, J.A., Gumperz, J., Smith, K.D., Lutz, C.T. & Parham, P. (1995), Low HLA-C expression at cell surfaces correlates with increased turnover of heavy chain mRNA. J. Exp. Med., 181, 2085-2095. Parham, P. (1985), Immunochemical analysis of histocompatibility antigens : Analysis of one immunological supergene with the products of another, in "Hybridomas in biotechnology and medicine" (T.A. Springer) (pp. 137-161). Plenum Press, New York. Parham, P., Adams, E.J. & Arnett, K.L. (~[995), The origins of HLA-A,B,C polymorphism. /mmunol. Rev., 143, 141-180. Parham, P. (1996), Functions for MHC clLass I carbohydrates inside and outside the cell. TIBS, 21,427-433. Trowsdale, J. (1995), "Both man & bird & beast": comparative organization of MHC genes. Imrnunogenetics, 41, 1-17. Wagtmann, N., Biassoni, R., Cantoni, C., Verdiani, S., Malnati, M.S., Vitale, M., Bottino, C., Moretta, L., Moretta, A. & Long, E.O. (1995), Molecular clones of the p58 NK cell receptor reveal immunoglobulinrelated molecules with diversity in both the extraand intracellular domains. Immunity, 2, 439-449. Watkins, D.I. (1995), The evolution of major histocompatibility class I genes in primates. CRC Crit. Rev. Immunol., 15, 1-29. Yokoyama, W.M. & Seaman, W.E. (1993), The Ly-49 and NKR-pl gene families encoding lectin-like receptors on natural killer cells the NK gene complex. Ann. Rev. lmmunol., 11,613-635. Zemmour, J., Gumperz, J.E., Hildebrand, W.H., Ward, F.E., Marsh, S.G.E., Williams, R.C. & Parham, P. (1992), The molecular basis for reactivity of antiC w l and anti-Cw3 alloantisera with HLA-B46 haplotypes. Tissue Antigens, 39, 249-257.
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Lopez-Botet, M. (1996), Functional analysis of otl[~l integrin in human natural killer cells. Eur. J. Immunol., 26, 2023-2029. Rabinowich, H., Lin, W., Manciulea, M., Herberman, R.B. & Whiteside, T. (1995), Induction of protein tyrosine phosphorylation in human natural killer cells by triggering via t~41~l or cz5[~l integrins. Blood, 85, 1858-1864. Rabinowich, H., Manciulea, M., Herberman, R.B. & Whiteside, T. (1996), 131 integrin-mediated activation of focal adhesion kinase and its association with fyn and zap-70 in human NK cells. J. Immunol., 157, 3860-3868. Raulet, D.H. (1996), Recognition events that inhibit and activate natural killer cells. Curt. Opin. Immunol., 8, 372-377. Ravetch, J.V. (1994), Fc receptors, rubor redux. Cell, 78, 553-560. Reth, M. (1989), Antigen receptor tail clue. Nature (Lond.), 338, 383-384. Ryan, J.C., Nienli, E.C., Nakamura, M.C. & Seaman, W.E. (1995), NKR-P1A is a target-specific receptor that activates natural killer cell cytotoxicity. J. Exp. Med., 181, 1911-1915.
Schwartz, M.A., Schaller, M.D. & Ginsberg, M.H. (1995), Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol., 11,549--599. Trinchieri, G. (1989), Biology of natural killer cells. Adv. lmmunol., 1989, 4, 187-376. Trinchieri, G. & Valiante, N. (1993), Receptors for the Fc fragment of IgG on natural killer cells. Nat. lmmun., 12, 218-234. Trotta, R., Kanakaraj, P. & Perussia, B. (1996), FcyRdependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-ct and early activation genes. J. Exp. Med., 184, 10271035. Yagita, H., Nakata, M., Kawasaki, A., Shinkai, Y. & Okumura, K. (1992), Role of pertorin in lymphocyte-mediated cytolysis. Adv. lmmunol., 51, 215242. Yokoyama, W.M. & Seaman, W.E. (1993), The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells : The NK gene complex. Annu. Rev. Immunol., 11,613-635. Yokoyama, W.M. (1995), Natural killer cell receptors, Curr. Opin. lmmunol., 7, 110-120.
