J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 https://doi.org/10.1186/s40425-019-0739-1 RESEARCH ARTICLE Open Access Extracellular NK histones promote immune cell anti-tumor activity by inducing cell clusters through binding to CD138 receptor B. Martín-Antonio1,2*, G. Suñe1,2, A. Najjar3, L. Perez-Amill1, A. Antoñana-Vildosola1, M. Castella1, S. León1, M. Velasco-de Andrés4, F. Lozano4,6,7, E. Lozano1, C. Bueno5, J. M. Estanyol8, C. Muñoz-Pinedo9, S. N. Robinson10 and A. Urbano-Ispizua1,2,11 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. Protected by copyright. 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 understand and exploit their anti-tumor activity. In this Natural killer (NK) cells are important anti-tumor cells of regard, NK cells present a wide array of activating and our innate immune system whose anti-tumor properties inhibitory receptors that interact with their ligands on led to anti-cancer, immune NK cell therapies under devel- tumor cells [4]. However, besides these receptor-ligands opment [1]. The majority of clinical studies infusing NK interactions, a cross-talk among different immune cells, cells worked mostly for acute myeloid leukemias but performed by pro-inflammatory molecules secreted by im- performed poorly in other malignancies [2, 3], suggesting mune cells, contributes to the final immune response [5]. that a deeper knowledge of NK cells is required to better The relevance of this cross-talk between immune cells is observed after microbial infection, where dendritic * Correspondence: bmartina@clinic.cat cells (DCs) activate NK cells through IL15 secretion 1 Department of Hematology, Hospital Clinic, IDIBAPS, Carrer Rosselló 149-153, 08036 Barcelona, Spain leading to T cell and monocyte activation [5–7]. The 2 Josep Carreras Leukaemia Research Institute, Carrer Rosselló 149-153, 08036 coordination of these immune responses requires the Barcelona, Spain creation of cellular clusters to enable intercellular cross- Full list of author information is available at the end of the article © 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. J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 2 of 16 talk between immune cells [7, 8]. We previously cultured in RPMI-1640 with 10% fetal bovine serum reported the relevance of this cell-cell contact as a (FBS) and U266 with 15% FBS. 293 T cells were cultured mechanism leading to a transmissible cytotoxicity from in DMEM with 10% FBS. CD138+ cells from MM cord blood derived NK cell (CB-NK) to neighboring patients were obtained by MACS selection (Miltenyi multiple myeloma (MM) cells, as CB-NK cytotoxicity is Biotec). transferred to ‘primary’ MM cells (1°MM) after contact; and afterwards, it is passed from 1°MM to adjacent ‘sec- TRANS-SILAC proteomics ondary’ MM cells (2°MM) unexposed to CB-NK [9]. Was performed culturing cells in their usual media lack- Interestingly, CB-NK perform Granzyme-B and Caspase- ing normal L-Arg, L-Leu and L-Lys, and supplemented 3 independent killing of MM cells [9], suggesting the with their corresponding heavy isotopic AA (hAA). involvement of other proteins in the CB-NK anti-MM ARP1-MM cells were expanded in this media for 21 activity. Moreover, whereas effector cytokines require days, and CB-NK during the 14 days of the usual CB-NK hours to be detected, cell cluster formations occur earl- in vitro expansion. After this period, both cell popula- ier, suggesting that other initiating molecules secreted at tions contained > 97% of hAA as determined by liquid early times of cell-cell contact will impact on the final Chromatography-Tandem Mass Spectrometry (LC-MS/ effector response. MS). Percentage of heavy proteins transferred to each These observations led us to hypothesize that novel cell population was analyzed by LC-MS/MS. cytotoxic molecules transferred from CB-NK to MM cells could be involved in the anti-MM CB-NK activity. Cytotoxicity assays