High-Resolution Analysis Identifies High Frequency of KIR-A Haplotypes and Inhibitory Interactions of KIR With HLA Class I in Zhejiang Han

Killer cell immunoglobulin-like receptors (KIR) interact with human leukocyte antigen (HLA) class I molecules, modulating critical NK cell functions in the maintenance of human health. Characterizing the distribution and characteristics of KIR and HLA allotype diversity across defined human populations is thus essential for understanding the multiple associations with disease, and for directing therapies. In this study of 176 Zhejiang Han individuals from Southeastern China, we describe diversity of the highly polymorphic KIR and HLA class I genes at high resolution. KIR-A haplotypes, which carry four inhibitory receptors specific for HLA-A, B or C, are known to associate with protection from infection and some cancers. We show the Chinese Southern Han from Zhejiang are characterized by a high frequency of KIR-A haplotypes and a high frequency of C1 KIR ligands. Accordingly, interactions of inhibitory KIR2DL3 with C1+HLA are more frequent in Zhejiang Han than populations outside East Asia. Zhejiang Han exhibit greater diversity of inhibitory than activating KIR, with three-domain inhibitory KIR exhibiting the greatest degree of polymorphism. As distinguished by gene copy number and allele content, 54 centromeric and 37 telomeric haplotypes were observed. We observed 6% of the population to have KIR haplotypes containing large-scale duplications or deletions that include complete genes. A unique truncated haplotype containing only KIR2DL4 in the telomeric region was also identified. An additional feature is the high frequency of HLA-B*46:01, which may have arisen due to selection pressure from infectious disease. This study will provide further insight into the role of KIR and HLA polymorphism in disease susceptibility of Zhejiang Chinese.


INTRODUCTION
Natural killer (NK) cells are innate immune effectors having multiple important roles in human health. NK cells are critical in controlling the outcome of infections, tumors and fetal implantation, as well as organ transplantation and immunotherapies (1)(2)(3)(4)(5). In these roles, NK cell functions are modulated by interactions of their cell surface receptors with ligands expressed by tissue cells (6,7). In humans, the interactions include polymorphic combinations of killer cell immunoglobulin-like receptors (KIR) with human leukocyte antigen (HLA) class I ligands (8,9). The extreme genetic variation of KIR and HLA class I directly impacts NK cell functions, and associates with multiple immune-mediated diseases (10,11). Also directly affected by combinatorial diversity, and influencing effector functions in health and disease, maturation of NK cells is guided through appropriate interactions of KIR with HLA class I (12)(13)(14).
The KIR locus of chromosome 19 (19q13.4) comprises up to 13 genes encoding receptors having distinct ligand specificities and inhibitory or activating functions (15,16). Encoded by each KIR haplotype are from one to four inhibitory receptors and up to five activating receptors specific for HLA-A, B or C. Four framework genes, present in most individuals, form the basic structure of the locus. Their arrangement divides the KIR locus into centromeric (KIR3DL3 to KIR3DP1) and telomeric (KIR2DL4 to KIR3DL2) oriented regions. Structural diversity of the KIR locus is formed through recombination events that duplicate or delete entire genes, or deletions that create in-frame gene fusions (17,18). This genetic variation thus has direct consequences for NK cell functions, which are further diversified by the alleles of KIR and their HLA class I ligands (19,20). Within HLA-A, B and C, only those allotypes that contain one of four amino acid motifs, termed A3/11, Bw4, C1 or C2, in the external-facing a-helices can be ligands for KIR. Polymorphism both within and outside these motifs diversifies the interactions of KIR with HLA and individualize NK cell functions (21,22). There are two broad forms of KIR haplotype, termed KIR-A and KIR-B (23). KIR-A haplotypes have fixed gene content, encoding four inhibitory KIR specific for HLA-A, B or C, as well as KIR2DS4, which is an activating receptor specific for some HLA-A and C allotypes (24). The majority of KIR2DS4 alleles are not expressed due to a deletion in exon 5 (25). KIR-B haplotypes contain a variable number of genes for activating KIR and fewer for inhibitory KIR. The two haplotype groups display distinct functional characteristics. For example, although KIR2DL2 and KIR2DL3 are encoded by the same locus, they differ in strength and specificity and characterize KIR-B and KIR-A haplotypes, respectively (26). Accordingly, KIR-A and KIR-B haplotypes also have distinct disease association profiles (27). KIR-A haplotypes associate with effective immunity against acute viral infections, but with increased risk of preeclampsia during pregnancy (28,29). KIR-B haplotypes protect women from this pregnancy disorder and improve the outcome of hematopoietic stem cell transplantation (30,31). Additional to the KIR-A and KIR-B haplotype designations, specific alleles that can be characteristic to the haplotypes associate with disease susceptibility or protection. A total of 1,110 alleles encoding 543 allotypes have been reported according to the IPD-KIR database release version 2.10.0 (32).
