Lisbeth A. Guethlein

Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia

Killer-cell immunoglobulin-like receptor (KIR) genes form a diverse, immunogenetic system. Group A and B KIR haplotypes have distinctive centromeric (Cen) and telomeric (Tel) gene-content motifs. Aiming to develop a donor selection strategy to improve transplant outcome, we compared the contribution of these motifs to the clinical benefit conferred by B haplotype donors. We KIR genotyped donors from 1409 unrelated transplants for acute myelogenous leukemia (AML; n = 1086) and acute lymphoblastic leukemia (ALL; n = 323). Donor KIR genotype influenced transplantation outcome for AML but not ALL. Compared with A haplotype motifs, centromeric and telomeric B motifs both contributed to relapse protection and improved survival, but Cen-B homozygosity had the strongest independent effect. With Cen-B/B homozygous donors the cumulative incidence of relapse was 15.4% compared with 36.5% for Cen-A/A donors (relative risk of relapse 0.34; 95% confidence interval 0.2-0.57; P < .001). Overall, significantly reduced relapse was achieved with donors having 2 or more B gene-content motifs (relative risk 0.64; 95% confidence interval 0.48-0.86; P = .003) for both HLA-matched and mismatched transplants. KIR genotyping of several best HLA-matched potential unrelated donors should substantially increase the frequency of transplants by using grafts with favorable KIR gene content. Adopting this practice could result in superior disease-free survival for patients with AML.

Donors with group B KIR haplotypes improve relapse-free survival after unrelated hematopoietic cell transplantation for acute myelogenous leukemia

Survival for patients with acute myeloid leukemia (AML) is limited by treatment-related mortality (TRM) and relapse after unrelated donor (URD) hematopoietic cell transplantation (HCT). Natural killer (NK)-cell alloreactivity, determined by donor killer-cell immunoglobulin-like receptors (KIRs) and recipient HLA, correlates with successful HCT for AML. Hypothesizing that donor KIR genotype (A/A: 2 A KIR haplotypes; B/x: at least 1 B haplotype) would affect outcomes, we genotyped donors and recipients from 209 HLA-matched and 239 mismatched T-replete URD transplantations for AML. Three-year overall survival was significantly higher after transplantation from a KIR B/x donor (31% [95% CI: 26-36] vs 20% [95% CI: 13-27]; P = .007). Multivariate analysis demonstrated a 30% improvement in the relative risk of relapse-free survival with B/x donors compared with A/A donors (RR: 0.70 [95% CI: 0.55-0.88]; P = .002). B/x donors were associated with a higher incidence of chronic graft-versus-host disease (GVHD; RR: 1.51 [95% CI: 1.01-2.18]; P = .03), but not of acute GVHD, relapse, or TRM. This analysis demonstrates that unrelated donors with KIR B haplotypes confer significant survival benefit to patients undergoing T-replete HCT for AML. KIR genotyping of prospective donors, in addition to HLA typing, should be performed to identify HLA-matched donors with B KIR haplotypes.

The Shaping of Modern Human Immune Systems by Multiregional Admixture with Archaic Humans

Viral defense and embryo implantation mechanisms have been shaped by contributions from Neandertal and Denisovan genes. Whole genome comparisons identified introgression from archaic to modern humans. Our analysis of highly polymorphic human leukocyte antigen (HLA) class I, vital immune system components subject to strong balancing selection, shows how modern humans acquired the HLA-B*73 allele in west Asia through admixture with archaic humans called Denisovans, a likely sister group to the Neandertals. Virtual genotyping of Denisovan and Neandertal genomes identified archaic HLA haplotypes carrying functionally distinctive alleles that have introgressed into modern Eurasian and Oceanian populations. These alleles, of which several encode unique or strong ligands for natural killer cell receptors, now represent more than half the HLA alleles of modern Eurasians and also appear to have been later introduced into Africans. Thus, adaptive introgression of archaic alleles has significantly shaped modern human immune systems.

Allelic Polymorphism Synergizes with Variable Gene Content to Individualize Human KIR Genotype

Killer Ig-like receptor (KIR) genes are a multigene family on human chromosome 19. KIR genes occur in various combinations on different haplotypes. Additionally, KIR genes are polymorphic. To examine how allelic polymorphism diversifies KIR haplotypes with similar or identical combinations of KIR genes, we devised methods for discriminating alleles of KIR2DL1, -2DL3, -3DL1, and -3DL2. These methods were applied to 143 individuals from 34 families to define 98 independent KIR haplotypes at the allele level. Three novel 3DL2 alleles and a chimeric 3DL1/3DL2 sequence were also identified. Among the A group haplotypes were 22 different combinations of 2DL1, 2DL3, 3DL1, and 3DL2 alleles. Among the B group haplotypes that were unambiguously determined were 15 distinct haplotypes involving 9 different combinations of KIR genes. A and B haplotypes both exhibit strong linkage disequilibrium (LD) between 2DL1 and 2DL3 alleles, and between 3DL1 and 3DL2 alleles. In contrast, there was little LD between the 2DL1/2DL3 and 3DL1/3DL2 pairs that define the two halves of the KIR gene complex. The synergistic combination of allelic polymorphism and variable gene content individualize KIR genotype to an extent where unrelated individuals almost always have different KIR types. This level of diversity likely reflects strong pressure from pathogens on the human NK cell response.