Therefore, TRANS-stable-isotope labeling of amino Were performed at 3 h by Europium Release Assays [9], acids in cell culture (TRANS-SILAC) [10] combined and at longer times by flow cytometry calculating % of with proteomic was undertaken to identify early mate- remaining live GFP+ tumor cells applying the formula: Protected by copyright. rials transferred between CB-NK and MM. Analysis % of target cell lysis = 100-(% of GFP+ cells at 24-48 h / revealed that histones are actively transferred between % of GFP+ cells at 0 h). CB-NK and MM and also released into the extracellular milieu after co-culturing CB-NK and MM. Released CB- In vivo myeloma murine model NK histones bind to CD138 in MM cells promoting the NOD/SCID IL-2Rcnull (NSG) mice were irradiated and formation of CB-NK/MM cell clusters which facilitates inoculated i.v. with GFP-Firefly Luciferase-transduced NK-MM contact and improves the anti-tumor NK ARP1 cells. Recombinant H2AZ (0.5 mg/kg) was given efficacy. Furthermore, NK-histones also promoted the i.v. on day 1 and day 7. Disease progression was moni- generation of cell clusters between T-cells and MM cells tored by bioluminescence using a Hamamatsu CDD increasing the T cell anti-MM activity and revealing a camera (Hamamatsu Photonics Sistems) following a 100 novel mechanism by which NK enhance the anti-tumor mL IP injection of D-luciferin (20 mg/mL), and measur- activity of T-lymphocytes. ing serum kappa light chain levels by ELISA (Bethyl La- boratories). Signal quantitation was performed with Methods ImageJ software. Cell cultures NK cells were isolated from CB and PB by magnetic Transfer of H2AZ-GFP transfer between cells depletion (Miltenyi Biotec). CB-NK expansion was Cells were co-cultured staining in blue (CMAC) the cell performed during 14 days as previously described [9] population of interest. Then, H2AZ-GFP transfer between using K562-based antigen presenting cells expressing cells was analyzed by flow cytometry gating on the CMAC+ membrane bound IL-21 (“Clone 9.mbIL21”). T cells population and analyzing the % of CMAC+GFP+ cells. were isolated from PB by magnetic depletion (Miltenyi Biotec) and expanded during 5 days with Dynabeads® Supernatant Containing Inflammatory Proteins (SIPs) Human T-Activator CD3/CD28 (Thermo-Fisher). IL2 analysis (Proleukin) was added at 100UI/mL every other day. To analyze released proteins to the extracellular milieu Culture NK and T cell media was comprised of 45% by each cell population, 30–40 min co-cultures experi- RPMI-1640 (Sigma-aldrich) and 45% Click’s (Irvine Sci- ments were performed collecting the supernatants and entific) with 10% AB human serum (Atlanta Biologicals). differentiating proteins of each cell population by their ARP1 cell line was provided by Multiple Myeloma previous hAA labeling (Additional file 1: Figure S1D). Research Center (Little Rock, AK). 293 T, K562, U266, RPMI-8226, Ramos and Jurkat cells were obtained from Reagents American Type Culture Collection (ATCC, Rockville, Caspase-1 inhibition was achieved with Y-VAD (50 μM) MD). K562, ARP1, RPMI, Ramos and Jurkat were addition. Heparinase III (Sigma-aldrich) treatment (0.01 J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 3 of 16 IU/mL) was used to remove HSGPG from MM cells the 1°MM to neighboring 2°MM cells. These proteins [11]. Recombinant H2AZ (Merck-Millipore) and H4 were thus originally derived from CB-NK, but via 1°MM (New England Biolabs) were added in cell culture at (Fig. 1a, Additional file 1: Table S2). As a consequence of 2 μM or 0.5 μM depending on the experiment. Heparin this transfer, 1°MM lost part of their labeled, previously (STEMCELL Technologies) was used at 20 IU/mL. acquired CB-NK proteome content that went down from DNAse I (D2) (Worthington Biochemical Corporation) 9.5 to 3.9% (Fig. 1a, Additional file 1: Table S3). These was used at 100 IU/mL. Antibodies used were CD138- data provided evidence for a primary-direct CB-NK prote- V421 