The development of next-generation sequencing (NGS) technologies for genotyping KIR and HLA at high-resolution has helped identify the substantial variation of KIR allotypes and their interactions with HLA ligands across human populations (33)(34)(35). It is therefore of great significance to characterize this combinatorial diversity across all human populations. Although China, as one of the four ancient civilizations of the world, accounts for approximately 20% of the human population, analysis of the KIR locus at high-resolution from East Asia is limited to two studies (36,37). In this study, we fully characterize the allele, haplotype and combinatorial diversity of KIR and HLA class I in the Zhejiang Han population from the Southeast coast of China (27°02' to 31°11' North, 118°01' to 123°10' East).

High-Resolution Sequencing for KIR and HLA
A biotinylated DNA probe-based capture method was applied to sequence KIR and HLA genes to high-resolution, as described previously (33). Briefly, 500 ng genomic DNA for each sample was randomly fragmented using enzyme digestion (New England Biolabs, Boston, MA) and then labeled with TruSeq DNA CD indexes (Illumina Inc, San Diego, CA). Fragments of 800-1,200 bp length were obtained by size selection and the individual samples pooled at equal quantity into one tube. Fragments containing target HLA and KIR genes were captured using specific probes, prepared to a final concentration of 12 pmol/L, as determined by Qubit instrument, then sequenced using a MiSeq instrument and a v3 Reagent Kit (600-cycle; Illumina Inc, San Diego, CA, USA).

Sequence Data Analysis
KIR gene content, copy numbers, and allele genotypes were determined using the Pushing Immunogenetics to the Next Generation pipeline (PING) as previously reported (33). The copy number was determined from the ratio of reads mapping to each KIR gene to those mapping to KIR3DL3, a reference gene present only one copy on each haplotype. Alleles for 13 KIR genes were analyzed, of which KIR2DL2 and KIR2DL3 are alleles at the same locus (KIR2DL2/3), as are KIR2DS3 and KIR2DS5 (KIR2DS3/5), and KIR3DL1 and KIR3DS1 (KIR3DL1/S1). KIR3DL3 alleles were determined using PHASE 2.1 (38). The HLA alleles were determined using TypeStream Visual NGS Analysis Software (TSV2.0, One Lambda Inc, Canoga Park, CA). The presence of KIR ligands, A3/11, Bw4, and C1/C2 was determined according to the HLA alleles.

Frequencies of Alleles, Genotypes, and Haplotypes
KIR genotypes were named based on the presence or absence of KIR genes according to the allelefrequencies.net database (39). Carrier frequencies of KIR genes and genotypes were calculated as their percentage of the total number of individuals. The frequencies of alleles or haplotypes were calculated by direct counting, and the number observed divided by 2N (alleles duplicated on a single haplotype were not included, and absence was counted as a distinct allele). The composition and frequencies of HLA haplotypes were determined using Arlequin 3.5.2.2 (40).

Statistical Analysis
Hardy-Weinberg equilibrium (HWE) was determined for each gene using Fisher's exact test implemented in the Arlequin software 3.5.2.2. The distributions of genes, alleles, haplotypes, and KIR/HLA interactions in the Zhejiang Han population were compared to other populations as described. Differences between populations were assessed using GraphPad software, by means of the c 2 test for categorical variables. The p values were calculated using Fisher's exact test (pf), applied when appropriate. Bonferroni correction for multiple comparisons was applied. p <0.05 was regarded as significant.

