Extracellular NK histones promote immune cell anti-tumor activity by inducing cell clusters through binding to CD138 receptor

RESEARCH ARTICLE Open Access Extracellular NK histones promote immune cell anti-tumor activity by inducing cell clusters through binding to CD138 receptor B. Martín-Antonio 1,2* , G. Suñe 1,2 , A. Najjar 3 , L. Perez-Amill 1 , A. Antoñana-Vildosola 1 , M. Castella 1 , S. León 1 , M. Velasco-de Andrés 4 , F. Lozano 4,6,7 , E. Lozano 1 , C. Bueno 5 , J. M. Estanyol 8 , C. Muñoz-Pinedo 9 , S. N. Robinson 10 and A. Urbano-Ispizua 1,2,11 Abstract Background: Natural killer (NK) cells are important anti-tumor cells of our innate immune system. Their anti-cancer activity is mediated through interaction of a wide array of activating and inhibitory receptors with their ligands on tumor cells. After activation, NK cells also secrete a variety of pro-inflammatory molecules that contribute to the final immune response by modulating other innate and adaptive immune cells. In this regard, external proteins from NK cell secretome and the mechanisms by which they mediate these responses are poorly defined. Methods: TRANS-stable-isotope labeling of amino acids in cell culture (TRANS-SILAC) combined with proteomic was undertaken to identify early materials transferred between cord blood-derived NK cells (CB-NK) and multiple myeloma (MM) cells. Further in vitro and in vivo studies with knock-down of histones and CD138, overexpression of histones and addition of exogenous histones were undertaken to confirm TRANS-SILAC results and to determine functional roles of this material transferred. Results: We describe a novel mechanism by which histones are actively released by NK cells early after contact with MM cells. We show that extracellular histones bind to the heparan sulfate proteoglycan CD138 on the surface of MM cells to promote the creation of immune-tumor cell clusters bringing immune and MM cells into close proximity, and thus facilitating not only NK but also T lymphocyte anti-MM activity. Conclusion: This study demonstrates a novel immunoregulatory role of NK cells against MM cells mediated by histones, and an additional role of NK cells modulating T lymphocytes activity that will open up new avenues to design future immunotherapy clinical strategies. Keywords: NK cells, Multiple myeloma, Cell-cell communication, Histones, Immunotherapy Introduction Natural killer (NK) cells are important anti-tumor cells of our innate immune system whose anti-tumor properties led to anti-cancer, immune NK cell therapies under development [1]. The majority of clinical studies infusing NK cells worked mostly for acute myeloid leukemias but performed poorly in other malignancies [2,3], suggesting that a deeper knowledge of NK cells is required to better understand and exploit their anti-tumor activity. In this regard, NK cells present a wide array of activating and inhibitory receptors that interact with their ligands on tumor cells [4]. However, besides these receptor-ligands interactions, a cross-talk among different immune cells, performed by pro-inflammatory molecules secreted by immune cells, contributes to the final immune response [5]. The relevance of this cross-talk between immune cells is observed after microbial infection, where dendritic cells (DCs) activate NK cells through IL15 secretion leading to T cell and monocyte activation [5–7]. The coordination of these immune responses requires the creation of cellular clusters to enable intercellular cross- © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: [email protected] 1 Department of Hematology, Hospital Clinic, IDIBAPS, Carrer Rosselló 149-153, 08036 Barcelona, Spain 2 Josep Carreras Leukaemia Research Institute, Carrer Rosselló 149-153, 08036 Barcelona, Spain Full list of author information is available at the end of the article Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 https://doi.org/10.1186/s40425-019-0739-1 Journal for ImmunoTherapy of Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. talk between immune cells [7,8]. We previously reported the relevance of this cell-cell contact as a mechanism leading to a transmissible cytotoxicity from cord blood derived NK cell (CB-NK) to neighboring multiple myeloma (MM) cells, as CB-NK