Human Mesenchymal Stromal Cells (MSCs) Suppress Cytotoxicity of Natural Killer (NK) Cell Lines and Can Be Targets for NK-Mediated Cytolysis
Authors: Hu Chia-Hsuan Donna , Kosaka Yoko , Keating Armand 
Published: April 2020

Abstract
MSCs are known to modulate immune responses and themselves are not immunogenic in some experimental systems. Given the increasing use of both MSCs and NK cells in cell therapy protocols, we investigated interactions between the 2 cell types using human NK cell lines KHYG-1 and NK-92 and human bone marrow-derived MSCs. NK lines were cultured in normal medium, in human MSC-conditioned medium, or in the presence of MSCs at NK:MSC ratio of 10:1 either with direct cell contact or in a transwell system for 96 hours. NK immunophenotype was assessed by flow cytometry. NK cytotoxicity against K562 or MSC target cells was measured by a 4-hour Cr release assay. Granule polarization in NK cells was analyzed by immunostaining of perforin.
Results: MSCs suppressed K562 lysis by KHYG-1 after direct cell contact co-culture: 24.8% ±14.4 (SD) versus control KHYG-1 without MSCs: 57.8%±1.4 (p=0.01). In contrast, cytotoxicity of NK-92 was unaffected by MSCs (81.3%±3.8 vs control 84.5%±1.2). We also found that NK-92 but not KHYG-1 lysed MSCs: 25.2%±11.7 vs −4%±4.1, respectively (p=0.001). Moreover, we found that while 57%±16.7 (n=10) of KHYG-1 cells had constitutively polarized granules not specifically directed toward MSCs upon cell-contact, MSCs induced polarization in NK-92 cells from 12.9%±8.8 to 50.5%±7.1 (p=0.000). Although lysis of MSCs by NK-92 may partially account for resistance to MSC-mediated suppression, incubation with MSC-conditioned medium was able to suppress NK-92 cytotoxicity: 49.6%±13.7 vs control 69.3%±8.8 (p=0.001). In transwell studies, K562 killing by KHYG-1 was suppressed (32.7%±9.7 vs control 44.7%±4.7; p=0.002), but such suppression was not observed by NK-92. These data indicate a factor-mediated mechanism of NK suppression with differential sensitivity. Among the possible candidates, we found that kynurenine inhibited KHYG-1 and NK-92 by 50% (p=0.028) and 30% (p=0.017), respectively. Furthermore, KHYG-1 cytotoxicity inhibited by MSCs in transwell system was partially reversed by addition of a nitric oxide synthase inhibitor (from 44.7%±4.7 to 57.2%±0.5; p=0.002) and a PGE2 synthase inhibitor (from 44.7%±4.7 to 49.7±0.9; p=0.013). Addition of an IDO inhibitor to the PGE2 synthase inhibitor had a synergistic effect on restoration (from 44.7%±4.7 to 62%±4.6; p=0.001). By further characterizing differences in NK-92 and KHYG-1, we found that NK-92 expressed 6-fold less activating receptor NKG2D (MFI: NK-92 4.4 vs KHYG-1 25.6) and 35-fold less activating receptor NKp44 (NK-92 1.1 vs KHYG-1 37.5) than KHYG-1, suggesting that NK-92 killing of MSCs was not mediated by these receptors. Greater killing of MSCs by NK-92 may be due to upregulation of CD94 and CD244 which were increased on NK-92 (CD94: 60.8±4.6 vs control 34.2±12.0, p=0.014; and CD244: 3.8±0.3 vs control 2.7±0.2, p=0.014), but not on KHYG-1, after MSC co-culture.
Conclusions: Our study shows that MSCs suppress the cytotoxic action of KHYG-1 and NK-92 via soluble factors, affect phenotypic expression, and change granule polarization. Of importance, MSCs can be lysed by NK-92 but not by KHYG-1. Previous studies demonstrated lysis of MSCs by human activated primary NK cells. Taken together, these observations must be considered when designing clinical protocols with MSCs or NK cell lines.