Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes.

Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric "half" was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family.

Donor-recipient combinations of group A and B KIR haplotypes and HLA class I ligand affect the outcome of HLA-matched, sibling donor hematopoietic cell transplantation.

The influence of donor and recipient killer immunoglobulin-like receptor (KIR) genotype on the outcome of hematopoietic cell transplantation between human leukocyte antigen (HLA)-matched siblings was investigated. Transplants were divided into four groups according to the combination of group A and B KIR haplotypes in the transplant donor and recipient. Overall survival of myeloid patients varied with KIR genotype combination. Best survival was associated with the donor lacking and the recipient having group B KIR haplotypes; poorest survival was associated with the donor having and the recipient lacking group B KIR haplotypes. The latter combination was also associated with increased relapse and acute graft-versus-host disease (GVHD). However, its detrimental effects were seen only for transplants where the recipient and donor were homozygous for the C1 KIR ligand and therefore lacked the C2 ligand. Presence of the Bw4 ligand was also associated with increased acute GVHD. In contrast presence of both KIR3DL1 and its cognate Bw4 ligand was associated with decreased nonrelapse mortality. Analysis of the KIR genes individually revealed KIR2DS3 as a protective factor for chronic GVHD. The results suggest how simple assessments of KIR genotype might inform the selection of donors for hematopoietic cell transplantation.

Multiple unit HLA-unmatched sex-mismatched umbilical cord blood transplantation for advanced hematological malignancy.

We investigated the effect of multiple-unit umbilical cord blood (UCB) transplantation on engraftment in the setting of severe human leukocyte antigen (HLA) mismatch. Ten poor-risk adult patients with hematological malignancy received multiple unit, HLA-unmatched, sex-mismatched, unrelated UCB transplantation after a reduced intensity-conditioning regimen (RICR) with engraftment as the primary endpoint. The median age of the patients was 55 years with a range of 28-67. Patients received one unit of UCB per 10 kg of recipient body weight (5-7 units). The median number of nucleated cells and CD34(+) cells per kilogram of recipient body weight infused was 6.3 x 10(7) (range 3.8-10.0) (NC/kg) and 5.7 x 10(5) (range 1.1-11.9) (CD34/kg), respectively. Three patients expired before day 28 and were not evaluable for engraftment. Five of the remaining 7 patients showed increasing neutrophil counts. Fluorescent in situ hybridization (FISH) for the Y chromosome or HLA-typing showed only donor cells in the peripheral blood. After engraftment, HLA typing was done on 3 patients and their infused UCB units. All revealed the presence of a single HLA type concordant with one of the infused units. Moreover, the order of infusion did not influence which UCB unit engrafted. The engrafting UCB units were infused first or second in one case and fourth in the other two. One patient transplanted for refractory acute lymphoblastic leukemia (ALL) survives in continuous complete remission 4 years after transplant. He engrafted with one UCB unit, is fully hematologically reconstituted, has no evidence of graft-versus-host disease (GVHD), and takes no immunosuppressive medication. HLA typing reveals that the recipient and the engrafted cord blood match at only one HLA-B locus using conventional 6 antigen typing (A, B, and DR). Although engraftment was not accelerated, it did occur in the majority of evaluable patients. Long-term disease-free survivorship without debilitating GVHD is possible in patients with refractory hematological malignancy who receive unmatched multiple unit UCB.

A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature.

How receptor acquisition correlates with the functional maturation of natural killer (NK) cells is poorly understood. We used quantitative real-time polymerase chain reaction (PCR) assays to compare NKG2 and killer immunoglobulin-like receptor (KIR) gene expression in NK cells from allogeneic transplant recipients and their donors. Marked differences were observed in the NK subsets of recipients who had 8-fold more CD56(bright) cells, diminished KIR expression (except 2DL4), and increased NKG2A. In normal blood not all CD56(dim) cells express KIR, and a novel subpopulation of cells committed to the NK-cell lineage was defined. These cells, which comprise 19.4% +/- 2.8% of the CD56(dim) NK population in healthy donors, express the activating NKG2D and NKG2E receptors but no KIR or NKG2A. Although the CD56(dim) NKG2A(-) KIR(-) NK cells lack "at least one" inhibitory receptor for autologous MHC class I, they are not fully responsive, but rather functionally immature cells with poor cytotoxicity and IFN-gamma production. Upon culture with IL-15 and a stromal cell line, CD56(dim) and CD56(bright) KIR(-) NK cells proliferate, express KIR, and develop cytotoxicity and cytokine-producing potential. These findings have implications for the function of NK cells reconstituting after transplantation and support a model for in vivo development in which CD56(bright) cells precede CD56(dim) cells.