cytotoxicity is transferred to ‘primary’MM cells (1°MM) after contact; and afterwards, it is passed from 1°MM to adjacent ‘secondary’MM cells (2°MM) unexposed to CB-NK [9]. Interestingly, CB-NK perform Granzyme-B and Caspase3 independent killing of MM cells [9], suggesting the involvement of other proteins in the CB-NK anti-MM activity. Moreover, whereas effector cytokines require hours to be detected, cell cluster formations occur earlier, suggesting that other initiating molecules secreted at early times of cell-cell contact will impact on the final effector response. These observations led us to hypothesize that novel cytotoxic molecules transferred from CB-NK to MM cells could be involved in the anti-MM CB-NK activity. Therefore, TRANS-stable-isotope labeling of amino acids in cell culture (TRANS-SILAC) [10] combined with proteomic was undertaken to identify early materials transferred between CB-NK and MM. Analysis revealed that histones are actively transferred between CB-NK and MM and also released into the extracellular milieu after co-culturing CB-NK and MM. Released CBNK histones bind to CD138 in MM cells promoting the formation of CB-NK/MM cell clusters which facilitates NK-MM contact and improves the anti-tumor NK efficacy. Furthermore, NK-histones also promoted the generation of cell clusters between T-cells and MM cells increasing the T cell anti-MM activity and revealing a novel mechanism by which NK enhance the anti-tumor activity of T-lymphocytes. Methods Cell cultures NK cells were isolated from CB and PB by magnetic depletion (Miltenyi Biotec). CB-NK expansion was performed during 14 days as previously described [9] using K562-based antigen presenting cells expressing membrane bound IL-21 (“Clone 9.mbIL21”). T cells were isolated from PB by magnetic depletion (Miltenyi Biotec) and expanded during 5 days with Dynabeads® Human T-Activator CD3/CD28 (Thermo-Fisher). IL2 (Proleukin) was added at 100UI/mL every other day. Culture NK and T cell media was comprised of 45% RPMI-1640 (Sigma-aldrich) and 45% Click’s (Irvine Scientific) with 10% AB human serum (Atlanta Biologicals). ARP1 cell line was provided by Multiple Myeloma Research Center (Little Rock, AK). 293 T, K562, U266, RPMI-8226, Ramos and Jurkat cells were obtained from American Type Culture Collection (ATCC, Rockville, MD). K562, ARP1, RPMI, Ramos and Jurkat were cultured in RPMI-1640 with 10% fetal bovine serum (FBS) and U266 with 15% FBS. 293 T cells were cultured in DMEM with 10% FBS. CD138 + cells from MM patients were obtained by MACS selection (Miltenyi Biotec). TRANS-SILAC proteomics Was performed culturing cells in their usual media lacking normal L-Arg, L-Leu and L-Lys, and supplemented with their corresponding heavy isotopic AA (hAA). ARP1-MM cells were expanded in this media for 21 days, and CB-NK during the 14 days of the usual CB-NK in vitro expansion. After this period, both cell populations contained > 97% of hAA as determined by liquid Chromatography-Tandem Mass Spectrometry (LC-MS/ MS). Percentage of heavy proteins transferred to each cell population was analyzed by LC-MS/MS. Cytotoxicity assays Were performed at 3 h by Europium Release Assays [9], and at longer times by flow cytometry calculating % of remaining live GFP+ tumor cells applying the formula: % of target cell lysis = 100-(% of GFP+ cells at 24-48 h / % of GFP+ cells at 0 h). In vivo myeloma murine model NOD/SCID IL-2Rcnull (NSG) mice were irradiated and inoculated i.v. with GFP-Firefly Luciferase-transduced ARP1 cells. Recombinant H2AZ (0.5 mg/kg) was given i.v. on day 1 and day 7. Disease progression was monitored by bioluminescence using a Hamamatsu CDD camera (Hamamatsu Photonics Sistems) following a 100 mL IP injection of D-luciferin (20 mg/mL), and measuring serum kappa light chain levels by ELISA (Bethyl Laboratories). Signal quantitation was performed with ImageJ software. Transfer of H2AZ-GFP transfer between cells Cells were co-cultured staining in blue (CMAC) the cell population of interest. Then, H2AZ-GFP transfer between cells was analyzed by flow cytometry gating on the CMAC+ population and analyzing the % of CMAC+GFP+ cells. Supernatant Containing Inflammatory Proteins (SIPs) analysis To analyze released proteins to the extracellular milieu by