Predominance of group A KIR haplotypes in Japanese associated with diverse NK cell repertoires of KIR expression

Genomic DNA from a panel of 41 healthy unrelated Japanese individuals was typed for the presence or absence of 16 KIR genes and pseudogenes. Only eight different KIR genotypes were found. Group A haplotypes outnumbered group B haplotypes in frequency by approximately 3:1, with individuals having two group A haplotypes accounting for 56% of the panel. The frequency of A haplotypes in the Japanese is higher than that observed in other populations. Flow cytometric comparison of KIR expression in 19 panel members showed considerable diversity in NK cell repertoire, which was also seen within the group of individuals having two A haplotypes. This diversity is likely due to allelic polymorphism in expressed genes of the A haplotype. In comparison to other populations, the Japanese appear less heterogeneous in KIR genotype as assessed by gene content.     

The impact of using related individuals for haplotype reconstruction in population studies

Recent studies have highlighted the dangers of using haplotypes reconstructed directly from population data for a fine-scale mapping analysis. Family data may help resolve ambiguity, yet can be costly to obtain. This study is concerned with the following question: How much family data (if any) should be used to facilitate haplotype reconstruction in a population study? We conduct a simulation study to evaluate how changes in family information can affect the accuracy of haplotype frequency estimates and phase reconstruction. To reconstruct haplotypes, we introduce an EM-based algorithm that can efficiently accommodate unrelated individuals, parent-child trios, and arbitrarily large half-sib pedigrees. Simulations are conducted for a diverse set of haplotype frequency distributions, all of which have been previously published in empirical studies. A wide variety of important results regarding the effectiveness of using pedigree data in a population study are presented in a coherent, unified framework. Insight into the different properties of the haplotype frequency distribution that can influence experimental design is provided. We show that a preliminary estimate of the haplotype frequency distribution can be valuable in large population studies with fixed resources.     

Evolution of killer cell Ig-like receptor (KIR) genes: definition of an orangutan KIR haplotype reveals expansion of lineage III KIR associated with the emergence of MHC-C

Orangutan (Pongo pygmaeus) MHC-C appears less evolved than human HLA-C: Popy-C is not fixed and its alleles encode only one (C1) of the two motifs for killer cell Ig-like receptor (KIR) ligands. To assess the structure and complexity of the orangutan KIR locus, the complete nucleotide sequence of an orangutan KIR haplotype was determined. The PopyKIR locus is flanked by LILR and FCAR and consists of seven genes and pseudogenes, two novel and five corresponding to known cDNA. Distinguishing all KIRs in this rapidly evolving KIR locus from the KIR3DX1 gene is an LTR33A/MLT1D element in intron 3. These two forms of KIR represent lineages that originated by duplication of a common ancestor. The conserved, framework regions of primate KIR loci comprise the 5' part of a lineage V KIR, the 3' part of a pseudogene, the complete 2DL4 gene, and the 3' part of a lineage II KIR. Although previously defined PopyKIR2DL4 alleles contain premature termination codons, the sequenced haplotype's PopyKIR2DL4 allele encodes a full-length protein. A model for KIR evolution is proposed. Distinguishing the orangutan KIR haplotype from the proposed common ancestor of primate KIR haplotypes is an increased number to give three lineage III KIR genes in the centromeric part of the locus, the site for most human lineage III genes encoding HLA-C specific KIR. Thus, expansion of lineage III KIR is associated with emergence of MHC-C.     

Independent control of Ly49g alleles: implications for NK cell repertoire selection and tumor cell killing