and CD56-V450 (BD Biosciences), H2AZ, H4, ome transfer to 1°MM, and a secondary-indirect CB-NK H1.5, Anti-Rabbit IgG-HRP, and Anti-Rabbit IgG-Alexa proteome transfer to 2°MM. Fluor-647 (Cell Signaling Technology). Analysis of CB-NK transferred proteins to MM cells showed a high number of CB-NK histones acquired by Cell cluster formation MM cells. In resting conditions, different histones were Area of cell clusters was visualized at different time present in CB-NK (Fig. 1b: CB-NK resting). However, points by measuring the GFP area from tumor cells after co-culturing with 1°MM, CB-NK lost their histone using ImageJ software. content (Fig. 1b: CB-NK after 1°MM), as indicated by absence of detection of Peptide Spectral Matches Ethics Statement (PSMs). Conversely, MM cells underwent an enrichment Research involving human materials was approved by in these histones (Fig. 1b), suggesting the selectivity of Ethical Committee of Hospital Clinic, Barcelona. CB this process. We observed that subsequently, CB-NK units and PB were obtained from healthy donors who histones from 1°MM cells were then transferred to gave informed consent. 2°MM cells (Fig. 1b: 2°MM), and as a consequence, the content of labeled, CB-NK histones in 1°MM Protected by copyright. Statistical analysis disappeared (Fig. 1b: 1°MM after 2°MM), suggesting a Mann-Whitney U test was used to analyze comparison continuous, dynamic and specific transfer of CB-NK between groups. Statistical analyses were performed with histones between MM cells. Of note, other NK proteins SPSS (IBM SPSS v. 23). detected in the proteomic data (YWHAZ and YWHAQ) LC-MS/MS, confocal fluorescence microscopy, flow did not show this pattern of continuous transfer cytometry, GFP-fused protein generation, lentivirus observed for histones (Fig. 1b). production and siRNA transfection are detailed in Additional file 1: Supplementary Methods. MM cells exposed to CB-NK increase their intercellular communication transferring proteins to CB-NK and to Results neighboring MM cells CB-NK histones are dynamically transferred from CB-NK In a complementary approach, MM cells were expanded to primary MM cells and subsequently to adjacent in vitro with hAA to identify 1°MM proteome transferred secondary MM cells to CB-NK and to 2°MM (Additional file 1: Figure S1B). As In order to identify cytotoxic CB-NK proteins trans- control, the transmission of MM proteome between MM ferred directly to MM cells (1°MM), and secondarily to cells under ‘resting’ conditions (absence of CB-NK) was neighboring MM cells (2°MM), TRANS-SILAC prote- also investigated (Additional file 1: Figure S1C). After CB- omic was performed to identify acquired proteome for NK exposure, CB-NK received 7.3% of MM proteome each cell population [10]. Co-culture experiments were (Fig. 1c, Additional file 1: Table S4). Moreover, whereas limited to 30–40 min to unravel early transferred pro- under ‘resting’ conditions 2.5% of MM proteome was teins between live cells responsible for initiating NK transferred between MM cells (Fig. 1c, Additional file 1: cytotoxicity. Table S5), after CB-NK, MM proteome transfer between CB-NK were labeled with heavy amino acids (hAA) to neighboring MM cells increased to 7.7% (Fig. 1c, allow for identification of ‘heavy’ CB-NK proteome Additional file 1: Table S6). These experiments suggested transferred to 1°MM (labeled with CMAC); and subse- that MM cells display a low constitutive transfer of their quently from 1°MM to 2°MM (unstained) (Additional proteome, which is increased after CB-NK exposure lead- file 1: Figure S1A). Proteomic data showed that after ing to a bidirectional exchange of proteome. CB-NK/1°MM co-culture, the 1°MM proteome contained 9.5% of proteins transferred from CB-NK CB-NK histones are also released into the extracellular (Fig. 1a) (Additional file 1: Table S1). Then, 1°MM were milieu after co-culture with MM cells co-cultured with fresh MM cells to