each cell population, 30–40 min co-cultures experiments were performed collecting the supernatants and differentiating proteins of each cell population by their previous hAA labeling (Additional file 1: Figure S1D). Reagents Caspase-1 inhibition was achieved with Y-VAD (50 μM) addition. Heparinase III (Sigma-aldrich) treatment (0.01 Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 2 of 16 Journal for ImmunoTherapy of Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. IU/mL) was used to remove HSGPG from MM cells [11]. Recombinant H2AZ (Merck-Millipore) and H4 (New England Biolabs) were added in cell culture at 2μM or 0.5 μM depending on the experiment. Heparin (STEMCELL Technologies) was used at 20 IU/mL. DNAse I (D2) (Worthington Biochemical Corporation) was used at 100 IU/mL. Antibodies used were CD138V421 and CD56-V450 (BD Biosciences), H2AZ, H4, H1.5, Anti-Rabbit IgG-HRP, and Anti-Rabbit IgG-Alexa Fluor-647 (Cell Signaling Technology). Cell cluster formation Area of cell clusters was visualized at different time points by measuring the GFP area from tumor cells using ImageJ software. Ethics Statement Research involving human materials was approved by Ethical Committee of Hospital Clinic, Barcelona. CB units and PB were obtained from healthy donors who gave informed consent. Statistical analysis Mann-Whitney U test was used to analyze comparison between groups. Statistical analyses were performed with SPSS (IBM SPSS v. 23). LC-MS/MS, confocal fluorescence microscopy, flow cytometry, GFP-fused protein generation, lentivirus production and siRNA transfection are detailed in Additional file 1: Supplementary Methods. Results CB-NK histones are dynamically transferred from CB-NK to primary MM cells and subsequently to adjacent secondary MM cells In order to identify cytotoxic CB-NK proteins transferred directly to MM cells (1°MM), and secondarily to neighboring MM cells (2°MM), TRANS-SILAC proteomic was performed to identify acquired proteome for each cell population [10]. Co-culture experiments were limited to 30–40 min to unravel early transferred proteins between live cells responsible for initiating NK cytotoxicity. CB-NK were labeled with heavy amino acids (hAA) to allow for identification of ‘heavy’CB-NK proteome transferred to 1°MM (labeled with CMAC); and subsequently from 1°MM to 2°MM (unstained) (Additional file 1: Figure S1A). Proteomic data showed that after CB-NK/1°MM co-culture, the 1°MM proteome contained 9.5% of proteins transferred from CB-NK (Fig. 1a) (Additional file 1: Table S1). Then, 1°MM were co-cultured with fresh MM cells to determine CB-NK proteins transferred secondarily between MM cells, revealing 7.2% of secondary transfer of NK proteome from the 1°MM to neighboring 2°MM cells. These proteins were thus originally derived from CB-NK, but via 1°MM (Fig. 1a, Additional file 1: Table S2). As a consequence of this transfer, 1°MM lost part of their labeled, previously acquired CB-NK proteome content that went down from 9.5 to 3.9% (Fig. 1a, Additional file 1: Table S3). These data provided evidence for a primary-direct CB-NK proteome transfer to 1°MM, and a secondary-indirect CB-NK proteome transfer to 2°MM. Analysis of CB-NK transferred proteins to MM cells showed a high number of CB-NK histones acquired by MM cells. In resting conditions, different histones were present in CB-NK (Fig. 1b: CB-NK resting). However, after co-culturing with 1°MM, CB-NK lost their histone content (Fig. 1b: CB-NK after 1°MM), as indicated by absence of detection of Peptide Spectral Matches (PSMs). Conversely, MM cells underwent an enrichment in these histones (Fig. 1b), suggesting the selectivity of this process. We observed that subsequently, CB-NK histones from 1°MM cells were then transferred to 2°MM cells (Fig. 1b: 2°MM), and as a consequence, the content of labeled, CB-NK histones in 1°MM disappeared (Fig. 1b: 1°MM after 2°MM), suggesting a continuous, dynamic and specific transfer of CB-NK histones between MM cells. Of note, other NK proteins detected in the proteomic data (YWHAZ and YWHAQ) did not show this pattern of continuous transfer observed for histones (Fig. 1b). MM cells exposed to CB-NK increase their intercellular communication transferring proteins to CB-NK and to neighboring MM cells