Donor-directed human leukocyte antigen (HLA)-specific allo-antibodies (DSAs) cause graft failure in animal models of hematopoietic stem cell transplantation (HCT). Archived pretransplantation sera from graft failure patients (n ‫؍‬ 37) and a matched case-control cohort (n ‫؍‬ 78) were tested to evaluate the role of DSAs in unrelated donor HCT. Controls were matched for disease, disease status, graft type, patient age, and transplantation year.Patients had acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, or myelodysplastic syndrome; 98% received myeloablative conditioning regimens 100% received T-replete grafts, 97% received marrow, 95% HLA-mismatched, and 97% received calcineurin-based graft-versus-host disease prophylaxis. Among the 37 failed transplantations, 9 (24%) recipients possessed DSAs against HLA-A, B, and/or DP, compared with only 1 (1%) of 78 controls. Therefore, the presence of DSAs was significantly associated with graft failure (odds ratio ‫؍‬ 22.84; 95% confidence interval, 3.57-ؕ; P < .001). These results indicate that the presence of pretransplantation DSAs in recipients of unrelated donor HCT is associated with failed engraftment and should be considered in HCT donor selection. (Blood. 2010; 115(13):2704-2708)

Purpose Disease relapse remains a major challenge to successful outcomes in patients who undergo allogeneic hematopoietic cell transplantation (HCT). Donor natural killer (NK) cell alloreactivity in HCT can control leukemic relapse, but capturing alloreactivity in HLA-matched HCT has been elusive. HLA expression on leukemia cells-upregulated in the post-HCT environment-signals for NK cell inhibition via inhibitory killer immunoglobulin-like (KIR) receptors and interrupts their antitumor activity. We hypothesized that varied strengths of inhibition among subtypes of the ubiquitous KIR3DL1 and its cognate ligand, HLA-B, would titrate NK reactivity against acute myelogenous leukemia (AML). Patients and Methods By using an algorithm that was based on polymorphism-driven expression levels and specificities, we predicted and tested inhibitory and cytotoxic NK potential on the basis of KIR3DL1/HLA-B subtype combinations in vitro and evaluated their impact in 1,328 patients with AML who underwent HCT from 9/10 or 10/10 HLA-matched unrelated donors. Results Segregated by KIR3DL1 subtype, NK cells demonstrated reproducible patterns of strong, weak, or noninhibition by target cells with defined HLA-B subtypes, which translated into discrete cytotoxic hierarchies against AML. In patients, KIR3DL1 and HLA-B subtype combinations that were predictive of weak inhibition or noninhibition were associated with significantly lower relapse (hazard ratio [HR], 0.72; P = .004) and overall mortality (HR, 0.84; P = .030) compared with strong inhibition combinations. The greatest effects were evident in the high-risk group of patients with all KIR ligands (relapse: HR, 0.54; P < .001; and mortality: HR, 0.74; P < .008). Beneficial effects of weak and noninhibiting KIR3DL1 and HLA-B subtype combinations were separate from and additive to the benefit of donor activating KIR2DS1. Conclusion Consideration of KIR3DL1-mediated inhibition in donor selection for HLA-matched HCT may achieve superior graft versus leukemia effects, lower risk for relapse, and an increase in survival among patients with AML.

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5∼KIR2DS3S5 and KIR2DS3S5∼KIR2DL1, and telomeric KIR3DL1∼KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis.

The HLA gene complex on human chromosome 6 is one of the most polymorphic regions in the human genome and contributes in large part to the diversity of the immune system. Accurate typing of HLA genes with short-read sequencing data has historically been difficult due to the sequence similarity between the polymorphic alleles. Here, we introduce an algorithm, xHLA, that iteratively refines the mapping results at the amino acid level to achieve 99-100% four-digit typing accuracy for both class I and II HLA genes, taking only ∼3 min to process a 30× whole-genome BAM file on a desktop computer.MHC | autoimmune diseases | transplantation

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expression of mutant ataxin-1 that contains an expanded polyglutamine tract. Overexpression of mutant ataxin-1 in Purkinje cells of transgenic mice results in a progressive ataxia and Purkinje cell pathology that are very similar to those seen in SCA1 patients. Two prominent aspects of pathology in the SCA1 mice are the presence of cytoplasmic vacuoles and dendritic atrophy. We found that the vacuoles in Purkinje cells seem to originate as large invaginations of the outer cell membrane. The cytoplasmic vacuoles contained proteins from the somatodendritic membrane, including mGluR1, GluRDelta1/Delta2, GluR2/3, and protein kinase C (PKC) gamma. Further examination of PKCgamma revealed that its sequestration into cytoplasmic vacuoles was accompanied by concurrent loss of PKCgamma localization at the Purkinje cell dendritic membrane and decreased detection of PKCgamma by Western blot analysis. In addition, the vacuoles were immunoreactive for components of the ubiquitin/proteasome degradative pathway. These findings present a link between vacuole formation and loss of dendrites in Purkinje cells of SCA1 mice and indicate that altered somatodendritic membrane trafficking and loss of proteins including PKCgamma, are a part of the neuronal dysfunction in SCA1 transgenic mice.

The killer cell immunoglobulin-like receptor (KIR) region of human chromosome 19 contains up to 16 genes for natural killer (NK) cell receptors that recognize human leukocyte antigen (HLA)/peptide complexes and other ligands. The KIR proteins fulfill functional roles in infections, pregnancy, autoimmune diseases and transplantation. However, their characterization remains a constant challenge. Not only are the genes highly homologous due to their recent evolution by tandem duplications, but the region is structurally dynamic due to frequent transposon-mediated recombination. A sequencing approach that precisely captures the complexity of KIR haplotypes for functional annotation is desirable. We present a unique approach to haplotype the KIR loci using single-molecule, real-time (SMRT) sequencing. Using this method, we have—for the first time—comprehensively sequenced and phased sixteen KIR haplotypes from eight individuals without imputation. The information revealed four novel haplotype structures, a novel gene-fusion allele, novel and confirmed insertion/deletion events, a homozygous individual, and overall diversity for the structural haplotypes and their alleles. These KIR haplotypes augment our existing knowledge by providing high-quality references, evolutionary informers, and source material for imputation. The haplotype sequences and gene annotations provide alternative loci for the KIR region in the human genome reference GrCh38.p8.

Here, we present results for DPA1 and DPB1 four-digit allele-level typing in a large (n = 5,944) sample of unrelated European American stem cell donors previously characterized for other class I and class II loci. Examination of genetic data for both chains of the DP heterodimer in the largest cohort to date, at the amino acid epitope, allele, genotype, and haplotype level, allows new insights into the functional units of selection and association for the DP heterodimer. The data in this study suggest that for the DPA1-DPB1 heterodimer, the unit of selection is the combined amino acid epitope contributed by both the DPA1 and DPB1 genes, rather than the allele, and that patterns of LD are driven primarily by dimer stability and conformation of the P1 pocket. This may help explain the differential pattern of allele frequency distribution observed for this locus relative to the other class II loci. These findings further support the notion that allele-level associations in disease and transplantation may not be the most important unit of analysis, and that they should be considered instead in the molecular context.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-012-0615-3) contains supplementary material, which is available to authorized users.

Seventy-two-hour storage of BM, PBSC, and PBMNC products at refrigerated temperature maintains optimal cell viability and recovery. Anticoagulation with ACD-A is preferred over heparin to reduce lactic acid accumulation in the product media.