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How receptor acquisition correlates with the functional maturation of natural killer (NK) cells is poorly understood. We used quantitative real-time polymerase chain reaction (PCR) assays to compare NKG2 and killer immunoglobulin-like receptor (KIR) gene expression in NK cells from allogeneic transplant recipients and their donors. Marked differences were observed in the NK subsets of recipients who had 8-fold more CD56bright cells, diminished KIR expression (except 2DL4), and increased NKG2A. In normal blood not all CD56dim cells express KIR, and a novel subpopulation of cells committed to the NK-cell lineage was defined. These cells, which comprise 19.4% ± 2.8% of the CD56dim NK population in healthy donors, express the activating NKG2D and NKG2E receptors but no KIR or NKG2A. Although the CD56dim NKG2A− KIR− NK cells lack “at least one” inhibitory receptor for autologous MHC class I, they are not fully responsive, but rather functionally immature cells with poor cytotoxicity and IFN-γ production. Upon culture with IL-15 and a stromal cell line, CD56dim and CD56bright KIR− NK cells proliferate, express KIR, and develop cytotoxicity and cytokine-producing potential. These findings have implications for the function of NK cells reconstituting after transplantation and support a model for in vivo development in which CD56bright cells precede CD56dim cells.

During mouse cytomegalovirus (CMV) infection, a population of Ly49H+ natural killer (NK) cells expands and is responsible for disease clearance through the induction of a “memory NK-cell response.” Whether similar events occur in human CMV infection is unknown. In the present study, we characterized the kinetics of the NK-cell response to CMV reactivation in human recipients after hematopoietic cell transplantation. During acute infection, NKG2C+ NK cells expanded and were potent producers of IFNγ. NKG2C+ NK cells predominately expressed killer cell immunoglobulin–like receptor, and self-killer cell immunoglobulin–like receptors were required for robust IFNγ production. During the first year after transplantation, CMV reactivation induced a more mature phenotype characterized by an increase in CD56dim NK cells. Strikingly, increased frequencies of NKG2C+ NK cells persisted and continued to increase in recipients who reactivated CMV, whereas these cells remained at low frequency in recipients without CMV reactivation. Persisting NKG2C+ NK cells lacked NKG2A, expressed CD158b, preferentially acquired CD57, and were potent producers of IFNγ during the first year after transplantation. Recipients who reactivated CMV also expressed higher amounts of IFNγ, T-bet, and IL-15Rα mRNA transcripts. Our findings support the emerging concept that CMV-induced innate memory-cell populations may contribute to malignant disease relapse protection and infectious disease control long after transplantation.

The killer cell immunoglobulin-like receptor (KIR) repertoire of natural killer (NK) cells determines their ability to detect infected or transformed target cells. Although epigenetic mechanisms play a role in KIR gene expression, work in the mouse suggests that other regulatory elements may be involved at specific stages of NK-cell development. Here we report the effects of the transcription factor c-Myc on KIR expression. c-Myc directly binds to, and promotes transcription from, a distal element identified upstream of most KIR genes. Binding of endogenous c-Myc to the distal promoter element is significantly enhanced upon interleukin-15 (IL-15) stimulation in peripheral blood NK cells and correlates with an increase in KIR transcription. In addition, the overexpression of c-Myc during NK-cell development promotes transcription from the distal promoter element and contributes to the overall transcription of multiple KIR genes. Our data demonstrate the significance of the 5′ promoter element upstream of the conventional KIR promoter region and support a model whereby IL-15 stimulates c-Myc binding at the distal KIR promoter during NK-cell development to promote KIR transcription. This finding provides a direct link between NK-cell activation signals and KIR expression required for acquisition of effector function during NK-cell education.

The effector function of human NK cells is down-regulated via ligation of inhibitory receptors (killer immunoglobulin-like receptors [KIR] and NKG2A) that recognize self MHC. Although most NK cells express “at least one” self MHC receptor, the mechanism producing self-tolerance is not yet understood. In order to precisely evaluate receptor expression to better understand this mechanism we developed and validated quantitative, real time RT-PCR (Q-RT-PCR) assays to measure mRNA levels from individual NKG2 and KIR genes. Comparison of subpopulations of NK cells sorted from blood showed a high expression of NKG2A on CD56+bright cells but virtually no KIR expression except for KIR2DL4. To further define KIR expression patterns on more mature cells we sorted the CD56+dim NK cells into KIR+ and KIR− subpopulations using an antibody cocktail that recognizes 6 KIR genes. Comparing KIR gene expression by Q-RT-PCR in these subsets showed much higher transcript levels for the KIR genes included in the sorting cocktail in the KIR+ subpopulation than in the KIR− cells (median expression ratio of 221.3; n=43; P=

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