Achim K. Moesta

Synergistic Polymorphism at Two Positions Distal to the Ligand-Binding Site Makes KIR2DL2 a Stronger Receptor for HLA-C Than KIR2DL3

Interactions between HLA-C ligands and inhibitory killer cell Ig-like receptors (KIR) control the development and response of human NK cells. This regulatory mechanism is usually described by mutually exclusive interactions of KIR2DL1 with C2 having lysine 80, and KIR2DL2/3 with C1 having asparagine 80. Consistent with this simple rule, we found from functional analysis and binding assays to 93 HLA-A, HLA-B, and HLA-C isoforms that KIR2DL1*003 bound all C2, and only C2, allotypes. The allotypically related KIR2DL2*001 and KIR2DL3*001 interacted with all C1, but they violated the simple rule through interactions with several C2 allotypes, notably Cw*0501 and Cw*0202, and two HLA-B allotypes (B*4601 and B*7301) that share polymorphisms with HLA-C. Although the specificities of the “cross-reactions” were similar for KIR2DL2*001 and KIR2DL3*001, they were stronger for KIR2DL2*001, as were the reactions with C1. Mutagenesis explored the avidity difference between KIR2DL2*001 and KIR2DL3*001. Recombinant mutants mapped the difference to the Ig-like domains, where site-directed mutagenesis showed that the combination, but not the individual substitutions, of arginine for proline 16 in D1 and cysteine for arginine 148 in D2 made KIR2DL2*001 a stronger receptor than KIR2DL3*001. Neither residue 16 or 148 is part of, or near to, the ligand-binding site. Instead, their juxtaposition near the flexible hinge between D1 and D2 suggests that their polymorphisms affect the ligand-binding site by changing the hinge angle and the relative orientation of the two domains. This study demonstrates how allelic polymorphism at sites distal to the ligand-binding site of KIR2DL2/3 has diversified this receptor’s interactions with HLA-C.

KIR2DS4 is a product of gene conversion with KIR3DL2 that introduced specificity for HLA-A*11 while diminishing avidity for HLA-C

Human killer cell immunoglobulin-like receptors (KIRs) are distinguished by expansion of activating KIR2DS, whose ligands and functions remain poorly understood. The oldest, most prevalent KIR2DS is KIR2DS4, which is represented by a variable balance between “full-length” and “deleted” forms. We find that full-length 2DS4 is a human histocompatibility leukocyte antigen (HLA) class I receptor that binds specifically to subsets of C1+ and C2+ HLA-C and to HLA-A*11, whereas deleted 2DS4 is nonfunctional. Activation of 2DS4+ NKL cells was achieved with A*1102 as ligand, which differs from A*1101 by unique substitution of lysine 19 for glutamate, but not with A*1101 or HLA-C. Distinguishing KIR2DS4 from other KIR2DS is the proline–valine motif at positions 71–72, which is shared with KIR3DL2 and was introduced by gene conversion before separation of the human and chimpanzee lineages. Site-directed swap mutagenesis shows that these two residues are largely responsible for the unique HLA class I specificity of KIR2DS4. Determination of the crystallographic structure of KIR2DS4 shows two major differences from KIR2DL: displacement of contact loop L2 and altered bonding potential because of the substitutions at positions 71 and 72. Correlation between the worldwide distributions of functional KIR2DS4 and HLA-A*11 points to the physiological importance of their mutual interaction.

Co-evolution of KIR2DL3 with HLA-C in a human population retaining minimal essential diversity of KIR and HLA class I ligands

