Natural Killer Cells Transfer Antimicrobial and Antitumoral Histone H2AZ to Kill Multiple Myeloma Cells Contributing to Transmissible Cytotoxicity
Authors: Martin-Antonio Beatriz , Suñe Guillermo , Najjar Amer , Perez-Amill Lorena , Velasco-de Andres Maria , Lozano Francisco , Lozano Ester , Muñoz-Punedo Cristina , Fernández de Larrea Carlos , Urbano-Ispizua Alvaro 
Published: October 2019

Abstract
Natural Killer (NK) cells are antitumoral, antiviral and antimicrobial cells. The first antitumor mechanism described for NK cells was the "missing self" recognition which happens between the inhibitory "Killer Immunoglobulin-like Receptors" (KIRs) on NK cells and the HLA-I present in all nucleated cells. Thus, down-regulation or absence of HLA-I in tumor cells leads to "missing self" recognition activating NK cells. As a consequence, NK cells deliver Granzyme-B activating apoptotic cell death. However, NK cells can mediate Granzyme-B and Caspase-independent cell death against tumor cells expressing HLA-I, such as multiple myeloma (MM) cells. The cytotoxic mediators mediating non-apoptotic cell death remain unknown. Mechanisms mediating NK cell antimicrobial activity have been less studied, being recognized the role of Granulysin mediating Caspase independent cell death. Cord blood (CB) derived NK cells (CB-NK) is a clinically applicable strategy for the generation of highly functional NK cells which can be used to treat MM and potentially other haematological malignancies. We have demonstrated that CB-NK cytotoxicity against MM is Granzyme B and Caspase-independent. This CB-NK cytotoxicity is transmissible among MM cells, meaning that the initial MM recipient cells (termed primary MM cells or 1ºMM cells)-which formed direct contact with CB-NK are able to transfer lipid-protein vesicles to neighbouring unexposed MM cells (termed secondary MM cells or 2ºMM cells) causing a transmissible cytotoxicity to 2ºMM cells, which have not been in direct contact with CB-NK. We aimed to determine the key cytotoxic mediators transferred from CB-NK to 1ºMM and afterwards to 2ºMM cells that mediate secondary cytotoxicity. To do that, we performed TRANS-SILAC proteomics to determine proteome trafficking between CB-NK and MM cells. TRANS-SILAC proteomics demonstrated increased cell-cell communication between MM cells after CB-NK exposure, with secondary communication between MM cells to transfer the previously received cytotoxic CB-NK material. This secondary transfer represents a dilution of the CB-NK cytotoxic material. At the same time, MM cells transferred their own proteome to neighboring MM cells. Histones were among the CB-NK transferred proteins to 1ºMM, and 2ºMM cells. We selected the Histone H2AZ variant 1 (H2AZ) for validation. Time lapse in vitro confocal microscopy with CB-NK over-expressing H2AZ confirmed dynamic transfer of H2AZ from CB-NK to MM cells. Transfer occurred through vesicles and large intercellular structures. Extracellular DNA staining confirmed that these structures with H2AZ were accompanied by DNA. At the extracellular level, Histones exhibit significant antimicrobial activity, being the main antimicrobial effectors of neutrophils. The multiple positive charges and hydrophobic residues present in Histones allow them to bind to and invade negatively-charged cell membranes which are present in both microbial pathogens and tumor cells and interact with the DNA. We determined the cytotoxic role of H2AZ. H2AZ over-expression caused Caspase independent cell death in four different MM cell lines (p<0.0001). No differences were found on K562 cells. H2AZ over-expression increased DNA damage in MM cell lines. Furthermore, MM cell lines over-expressing H2AZ transferred to neighboring MM cells both H2AZ and the DNA damage. H2AZ transfer and DNA damage transfer also translated into a transmissible cytotoxicity between MM cells, as MM cells over-expressing H2AZ transferred cytotoxicity to neighboring MM cells who received this H2AZ. H2AZ involvement in CB-NK cytotoxicity against MM cells was demonstrated as knock-down and over-expression of H2AZ in CB-NK reduced and increased, respectively, CB-NK cytotoxicity against MM cell lines (p<0.05). For K562, no impact was observed confirming again a different CB-NK cytotoxicity mechanism for MM cell lines. Knock-down and over-expression of H2AZ in CB-NK reduced and increased, respectively, CB-NK antimicrobial activity against Candida albicans and Escherichia coli, demonstrating the antimicrobial role of H2AZ. Last, the antitumoral and antimicrobial properties of the recombinant protein H2AZ against MM cell lines and against bacteria were also demonstrated. Taken together, we demonstrate for first time the antitumoral and antimicrobial role of CB-NK Histones, revealing new therapeutic targets

Disclosures
No relevant conflicts of interest to declare.