determine CB-NK A third experiment co-culturing hAA-labeled MM cells proteins transferred secondarily between MM cells, re- and CB-NK was performed to analyze released SIPs (Add- vealing 7.2% of secondary transfer of NK proteome from itional file 1: Figure S1D). Proteomic analysis showed that J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Protected by copyright. Page 4 of 16 (2019) 7:259 Martín-Antonio et al. Journal for ImmunoTherapy of Cancer Fig. 1 (See legend on next page.) J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 5 of 16 (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. a and 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 co- culture 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 SIPs contained 30% of CB-NK proteins and 70% of proteins detected after co-culturing CB-NK with primary CD138+ from hAA-labeled MM cells (Fig. 1d, Additional file 1: cells from MM patients (Additional file 1: Figure S2B). Tables S7 and S8). Clustering analysis of this 30% of We next analyzed whether CB-NK could transfer H2AZ CB-NK proteins by using STRING-database showed the to other cells besides MM cells. Co-culturing CB-NK- presence of histones in this released NK material. His- H2AZ-GFP with MM and non-MM (K562) cell lines, tones are highly involved in inflammation and coagula- showed that a fraction of all tumor cell lines expressed tion mechanisms known as ‘immunothrombosis’ [12]. H2AZ-GFP after 24 h (Fig. 2d). To confirm that H2AZ As shown in Fig. 1d, most of the other NK proteins de- transfer was an active and regulated mechanism, the same tected in the same cluster of histones (red cluster) are experiment was performed in parallel at 4 °C and 37 °C, also involved in inflammation, coagulation and/or cell confirming an increased and active H2AZ transfer from Protected by copyright. migration processes. CB-NK to tumor cells at 37 °C, and that the degree of transfer was lower for non-MM K562 (Fig. 2e). CB-NK histones are actively transferred through MM cells Since H2AZ was transferred from CB-NK to MM cells Proteomic data suggested a dynamic movement of CB- and then, secondarily between MM cells (Fig. 1b), we NK histones through different MM cells, as 1°MM lost next analyzed whether tumor (MM and non-MM K562) their CB-NK histones by passing them to 2°MM. As his- cells over-expressing H2AZ could transfer this protein tones show antimicrobial [13, 14] and anti-tumor prop- to neighboring tumor cells in the absence of CB-NK. In- erties [15], further studies were undertaken to confirm deed, transmission of H2AZ-GFP to neighboring tumor their cell-cell transfer and impact on MM cells. Histone cells was observed in the absence of CB-NK (Fig. 2f). variant H2AZ1 (H2AZ) was first selected due to the high The kinetics of H2AZ-GFP transfer between neighboring number of PSMs detected and to its presence in 1°MM MM cells was monitored from 1 to 18 h (Fig. 2g) dem- and 2°MM cells. In addition, YWHAZ and YWHAQ onstrating that the rate of H2AZ transfer occurred at a were also selected for further analysis since these CB- much lower rate than in the presence of CB-NK (Fig. NK proteins were identified either in 1°MM or 2°MM 2e) and indicating that histone NK transfer to MM cells cells and are involved in tumor cell survival [16]. is an active process. Different rates of H2AZ-GFP trans- H2AZ, YWHAZ and YWHAQ fused to green fluores- fer were observed for each cell line. Thus, although for cent protein (GFP) were overexpressed in MM cells. RPMI cells, the initial rate of H2AZ-GFP transfer was While YWHAZ-GFP and YWHAQ-GFP over- relatively high, it increased less with extended time in expression had no effect on the in vitro proliferation of culture than for ARP1 and U266 cells where the initial ARP1 cells, H2AZ-GFP over-expression significantly de- rate of H2AZ-GFP transfer was lower (Fig. 2g). creased ARP1 proliferation (Fig. 2a), and this inhibitory To investigate whether the