A novel murine NK cell-reactive mAb, AT8, was generated. AT8 recognizes Ly49G from 129/J, BALB/c, and related mouse strains, but does not bind to Ly49G(B6). Costaining with AT8 and a Ly49G(B6)-restricted Ab (Cwy-3) provides the first direct evidence that Ly49G protein is expressed from both alleles on a significant proportion of NK cells from four different types of F(1) hybrid mice. The observed level of biallelic Ly49G expression reproducibly followed the product rule in both freshly isolated and cultured NK cells. Surprisingly, the percentage of NK cells expressing both Ly49G alleles could be dramatically increased in vitro and in vivo through IL-2R- and IFN receptor-dependent signaling pathways, respectively. Unexpectedly, Ly49G(B6+) NK cells in an H-2(d), but not H-2(b), background were more likely to lyse D(d+) and Chinese hamster ovary tumor cells than Ly49G(BALB/129+) NK cells. Furthermore, Ly49G(B6+) NK cells also proliferated to a higher degree in response to poly(I:C) than NK cells expressing a non-Ly49G(B6) allele in an H-2(d), but not H-2(b), background. These results suggest that Ly49G(B6) has a lower affinity for H-2D(d) than Ly49G(BALB/129), and the genetic background calibrates the responsiveness of NK cells bearing self-specific Ly49. Other H-2D(d) receptors on the different Ly49G(+) NK cell subsets were unequally coexpressed, possibly explaining the disparate responses of Ly49G(B6+) NK cells in different hybrid mice. These data indicate that the stochastic mono- and biallelic expression of divergent Ly49G alleles increases the range of MHC affinities and the functional potential in the total NK cell population of heterozygous mice.     

A distinct subset of human CD4+ cells with a limited alloreactive T cell receptor repertoire

We describe a subset of CD4+/CD3+ human T lymphocytes that demonstrated a remarkably limited TCR repertoire responding to alloantigen stimulation. These cells have been characterized previously by their granular morphology and expression of CD11b but not CD28. Whereas multiple CD28+/CD4+ alloproliferative cloned cell lines generated by culture at limiting dilution immediately after isolation from peripheral blood each had a unique TCR-beta gene rearrangement, 19 of 21 CD11b+/CD4+ clones showed identical TCR-beta, and gamma gene rearrangements. In conventional MLR, the CD11b+/CD4+ cells responded poorly after stimulation with some HLA-class II Ag, and staining with a TCR Id-specific antibody and DNA blot hybridization suggested that the responding CD11b+/CD4+ cells typically contained predominant clonal populations. Clones of CD11b+/CD4+ cells with different TCR gene rearrangements showed closely similar patterns of responses when stimulated by a panel of allogeneic PBMC, but the response pattern did not correspond to that of any known HLA-class II Ag. These findings indicate that CD11b+/CD4+ cells have a limited alloproliferative repertoire characterized by predominant recognition of a limited number of undefined determinants that appear to be expressed in association with multiple distinct HLA-class II Ag. Our results suggest that CD11b+/CD4+ cells are selected for clonal reactivity by processes distinct from those for CD28+/CD4+ cells.     

Prevalence of diabetes mellitus in individuals heterozygous and homozygous for sickle cell anemia

Colorimetric determinations of glycosylated Hb were carried out in a sample (n = 97) of sickle cell anemia patients, and in an age- and sex-matched group of individuals (n = 45) heterozygous for sickle cell anemia, from the Eastern Province of Saudi Arabia. A statistically significant increase in the value of glycosylated Hb was found in sickle cell trait (HbAS) group, when compared with those of sickle cell anemia (HbSS) and normal (HbAA) groups. Since glycosylated Hb is considered a valid indicator of long-term blood glucose, and assuming normal red blood cell survival in HbAS carriers, the increased value of glycosylated Hb may suggest that there exists a higher incidence of undiagnosed diabetes mellitus in individuals with heterozygous inheritance for sickle cell hemoglobin than homozygous sickle cell patients and normal individuals. The mechanism underlying this observation remains to be defined.     

Generation of the alloreactive T cell repertoire: K region homology between H-2b T cell precursors and T cell maturation environment is required for the generation of the Kbm6-specific cytotoxic T cell repertoire

The influence of T cell genotype and T cell maturation environment on the generation of the T cell alloreactive repertoire was evaluated in the H-2b cytotoxic T lymphocyte response to Kb mutant determinants expressed by the strain B6-H-2bm6. Specifically, by constructing radiation bone marrow chimeras with B6 or B10 (H-2b) donor cells and B10.BR, B10.A(4R), B10.MBR, and B6.C-H-2bm1 irradiated mice as recipients, it was possible to investigate the major histocompatibility complex (MHC)-encoded gene products of the host environment required for the generation of a bm6-specific H-2b CTL response. The results of such experiments confirmed the previous finding that the alloreactive T cell repertoire is influenced both by T cell MHC genotype and by the MHC gene products of the T cell maturation environment. In addition, the results of the present study further demonstrated that in the chimeric donor and host genetic combinations used, it was both necessary and sufficient that there be a homology of K region-encoded determinants for the generation of a bm6-specific CTL response. Experiments utilizing a mixed responder population of unresponsive B6----B10.D2 spleen cells and responsive Lyt-2 congenic B6.Lyt-2.1 spleen cell suggested that the cellular defect(s) underlying the unresponsiveness of the chimeric cells to bm6-encoded determinants was at the level of the CTL precursor. Together, these findings indicate that an interaction of the K region-encoded gene products of the T cell and its maturation environment play a critical role in the generation of the CTL repertoire specific for bm6 mutant determinants. We discuss here the possibility that this interaction may reflect a requirement that T cells recognize such mutant allodeterminants in association with self restriction elements present on the same mutant K region-encoded molecule.     