In a complementary approach, MM cells were expanded in vitro with hAA to identify 1°MM proteome transferred to CB-NK and to 2°MM (Additional file 1:FigureS1B).As control, the transmission of MM proteome between MM cells under ‘resting’conditions (absence of CB-NK) was also investigated (Additional file 1: Figure S1C). After CBNK exposure, CB-NK received 7.3% of MM proteome (Fig. 1c, Additional file 1: Table S4). Moreover, whereas under ‘resting’conditions 2.5% of MM proteome was transferred between MM cells (Fig. 1c, Additional file 1: Table S5), after CB-NK, MM proteome transfer between neighboring MM cells increased to 7.7% (Fig. 1c, Additional file 1: Table S6). These experiments suggested that MM cells display a low constitutive transfer of their proteome, which is increased after CB-NK exposure leading to a bidirectional exchange of proteome. CB-NK histones are also released into the extracellular milieu after co-culture with MM cells A third experiment co-culturing hAA-labeled MM cells and CB-NK was performed to analyze released SIPs (Additional file 1: Figure S1D). Proteomic analysis showed that Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 3 of 16 Journal for ImmunoTherapy of Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. Fig. 1 (See legend on next page.) Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 4 of 16 Journal for ImmunoTherapy of Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. SIPs contained 30% of CB-NK proteins and 70% of proteins from hAA-labeled MM cells (Fig. 1d, Additional file 1: Tables S7 and S8). Clustering analysis of this 30% of CB-NK proteins by using STRING-database showed the presence of histones in this released NK material. Histones are highly involved in inflammation and coagulation mechanisms known as ‘immunothrombosis’[12]. AsshowninFig.1d, most of the other NK proteins detected in the same cluster of histones (red cluster) are also involved in inflammation, coagulation and/or cell migration processes. CB-NK histones are actively transferred through MM cells Proteomic data suggested a dynamic movement of CBNK histones through different MM cells, as 1°MM lost their CB-NK histones by passing them to 2°MM. As histones show antimicrobial [13,14] and anti-tumor properties [15], further studies were undertaken to confirm their cell-cell transfer and impact on MM cells. Histone variant H2AZ1 (H2AZ) was first selected due to the high number of PSMs detected and to its presence in 1°MM and 2°MM cells. In addition, YWHAZ and YWHAQ were also selected for further analysis since these CBNK proteins were identified either in 1°MM or 2°MM cells and are involved in tumor cell survival [16]. H2AZ, YWHAZ and YWHAQ fused to green fluorescent protein (GFP) were overexpressed in MM cells. While YWHAZ-GFP and YWHAQ-GFP overexpression had no effect on the in vitro proliferation of ARP1 cells, H2AZ-GFP over-expression significantly decreased ARP1 proliferation (Fig. 2a), and this inhibitory effect was not observed in CB-NK (Fig. 2a). Moreover, when CB-NK were transduced with these GFP-fused proteins and co-cultured with MM cells for 30 min, transfer of these proteins from CB-NK to MM cells was confirmed. While YWHAQ-GFP and YWHAZ-GFP were transmitted from CB-NK to MM cells in vesicles (Fig. 2b), H2AZ-GFP was transmitted by both vesicles (Fig. 2c) and large intercellular structures co-localizing with DNA (Additional file 1: Figure S2A). Moreover, H2AZ-GFP also appeared to adhere to MM surface (Fig. 2c). In addition, H2AZ-intercellular structures were also detected after co-culturing CB-NK with primary CD138 + cells from MM patients (Additional file 1: Figure S2B). We next analyzed whether CB-NK could transfer H2AZ to other cells besides MM cells. Co-culturing CB-NKH2AZ-GFP with MM and non-MM (K562) cell lines, showed that a fraction of all tumor cell lines expressed H2AZ-GFP after 24 h (Fig. 2d). To confirm that H2AZ transfer was an active and regulated mechanism, the same experiment was performed in parallel at 4 °C and 37 °C, confirming an increased and active H2AZ transfer from CB-NK to tumor cells at 37 °C, and that the degree of transfer was lower for non-MM K562 (Fig. 2e). Since H2AZ was transferred from CB-NK to MM cells and then, secondarily between MM cells (Fig. 