High Frequency of KIR-A Haplotypes in Zhejiang Han
To analyze the KIR locus of the Zhejiang Han at high resolution, we first established which KIR genes are present, and in how many copies, for every individual. The four framework genes [KIR3DL3, KIR2DL4, KIR3DP1 and KIR3DL2 (15)] were detected in all 176 individuals of the cohort ( Figure 1A). KIR2DL4 and KIR3DP1 were each observed with one to three copies and one individual in the cohort has only one copy of KIR3DL2 ( Figure 1A). Only four individuals have less than two copies of any of these framework genes, six individuals harbor three copies, and none were observed with four copies. These observations indicate that, although structural diversity of the KIR locus is present, it is rare in the Zhejiang Han population. KIR2DL1, KIR2DL3, KIR3DL1, KIR2DS4, and KIR2DP1 were all observed at a frequency greater than 90% and at two copies in at least 64.8% of the population ( Figures 1A, B). These five genes are not present on every KIR haplotype, and when they occur together, they characterize the KIR A haplotype (15,51). KIR2DL3, which occurs in the centromeric KIR region, showed higher frequency than KIR3DL1 and KIR2DS4, which are located in the telomeric KIR region (81.8% vs 64.8%), indicating a higher frequency of centromeric KIR-A haplotype motifs (Cen-A) than telomeric KIR-A motifs (Tel-A). Accordingly, the genes characteristic of KIR-B haplotypes have frequencies less than 25% in centromeric and approximately 35% in telomeric KIR region ( Figure 1B). We compared the distribution of KIR genes of Zhejiang Han with populations representing Asia (37,43,46,(52)(53)(54), Europe (47,48,55,56), Africa (49,57), America (50) and Oceania (58, 59) (Supplementary Table 1). As a control, this analysis showed the KIR gene frequencies to be similar to our previous study of an independent Zhejiang Han cohort that was performed at lower resolution (60). The KIR gene frequencies are also similar to those of other populations from East Asia including Japan (37), South Korea (43) and Singapore (53), but distinct from Southeast Asians (46) and populations from other continents (Supplementary Table 1). Accordingly, the frequencies of Cen-B specific KIR genes (KIR2DS2, KIR2DL2, KIR2DL5B) in the Zhejiang Han are significantly lower than all populations outside East Asia (Supplementary Table 1).
As determined by the copy number of each of the 13 KIR genes, twenty-three distinct genotypes were observed ( Figure  1C). Of these genotypes, KIR-AA is the most frequent (53.4%) in Zhejiang Han ( Figure 1C). The KIR-AA genotype is common across East Asia, also being observed frequently in other Chinese Southern Han (36), Taiwan Chinese (44), South Korean (43), and Japanese (37). The KIR-AA genotype is less frequent outside East Asia, as illustrated by populations representing Southeast Asia, Europe, South America, and sub-Saharan Africa ( Figure  1D). The other 22 KIR genotypes observed in Zhejiang Han are designated KIR-Bx, the distributions of which are also similar among East Asians and distinct from populations of other continents ( Figure 1D). For example, Bx2, which is the most frequent KIR-B genotype in Zhejiang Han, was not observed in sub-Saharan Africans, whereas Bx5 is frequent in sub-Saharan Africans, but very rare in Zhejiang Han ( Figure 1D). Another example is Bx3, which was observed with high frequency in Amerindians, but almost absent from all other populations ( Figure 1D). These examples of distinct genotype profiles characterizing major human population groups illustrate the rapid evolution of the KIR locus (27,61).   (Figure 2A and Supplementary Table 2) and no novel alleles were identified. The allele frequency distributions for all the KIR genes were in accordance with Hardy-Weinberg equilibrium. The inhibitory KIR are more diverse and polymorphic (88 alleles in total) than the activating KIR (14 alleles). The most diverse gene encoding an activating KIR is KIR2DS4, which is the only activating KIR carried by the KIR-A haplotype, with five alleles observed ( Figure 2B). KIR2DS4*00101 is the most frequent allele and is the only one that can encode a functional allotype on the cell surface, whereas the remainder are non-expressed alleles because of a 22bp deletion in exon 5 (25 (62), the non-expressed KIR2DS4 alleles predominate. Among genes encoding inhibitory KIR, those with three domains show greatest variability (25,32). We observed 23 alleles each of KIR3DL3 and KIR3DL2 and 11 alleles of KIR3DL1/S1, which encode 16, 17, and 11 allotypes, respectively (Supplementary Table 2). Thus, each allele of KIR3DL1 we observed here ( Figure 2C) encodes a distinct allotype and the most abundant have high-expressing, highbinding, phenotypes (37,63,64). KIR3DL1*015 is the most frequent KIR3DL1 allotype in Zhejiang Han. Comparison with other representative populations shows that KIR3DL1*015 is present worldwide, reaching highest frequency in East Asians ( Figure 3). By contrast, KIR3DL1*004, which is a poorly expressed phenotype found in Europeans and Africans, was not observed in Zhejiang Han ( Figure 3) and neither was it observed in another Chinese Southern Han group (36). We previously reported KIR3DL1 allele frequencies from an independent cohort of Zhejiang Han (66). Because KIR3DS1 was not analyzed in that cohort, the reported KIR3DL1 allele frequencies are higher than in the present cohort, however, the frequencies are similar when KIR3DS1 is considered. The present analysis also showed that KIR3DL3 has the most extensive crosspopulation diversity. Here, KIR3DL3*010 clearly characterizes East Asian populations, whereas 3DL3*003 for example characterizes Amerindians, but is rare in East Asians (Figure 3).