transfer of materials be- effect was not observed in CB-NK (Fig. 2a). Moreover, tween cells was unique to toxic proteins, or applic- when CB-NK were transduced with these GFP-fused able to all proteins, we compared the transfer proteins and co-cultured with MM cells for 30 min, kinetics of H2AZ-GFP and GFP. Although the actual transfer of these proteins from CB-NK to MM cells was rates of transfer and proportion of protein trans- confirmed. While YWHAQ-GFP and YWHAZ-GFP ferred differed between the different cell lines, simi- were transmitted from CB-NK to MM cells in vesicles lar rates of transfer were observed for both (Fig. 2b), H2AZ-GFP was transmitted by both vesicles molecules (H2AZ-GFP and GFP) for each cell line (Fig. 2c) and large intercellular structures co-localizing (Fig. 2h). Altogether, our results indicate that tumor with DNA (Additional file 1: Figure S2A). Moreover, cells transfer proteins between them, and that the H2AZ-GFP also appeared to adhere to MM surface (Fig. presence of CB-NK cells greatly increases the trans- 2c). In addition, H2AZ-intercellular structures were also fer rate. J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Protected by copyright. Page 6 of 16 (2019) 7:259 Martín-Antonio et al. Journal for ImmunoTherapy of Cancer Fig. 2 (See legend on next page.) J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 7 of 16 (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 c indicate 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. f to 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 Histones are involved in CB-NK anti-MM activity Importantly, addition of recombinant H2AZ induced anti- To test whether H2AZ was involved in NK cytotoxicity, MM activity (Fig. 4b), a finding also observed for recombin- we performed knock-down (siRNA) and over-expression ant H4 (Additional file 1: Figure S4). In these experimental of H2AZ in CB-NK. H2AZ knock-down in CB-NK conditions, caspase-1 inhibition also reduced H2AZ- reduced cytotoxicity against MM cells but not against mediated cytotoxicity (Fig. 4b). K562 cells (Fig. 3a), suggesting that although H2AZ is To assess the anti-MM and inflammatory role for transferred from CB-NK to K562 (Fig. 2d) it has no role histones in vivo, NSG-mice receiving ARP1 and treated in the killing of K562. Conversely, H2AZ over- with recombinant H2AZ showed that administration of Protected by copyright. expression in CB-NK increased their cytotoxicity against H2AZ was associated with a remarkable delay in the MM cells in a different degree depending on the MM progression of MM (Fig. 4c-e). However, abnormally cell line (Fig. 3b). No effect was detected for K562, con- enlarged lung and spleen tissues were observed, which sistent with previous studies reporting that NK- could result from inflammatory damage induced by cytotoxicity against K562 is mainly mediated through H2AZ (Fig. 4f). Interestingly, the phenotype of MM cells Granzyme B and Caspase-3 [9]. in bone marrow and spleen from, H2AZ-treated mice The role of H2AZ in the killing of MM cells by NK presented decreased intensity of CD138 (Fig. 4g), a was further confirmed by the assessment of peripheral marker highly expressed on MM cells. blood (PB)-NK. Although PB-NK expressed lower H2AZ levels than CB-NK (Fig. 3c), H2AZ knock-down reduced NK-histones specifically bind to CD138 on MM cell surface PB-NK cytotoxicity against MM cells, a finding not ob- We noticed that NK-histones adhered to MM cell served against K562 (Fig. 3d), confirming also H2AZ in- surface (Fig. 2c) and a decreased CD138 expression volvement in PB-NK anti-MM activity. in vivo in MM cells after H2AZ treatment (Fig. 4g). Finally, the impact of other histones (H4 and H1.5) in In this regard, histones are cationic proteins that can CB-NK cytotoxicity was also assessed. Individual knock- be neutralized with anionic proteins such as heparin down of H2AZ, H4 and H1.5 in CB-NK (Additional file 1: [20]. Interestingly, CD138 is a highly anionic type of Figure S3) decreased CB-NK cytotoxicity against MM HSPG [21] greatly abundant in the surface of MM cells, and not against K562 cells (Fig. 3e), confirming that, cells, required for MM tumor growth, vascularization, as suggested in the proteomic data, histones are involved and metastasis, being essential for MM cells [22, 23]. in CB-NK anti-MM activity. Therefore, we hypothesized that cationic histones would bind to anionic CD138 in tumor cells. After CB-NK and histones promote pyroptosis with in vivo MM confirming CD138 expression in MM cells (Fig. 5a), cell death and concomitant inflammation MM cells were treated with either recombinant H2AZ Extracellular histones are highly pro-inflammatory and acti- or H4 and stained for CD138 and CD56, two markers vate the inflammasome leading to pyroptosis, an inflamma- of MM cells. Histone treatment decreased only tory form of cell death caspase-1-dependent [13, 17, 18]. CD138 expression (Fig. 5b) suggesting that histones Since NK cells show pro-inflammatory activity [19], and we were binding to CD138. Confocal fluorescence mi- had previously discarded apoptosis as the form of cytotox- croscopy demonstrated a high colocalization of icity [9], we hypothesized that CB-NK-associated histones CD138 and H2AZ (Fig. 5c and d). Moreover, co- might induce pyroptosis to kill MM cells. Indeed, we found culture of CB-NK overexpressing H2AZ-GFP with that inhibition of Caspase-1 decreased CB-NK anti-MM ac- MM cells also demonstrated colocalization of CD138 tivity, an effect not detected in K562 cells (Fig. 4a). and H2AZ (Fig. 5e). J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 8 of 16 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 Protected by copyright. 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 (PB- NK 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) In addition, MM cells were co-cultured with either CB- (Fig. 5f). In both cases, CB-NK siRNA ctrl (Fig. 5g) and NK control or CB-NK where histones (H2AZ, H4 and SIPs siRNA ctrl (Fig. 5h) decreased CD138 expression in H1.5) had been knocked-down (CB-NK siRNA Hist), and MM cells, an effect which was reversed after knock-down CD138 expression was analyzed. Moreover, SIPs from of histones (Fig. 5g and h). Additionally, SIPs did not these co-cultures were taken and added into MM cells impact CD56 expression (Fig. 5h) further supporting the alone to analyze also their impact in CD138 expression specific binding of CB-NK histones to CD138. J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 9 of 16 Protected by copyright. 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. Cell proliferation was measured by viable cell count. c to g: Anti-MM and pro-inflammatory in vivo activity of H2AZ. NSG mice received ARP1 cells and were treated with recombinant H2AZ. Weekly bioluminescence (c and d) images and kappa ELISA light chains measurements (e) were performed. f. Lung and tissues of mice untreated (MM) or treated with H2AZ (MM + H2AZ). g: CD138 expression in MM cells of mice tissues. *p < 0.05 NK-histones promote cell clustering formation required represent 1–6% of peripheral blood leukocytes, therefore for NK cell anti-MM activity a process that can promote a high localized E:T ratio Proteomic data showed a high number of released NK bringing NK and MM cells into close proximity would inflammatory proteins in the SIPs, including histones, be physiologically relevant. Therefore, we hypothesized which were also involved in coagulation (Fig. 1d). The that histones were required for NK to promote cell clus- innate immune system has the ability to initiate a tering thereby improving MM cell killing. It is known process termed ‘immunothrombosis’, whereby the re- that heparin inhibits immunothrombus formation, neu- lease of inflammatory proteins, including histones, a tralizes cationic histones [20] and also competes with local intravascular