Natural killer (NK) cells contribute to immunity and reproduction. Guiding these functions, and NK cell education, are killer cell Ig-like receptors (KIR), NK cell receptors that recognize HLA class I. In most human populations, these highly polymorphic receptors and ligands combine with extraordinary diversity. To assess how much of this diversity is necessary, we studied KIR and HLA class I at high resolution in the Yucpa, a small South Amerindian population that survived an approximate 15,000-year history of population bottleneck and epidemic infection, including recent viral hepatitis. The Yucpa retain the three major HLA epitopes recognized by KIR. Through balancing selection on a few divergent haplotypes the Yucpa maintain much of the KIR variation found worldwide. HLA-C*07, the strongest educator of C1-specific NK cells, has reached unusually high frequency in the Yucpa. Concomitantly, weaker variants of the C1 receptor, KIR2DL3, were selected and have largely replaced the form of KIR2DL3 brought by the original migrants from Asia. HLA-C1 and KIR2DL3 homozygosity has previously been correlated with resistance to viral hepatitis. Selection of weaker forms of KIR2DL3 in the Yucpa can be seen as compensation for the high frequency of the potent HLA-C*07 ligand. This study provides an estimate of the minimal KIR-HLA system essential for long-term survival of a human population. That it contains all functional elements of KIR diversity worldwide, attests to the competitive advantage it provides, not only for surviving epidemic infections, but also for rebuilding populations once infection has passed.

Human-Specific Evolution and Adaptation Led to Major Qualitative Differences in the Variable Receptors of Human and Chimpanzee Natural Killer Cells

Natural killer (NK) cells serve essential functions in immunity and reproduction. Diversifying these functions within individuals and populations are rapidly-evolving interactions between highly polymorphic major histocompatibility complex (MHC) class I ligands and variable NK cell receptors. Specific to simian primates is the family of Killer cell Immunoglobulin-like Receptors (KIR), which recognize MHC class I and associate with a range of human diseases. Because KIR have considerable species-specificity and are lacking from common animal models, we performed extensive comparison of the systems of KIR and MHC class I interaction in humans and chimpanzees. Although of similar complexity, they differ in genomic organization, gene content, and diversification mechanisms, mainly because of human-specific specialization in the KIR that recognizes the C1 and C2 epitopes of MHC-B and -C. Humans uniquely focused KIR recognition on MHC-C, while losing C1-bearing MHC-B. Reversing this trend, C1-bearing HLA-B46 was recently driven to unprecedented high frequency in Southeast Asia. Chimpanzees have a variety of ancient, avid, and predominantly inhibitory receptors, whereas human receptors are fewer, recently evolved, and combine avid inhibitory receptors with attenuated activating receptors. These differences accompany human-specific evolution of the A and B haplotypes that are under balancing selection and differentially function in defense and reproduction. Our study shows how the qualitative differences that distinguish the human and chimpanzee systems of KIR and MHC class I predominantly derive from adaptations on the human line in response to selective pressures placed on human NK cells by the competing needs of defense and reproduction.

Humans Differ from Other Hominids in Lacking an Activating NK Cell Receptor That Recognizes the C1 Epitope of MHC Class I

Modulation of human NK cell function by killer cell Ig-like receptors (KIR) and MHC class I is dominated by the bipartite interactions of inhibitory lineage III KIR with the C1 and C2 epitopes of HLA-C. In comparison, the ligand specificities and functional contributions of the activating lineage III KIR remain poorly understood. Using a robust, sensitive assay of KIR binding and a representative panel of 95 HLA class I targets, we show that KIR2DS1 binds C2 with ~50% the avidity of KIR2DL1, whereas KIR2DS2, KIR2DS3, and KIR2DS5 have no detectable avidity for C1, C2, or any other HLA class I epitope. In contrast, the chimpanzee has activating C1- and C2-specific lineage III KIR with strong avidity, comparable to those of their paired inhibitory receptors. One variant of chimpanzee Pt–KIR3DS2, the activating C2-specific receptor, has the same avidity for C2 as does inhibitory Pt–KIR3DL4, and a second variant has ~73% the avidity. Chimpanzee Pt–KIR3DS6, the activating C1-specific receptor, has avidity for C1 that is ~70% that of inhibitory Pt–KIR2DL6. In both humans and chimpanzees we observe an evolutionary trend toward reducing the avidity of the activating C1- and C2-specific receptors through selective acquisition of attenuating substitutions. However, the extent of attenuation has been extreme in humans, as exemplified by KIR2DS2, an activating C1-specific receptor that has lost all detectable avidity for HLA class I. Supporting such elimination of activating C1-specific receptors as a uniquely human phenomenon is the presence of a high-avidity activating C1-specific receptor (Gg–KIR2DSa) in gorilla.