1b), we next analyzed whether tumor (MM and non-MM K562) cells over-expressing H2AZ could transfer this protein to neighboring tumor cells in the absence of CB-NK. Indeed, transmission of H2AZ-GFP to neighboring tumor cells was observed in the absence of CB-NK (Fig. 2f). The kinetics of H2AZ-GFP transfer between neighboring MM cells was monitored from 1 to 18 h (Fig. 2g) demonstrating that the rate of H2AZ transfer occurred at a much lower rate than in the presence of CB-NK (Fig. 2e) and indicating that histone NK transfer to MM cells is an active process. Different rates of H2AZ-GFP transfer were observed for each cell line. Thus, although for RPMI cells, the initial rate of H2AZ-GFP transfer was relatively high, it increased less with extended time in culture than for ARP1 and U266 cells where the initial rate of H2AZ-GFP transfer was lower (Fig. 2g). To investigate whether the transfer of materials between cells was unique to toxic proteins, or applicable to all proteins, we compared the transfer kinetics of H2AZ-GFP and GFP. Although the actual rates of transfer and proportion of protein transferred differed between the different cell lines, similar rates of transfer were observed for both molecules (H2AZ-GFP and GFP) for each cell line (Fig. 2h). Altogether, our results indicate that tumor cells transfer proteins between them, and that the presence of CB-NK cells greatly increases the transfer rate. (See figure on previous page.) Fig. 1 Cord blood derived NK cells (CB-NK) increase cell-cell communication between CB-NK and MM cells, leading to enhanced proteome transfer, including a high number of histones. aand c: Percentage of heavy-labeled (transferred) proteins from the total cell proteome in each cell population after labeling either CB-NK (a) or MM cells (c) with heavy amino acids (hAA). Each cell population was obtained after co-culturing and FACS sorting according to Diagram shown in Additional file 1: Figure S1. b: Schematic design of the cell populations analyzed which are shown in the Table below to present trafficking of CB-NK histones and other NK proteins through MM cells (Additional file 1: Tables S1, S2 and S3). Numbers in the table indicate number of PSMs (peptide spectral match) detected, indicating the relative abundance of proteins. Scheme shows CB-NK in resting conditions and after co-culture with MM cells (1°MM). Afterwards, 1°MM cells transfer CB-NK histones secondarily to neighboring MM cells (2°MM), with subsequent loss of CB-NK histones in 1°MM cells. d. Analysis of released proteins after CB-NK/MM cell coculture termed Supernatant containing Inflammatory Proteins (SIPs). See diagram shown in Additional file 1: Figure S1D. CB-NK proteins from SIPs are shown in the diagram, and proteins of the red cluster, which includes histones (in a red circle), are detailed. See also Additional file 1: Tables S1-S8 for list of transferred proteins Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 5 of 16 Journal for ImmunoTherapy of Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. Fig. 2 (See legend on next page.) Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 6 of 16 Journal for ImmunoTherapy of Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. Histones are involved in CB-NK anti-MM activity To test whether H2AZ was involved in NK cytotoxicity, we performed knock-down (siRNA) and over-expression of H2AZ in CB-NK. H2AZ knock-down in CB-NK reduced cytotoxicity against MM cells but not against K562 cells (Fig. 3a), suggesting that although H2AZ is transferred from CB-NK to K562 (Fig. 2d) it has no role in the killing of K562. Conversely, H2AZ overexpression in CB-NK increased their cytotoxicity against MM cells in a different degree depending on the MM cell line (Fig. 3b). No effect was detected for K562, consistent with previous studies reporting that NKcytotoxicity against K562 is mainly mediated through Granzyme B and Caspase-3 [9]. The role of H2AZ in the killing of MM cells by NK was further confirmed by the assessment of peripheral blood (PB)-NK. Although PB-NK expressed lower H2AZ levels than CB-NK (Fig. 3c), H2AZ knock-down reduced PB-NK cytotoxicity against MM cells, a finding not observed against K562 (Fig. 3d), confirming also H2AZ involvement in PB-NK anti-MM