Although KIR2DL1 is the most polymorphic two-domain KIR, having 173 known alleles [IPD-IMGT/KIR Database version 2.10.0, December 2020 (32)], it has low diversity in Zhejiang Han, with five alleles observed (Figure 2A). The most prominent of these alleles is KIR2DL1*00302 at an allele frequency of 77.8% (Supplementary Table 2), corresponding with a similar frequency of KIR2DL3*00101. This strong linkage disequilibrium between the two alleles is shared across multiple populations worldwide (34,37,45,50,62,65). KIR2DL4, which can be an activating receptor specific for HLA-G (67, 68), has six alleles in Zhejiang Han (Supplementary Table 2). Of 107 KIR2DL4 alleles, encoding 54 distinct polypeptide sequences, 18 do not express a receptor on the cell surface due to a single nucleotide deletion at position 811 in Exon 7 (69). The non-expressed alleles include KIR2DL4*008 and KIR2DL4*011, which were observed at a total frequency of 20.2% in Zhejiang Han ( Figure 2B). The four expressible   Figure 3).

KIR-A Haplotypes Are More Frequent Than KIR-B Haplotypes Both in Centromeric and Telomeric Regions
Based on the copy number and allele information obtained from high resolution analysis, 54 centromeric haplotypes and 37 telomeric haplotypes were observed in Zhejiang Han (Supplementary Table 3). As defined by gene content, three common centromeric haplotype groups, cA01, cB01, and cB02, and two telomeric groups, tA01 and tB01, were observed, as well as rarer forms having duplicated or deleted segments (Figure 4). When accounting for allele diversity, there are 41 haplotypes in the cA01 group (Supplementary Table 3A), and 23 in the tA01 group (Supplementary Table 3B). The most frequent cA01 h a p l oty p e a t t he a ll e le l e ve l in Z h e j i a n g Ha n i s KIR3DL3*01002-2DL3*00101-2DP1*00201-2DL1*00302, with a frequency of 19.03% ( Figure 5A). The five most common cA01 haplotypes (64.5% in total) differ only in their KIR3DL3 alleles ( Figure 5A). The high polymorphism of KIR3DL3 alleles diversifies the centromeric region, and the number of the centromeric haplotypes would decrease from 54 to 19 if KIR3DL3 alleles were excluded (Supplementary Table 3A).
