CD137L Reverse the Immunological Synapse Defects of Natural Killer Cells in Acute Myeloid Leukemia
Authors: Xing Dongxia , Ramsay Alan G. , Decker William , Lee Dean A. , Robinson Simon , Shah Nina , Parmar Simrit , Bollard Catherine M. , Hosing Chitra M , Champlin Richard , Gribben John G. , Shpall Elizabeth J 
Published: October 2019

Abstract
Abstract 246
Natural killer (NK) cells are an innate component of immune system that can produce a graft vs. leukemia (GVL) effect after stem cell transplantation. NK cells derived from acute myeloid leukemia (AML) patients are defective in their cytolytic function against leukemic cells. In order to better understand the mechanism of this defect, we performed functional assays examining immunological synapse formation of AML patient NK cells with autologous and allogeneic primary AML cells acting as antigen-presenting cells (APCs). Confocal microscopy was used to image and score F-actin polymerization at the immunological synapse between patient NK cells and leukemic cells. Accumulation of F-actin beneath the area of the NK: APC contact site is a hallmark of NK lytic synapses and allows signaling molecules to regulate appropriate activation and effector function. AML patient derived NK cells (AML-NK cells) formed significantly fewer synapses with autologous leukemia cells than healthy donor NK cells (12% versus 30%, n = 16. p > 0.001). Moreover, AML-NK cells were defective in their ability to recruit the key receptor NKG2D and the signaling molecule phosphotyrosine to immunological synapse contact sites. Signaling through the costimulatory ligand4-1BB-L (CD137L) has been shown to activate T cells, enhance antitumor responses and has multiple immunomodulatory effects on dendritic cells and NK cells. We postulated that AML-NK cells could be activated for enhanced cytolytic activity using artificial APCs generated to express CD137L. To test this, we setup co-culture assays using AML-NK cells and artificial CD137L-APCs before subsequent examination of immunological synapse function with AML blasts. Stimulated AML-NK cells that formed cell conjugate interactions with AML blasts, showed a significant increase in formation of immunological synapses compared to unstimulated AML-NK cells. The number of AML-NK/AML blast immunological synapses increased 16 hours after stimulation and peaked at approximately 72 hours. CD137L stimulation of AML-NK cells was also associated with increased cytotoxic function against primary AML cells (n = 6, p <0.01). Furthermore, CD137L stimulation increased recruitment of tyrosine-phosphorylated proteins at AML-NK immunological synapses compared with unstimulated control experiments (RRI 4.1 versus 2.3, n = 3, p < 0.01). Taken together, our data suggests that immune functional suppression of AML-NK cells in leukemia patients can be reversed by CD137L activation signaling, resulting in enhanced F-actin synapse formation, phosphotyrosine signaling, and cytolytic function. Thus, enhanced recruitment of signaling molecules to the NKIS may represent a novel immunomodulatory function of CD137L in the NK cell–mediated killing of AML cells. These findings should aid development of new immune based therapies for leukemia.

Disclosures:
Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.