C-MYC Induces KIR Expression Via a Novel Control Region Upstream of the Conventional Adult KIR Promoter.

KIR determine whether NK cells will be alloreactive against targets. Although epigenetic control is thought to play a role in KIR expression, the mechanism of KIR repertoire formation is unknown. Recently, a probabilistic transcriptional mechanism has been shown to control the acquisition of mouse Ly49 receptors using a switch region upstream of each Ly49 promoter (S. Anderson). Based on this information, the DNA upstream of known KIR genes was scanned for similar motifs to test the hypothesis that similar control mechanisms are operant in human NK cells. A putative c-myc/AML binding site was found approximately 1Kb upstream of the traditional KIR promoters. Initiation of transcription in this region was observed by RNAse protection, and spliced KIR mRNAs originating in the upstream region were cloned. To look at the effects of c-myc on KIR expression, we transduced NK92 cells using retroviral murine stem cell virus vectors containing either c-myc/eGFP or eGFP alone. Cells expressing high levels of c-myc were 18% KIR positive as compared to 0.9% of control cells. To further look at the effect of c-myc on KIR from primary cells, we transduced CD34⁺ progenitors isolated from umbilical cord blood with c-myc/eGFP and eGFP vectors. eGFP+ cells were plated on the murine fetal liver line AFT024 and cultured with IL-15, IL-7, IL-3, Flt3 ligand, and c-kit ligand, all known to induce NK cell differentiation. Although c-myc is an oncogene, it did not promote autonomous growth of NK cells in the absence of exogenous cytokines. Proliferation was significantly increased in single cell progenitors over-expressing c-myc with 739.3x10³ NK cell progeny compared to 0.56x10³ from each control cell after 21 days (p<0.0001). At this early time point, 21.68 ± 3.25% of c-myc cells were KIR positive while 0.0% of control cells were KIR positive (p<0.0001) suggesting that c-myc targets KIR expression. Furthermore, bulk-transduced cells over expressing c-myc had 3.9 times the level of early non-coding transcripts originating from the upstream promoter as compared to eGFP transduced cells. A direct effect of c-myc on the upstream promoter was supported by ChIP assays demonstrating increased binding to the c-myc/AML site. The presence of these upstream transcripts directly correlates with increased conventional KIR transcripts as shown by Q-RT-PCR and an increase of KIR expression as shown by flow cytometry. These data show that c-myc play an important role in the differentiation of NK cells and in KIR acquisition. Experiments are currently in progress to look at the effects of the competing AML site on KIR expression and to investigate how non-coding RNA affects adult KIR expression. Understanding the mechanisms of KIR expression may lead to development of novel strategies to manipulate NK cells for therapeutic benefit.