Immunophenotypic and functional characterisation of Mesodermal Killer (MK) cells: a novel cell type and potential cellular therapy for cancer

Background & Aim We have identified a novel cell type, named mesodermal killer (MK) cells, derived from adult bone marrow under proprietary culture conditions. This study characterized the MK cells by immunophenotype, cytokine profile, immunomodulatory function and cytotoxicity. Methods, Results & Conclusion Methods: We investigated MK cells before and after activation with TNFα or IFNγ for immunophenotype using high throughput-FACS, for secretome and for inflammatory response in the murine air pouch assay. We evaluated MK cell cytotoxicity using the chromium release assay and immunomodulatory function by co-culture with natural killer (NK) cells in transwells. We used immunocytochemistry, flow cytometry and RT-PCR to detect expression of granzyme/perforin genes and proteins and investigated death pathways by siRNA silencing of FasL and TRAIL. Results MK cells displayed a novel immunophenotype (and different from BM-MSCs) including expression of CD16, CD96, CD112, CD137L, FasL, TRAIL and CD277 and some NK cell markers. In contrast to NK cells, MK cells lacked CD56 expression. TNFα and IFNγ activation increased FasL expression. MK cells secreted GROa, IL-12, IL-2Ra, IL-8, TRAIL and IL-6, and GROa and IL-8 secretion was increased by TNFα. MK cell cytotoxicity was observed against leukemia (K562), multiple myeloma (RPMI-8226) and breast cancer (MCF7) cell lines. MK cell conditioned media had no effect on MCF7 and antibody dependent cell mediated cytotoxicity (ADCC) experiments also proved negative. MK cells expressed granzymes and perforin and these were upregulated following exposure to RPMI-8226 (in contrast to BM-MSCs). Pre-treatment of MK cells with EGTA (a non-specific inhibitor of granule exocytosis) demonstrated >50% decrease in cytotoxicity against MCF7 suggesting a partial role of granule exocytosis in MK cell cytotoxicity. Inhibition of TRAIL expression by siRNA led to a significant reduction in MK cell cytotoxicity suggesting TRAIL may also be involved. In contrast to BM-MSCs, MK cells enhanced the cytotoxicity of primary NK cells against K562, RPMI-8226 and U226 (plasma cell myeloma) and IFNγ-activated MK cells enhanced migration of NK cells and monocytes into the murine air pouch suggesting a pro-inflammatory response. Conclusion MK cells exhibit cytotoxicity against a variety of cancer cell lines, are capable of priming NK cells in vitro and are pro-inflammatory in vivo. These results suggest the clinical translation of the MK cells is warranted and animal efficacy studies will begin shortly.