The most frequent tA01 haplotype at the allele level in Zhejiang Han is KIR2DL4*00102-3DL1*01502-2DS4*00101-3DL2*00201 ( Figure 5B), with a frequency of 45.5%. Within the telomeric haplotypes, KIR2DS3/5t and KIR2DL5A alleles show strong linkage disequilibrium, in which KIR2DS5*00201 and KIR2DL5A*00101 are always found together, as are KIR2DS3*00201 and KIR2DL5A*00501. These patterns of linkage disequilibrium are the same as those found in other populations outside of Africa. Six individuals having an insertion/duplication of KIR3DP1-2DL4-3DS1 were observed in Zhejiang Han ( Figure 5C). The corresponding haplotypes comprised two distinguished at the gene content level, and three as determined by allele content. We also identified four individuals with haplotypes lacking KIR3DP1-2DL4-3DL1/S1 ( Figure 5D), with three of these haplotypes (individuals 1, 2 and 4: Figure 5D) distinguished only by their alleles of KIR3DL3. These haplotypes likely formed by uneven exchange during meiotic recombination, and have also been observed in other populations (17,45,70,71). Although it remains to be confirmed through full haplotype sequence analysis, the insertions/duplications and the deletions occur in approximately the same location 5' from KIR3DP1, suggesting the crossovers can occur frequently through shared mechanisms (72). In addition, we identified a unique haplotype that lacks any KIR genes in the telomeric region 3' from KIR2DL4, leaving only six genes on the entire KIR haplotype (individual 5; Figure 5D). That the allele is KIR2DL4*00102 suggests the parental haplotype that lost these genes was similar to the most frequent KIR-A in Zhejiang Han ( Figure 5B).  (39). C1 + HLA-C and C1 + HLA-B were both observed at higher frequencies than non-Asian populations ( Figure 6C). Accordingly, HLA-C2 shows extremely low frequency in Zhejiang Han, and significantly lower than other populations ( Figure 6C, Supplementary Table 5). Moreover, A3/11, specifically encoded by A*11:01, was observed at higher frequency in Zhejiang Han than any other populations. We identified 166 HLA haplotypes (Supplementary Table 4).
Most of the observed haplotypes (77.3%) encode more than two KIR ligands, including A*11:01, which was detected in five of the ten most frequent haplotypes ( Figure 6D). These findings are similar to those recently reported for Chinese Southern Han from Shenzhen (36). The high frequency of the KIR-A haplotype, which encodes four inhibitory receptors able to interact with HLA class I ligands (KIR2DL3, KIR2DL1, KIR3DL1 and KIR3DL2), indicates inhibitory KIR-HLA interactions predominate in Zhejiang Han. Here, KIR2DL1*00302, KIR2DL3*00101, KIR3DL1*01502 and KIR3DL2*00201 are the major alleles carried by the KIR-A haplotype, and all the allotypes they encode have strong interactions with their HLA ligands compared to other allotypes (20,37,41,73,74). Because of the high frequency of KIR-A, Zhejiang Han are rich in KIR-AA genotypes, and each of these individuals therefore has eight strong inhibitory KIR allotypes that can interact with HLA. The mean number of inhibitory KIR-HLA pairs per individual was analyzed, revealing that the mean interaction of KIR2DL3-C1 + HLA per individual (2.2) is higher than other populations ( Figure 6E). This high frequency is caused by the high frequencies of KIR2DL3 and C1 + HLA-C, and the extra C1 + HLA-B allotype present ( Figure 6A). By contrast, due to the low frequency of the C2 + HLA-C allotypes, the frequency of interactions with C2 + HLA is low in Zhejiang Han ( Figure 6E). In these regards the Zhejiang Han also show very similar repertoires to the Shenzhen Chinese Southern Han (36). Further diversifying NK cell interactions with polymorphic HLA, conserved HLA-E can present peptides derived from HLA-A, C and some B allotypes. The peptides are derived from allotypes possessing methionine at the second residue of the leader sequence (-21M). HLA-E is recognized by inhibitory CD94:NKG2A expressed by NK cell and T cell subsets (75,76). In Zhejiang Han, the total frequency of HLA-B alleles encoding -21M is 11.6% ( Figure 6F). Among them, only HLA-B*38 also encodes a Bw4 ligand for KIR and is the most frequent at 3.7%.