Events in the adaptation of natural killer cell receptors to MHC class I polymorphisms E Parham
Departments o f Structural Biology and Microbiology and Immunology, Sherman Fairchild Building, Stanford University, Stanford, CA 94305-5400 (USA)
It was experiments in transplantation which led to discovery of the major histocompatibility complex (MHC) and its highly p o l y m o r p h i c class I genes. Tissue transplantation has since been perf o r m e d on a wide range o f multicellular organisms and the phenomena o f rejection observed in both invertebrate and vertebrate species (Hildemann, 1977). In pursuing these observations with m o l e c u l a r analysis, class I g e n e s were a l m o s t always found in the vertebrates but never in the invertebrates. This situation could be viewed in
Received April 21, 1997.
two rather different ways. First is that invertebrates do not possess MHC class I genes and that tissue rejections in vertebrates and invertebrates are separately e v o l v e d p h e n o m e n a w h i c h have c o n v e r g e d to appearing similar. S e c o n d is that class I genes exist in invertebrates, but the right e x p e r i m e n t s h a v e y e t to be d o n e . This o l d e r debate bears revisiting in the context of the explanations offered for the divergent class I receptors o f rodent and primate natural killer (NK) cells (Gumperz and Parham, 1995).
NK CELL FUNCTIONS
That molecules resembling either lectins or immunoglobulins are both class I receptors of mmnmalian NK cells is indisputably the result of convergent evolution. The alternative that these two classes of molecule share a common ancestor that was itself a class I receptor of N K cells seems improbable at best. Thus, Ly-49, CD94/NKG2 and other lectin-like receptors derive from an ancestral lectin that was not an MHC class I receptor; likewise, the killer cell inhibitory receptors (KIRs) derive from an immunoglobulin superfamily molecule that was not a class I receptor. Assuming convergent evolution, the question shifts to when and where these two molecular families were co-opted into the world of NK cells. In the remainder of this article, I will review chosen observations that bear on these questions.
Lectin r e c e p t o r s c a m e first It is now a matter of fact that humans and mice both have lectin-like molecules that serve as M H C class I receptors for N K cells. In mice they comprise the Ly-49 family of molecules ( Y o k o y a m a and Seaman, 1993), while in humans they are the C D 9 4 / N K G 2 molecules (Houchins e t al., 1991 ; L r p e z - B o t e t et al., 1997). In contrast, class I KIRs are m e m b e r s of the immunoglobulin superfamily that have only been found in the primates (Wagtmann et al., 1995). A parsimonious interpretation of these observations is that the application of lect i n - l i k e m o l e c u l e s to the j o b o f b i n d i n g M H C class I by N K cells predates that of the immunoglobulin-like m o l e c u l e s ( G u m p e r z and Parham, 1995). This evolutionary scheme is consistent with the H L A class I specificity of the CD94/NKG2 molecule. Although investigators disagree on the precise details as to which allotypes do and do not engage this receptor, what is clear is that it has a broad specificity for HLA-A, B and C allotypes (Lrpez-Botet et al., 1997). HLA-A, B and C are divergent loci which, unlike their m o u s e c o u n t e r p a r t s , rarely exchange information through the agency of gene conversion (Parham et al., 1995). With this history, the fact that CD94/NKG2 engages allotypes of all three loci argues for the C D 9 4 / N K G 2 interaction having predated the divergence of HLA-A, -B and -C from a common ancestral class I molecule. This divergence would have started on the lineage leading to m o d e m humans, apes and old world monkeys, after separation of the new world m o n k e y s . The MHC class I genes of modern species of new world monkey do not resemble HLA-A, -B, or -C genes and it therefore seems likely that this lineage of primates never had such genes (Watkins, 1995). The H L A class I specificities of the receptors of human NK cells have similarities in kind to the epi-