Different NK Cell Surface Phenotypes Defined by the DX9 Antibody Are Due to KIR3DL1 Gene Polymorphism

KIR3DL1 and KIR3DL2 are NK cell receptors for polymorphic HLA-B and -A determinants. The proportion of NK cells that bind anti-KIR3DL1-specific Ab DX9 and their level of binding vary between individuals. To determine whether these differences are due to KIR polymorphism, we assessed KIR3D gene diversity in unrelated individuals and families. Both KIR3DL1 and KIR3DL2 are highly polymorphic genes, with KIR3DS1 segregating like an allele of KIR3DL1. A KIR haplotype lacking KIR3DL1 and KIR3DS1 was defined. The two KIR3DL1 alleles of a heterozygous donor were expressed by different, but overlapping, subsets of NK cell clones. Sequence variation in KIR3DL1 and KIR3DL2 appear distinct; recombination is more evident in KIR3DL1, and point mutation is more evident in KIR3DL2. The KIR3DL1 genotype correlates well with levels of DX9 binding by NK cells, but not with the frequency of DX9-binding cells. Different KIR3DL1 alleles determine high, low, and no binding of DX9 Ab. Consequently, heterozygotes for high and low binding KIR3DL1 alleles have distinct subpopulations of NK cells that bind DX9 at high and low levels, giving characteristic bimodal distributions in flow cytometry. The Z27 Ab gave binding patterns similar to those of DX9. Four KIR3DL1 alleles producing high DX9 binding phenotypes were distinguished from four alleles producing low or no binding phenotypes by substitution at one or more of four positions in the encoded protein: 182 and 283 in the extracellular Ig-like domains, 320 in the transmembrane region, and 373 in the cytoplasmic tail.

Genetic and Environmental Determinants of Human NK Cell Diversity Revealed by Mass Cytometry

Both genetics and environment contribute to human NK cell diversity. NK Cell Nature Versus Nurture Natural killer (NK) cells were first discovered because of their ability to kill tumor cells without any previous exposure. However, this population is actually quite heterogeneous: Different subgroups of NK cells express different combinations of activating and inhibiting receptors that govern their specificity. Now, Horowitz et al. use mass cytometry to examine NK cell diversity in humans. The authors examined 35 parameters simultaneously in 5 sets of monozygotic twins as well as 12 unrelated donors. They found up to 30,000 phenotypic NK cell populations in a given individual. What’s more, by comparing the twins versus unrelated donors, they determined that although genetics primarily determined inhibitory receptor expression, activating receptors were controlled by the environment. These data suggest that inhibitory receptors may contribute more to NK cell self-tolerance, whereas activating receptors may guide response to pathogens and tumors. Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.

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

Abstract. 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.

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.

Different patterns of evolution in the centromeric and telomeric regions of group A and B haplotypes of the human killer cell Ig-like receptor locus.

The fast evolving human KIR gene family encodes variable lymphocyte receptors specific for polymorphic HLA class I determinants. Nucleotide sequences for 24 representative human KIR haplotypes were determined. With three previously defined haplotypes, this gave a set of 12 group A and 15 group B haplotypes for assessment of KIR variation. The seven gene-content haplotypes are all combinations of four centromeric and two telomeric motifs. 2DL5, 2DS5 and 2DS3 can be present in centromeric and telomeric locations. With one exception, haplotypes having identical gene content differed in their combinations of KIR alleles. Sequence diversity varied between haplotype groups and between centromeric and telomeric halves of the KIR locus. The most variable A haplotype genes are in the telomeric half, whereas the most variable genes characterizing B haplotypes are in the centromeric half. Of the highly polymorphic genes, only the 3DL3 framework gene exhibits a similar diversity when carried by A and B haplotypes. Phylogenetic analysis and divergence time estimates, point to the centromeric gene-content motifs that distinguish A and B haplotypes having emerged ∼6 million years ago, contemporaneously with the separation of human and chimpanzee ancestors. In contrast, the telomeric motifs that distinguish A and B haplotypes emerged more recently, ∼1.7 million years ago, before the emergence of Homo sapiens. Thus the centromeric and telomeric motifs that typify A and B haplotypes have likely been present throughout human evolution. The results suggest the common ancestor of A and B haplotypes combined a B-like centromeric region with an A-like telomeric region.

Modes of Salmonid MHC Class I and II Evolution Differ from the Primate Paradigm

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15–20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II β-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles α1 and α2 domains into different combinations.