STAT5A Overexpression Enhances Killer Immunoglobulin Receptor (KIR) Expression in Developing NK Cells and Is Associated with a Loss of Reverse Transcription from the Proximal KIR Promoter.

Down-regulation of HLA-class I molecules is commonly observed in virally infected and malignantly transformed cells and can contribute to the ability of these cells to escape recognition by the adaptive immune system. NK cells are able to detect the loss of even single HLA alleles on potential target cells through their clonally distributed HLA-class I-specific inhibitory receptors (KIR). While it is well established that individual KIR gene expression is strongly correlated with the DNA methylation status of CpG dinucleotides in areas surrounding the transcription initiation region, the mechanisms that regulate variegated KIR expression are largely unknown. The manipulation of KIR expression is of considerable interest due to the widely reported correlation between KIR ligand status and patient survival in hematopoietic stem cell transplant settings. Because the transcription factor STAT5 is activated downstream of the BCR/ABL oncogene, which we have previously shown to substantially augment KIR expression levels in primary NK cells, we hypothesized that STAT5 could directly influence KIR expression. To test this hypothesis, we purified CD34⁺ hematopoietic progenitor cells from umbilical cord blood and retrovirally transduced these cells with either a control murine stem cell virus (MSCV) vector encoding eGFP alone or an MSCV vector encoding both eGFP and a constitutively active form of STAT5A, termed STAT5A 1*6. CD34/eGFP-positive cells were then purified and cultured on the EL08-D1 stroma line, known to support NK cell development. After a period of 28 days in culture, 6.25±2.73% of the NK cells in eGFP control cultures were KIR-positive compared with 29.3±4.27% of STAT5A 1*6 transductants. To more closely examine individual KIR expression at the transcriptional level, we carried out a quantitative RT-PCR analysis with probe sets previously validated for amplification of individual KIR (Cooley et al., Blood). We observed a general increase in the mRNA expression levels of both inhibitory and activating KIR in STAT5A 1*6-transduced cells. In order to investigate the mechanism underlying the increased KIR expression observed in the STAT5A 1*6-transductants, we performed an RT-PCR specific for the reverse transcript originating from the proximal promoter of the KIR3DL1 gene. In a model proposed by Anderson et al., reverse transcription from the bidirectional proximal promoter of variegated KIR genes may be responsible for the silencing of the KIR locus through the production of dsRNA, which then induces DNA methylation in a siRNA-dependent manner. Our RT-PCR results from multiple donors clearly show an absence of reverse proximal transcript in cells transduced with the STAT5A 1*6 construct in contrast to high levels of transcript in eGFP controls. These results provide evidence for a role of STAT5A in the induction of KIR expression and support a model of KIR regulation involving intergenic transcription and possibly siRNA-mediated gene silencing. A better understanding of how to manipulate these KIR expression patterns may be of benefit to exploit the potential of NK cell therapy to improve transplant outcomes.