Diversity of NK cell receptor repertoire in adult and neonatal mice.

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.     

KIR2DS1-positive NK cells mediate alloresponse against the C2 HLA-KIR ligand group in vitro

The inhibitory 2DL1 and activating 2DS1 killer Ig-like receptors (KIR) both have shared ligand specificity for codon sequences in the C2 group HLA-Cw Ags. In this study, we have investigated NK cell activation by allogeneic target cells expressing different combinations of the HLA-KIR ligand groups C1, C2, and Bw4. We demonstrate that fresh NK cells as well as IL-2-propagated NK cells from 2DS1-positive donors that are homozygous for the C1 ligand group are activated in vitro by B lymphoblastoid cell lines expressing the C2 group. This response is, in part, due to the absence of C1 group recognition mediated by the inhibitory receptor 2DL2/3. This "missing self" alloresponse to C2, however, is rarely observed in NK cells from donors lacking 2DS1. Even in presence of 2DS1, the NK alloresponse is dramatically reduced in donors that have C2 group as "self." Analysis of selected NK clones that express 2DS1 mRNA and lack mRNA for 2DL1 demonstrates that activation by the C2 ligand and mAb cross-linking of 2DS1 in these clones induces IFN-gamma. Furthermore, this C2 group-induced activation is inhibited by Abs to both HLA class I and the receptor. Collectively, these studies demonstrate that NK cells from 2DS1-positive donors are activated by target cells that express the C2 group as an alloantigen. This leads to increased IFN-gamma-positive fresh NK cells and induces NK allocytotoxicity in IL2-propagated polyclonal NK cells and NK clones. This study also provides support for the concept that incompatibility for the HLA-KIR ligand groups C1, C2, and Bw4 dominates NK alloactivation in vitro.     

A novel approach of homozygous haplotype sharing identifies candidate genes in autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (~90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data.     

Multilevel selection 3: modeling the effects of interacting individuals as a function of group size

Bijma et al. (2007a,b) presented a quantitative genetic theory of multilevel selection and showed how to estimate the relevant parameters using standard restricted maximum-likelihood (REML) methodology. Extending their results we develop a wider class of models that provide a more realistic framework for capturing the effects of interacting individuals. These models also make use of standard REML techniques and include the original model as a special case.     

Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination.

Viewing the immune system as a molecular recognition device designed to identify “foreign shapes”, we estimate the probability that an immune system with N_Ab monospecific antibodies in its repertoire can recognize a random foreign antigen. Furthermore, we estimate the improvement in recognition if antibodies are multispecific rather than monospecific. From our probabilistic model we conclude: (1) clonal selection is feasible, i.e. with a finite number of antibodies an animal can recognize an effectively infinite number of antigens; (2) there should not be great differences in the specificities of antibody molecules among different species; (3) the region of a foreign molecule recognized by an antibody must be severely limited in extent; (4) the probability of recognizing a foreign molecule, P, increases with the antibody repertoire size N_Ab; however, below a certain value of N_Ab the immune system would be very ineffectual, while beyond some high value of N_Ab further increases in N_Ab yield diminishing small increases in P; (5) multispecificity is equivalent to a modest increase (probably less than 10) in the antibody repertoire size N_Ab, but this increase can substantially improve the probability of an immune system recognizing a foreign molecule.Besides recognizing foreign molecules, the immune system must distinguish them from self molecules. Using the mathematical theory of reliability we argue that multisite recognition is a more reliable method of distinguishing between molecules than single site recognition. This may have been an important evolutionary consideration in the selection of weak non-covalent interactions as the basis of antigen-antibody bonds.     