Diverse functionality among human NK cell receptors for the C1 epitope of HLA-C: KIR2DS2, KIR2DL2, and KIR2DL3.

Interactions between killer immunoglobulin-like receptors (KIRs) and their HLA-A, -B, and -C ligands diversify the functions of human natural killer cells. Consequently, combinations of KIR and HLA genotypes affect resistance to infection and autoimmunity, success of reproduction and outcome of hematopoietic cell transplantation. HLA-C, with its C1 and C2 epitopes, evolved in hominids to be specialized KIR ligands. The system’s foundation was the C1 epitope, with C2 a later addition, by several million years. The human inhibitory receptor for C1 is encoded by KIR2DL2/3, a gene having two divergent allelic lineages: KIR2DL2 is a B KIR haplotype component and KIR2DL3 an A KIR haplotype component. Although KIR2DL2 and KIR2DL3 exhibit quantitative differences in specificity and avidity for HLA-C, they qualitatively differ in their genetics, functional effect, and clinical influence. This is due to linkage disequilibrium between KIR2DL2 and KIR2DS2, a closely related activating receptor that was selected for lost recognition of HLA-C.

Coevolution of killer cell Ig-like receptors with HLA-C to become the major variable regulators of human NK cells.

Interactions between HLA class I and killer cell Ig-like receptors (KIRs) diversify human NK cell responses. Dominant KIR ligands are the C1 and C2 epitopes of MHC-C, a young locus restricted to humans and great apes. C1- and C1-specific KIRs evolved first, being present in orangutan and functionally like their human counterparts. Orangutans lack C2 and C2-specific KIRs, but have a unique C1+C2-specific KIR that binds equally to C1 and C2. A receptor with this specificity likely provided the mechanism by which C2–KIR interaction evolved from C1–KIR while avoiding a nonfunctional intermediate, that is, either orphan receptor or ligand. Orangutan inhibitory MHC-C–reactive KIRs pair with activating receptors of identical avidity and specificity, contrasting with the selective attenuation of human activating KIRs. The orangutan C1-specific KIR reacts or cross-reacts with all four polymorphic epitopes (C1, C2, Bw4, and A3/11) recognized by human KIRs, revealing their structural commonality. Saturation mutagenesis at specificity-determining position 44 demonstrates that KIRs are inherently restricted to binding just these four epitopes, either individually or in combination. This restriction frees most HLA-A and HLA-B variants to be dedicated TCR ligands, not subject to conflicting pressures from the NK cell and T cell arms of the immune response.

Primate-Specific Regulation of Natural Killer Cells

Natural killer (NK) cells are circulating lymphocytes that function in innate immunity and placental reproduction. Regulating both development and function of NK cells is an array of variable and conserved receptors that interact with major histocompatibility complex (MHC) class I molecules. Families of lectin‐like and immunoglobulin‐like receptors are determined by genes in the natural killer complex (NKC) and leukocyte receptor complex (LRC), respectively. As a consequence of the strong, varying pressures on the immune and reproductive systems, NK cell receptors and their MHC class I ligands evolve rapidly, are highly diverse and exhibit dramatic species‐specific differences. The variable, polymorphic family of killer cell immunoglobulin‐like receptors (KIR) that regulate human NK cell development and function arose recently, from a single‐copy gene during the evolution of simian primates. Our studies of KIR and MHC class I genes in representative species show how these two unlinked but functionally intertwined genetic complexes have co‐evolved. In humans, combinations of KIR and HLA class I factors are associated with infectious diseases, including HIV/AIDS, autoimmunity, reproductive success and the outcome of therapeutic transplantation. The extraordinary, and unanticipated, divergence of human NK cell receptors and MHC class I ligands from their mouse counterparts can in part explain the difficulties experienced in finding informative mouse models for human diseases. Non‐human primate models have far greater potential, but to realize their promise will first require more complete definition of the genetics and function of KIR and MHC variation in non‐human primate species, at a level comparable to that achieved for the human species.

