Aberrant NK Receptor Signaling Associated with Large Granular Lymphoycte Leukemia and Primary Pulmonary Hypertension.

INTRODUCTION: Large granular lymphocyte (LGL) leukemia is associated with increased numbers of circulating NK or T-LGL cells with only 5% of these cases related to NK-cell expansion. NK-LGL leukemia is primarily characterized by chronic anemia in association with low to normal numbers of erythroid precursors in the bone marrow and chronic neutropenia. Here, we explore the mechanism of pathogenesis in the first case report of a patient with NK-LGL leukemia in association with primary pulmonary hypertension (PPH). This patient displayed a diffuse pulmonary infiltrate with sustained elevated mean pulmonary artery pressure at rest confirmed by right-sided cardiac catheterization meeting the clinical diagnostic criteria for PPH. Association between T-LGL leukemia and PPH has been reported previously suggesting a causitive link between LGL expansion and PPH pathogenesis.

METHODS: Cytotoxicity of a normal pulmonary endothelial cell line (CRL-2598) was determined by 5-hr ⁵¹-Cr-release assays using NK-cells from this NK-LGL patient compared to healthy controls. Specific NK receptors were blocked by antibodies added to the effector cells for 30 min prior to the cytotoxicity assay. Production of TNFα was assessed by intracellular cytokine staining with detection by flow cytometry. To block NK receptor signals, dominant negative adaptor proteins, DAP10 and DAP12, were constructed and expressed in recombinant vaccinia viruses. These viruses were engineered to carry a single Y-A mutation at Y102 or a double mutation at both Y9 and Y102 within the YxxM activating ITAM motif of the adaptor protein. Perforin granule mobilization was determined by immunofluorescence microscopy.

RESULTS: Sixty-eight % of peripheral blood and bone marrow mononuclear cells from this patient were phenotypically NK-LGLs with skewed reactivity to the anti-CD158a antibody (KIR2DS1/2DS2), which was shown by mRNA analysis to consist primarily of activating KIR2DS1. We examined the susceptibility of CRL-2598 cells to NK lysis and found that patient-derived NK-cells produced greater lysis without prior activation than healthy controls. Furthermore, NK-cells from this patient produced more TNF-α in response to paraformaldehyde-fixed CRL-2598 cells and displayed perforin granule mobilization to the contact site between effector and target cells. Addition of blocking antibodies against the activating receptors NKG2D and KIR2DS1/KIR2DL1 (anti-CD158a) produced a blockade in this process showing that these receptors were responsible for lysis. When DAP10 and DAP12 adaptor signaling steps were blocked, killing and perforin granule redistribution toward the contact site of the conjugated targets was suppressed.

CONCLUSION: This study shows that the presence of activating NK-receptors and DAP10/DAP12 adaptors contibuted to lysis of normal endothelial cells in this patient. DAP10 and DAP12 are key initiators for both NK− and T−cell function suggesting that targeted disruption of these signaling events may prove beneficial for autoimmune inflammatory diseases such as PPH and LGL leukemia.