Missing KIR ligands are associated with less relapse and increased graft-versus-host disease (GVHD) following unrelated donor allogeneic HCT.

Natural killer (NK) cells can alter the outcome of hematopoietic cell transplantation (HCT) if donor alloreactivity targets the recipient. Since most NK cells express inhibitory killer-immunoglobulin receptors (KIRs), we hypothesized that the susceptibility of recipient cells to donor NK cell-mediated lysis is genetically predetermined by the absence of known KIR ligands. We analyzed data from 2062 patients undergoing unrelated donor HCT for acute myeloid leukemia (AML; n = 556), chronic myeloid leukemia (CML; n = 1224), and myelodysplastic syndrome (MDS; n = 282). Missing 1 or more KIR ligands versus the presence of all ligands protected against relapse in patients with early myeloid leukemia (relative risk [RR] = 0.54; n = 536, 95% confidence interval [CI] 0.30-0.95, P = .03). In the subset of CML patients that received a transplant beyond 1 year from diagnosis (n = 479), missing a KIR ligand independently predicted a greater risk of developing grade 3-4 acute graft-versus-host disease (GVHD; RR = 1.58, 95% CI 1.13-2.22; P = .008). These data support a genetically determined role for NK cells following unrelated HCT in myeloid leukemia.

The protein made from a common allele of KIR3DL1 (3DL1*004) is poorly expressed at cell surfaces due to substitution at positions 86 in Ig domain 0 and 182 in Ig domain 1.

KIR3DL1 is an inhibitory HLA-B receptor of human NK and T cells that exhibits genetic and phenotypic polymorphism. KIR3DL1*004, a common allotype, cannot be detected on the surface of PBLs using the KIR3DL1-specific Ab DX9. The nature of this phenotype was investigated through comparison of 3DL1*004 with 3DL1*002, an allele giving high DX9 binding to cell surfaces. Analysis of Jurkat T cell transfectants with 3DL1*004 cDNA showed that 3DL1*004 is poorly expressed at the cell surface, but detectable intracellularly. Analysis of recombinant mutants made between 3DL1*004 and 3DL1*002 showed that polymorphism in Ig domains 0 and 1 (D0 and D1) causes the intracellular retention of 3DL1*004. Reciprocal point mutations were introduced into 3DL1*004 and 3DL1*002 at positions 44 and 86 of the D0 domain, where 3DL1*004 has unique residues, and at position 182 of the D1 domain, where 3DL1*004 resembles 3DL1*005, an allotype giving low DX9-binding phenotype. Leucine 86 in 3DL1*004 is the principal cause of its intracellular retention, with a secondary and additive contribution from serine 182. By contrast, glycine 44, which is naturally present in 3DL1*004, slightly increased cell surface expression when introduced into 3DL1*002. In 3DL1*004, the presence of leucine at position 86 corrupts the WSXPS motif implicated in proper folding of the KIR D0 Ig-like domain. This study demonstrates how a difference between KIR3DL1 allotypes in the D0 domain profoundly affects cell surface expression and function.

Activation of a subset of human NK cells upon contact with Plasmodium falciparum-infected erythrocytes.

Human NK cells are the earliest source of the protective cytokine IFN-gamma when PBMC from nonimmune donors are exposed to Plasmodium falciparum-infected RBC (iRBC) in vitro. In this study, we show that human NK cells form stable conjugates with iRBC but not with uninfected RBC and that induction of IFN-gamma synthesis is dependent on direct contact between the NK cell and the iRBC. NK cells respond to iRBC only in the presence of a source of IL-12/IL-18 and the subset of NK cells that preferentially respond to iRBC express high levels of the lectin-like receptor CD94/NKG2A. There is heterogeneity between donors in their ability to respond to iRBC. DNA analysis has revealed considerable heterogeneity of killer Ig-like receptor (KIR) genotype among the donor population and has identified 21 new KIR allelic variants in the donors of African and Asian descent. Importantly, we find evidence for significant associations between KIR genotype and NK responsiveness to iRBC. This emphasizes the need for large-scale population-based studies to address associations between KIR genotype and susceptibility to malaria.