Estimating the proportion of identifiable individuals and group sizes in photographic identification studies

Simple Bayesian statistical models are introduced to estimate the proportion of identifiable individuals and group sizes in photographic identification, or photo‐ID, studies of animals that are found in groups. The models require a simple random photographic sampling of animals, where the photographic captures are treated as sampling with replacement within each group. The total number of images, including those that cannot be identified, and the number of images that contain identifiable individuals are used to make inference about the proportion of identifiable individuals within each group and as the population when a number of groups are sampled. The numbers of images for individuals within each group are used to make inference about the group size. Based on analyses of simulated and real data, the models perform well with respect to accuracy and precision of posterior distributions of the parameters. Widths of posterior intervals were affected by the number of groups sampled, sampling duration, and the proportion of identifiable individuals in each group that was sampled. The structure of the models can accommodate covariates, which may affect photographic efficiency, defined in this study as the probability of photographically capturing individuals.     

Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals

The deterioration in immune function with aging is thought to make a major contribution to the increased morbidity and mortality from infectious disease in old age. One aspect of immune senescence is the reduction in CD8 T cell repertoire as due to the accumulation of oligoclonal, memory T cells and a reduction in the naive T cell pool. CD8 T cell clonal expansions accumulate with age, but their antigenic specificity remains unknown. In this study, we show that in elderly individuals seropositivity for human CMV leads to the development of oligoclonal populations of CMV-specific CTL that can constitute up to one-quarter of the total CD8 T cell population. Furthermore, CMV-specific CTL have a highly polarized membrane phenotype that is typical of effector memory cells (CD28(-), CD57(+), CCR7(-)). TCR analyses show that CMV-specific CTL have highly restricted clonality with greater restriction in the larger expansions. Clonal analysis of the total CD8 T cell repertoire was compared between CMV-seropositive and CMV-seronegative donors. Thirty-three percent more clonal expansions were observed in CMV-seropositive donors in comparison with seronegative individuals. These data implicate CMV as a major factor in driving oligoclonal expansions in old age. Such a dramatic accumulation of virus-specific effector CTL might impair the ability to respond to heterologous infection and may underlie the negative influence of CMV seropositivity on survival in the very elderly.     

Rescue of embryonic cells homozygous for a lethal haplotype of the T/t complex: tw12

A series of recessive mutations which arrest embryonic development are located within the T/t region of chromosome 17 in the mouse. To assess whether these mutations cause death in specific differentiating cells or in all cells of the embryo, we removed the embryonic cells from normal developmental constraints and attempted to grow them ectopically in vivo and in vitro. We have succeeded in producing teratomas and teratocarcinomas by transplantation of inner cell masses from blastocysts of $\text{t}{}^{\mathrm{w12}}\text{+}$ and $\text{t}{}^{\mathrm{w12}}\text{t}{}^{\mathrm{w12}}$ genotypes. The ability of embryonic cells to grow as tumors was not affected by their genotype; 7 of the 17 tumors were homozygous for t^w12, 7 were heterozygous, and 3 could not be analyzed. Virtually all the tumors of both genotypes contained derivatives of all three germ layers. Neuroepithelial and mature nervous tissue was present in all homozygous tumors and all except one heterozygous tumor. However, no cartilage or bone was found in 5 of 5 t^w12 homozygous tumors, while both tissues were present in 3 of 4 t^w12 heterozygous tumors. This observation is compatible with the abnormalities characteristic of $\text{t}{}^{\mathrm{w12}}\text{t}{}^{\mathrm{w12}}$ embryos, which show very localized effects in nervous tissue and more general effects on bone and cartilage formation. Cells derived from homozygous tumors were capable of at least limited growth in culture and a cell line has been derived from one of them. The p63/6.9a marker protein was used to determine the presence of the t^w12 haplotype in the tumor and cultured cells. We conclude that the lethality associated with the t^w12 haplotype is due to lethality of specific cells, and not all cell types.     