Mutation at Positively Selected Positions in the Binding Site for HLA-C Shows That KIR2DL1 Is a More Refined but Less Adaptable NK Cell Receptor Than KIR2DL3

Through recognition of HLA class I, killer cell Ig-like receptors (KIR) modulate NK cell functions in human immunity and reproduction. Although a minority of HLA-A and -B allotypes are KIR ligands, HLA-C allotypes dominate this regulation, because they all carry either the C1 epitope recognized by KIR2DL2/3 or the C2 epitope recognized by KIR2DL1. The C1 epitope and C1-specific KIR evolved first, followed several million years later by the C2 epitope and C2-specific KIR. Strong, varying selection pressure on NK cell functions drove the diversification and divergence of hominid KIR, with six positions in the HLA class I binding site of KIR being targets for positive diversifying selection. Introducing each naturally occurring residue at these positions into KIR2DL1 and KIR2DL3 produced 38 point mutants that were tested for binding to 95 HLA- A, -B, and -C allotypes. Modulating specificity for HLA-C is position 44, whereas positions 71 and 131 control cross-reactivity with HLA-A*11:02. Dominating avidity modulation is position 70, with lesser contributions from positions 68 and 182. KIR2DL3 has lower avidity and broader specificity than KIR2DL1. Mutation could increase the avidity and change the specificity of KIR2DL3, whereas KIR2DL1 specificity was resistant to mutation, and its avidity could only be lowered. The contrasting inflexibility of KIR2DL1 and adaptability of KIR2DL3 fit with C2-specific KIR having evolved from C1-specific KIR, and not vice versa. Substitutions restricted to activating KIR all reduced the avidity of KIR2DL1 and KIR2DL3, further evidence that activating KIR function often becomes subject to selective attenuation.

Chimpanzees Use More Varied Receptors and Ligands Than Humans for Inhibitory Killer Cell Ig-Like Receptor Recognition of the MHC-C1 and MHC-C2 Epitopes

Humans and chimpanzees have orthologous MHC class I, but few orthologous killer cell Ig-like receptors (KIR). Most divergent are lineage III KIR, which in humans include the inhibitory KIR2DL1 and 2DL2/3 specific for HLA-C. Six lineage III chimpanzee KIR were identified as candidate inhibitory MHC-C receptors and studied using cytolytic assays, to assess the capacity of a defined KIR to function with a defined MHC class I allotype, and direct binding assays with KIR-Fc fusion proteins. Pt-KIR2DL6 and 2DL8 were demonstrated to be inhibitory C1 receptors with a specificity and specificity-determining residue (lysine 44) like KIR2DL3. Analogously, Pt-KIR2DL7 is like KIR2DL1, an inhibitory C2 receptor having methionine 44. Pt-KIR3DL4 and 3DL5 are unusual lineage III KIR with D0 domains, which are also inhibitory C2 receptors with methionine 44. Removal of D0 from KIR3DL, or its addition to KIR2DL, had no effect on KIR function. Pt-KIR2DL9, a fourth inhibitory C2 receptor, has glutamate 44, a previously uncharacterized specificity-determining residue that is absent from human KIR. Reconstruction of the ancestral hominoid KIR sequence shows it encoded lysine 44, indicating that KIR having methionine 44 and glutamate 44 subsequently evolved by independent point substitutions. Thus, MHC-C2-specific KIR have evolved independently on at least two occasions. None of the six chimpanzee KIR studied resembles KIR2DL2, which interacts strongly with C1 and cross-reacts with C2. Whereas human HLA-B allotypes that have functional C1 epitopes are either rare (HLA-B*73) or geographically localized (HLA-B*46), some 25% of Patr-B allotypes have the C1 epitope and are functional KIR ligands.