Journal: Leukemia

Article Title: Identifying candidate allogeneic NK-cell donors for hematopoietic stem-cell transplantation based on functional phenotype

doi: 10.1038/leu.2010.19

Figure Lengend Snippet: NK cells were KIR-typed using two methodologies testing for the presence or absence of KIR genes. This analysis also distinguished two groups of 2DS4 alleles, those carrying a deletion of 22 nucleotides (003, 004, 006, 007), and alleles carrying full length allele. Genomic DNA was extracted from nucleated cells utilizing the QiaGen DNA purification kit EZ1 DNA Tissue Card (Qiagen, Germantown, MD, USA). KIR typing was performed utilizing PCR amplification combined with hybridization with oligonucleotide probes (PCR-SSOP). For SSOP KIR genotyping we used KIR SSO genotyping kits from One Lambda Inc. (Canoga Park, CA, USA). DNA was amplified with specific primers and products were incubated with beads labeled by specific oligonucleotide probes. Beads were collected by the Luminex 100 platform (Luminex Corporation, Austin, TX, USA) and analyzed using HLA Visual 2.2 (One Lambda Inc.) software. KIR typing was also performed by PCR amplification utilizing sequence specific primers (SSP) using the Pel-Freeze Genotyping SSP KIR kit (Invitrogen/Pel-Freeze, Brown Deer, WI, USA) containing 21 locus-specific primer mixes. Donors were typed for alleles at HLA-A, B, C, DRB1, DRB3/4/5/ and DQB1 by PCR amplification and oligonucleotide hybridization by molecular methods utilizing commercial kits (Invitrogen, Carlsbad, CA, USA and One Lambda) that achieve intermediate resolution. The donors were also typed for these loci by high resolution methods utilizing PCR amplification and nucleotide sequencing (Abbott Laboratories, Abbott Park, IL, USA). Additional high resolution tests for selected loci were performed in donors in whom an allele level mismatch could not be ruled out. HLA-KIR ligands were identified by analyzing the intermediate resolution and high resolution results of HLA-B and C loci in the donors. The polymorphism at residue 80 of HLA-B and C loci was analyzed to classify for the presence of the KIR ligands. The alleles of HLA-B having a codon encoding for 'threonine' (T) or isoleucine (I) at codon 80 were classified as 'Bw4-positive' being the ligands of the KIR receptor 3DL1. The alleles of HLA-C having a codon encoding for 'asparagine' (N) at codon 80 were classified belonging to the HLA-C 'Group-1' being the ligands of the KIR receptors 2DL2 and 2DL3. The alleles of HLA-C having a codon encoding for 'lysine' (K) at codon 80 were classified belonging to the HLA-C 'Group-2' being the ligands of the KIR receptor 2DL1. The assignment into KIR ligand groups was made from the interpretation of the high resolution results or from the reactivity of oligonucleotide probes detecting sequence polymorphisms at codon 80. The (+) or (−) symbols denote the presence or absence of KIR ligand on donor-derived NK cells. Observed inhibition was deemed as: Low – less than 25%, Moderate (Mod) – 25% to 60%, and High – greater than 60% relative NK-cell inhibition. Blue boxes indicate disparity between predicted inhibition and observed inhibition.

Article Snippet: KIR typing was also performed by PCR amplification utilizing sequence specific primers (SSP) using the Pel-Freeze Genotyping SSP KIR kit (Invitrogen/Pel-Freeze, Brown Deer, WI, USA) containing 21 locus-specific primer mixes.

Techniques: DNA Purification, Amplification, Hybridization, Incubation, Labeling, Luminex, Software, Sequencing, Inhibition