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tope specificities of antibodies specific for polymorphic class I determinants (Parham, 1985). There are essentially two categories of complementmTy determinant: one that is due to new substitutions and is defined positively by being on a subset of allotypes, and a second that is due to the ancestral structure, can cross between loci, and is present on many allotypes. The specificity of the KIRs fits into the first category, while that of C D 9 4 / N K G 2 fits into the second. C D 9 4 / N K G 2 appears to target a structure that might once have been a m o n o m o r p h i c feature o f ancestral primate class I. That CD94/NKG2 are lectin-like subunits suggests some sort of involvement with the single, invariant carbohydrate of H L A class I heavy chains which is attached at asparagine 86. Residues 84-89 are invariant in H L A - A , El and C molecules (Parham e t al., 1995) and the oligosaccharide structure itself has little structural heterogeneity or variation with protein allotype (Barber et al., 1996; Parham, 1996). This patch of conserved protein and carbohydrate, which encompa,;ses the carboxyl-terminus of the al helix and the following loop to which the carbohydrate is attached, could p r o v i d e the c o m m o n basis for the C D 9 4 / N K G 2 binding site. Allotypic specificity would then be the result o f effects due to protein p o l y m o r p h i s m in neighbouring regions of the antigen recognition site where variation is rife. I would argue that the: principal effects of these p o l y m o r p h i s m s is to reduce affinity of CD98/NKG2 for particular allotypes or subsets of allotypes. During the last 50 million years or so, the A, B and C loci as we know them have formed and their complicated polymorphisms elaborated. DmSing this time the CD94/NKG2 interaction, which hzLd been established much earlier, has not adapted to all the nuances. The long-term effect of this progression is that the proportion of class I allotypes that can regulate lectin receptors has decreased. In that situation, there could be benefit to be gained from the evolution of additional new receptors that interact with allotypes no longer working well with the lectins. The KIRs of the immunoglobulin superfami]y have properties that fit this bill.
I m m u n o g i o b u l i n - l i k e receptors are new, like HLA-C Three KIR specificities for H L A class I epitopes have been defined and they resemble the fir,;t category of antibody-defined epitope to which I alluded above. They are defined positively by small numbers of substitutions found on a subset of allotypes. More importantly each of the three specificities is confined to the allotypes of a single locus - - two to HLA-C and one to HLA-B - - suggesting that these
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receptor specificities have evolved subsequent to the divergence of the HLA-A, -B and -(3 loci. This in itself is evidence for the K I R type of receptor specificity being newer than that of the lectins. The H L A class I locus most committed to interaction with KIRs is HLA-C. All H L A - C allotypes interact with a K I R and these l i g a n d - r e c e p t o r s divide into two g r o u p s b a s e d upon a m i n o acid substitutions at positions 77 and 80 of the c t t h e l i x ( C o l o n n a et al., 1993). T h u s , f o r H L A - C , the interaction with K I R is fixed: every human being possesses at least one H L A - C ligand that can bind to a KIR. Only one third o f H L A - B alleles are k n o w n to i n t e r a c t w i t h K I R s w h i c h t a r g e t sequence motifs at residues 77-83 of the a t helix that determine the Bw4 serological epitope (Celia et al., 1994; Gumperz et al., 1995). Within populations, B w 4 epitopes are present at c o m p a r a b l e frequencies. Italians, for e x a m p l e , h a v e a B w 4 gene frequency of about 35 %, such that 42 % of Italians do not possess a Bw4 ligand for the H L A B-specific KIR (Imanishi et al., 1992). The conclusion to be drawn f r o m these o b s e r v a t i o n s is t h a t H L A - C i n t e r a c t i o n s w i t h K I R are m o r e i m p o r t a n t than H L A - B i n t e r a c t i o n s with K I R , and/or the H L A - B interactions with KIR are more recently evolved than those of H L A - C and have yet to b e c o m e fixed. For HLA-A, -B and -C, a hierarchy emerges with regard to their i n v o l v e m e n t with KIR. H L A - C is fully c o m m i t t e d , H L A - B is e x p e r i m e n t i n g and H L A - A appears diffident, if not totally detached. The functional segregation of the three H L A class I loci with regard to K I R interaction reflects their structural relationships: H L A - B and C are much more similar to each other than either is to HLA-A. Combining this observation with the close proximity of the HLA-B and HLA-C genes, and their distance from HLA-A (Trowsdale, 1995), makes a good case for H L A - B and H L A - C having e v o l v e d by gene duplication from a common ancestor, long after that ancestor had diverged from HLA-A. When did the gene duplication that produced HLA-B and C occur? Information addressing this question has emerged from the study of M H C class I genes in n o n - h u m a n primate species (Watkins, 1995). R e c o g n i z a b l e A, B and C genes are only found in humans, apes and old world monkeys. Thus, the features that distinguish these loci have evolved since divergence of old and new world monkeys. A recognizable C gene has only been found in humans and apes. That no species of monkey has been found to express HLA-C shows that evolution of the characteristics that distinguish m o d e r n - d a y B and C occurred subsequent to the divergence of old world monkeys and apes, some 30-40 million years ago. Recognizable B-like genes are, however, present in old world monkeys, indicating that HLA-B is more
