Myeloma Patient-Derived Bone Marrow Serum Negatively Regulates Natural Killer Cell Activity
Authors: Garg Tarun K. , Stone Katie , Wang Hongwei , Hoering Antje , Walker Brian A , Zangari Maurizio , Thanendrarajan Sharmilan , Schinke Carolina D. , Davies Faith E. , Morgan Gareth , van Rhee Frits 
Published: October 2019

Abstract
Introduction
Highly activated expanded NK cells (ENKs) can be generated in large numbers from the PBMCs of healthy donors or myeloma patients by coculture with K562 transfectants (cells expressing membrane-bound interleukin (IL)15 and 4-1BBL). These ENKs expand, persist, and retain their cytolytic ability further in vivo post adoptive transfer (Garg et al. 2012, Szmania et al. 2015). We have previously treated high-risk multiple myeloma (MM) patients in frank relapse with ENKs. These cells expanded in the patients after adoptive transfer, but rapidly lost their activation state. We hypothesized that the ENKs were adversely affected in the myeloma micro-environment, which contains a wide range of immunosuppressive soluble factors, such as shed NK ligands, cytokines and myeloma-derived exosomes. We therefore studied the effect of patient-derived bone marrow serum on activating ENK cell surface repertoire, immune synapse formation and cytolytic ability.
Methods
ENKs were generated from healthy donors (n=7) and incubated either with myeloma patients' pooled serum (PT-P), fetal bovine serum (FBS) or normal human AB serum (AB). Subsequently, the ENKs were evaluated for their viability, immune synapse (IS) formation and cytolytic ability. Immunophenotypic expression of these cells was assessed by flow cytometry to evaluate the receptors/markers for activation (NKp46, NKp30, NKp44, NKG2D), costimulation (2B4, NTB-A, NKp80, DNAM-1, CD26, CD69), adhesion (LFA-1, CD54) as well as molecules related to cytotoxicity (perforin, granzyme B, TRAIL). Global gene expression profile (GEP) of ENKs (n=7 pairs) was performed using U133 Plus 2.0 microarrays.
Results
ENKs exposed to PT-P exhibited a significant drop in the viability compare to FBS or AB in a time- and dose-dependent manner in MTT assays (range 34%-53.8% reduction, p<0.001) and in trypan blue counts (range 11%-34.7% reduction, p<0.0001). This drop in viability was accompanied by induction of apoptosis in ENKs treated with PT-P (range 14.9%-31.7%, p<0.01). Flow cytometric analysis showed that activation markers (CD26, CD69, CD70), and receptors for activation (NKp46, NKp30, NKp44, NKG2D), costimulation (2B4, DNAM-1), adhesion (LFA-1, CD54) as well as molecules related to cytotoxicity (perforin, granzyme B, TRAIL) were down-regulated in the ENK cells incubated with PT-P. Imaging flow cytometry (ImageStream) revealed that PT-P treated ENKs formed significantly fewer immune synapses with OPM2, U266, JJN3 and K562 cells (range 12%-41% down, p<0.001). Blocking of LFA1, a receptor for the adhesion molecules ICAM-1, -2 and -3, on ENK cells in cytotoxicity assays showed reduced killing further confirming that immune synapse formation is critical to NK-mediated lysis. PT-P treated ENKs demonstrated a significant decrease in specific lysis of the MM cell lines OPM2, U266 and JJN3 in cytotoxicity assays compared to ENKs treated with FBS/AB at different effector to target ratios (14.2%-41.9%, p<0.05). Adding IL2 (300U/ml) in media augmented ENK-mediated specific lysis >1.6 to 3.6 fold compare to PT-P treated ENKs in myeloma cells (OPM2, JJN3 and U266).
Microarray analysis revealed that ENK cells incubated with PT-P led to the expression of a unique set of 38 probes compare to AB+FBS (35 up and 3 down with FDR<0.15 cutoff). Genes encoding cell cycle, cell growth, survival, and motility (ANAPC16, AREG, DDIT4, TSC22D3, CXCR4), intracellular signaling molecules (FKBP5, ISG20, DEFB132, ZFP36L2), cell energy and metabolism (RAP1GDS1, GALM, PARP8) are up-regulated in PT-P treated ENKs as a response to PT-P serum. Over-expression of TSC22D3 is linked to anti-apoptosis and may be up-regulated as a protection from PT-P-induced apoptosis in these cells. Proteomics on patient serum to characterize soluble factors responsible for the down modulation of ENKs is in progress and the data will be presented.
Conclusion
Myeloma bone marrow serum derived soluble factors are immunosuppressive and induce apoptosis in ENKs. In surviving ENKs, there is adverse modulation of the cell surface receptor repertoire, and impaired immune synapse formation resulting in significantly reduced anti-myeloma cytolytic ability. Further characterization of patient serum by proteomics followed by functional analysis is in progress to pinpoint the individual immunosuppressive factors.

Disclosures
Davies: Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.