activity. Finally, the impact of other histones (H4 and H1.5) in CB-NK cytotoxicity was also assessed. Individual knockdown of H2AZ, H4 and H1.5 in CB-NK (Additional file 1: Figure S3) decreased CB-NK cytotoxicity against MM cells, and not against K562 cells (Fig. 3e), confirming that, as suggested in the proteomic data, histones are involved in CB-NK anti-MM activity. CB-NK and histones promote pyroptosis with in vivo MM cell death and concomitant inflammation Extracellular histones are highly pro-inflammatory and activate the inflammasome leading to pyroptosis, an inflammatory form of cell death caspase-1-dependent [13,17,18]. Since NK cells show pro-inflammatory activity [19], and we had previously discarded apoptosis as the form of cytotoxicity [9], we hypothesized that CB-NK-associated histones might induce pyroptosis to kill MM cells. Indeed, we found that inhibition of Caspase-1 decreased CB-NK anti-MM activity, an effect not detected in K562 cells (Fig. 4a). Importantly, addition of recombinant H2AZ induced antiMM activity (Fig. 4b), a finding also observed for recombinant H4 (Additional file 1:FigureS4).Intheseexperimental conditions, caspase-1 inhibition also reduced H2AZmediated cytotoxicity (Fig. 4b). To assess the anti-MM and inflammatory role for histones in vivo, NSG-mice receiving ARP1 and treated with recombinant H2AZ showed that administration of H2AZ was associated with a remarkable delay in the progression of MM (Fig. 4c-e). However, abnormally enlarged lung and spleen tissues were observed, which could result from inflammatory damage induced by H2AZ (Fig. 4f). Interestingly, the phenotype of MM cells in bone marrow and spleen from, H2AZ-treated mice presented decreased intensity of CD138 (Fig. 4g), a marker highly expressed on MM cells. NK-histones specifically bind to CD138 on MM cell surface We noticed that NK-histones adhered to MM cell surface (Fig. 2c) and a decreased CD138 expression in vivo in MM cells after H2AZ treatment (Fig. 4g). In this regard, histones are cationic proteins that can be neutralized with anionic proteins such as heparin [20]. Interestingly, CD138 is a highly anionic type of HSPG [21] greatly abundant in the surface of MM cells, required for MM tumor growth, vascularization, and metastasis, being essential for MM cells [22,23]. Therefore, we hypothesized that cationic histones would bind to anionic CD138 in tumor cells. After confirming CD138 expression in MM cells (Fig. 5a), MM cells were treated with either recombinant H2AZ or H4 and stained for CD138 and CD56, two markers of MM cells. Histone treatment decreased only CD138 expression (Fig. 5b) suggesting that histones were binding to CD138. Confocal fluorescence microscopy demonstrated a high colocalization of CD138 and H2AZ (Fig. 5candd).Moreover,coculture of CB-NK overexpressing H2AZ-GFP with MM cells also demonstrated colocalization of CD138 and H2AZ (Fig. 5e). (See figure on previous page.) Fig. 2 CB-NK histones are actively transferred through MM cells. a: ARP1 and CB-NK cell proliferation during four days after over-expression of YWHAQ-GFP, YWHAZ-GFP and H2AZ-GFP vs control, measured by viable cell count. b-c: Transfer of YWHAZ and YWHAQ (b), and H2AZ (c) from CB-NK to ARP1 cells. CB-NK transduced with the corresponding protein fused to GFP are co-cultured with ARP1 cells for 30 min. ARP1 cells in blue (CMAC) and CB-NK show in green the corresponding GFP-fused protein. Arrows in cindicate H2AZ-vesicles and H2AZ adhered to the surface of MM cells. d: Transfer of H2AZ-GFP from CB-NK to MM (ARP1, RPMI and U266) and non-MM K562 cells after 24 h of co-culture. Target cells are shown in blue (CMAC) in plot 1, and plot 2 corresponds to the gate of CMAC+ cells. e. Transfer of H2AZ-GFP from CB-NK to MM and non-MM K562 cells after 24 h of co-culture performed in parallel at 37 °C and 4 °C. fto h: H2AZ can be transferred between tumor cells in a CB-NK independent manner. f: Transfer of H2AZ from tumor cells over-expressing H2AZ-GFP to neighboring tumor cells stained in blue (CMAC) after 30 min of co-culture. Arrows indicate H2AZ-vesicles and H2AZ-intercellular structure being transferred to neighboring tumor cells. g: H2AZ transfer from MM cells over-expressing H2AZ-GFP to neighboring MM cells in blue (CMAC) at different