like the common ancestor, while HLA-C has derived novel characteristics. It therefore seems most likely that the m o d e m B and C genes derive from an ancestral B-like gene w h i c h duplicated. This d u p l i c a t i o n w o u l d h a v e given rise to two identical B-like genes, and with time these two genes diverged. Selection on one of the genes (that we now call B) would have tended to preserve ancestral characteristics, while the second gene (that we now call C) acquired new features that placed it under a different type of' selection. The duplication may have occurred prior to the diverg e n c e o f a p e s and old w o r l d m o n k e y s , but the acquisition of C-like character in one of the daughter g e n e s p r o b a b l y o c c u r r e d s u b s e q u e n t to that divergence. Within this scheme for the evolution of the HLAB and -C loci, I hypothesize that the selection which drove the divergence of C from B was that coming from interactions with NK cells and competing with the pre-existing pressures from cytotoxic T cells. With gene duplication, one locus could still fulfill the r e q u i r e m e n t s o f C T L while the other c o u l d change to accommodate N K cells. At the heart of this accommodation was the development of a functional interaction with the ancestral KIR molecule.
A d a p t a t i o n of H L A class I to K I R Today, HLA-B is expressed at high levels on the cell surface, while HLA-C is expressed at low levels (McCutcheon et al., 1995). StructuraJ[ comparisons show that HLA-B retains ancestral qualities, whereas H L A - C has acquired new traits. If this is also true for the level of expression, then we can conclude that reduced expression is an adaptation to KIR interaction and the control of NK cells. This could stem from the relative independence of K1R interaction from the particular species o f peptide bound to a class I molecule. Whereas, for a T-cell receptor, the target is limited to the minute subset of class I molecules carrying a particular peptide, that is clearly not the case for KIR and thus functional r e c e p t o r - l i g a n d interaction can, in principle, be developed with lower levels of expression, and may indeed be desirable. The alternative KIR binding sites of' H L A - C allotypes are defined by having either serine 77 and asparagine 80 (group 1) or aspamgine 77 and lysine 80 (group 2). Experiments in mutation show that the group 1 specificity can be introduced into a group 2 molecule by replacement of asparagine for serine at residue 77 and that the group 2 specificity can be introduced into a group 1 molecule by substitution of lysine for asparagine at position 80 (Biassoni et al., 1995). Within the context of the H L A - C locus, the difference between the two specificities is trivial
NK CELL FUNCTIONS
in terms of evolutionary divergence. These do not s e e m to be alternative structures crafted by the steady accumulation of ever-refining change. One or two substitutions is all it took ! O f the four amino acids that define the group 1 and 2 H L A - C determinants, only lysine 70 of the group 2 motif is unique to the HLA-C locus, suggesting that group 2 specificity is the most recently derived. Consistent with this model is the fact that many HLA-B allotypes have the group 1 sequence motif, although, with one exception, they do not interact with KIR. The exceptional H L A - B allotype that interacts with KIR is HLA-B*4601. This allele is one of few that have been formed by a gene conversion between two HLA class I loci. The recipient gene in the conversion was HLA-B*1501, which has serine 77 and asparagine 80 but does not interact with group 1 KIR. The donor gene in the conversion was HLACw*0102, which has serine 77 and asparagine 80 and does interact with group 1 KIR. The effect of the gene conversion was to replace residues 66-76 in B * 1 5 0 1 w i t h the h o m o l o g o u s s e q u e n c e o f Cw*0102. This change involves a total of six amino acid substitutions, including the residues lysine 66, arginine 69 and valine 76 which are fixed in HLA-C and absent in HLA-B (Zemmour et al., 1992). The single gene conversion event that created B ' 4 6 0 1 f r o m B*1501 and C w * 0 1 0 2 appears, in terms of functional immunology, to have reversed the effect of millions of years of divergence of the HLA-B and C loci. Although still an allele of HLAB, the functional properties of B ' 4 6 0 1 are those of an HLA-C allele: it interacts with group-l-specific KIR, it stimulates cytotoxic T cells that cross-react with HLA-Cw*0102 not B*1501, and its pattern of glycosylation is characteristic of HLA-C (Barber et al., 1996).