scaffold is provided to immobilize, anionic HSPG for their binding to cationic ligands [11], contain and destroy pathogenic microorganisms [12, 24]. such as histones. Therefore, 24 h cytotoxicity assays with Interestingly, NK cells require high effector (E):target (T) heparin were performed at low E:T ratios, and the area ratio for the NK and target cells to be in close proximity of cell clusters were measured at 2.5 h and at 24 h. Hep- for optimal anti-tumor efficacy. However, NK cells arin inhibited cell cluster formation (Additional file 1: J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 10 of 16 Protected by copyright. Fig. 5 NK-histones specifically bind to CD138 on MM cell surface. a. CD138 expression in MM and non-MM K562 cells. b: MM cells incubated with either H2AZ or H4 for 1 h and stained for CD138 and CD56. c-d: confocal microscopy images (c) and colocalization analysis (d) of CD138 and H2AZ after addition of recombinant H2AZ to ARP1 MM cells. e. CB-NK overexpressing H2AZ-GFP co-cultured with MM cells showing co- localization of H2AZ-GFP and CD138. f. MM cells and CB-NK (either ctrl or with knock-down of histones H2AZ, H4 and H1.5) were co-cultured for 3 h with MM cells and CD138 expression was analyzed (g). h. SIPs from co-cultures in f were added into MM cells alone for 40 min to analyze CD138 and CD56 expression. *p < 0.05 Figure S6A and Fig. 6a) and reduced the anti-MM CB- absence of HLA-I expression by K562 cells makes these NK activity (Fig. 6b), indicating that CB-NK requires cells strong targets for NK even in the absence of the early cluster formation to perform anti-MM activity, as improved cell-cell contact provided by cell clustering. it has been observed for NK antimicrobial activity [7]. Moreover, knock-down of histones in CB-NK also For K562, the effect of heparin was minimal. The decreased cell clusters formation at 4 h of co-culturing J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Protected by copyright. Page 11 of 16 (2019) 7:259 Martín-Antonio et al. Journal for ImmunoTherapy of Cancer Fig. 6 (See legend on next page.) J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 12 of 16 (See figure on previous page.) Fig. 6 NK histones promote cell clustering formation required for NK cells anti-MM activity. a: Cell clustering formation between CB-NK and MM- GFP and non-MM K562-GFP cells at 2.5 h and 24 h analyzing in parallel the impact of heparin (Hep) (see also Additional file 1: Figure S6A). b: 24 h cytotoxicity assays values obtained from co-cultures in a. c and d: Cell clustering formation at 2.5 h of co-culturing MM-GFP and non-MM K562- GFP cells with either CB-NK ctrl (siRNA ctrl) or CB-NK with knock-down of histones (siRNA Hist). Bar size indicates 100 μm. e and f: Impact of DNase and absence of Fetal bovine serum (FBS) in cell culture media on cell clustering formation (e) and CB-NK cytotoxicity (f) against MM and non-MM K562 cells. Heparin was added in parallel as control. g-j: Impact of recombinant H2AZ (g and h) and H4 (i and j), in the formation of cell clustering and in CB-NK cytotoxicity against MM and non-MM K562 cells. Cell clustering analysis was measured at 2.5 h (g and i) and cytotoxicity at 24 h (h and j). See also Additional file 1: Figure S6B. k-o: CD138 impact on histones activity. k-l: CD138 expression in MM and K562 cells after Heparinase III treatment for 1-2 h (k) and after CD138 knock-down with siRNAs (l). m. Tumor cells ctrl, after Heparinase III treatment (HSPG-) and after CD138 knock-down (CD138−) were co-cultured with CB-NK adding exogenous H2AZ or H4 and cell clustering formation was evaluated at 2 h (n) and cytotoxicity at 6 h (o) (See also Additional file 1: Figure S6C). *p < 0.05. ** p < 0.001 CB-NK and MM cells at low E:T ratios, an effect not ob- anti-MM activity (Fig. 6n and o) without changes in served for K562 (Fig. 6c and d), confirming that CB-NK cluster formation and cytotoxicity against K562 