Alloreactive killer cells: hindrance and help for haematopoietic transplants.

Haematopoietic-cell transplantation is a treatment for leukaemia and lymphoma. To reduce the incidence of graft-versus-host disease (GVHD) caused by transplanted T cells, donors and recipients are HLA matched. For patients for whom a matched donor is not available, one option is transplantation from an HLA-mismatched relative who shares one HLA haplotype. This procedure is distinguished by the use of a stronger conditioning regimen for the patient and of a T-cell-depleted graft containing numerous stem cells. After transplantation, natural killer cells are prevalent, and they can include alloreactive cells that kill tumour cells and prevent GVHD. The alloreactions seem to be determined by the mismatched HLA class I ligands and their killer-cell immunoglobulin-like receptors.

Reconstitution of NK cell receptor repertoire following HLA-matched hematopoietic cell transplantation.

Interactions between killer immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands influence development of natural killer (NK) cell repertoire and response to infection, cancer, and allogeneic tissue. As KIRs and HLA class I molecules are highly polymorphic, clinical allogeneic hematopoietic cell transplantation is predicted to frequently involve KIR mismatch, and thus to provide a unique system for study of human NK cell receptor repertoire development. Eighteen leukemia patients undergoing HLA-matched transplantation and their donors were analyzed for KIR genotype. Ten of 13 HLA-identical donor-patient pairs were KIR mismatched and 3 were matched; all HLA-matched unrelated pairs were KIR mismatched. Reconstitution of recipient NK cell repertoire following transplantation was examined using flow cytometry and monoclonal antibodies specific for KIR and CD94:NKG2A. These data form 3 groups. Six to 9 months after transplantation, 8 patients (group 1) reconstituted an NK cell repertoire resembling that of their donor, and for KIR-mismatched transplants, distinct from the recipient before transplantation. In the first year after transplantation, 5 patients (group 2) exhibited a generally depressed frequency of KIR-expressing NK cells and concomitant high frequency of CD94:NKG2A expression. By 3 years after transplantation, the frequency of KIR-expressing NK cells had increased to donor values, in the 3 patients from group 2 analyzed for this period. The remaining 5 patients experienced severe clinical complications following transplantation and displayed unique features in their NK cell receptor reconstitution. These results demonstrate that a majority of HLA-matched hematopoietic cell transplantations involve KIR mismatch and reveal differences in NK cell repertoire having potential impact for immune responsiveness and transplantation outcome.

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.

Variable receptors controlling activation and inhibition of NK cells.

NK cells are important effector lymphocytes of innate immunity; they kill infected cells and produce cytokines that stimulate other immune effects. Once considered relatively homogeneous, NK cells are now seen to be highly diverse. Within an individual, expression of different combinations of inhibitory and stimulatory receptors creates a diverse NK cell repertoire, which exhibits specificity in the immune response. Rapid evolution of NK cell receptor gene families distinguishes members of a species and causes substantial species-specific differences in NK cell receptor systems. All known ligands for these diverse receptors are MHC class I molecules, or molecules of host or pathogen origin that are homologous to MHC class I.

Evolution of NK receptors: a single Ly49 and multiple KIR genes in the cow.

Natural killer (NK) cell receptors for classical MHC class I molecules are encoded by the killer Ig-like receptor (KIR) multigene family in humans and other primates. Mouse NK cells, however, employ a completely different multigene family, the C-type lectin-like Ly49 genes, to perform the same function. This example of functional convergent evolution raises the question of what type of receptors are found in non-primate and non-rodent mammals. By screening a bovine spleen cDNA library, we isolated an Ly49 gene from the cow (Bos Taurus) and show by genomic Southern blotting that it is likely a single copy gene in this species. The coding region is intact and has an immunoreceptor tyrosine-based inhibition motif (ITIM) in the cytoplasmic domain, suggesting a role as an inhibitory receptor. We have also identified several bovine cDNA clones related to KIR and show that at least one has an intact open reading frame with two ITIM. Evidence for multiple KIR-like genes in the cow was obtained by Southern blotting and we found that at least two of these genes contain an ancient retro-element present in all human KIR genes. These results suggest that the cow and primate KIRgene families arose from a common ancestral gene but amplified independently. Furthermore, these findings indicate that the existence of multiple Ly49 genes may be a phenomenon unique to rodents.