times (1 h, 5 h, 18 h) of co-culture. Statistical analysis shown is performed for each cell line vs 1 h time point. h: Transfer of GFP (plot on the left) and H2AZ-GFP (plot on the right) from MM and non-MM K562 cells over-expressing these proteins to neighboring MM and non-MM K562 cells after 24 h of co-culture. Representative images from at least three independent experiments Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 7 of 16 Journal for ImmunoTherapy of Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. In addition, MM cells were co-cultured with either CBNK control or CB-NK where histones (H2AZ, H4 and H1.5) had been knocked-down (CB-NK siRNA Hist), and CD138 expression was analyzed. Moreover, SIPs from these co-cultures were taken and added into MM cells alone to analyze also their impact in CD138 expression (Fig. 5f). In both cases, CB-NK siRNA ctrl (Fig. 5g) and SIPs siRNA ctrl (Fig. 5h) decreased CD138 expression in MM cells, an effect which was reversed after knock-down of histones (Fig. 5g and h). Additionally, SIPs did not impact CD56 expression (Fig. 5h) further supporting the specific binding of CB-NK histones to CD138. Fig. 3 Histones are involved in CB-NK anti-MM activity. a. 3 h cytotoxicity assays comparing CB-NK control (CB-NK siRNA ctrl) vs CB-NK with knockdown of H2AZ (CB-NK siRNA H2AZ). b. 3 h cytotoxicity assays comparing CB-NK control (CB-NK GFP) vs CB-NK over-expressing H2AZ (CB-NK H2AZ). c. H2AZ levels in peripheral blood NK cells (PB-NK) vs CB-NK, analyzed by confocal fluorescence microscopy. Representative image of H2AZ levels is shown on the right. d. 3 h cytotoxicity assays comparing PB-NK control (PB-NK siRNA ctrl) vs PB-NK with knockdown of H2AZ (PBNK siRNA H2AZ). e. 3 h cytotoxicity assays comparing CB-NK (CB-NK siRNA ctrl) with CB-NK where histones H2AZ, H4 and H1.5 were knockdown. Assays were performed at least in three independent experiments. a: all groups analyzed compared to CB-NK siRNA ctrl are different (p< 0.05). b: at least one group analyzed compared to CB-NK siRNA ctrl is different (p< 0.05). *p< 0.05. ** p< 0.001. Efficiency of knockdown of H2AZ was confirmed by Western Blot and by flow cytometry (Additional file 1: Figure S3) Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 8 of 16 Journal for ImmunoTherapy of Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. NK-histones promote cell clustering formation required for NK cell anti-MM activity Proteomic data showed a high number of released NK inflammatory proteins in the SIPs, including histones, which were also involved in coagulation (Fig. 1d). The innate immune system has the ability to initiate a process termed ‘immunothrombosis’, whereby the release of inflammatory proteins, including histones, a local intravascular scaffold is provided to immobilize, contain and destroy pathogenic microorganisms [12,24]. Interestingly, NK cells require high effector (E):target (T) ratio for the NK and target cells to be in close proximity for optimal anti-tumor efficacy. However, NK cells represent 1–6% of peripheral blood leukocytes, therefore a process that can promote a high localized E:T ratio bringing NK and MM cells into close proximity would be physiologically relevant. Therefore, we hypothesized that histones were required for NK to promote cell clustering thereby improving MM cell killing. It is known that heparin inhibits immunothrombus formation, neutralizes cationic histones [20] and also competes with anionic HSPG for their binding to cationic ligands [11], such as histones. Therefore, 24 h cytotoxicity assays with heparin were performed at low E:T ratios, and the area of cell clusters were measured at 2.5 h and at 24 h. Heparin inhibited cell cluster formation (Additional file 1: Fig. 4 CB-NK and histones promote pyroptosis with in vivo MM cell death and concomitant inflammation. a. 3 h cytotoxicity assays of CB-NK against MM and non-MM K562 cells, adding Caspase-1 inhibitor to analyze the impact on pyroptotic cell death. b. Impact of recombinant H2AZ on viability of MM and non-MM K562 cells. HSA: Human Serum Albumin (2 μM) was added as protein control in parallel with H2AZ (2 μM). Y-VAD was added to analyze the impact on H2AZ effect. 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