Integrated Genotyping and mRNA Expression Profiling of Killer Immunoglobulin-Like Receptors.

Abstract

Natural killer (NK) cells belong to a subgroup of lymphocytes (CD3-CD56+) which play an important role in the cellular immune response against virus-infected cells and tumors. The activity of NK cells is regulated by a balance of triggering and inhibitory receptors, including Killer Ig-like Receptor (KIR) molecules which interact with specific HLA class I molecules, predominantly HLA-C, on target cells. The 17 known KIR genes are divided into two classes: activating KIRs and inhibitory KIRs. There is strong evidence that inhibitory KIR mismatch between donor and recipient improves the outcome of haploidentical hematopoietic stem cell transplantation (HSTC) in leukemia patients (Ruggeri et al. 2002). In addition, the KIR-HLA constellation is assumed to have an influence on the severity of graft versus host disease (GvHD). Whether these activities of NK cells are clinically important and to what extent these processes are mediated only by KIR-HLA class I interactions remains to be determined. In human populations, KIR gene haplotypes vary in the number and type of KIR genes they contain. Further diversification is observed by expanded allelic polymorphism at the individual genes. In general, KIR haplotypes contain 7–12 genes plus 2 pseudogenes. Extra KIR heterogeneity is provided at the expression level: different subsets of NK cells within an individual express different KIRs. Recently, it was shown that KIR genotyping alone does not seem to be sufficient for donor KIR assessment because of the lack of gene expression in approximately one-fourth of the individuals for one of the inhibitory KIRs that recognize the three major groups of MHC class I ligands (Leung et al. 2005). KIR phenotyping by flow cytometry using monoclonal antibodies is insufficient due to the lack of specific monoclonal antibodies. For trustworthy analysis, one has to combine KIR genotyping with mRNA expression profiling and flow cytometry. Therefore, we developed a new set of sequence-specific primers (SSP). This primer set can be applied to perform either KIR genotyping or mRNA expression profiling despite the high degree of identity of the genes (80–90%, sometimes more than 95%). The primers of each KIR gene (15 genes and 2 pseudogenes) cover all allelic variants annotated by the IPD KIR Sequence Data Base (status quo July 05). Using this primer set, we genotyped 25 individuals, and compared the results with other sets of KIR primers published elsewhere. Additionally, we show the mRNA expression profile employing the same set of new primers. We confirmed these results on the protein level by flow cytometry.