Abstract
The impact of natural killer (NK) cell alloreactivity on hematopoietic stem cell transplantation (HSCT) outcome is still debated due to the complexity of graft parameters, HLA class I environment, the nature of killer cell immunoglobulin-like receptor (KIR)/KIR ligand genetic combinations studied, and KIR+ NK cell repertoire size. KIR genes are known to be polymorphic in terms of gene content, copy number variation, and number of alleles. These allelic polymorphisms may impact both the phenotype and function of KIR+ NK cells. We, therefore, speculate that polymorphisms may alter donor KIR+ NK cell phenotype/function thus modulating post-HSCT KIR+ NK cell alloreactivity. To investigate KIR allele polymorphisms of all KIR genes, we developed a next-generation sequencing (NGS) technology on a MiSeq platform. To ensure the reliability and specificity of our method, genomic DNA from well-characterized cell lines were used; high-resolution KIR typing results obtained were then compared to those previously reported. Two different bioinformatic pipelines were used allowing the attribution of sequencing reads to specific KIR genes and the assignment of KIR alleles for each KIR gene. Our results demonstrated successful long-range KIR gene amplifications of all reference samples using intergenic KIR primers. The alignment of reads to the human genome reference (hg19) using BiRD pipeline or visualization of data using Profiler software demonstrated that all KIR genes were completely sequenced with a sufficient read depth (mean 317× for all loci) and a high percentage of mapping (mean 93% for all loci). Comparison of high-resolution KIR typing obtained to those published data using exome capture resulted in a reported concordance rate of 95% for centromeric and telomeric KIR genes. Overall, our results suggest that NGS can be used to investigate the broad KIR allelic polymorphism. Hence, these data improve our knowledge, not only on KIR+ NK cell alloreactivity in HSCT but also on the role of KIR+ NK cell populations in control of viral infections and diseases.
Highlights
Hematopoietic stem cell transplantation (HSCT) provides a curative therapy for many patients with hematological malignancies [1]
A robust LR amplification of killer cell immunoglobulin-like receptor (KIR) genes was obtained for all samples as illustrated for three representative International Histo compatibility Workshop (IHW) samples (Figure 1B)
One specific band between 4 and 5 kb for the KIR3DP1 pseudogene and another specific band between 9 and 17 kb corresponding to a cluster of all other KIR genes were observed, irrespective of KIR AA or AB genotype (Figure 1B) as KIR genomic length varies depending on KIR genes (Table S2 in Supplementary Material)
Summary
Hematopoietic stem cell transplantation (HSCT) provides a curative therapy for many patients with hematological malignancies [1]. Donors for HSCT are currently selected based on the level of matching for HLA-A, -B, -C, -DRB1, and -DQB1 loci. Ruggeri et al [7] were first to report the beneficial effect of KIR ligand mismatched donor NK cell alloreactivity after T cell-depleted HLA haplo-identical HSCT resulting in less relapse, less GvHD, and better overall survival in patients with acute myeloid leukemia. The impact of KIR+ NK cell alloreactivity on HSCT outcome is still controversial due to the heterogeneity of graft parameters, HLA class I environment, nature of KIR/KIR ligand genetic combinations studied, and KIR+ NK cell repertoire size [8,9,10,11,12]
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