DISCUSSION
Specific genetic variants of KIR, HLA, and their combinations are associated with susceptibility and progression of multiple immune-mediated diseases, including viral infections, autoimmunity, cancer and reproductive disorders (10,11,13). Critical to understanding these disease mechanisms are systematic    Table 4). more than 90% of centromeric and 80% telomeric haplotypes have the KIR-A configuration. Previous whole-genome, as well as analyses focused on the KIR genomic region, have shown that the centromeric KIR-A haplotypes are subject to positive natural selection specifically in East Asians (37,78,79). By contrast, other studies continue to identify a balance between KIR-A and KIR-B haplotypes across populations worldwide, likely due to competing influences from infection control and reproduction (27,80). Another outcome of selection favoring KIR-A in East Asia is the low frequency of KIR-B haplotypes, specifically those that contain KIR2DL5B and KIR2DS3. In Amerindians, a complete loss of KIR2DS3 is observed (50), and this may be tolerated because the common 2DS3*00102 allele does not form a functional receptor (81). In addition to gene content, KIR haplotypes characteristic to human populations are distinguished by the alleles they carry (34,45,50,82 (27). Chinese Han have a high frequency of HLA-B*46:01, which is only common in Asia and formed by recombination between HLA-B*15:01 and HLA-C*01:02 (83). HLA-B*46:01 encodes an exceptional HLA-B allotype that can react with KIR2DL2/3, due to possession of a C1 ligand (26), and has risen to high frequency in this geographic region through admixture and natural selection (36,84). Candidates for this selection include endemic bacterial or viral infections (10,29,(85)(86)(87)(88)(89). An alternative explanation for selection acting on B*46 is that it provides a KIR ligand, whereas B*15:01, from which B*46:01 is derived, does not carry a KIR ligand (36). Co-evolution of KIR with HLA has been driven under natural selection, leading to frequency variation across populations for specific KIR-HLA interactions (49,50,90). Our analysis identified many similarities in immunogenetic diversity between the Zhejiang Han and the closely related Shenzhen Han, from Southern China (36). Distinguishing the Zhejiang Han is a high frequency of KIR-A and C1 + HLA-C, which is a genetic combination associated with preeclampsia (28). This serious multisystem disorder of pregnancy is most frequent in women homozygous both for KIR-A and C1 + HLA-C, if the fetus carries C2 + HLA-C (28). However, there is a substantially lower incidence of preeclampsia in China compared with western countries (91)(92)(93), which is likely due to the lower frequency C2 + ligands. Moreover, HLA-G expression by extravillous trophoblasts, which promotes vascular remodeling, may help ensure successful placentation thus preventing preeclampsia (94). HLA-G is a ligand for KIR2DL4 (67), and in Zhejiang Han the majority of KIR2DL4 alleles express a functional allotype.
Uneven exchange during recombination events has been a frequent occurrence during KIR gene evolution, creating or amplifying structural diversity of the locus (25,95). Although our previous analysis of an independent cohort from Zhejiang Han identified similar frequencies of individual KIR genes (60), it was not previously possible to detect any additional structural diversity. In the present analysis of Zhejiang Han, which accounted for gene copy number in addition to gene presence, haplotypes carrying a KIR3DP1-2DL4-3DL1/S1 insertion were observed in six individuals. Acquisition of full sequence data also permitted inference of the allele composition of these haplotypes. Similar haplotypes to those characterized here have been identified in European, Oceanian and other Asian populations, but are very rare in Africans (17). We identified a unique contracted haplotype lacking the telomeric segment, retaining only KIR2DL4 as the 3' terminal gene. The capture probes we used can tolerate up to 20% sequence divergence (33) and their high density in the target region would imply that we did not miss some highly divergent sequences from this individual. That the allele is KIR2DL4*00102, indicates that this contracted haplotype was formed from the most frequent KIR-A haplotype observed in the Zhejiang Han. As these recombination events most likely occur during meiosis, it remains unlikely that we will observe the reciprocal haplotype formed during that recombination event. That the more frequent structurally divergent haplotypes we observed are all characterized by recombination in approximately similar locations to each other, therefore suggests similar mechanisms or additional recombination hotspots abound in the KIR locus.
In conclusion, characterizing combinatorial diversity of KIR and HLA at high resolution in discrete populations is an important step toward understanding the roles of their polymorphism in disease and in human evolution. The mean number and diversity of KIR and HLA interactions in Zhejiang Han is similar to that of other East Asians, yet highly distinct from African, European and Oceanic populations. These findings will be important for analyzing diseases with susceptibilities that vary across populations and will form a baseline for individualizing immune-based therapies, including transplantation, in Southeastern China.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Blood Center of Zhejiang Province. The patients/ participants provided their written informed consent to participate in this study.

ACKNOWLEDGMENTS
We thank all the participants of this study for their generous donation of DNA to facilitate genetic research. Supplementary Table 4 | Distribution of (A) HLA-A, -B, -C alleles and (B) HLA-A-B-C haplotypes in 176 Zhejiang Han individuals. KIR ligands are colored according to Figure 6A, and those expressing -21M are indicated with orange shading.