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linked to C w l , 77.5% are linked to both Cw*0102 and A2, and 47.7% are linked to C w l , A2, DR9 and D Q 3 ( I m a n i s h i e t a l . , 1992). T h e s t r o n g a n d extended linkage disequilibrium, which is reiterated in Thais and other southeast Asian populations, suggests that recombination within this haplotype has been suppressed either by selection or by being physically frozen, perhaps as a consequence of the gene conversion.
Conclusion In this article, I have attempted to develop some order to the historical events that formed the set of H L A class I receptors of N K cells that now function in the h u m a n i m m u n e response. The lectins with their broad cross-locus specificity for H L A class I d e t e r m i n a n t s c a m e first, to be f o l l o w e d b y the i m m u n o g l o b u l i n - l i k e K I R m o l e c u l e s with their tighter intra-locus specificities. Both the HLA class I oligosaccharide, to which a lectin could bind, and the protein polymorphisms that determine KIR specificities, localize to the same area of the class I structure, providing further unity of target to these structurally different receptors. The development of KIR specificity and the gene duplication and divergence that produced distinctive H L A - B and C loci p r o b a b l y occurred c o n c o m i tantly, under selection imposed by NK cells. Gene duplication allowed the HLA-C locus to commit to N K cell regulation at the expense of its contribution to the T-cell response. This is manifest in hs lower cell surface expression which reflects the relative indifference of K I R receptors to specific peptides bound by class I.
These observations are particularly striking when one considers that the minimum difference between an H L A - B and an H L A - C allele is 55 nucleotide substitutions or 24 amino acid substitutions (Parham et al., 1995). The inference is that much of this considerable d i v e r g e n c e is of little or m i n o r consequence to the basic immunological functions.
As a consequence of the focus of HLA-C upon the simple ligands that bind to KIR, polymorphism at HLA-C is lower than at HLA-B. Interallelic conversion events have been less common in the evolution of HLA-C alleles and it is striking that there is only one example of a conversion targeted to residues 77-80 defining the KIR ligands. In contrast, the Bw4 motif recognized by HLA-B-specific KIR is the most common target for interallelic HLA-B conversion.
Although the product of a rare intergenic conversion event, B ' 4 6 0 1 itself is h a r d l y rare. H L A B ' 4 6 0 1 is predominantly localized to Asian populations, although the recent patterns of trade and human migration have carried it elsewhere. In Italians, of whom Marco Polo founded the China trade, HLA-B*4601 has a frequency of 0.1% compared to 0.0% in their rivals, the Spanish and Portuguese, and 15.1% in the Singapore Chinese. The linkage disequilibrium of HLA-B*4601 with alleles at other H L A loci is very striking. From the total number of B ' 4 6 0 1 alleles in the Singapore Chinese: 97..4% are
Both experiments of nature and mutagenesis in the laboratory show that the H L A class I epitopes recognized by K I R are rather simple entities that can be removed or replaced by single events that commonly contribute to H L A class I evolution. Similarly, it appears that the functional diversification of the HLA-B and C loci is simpler than structural differences would suggest. These properties are all consistent with the relative youth of the inte,ractions between H L A class I molecules and KIR, suggesting there may be considerable potential for their future improvement.
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References
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