KIR Genotype and KIR Ligand Phenotypes That Improve the in Vitro Cord Blood or Autologous NK Cell Mediated Cytotoxicity Against Myeloma Marrow Plasma Cells

Abstract

<h3>Instruction</h3> Killer Immunoglobulin-like Receptor (KIR) inhibition is known to influence the cytotoxic effects of natural killer (NK) cells against cancer cells. KIRs can be protective against relapse in myeloma (MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011) by NK cell licencing <i>or through</i> blocking of the inhibitory KIR receptors such as NKG2A (Mahaweni et al, 2018). <h3>Objectives</h3> In this study, the role of KIR geno/phenotypes and their ligands on autologous and cord blood (CB) derived NK cell mediated cytotoxicity against MM plasma cells (PC) were investigated. <h3>Methods</h3> Peripheral blood NK cells and marrow PCs were isolated from 20 patients with relapsed MM. PC were isolated from fresh BM aspirates using EasySep Human CD138 Positive Selection Kit (StemCell Technologies) and were subjected to cytotoxicity assays with either autologous or CB derived NK cells isolated (Human NK cell Enrichment Cocktail, Stemcell Technologies) and cultured in the presence of IL-15 (10ng/ml) for 2-8 days. KIR/KIR ligand genotyping were performed by Olerup SSP KIR Genotyping Kit (Olerup, Stockholm, Sweden) in order to detect KIR genotypes; KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A/B, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, and KIR ligands; HLA-ABw4+, HLA-BBw4+Thr80, HLA-BBw4+Ile80, HLA-BBw4+Asp77,Thr80, HLA-CAsn80, and HLA-CLys80. Effector cells were stained with antibodies against NKG2A (CD159a) and CD158i (KIR2DS4) and analyzed using a Beckman Coulter Flow Cytometer. <i>Cytotoxicity assays</i>: Using <i>3,3</i> (DiO) as an indicator and 1:10 target:effector cell ratio PC death was evaluated after 120 mins of incubation, using flow cytometry. <h3>Results</h3> 20 MM patients were tested in vitro. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.0% (11.4%-51.2%). While autologous NK mediated cytotoxicity was median 17.3% (11.4%-43.6%), CB-NK cell mediated cytotoxicity was 23.2% (range: 11.5%-51.2%). Highest cytotoxicity was achieved with CB-NK cells (CB7) with a KIR AB haplotype, carrying 8 inhibitory KIR genes against PC from MM12. Effector cells with the iKIR gene number above 6 were found to cause higher cytotoxicity (13/19; p=0.031). Additionally, higher (>30%) expression of NKG2A on CB-NK cells were found to be associated with lack of cytotoxicity. <h3>Conclusion</h3> CB-derived NK cells were found to be able to induce higher PC death than autologous PB-NK cells. Also higher number of iKIR genotypes and lacking KIR2DS4 receptor was found to be associated with higher cytotoxicity. In our current study we are reporting KIR NKG2A which binds with HLA-E to play an additional role. Allogeneic NK cell mediated immunotherapies are promising due their clinical advantages ie no need for HLA matching. Furthermore certain KIR haplotypes (ie KIR2DS4⁺ or NKG2A⁻) may offer better therapeutic efficacy if confirmed by others as well.