Homology between an alloantigen and a self MHC allele calibrates the avidity of the alloreactive T cell repertoire independent of TCR affinity

The self-restricted T cell repertoire exhibits a high frequency of alloreactivity. Because these alloreactive T cells are derived from the pool of cells selected on several different self MHC alleles, it is unknown how development of the alloantigenic repertoire is influenced by homology between a self MHC allele and an alloantigen. To address this, we used the 2C transgenic TCR that is selected by K(b), is alloreactive for L(d), and cross-reacts with L(q). L(q) is highly homologous to L(d) and binds several of the same peptide ligands, including p2Ca, the peptide recognized by 2C. We find that L(d)/p2Ca is a high avidity agonist ligand, whereas L(q)/p2Ca is a low avidity agonist ligand for 2C T cells. When mice transgenic for the 2C TCR are bred to L(q)-expressing mice, 2C(+) T cells develop; however, they express lower levels of either the 2C TCR or CD8 and require a higher L(d)/p2Ca ligand density to be activated than 2C(+) T cells selected by K(b). Furthermore, the 2C T cells selected in the presence of L(q) fail to detect L(q)/p2Ca complexes even at high ligand density. Thus, despite possessing the identical TCR, there is a functional avidity difference between 2C(+) T cells selected in the presence of L(q) vs K(b). These data provide evidence that homology between the selecting ligand and an alloantigen can influence the avidity of the T cell repertoire for the alloantigen, and suggest that thymic selection can fine tune T cell avidity independent of intrinsic TCR affinity.     

The impact of thymic antigen diversity on the size of the selected T cell repertoire

The TCR repertoire of a normal animal is shaped in the thymus by ligand-specific positive- and negative-selection events. These processes are believed to be determined at the single-cell level primarily by the affinity of the TCR-ligand interactions. The relationships among all the variables involved are still unknown due to the complexity of the interactions and the lack of quantitative analysis of those parameters. In this study, we developed a quantitative model of thymic selection that provides estimates of the fractions of positively and negatively selected thymocytes in the cortex and in the medulla, as well as upper-bound ranges for the number of selecting ligands required for the generation of a normal diverse TCR repertoire. Fitting the model to current estimates of positive- and negative-selected thymocytes leads to specific predictions. The results indicate the following: 1) the bulk of thymocyte death takes place in the cortex, and it is due to neglect; 2) the probability of a thymocyte to be negatively selected in the cortex is at least 10-fold lower than in the medulla; 3) <60 ligands are involved in cortical positive selection; and 4) negative selection in the medulla is constrained by a large diversity of selecting ligands on medullary APCs.     

Plasmodium berghei NK65: studies on the effect of treatment duration and inoculum size on recrudescence

Recrudescence of Plasmodium berghei NK65 infection was studied to examine factors affecting recrudescence. Treatment with a high dose of chloroquine did not prevent recrudescence, but an extended duration of treatment suppressed the frequency of recrudescence. Infection with a larger number of parasites also resulted in more frequent recrudescences. Recrudescent parasites were as sensitive to chloroquine as those before treatment. Splenectomized mice were administered carbon particles, infected, and treated with chloroquine. Recrudescence was significantly more frequent in these mice than in mice given a sham operation and PBS. The results do not suggest that merozoite stages escape the effect of chloroquine by 'hiding' in phagocytes, but that latent parasites such as dormant ring stages may cause recrudescence.     

Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes

The segregation of killer cell immunoglobulin-like receptor ( KIR) genes was determined for a panel of 21 Caucasoid families: 23 different KIR gene patterns were found and could be assigned to combinations of 16 different haplotypes. Four loci were held in common by all haplotypes: KIR2DL4, KIR3DL2, the putative pseudogene KIR3DL3 and KIR2DL2/KIR2DL3, the latter likely being alleles of one gene. Group A haplotypes, which have a unique combination of seven KIR genes, were found at 80% frequency in the family panel, the polygenic group B haplotypes at 65% frequency. KIR gene segregation was fully determined for the nine group B haplotypes, which occurred at highest frequencies in both the family panel and a panel of unrelated individuals. The group B haplotypes carried between seven and 11 KIR genes and encoded inhibitory KIR for one, two, or all three major HLA class I epitopes. Analysis of human leucocyte antigen (HLA) class I genotypes revealed that most, but not all, individuals possess an inhibitory KIR for a self HLA class I epitope. The number of stimulatory KIR genes in group B haplotypes varied considerably between one and five. The data show that group B haplotypes possess a broad spectrum of KIR gene patterns, which is largely complementary to the KIR gene set of group A haplotypes. The results suggest that rapid diversification of group B haplotypes is the result of pathogen-mediated selection for KIR genotypes that have more than the set of KIR genes provided by the group A haplotype.     

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.