cells histones are involved in the formation of these cell clus- (Additional file 1: Figure S6C). ters which are inhibited with heparin. Furthermore, histones co-localize with DNA and were Released NK-histones also promote T cell/MM cell found in the SIPs with other pro-inflammatory and pro- clustering increasing T cell anti-MM activity coagulant proteins (Fig. 1d), some of which may origin- As NK-histones were detected in the SIPs (Fig. 1d) and ate from serum in the culture medium. Therefore, the given that NK cells can shape the antitumor activity of T effects of both DNAse treatment and removal of serum lymphocytes [25–28], we hypothesized that released NK- were tested. Removal of serum reduced cell clustering histones would also increase T cell/MM cell cluster Protected by copyright. formation (Fig. 6e) and CB-NK cytotoxicity against MM formation facilitating anti-tumor T cell activity. There- (Fig. 6f). The serum impact was also observed for K562 fore, SIPs obtained from CB-NK/MM co-cultures (Fig. 6e and f), suggesting that the serum provides other (Fig. 7a) added into cytotoxicity assays of CD3 T lym- relevant proteins in addition to CB-NK histones. DNAse phocytes against MM cells resulted in specifically treatment, on the other hand, did not impact CB-NK increased anti-MM T cell activity (Fig. 7b). cytotoxicity against MM or K562 cells (Fig. 6e and f) To further investigate whether histones contained in suggesting a histone activity independent of DNA. Inter- the SIPs were responsible for the improved T-cell estingly, DNAse treatment increased cell clustering in killing of MM, H2AZ, H4 and H1.5 were knocked some cases (U266 in Fig. 6e). down in CB-NK that were co-cultured with MM cells. In addition, same experiments were performed adding SIPs obtained from these co-cultures were added into recombinant H2AZ or H4 at a non-toxic dose neither 48 h cytotoxicity assays with T cells at low E:T ratios, for CB-NK nor for target cells (Additional file 1: Figure measuring area of cell clusters at 4 h (Fig. 7c). S5A-C). The addition of H2AZ or H4 increased the size Whereas SIPs from NK cells with siRNA control in- of cell clusters (Additional file 1: Figure S6B, Fig. 6g and creased the area of T cell/MM cell clusters (Fig. 7d i) and the CB-NK anti-MM activity (Fig. 6h and j), being and e) as well as T cell anti-MM activity (Fig. 7f); this effect abrogated by the addition of heparin (Fig. 6g SIPs from NK with silenced histones showed dimin- to j). Consistently, heparin, H2AZ and H4 impact in the ished effects (Fig. 7d-f), confirming that histones CB-NK cytotoxicity against K562 was much lower or released from NK cells increase also T cell-MM cell non-detectable (Fig. 6g to j). clustering and T cell anti-MM activity. For K562, Last, to confirm interaction of CD138 and histones in even though SIPs increased cell cluster formation be- cell cluster formation and in CB-NK cytotoxicity, the ef- tween T cells and K562 cells, they could not increase fect of histones was evaluated in the absence of CD138 T cell cytotoxicity since K562 cells are not a target after Heparinase III treatment [11] or knock-down of for T cells. CD138 by siRNAs. Both heparinase III and siRNA treat- We also tested whether recombinant H2AZ and H4 ment reduced CD138 expression in MM cells (Fig. 6k could also promote clustering of T cells with MM. In and l). Tumor cells (ARP1 and K562) after Heparinase both cases, the area of T cell/MM cell clusters (Fig. 7g, h III treatment (ARP1HSPG- and K562HSPG-) and MM cells and j) and the anti-MM T-cell activity were increased after knock-down of CD138 (siRNA) were co-cultured (Fig. 7i and k) in the presence of histones H2AZ and in parallel with CB-NK, adding exogenous recombinant H4. Heparin addition reversed the enhanced clustering H2AZ and H4 at a non-toxic dose (Fig. 6m). Heparinase (Fig. 7g, h and j) and tumor cell killing (Fig. 7i and k). III treatment and CD138 knock-down abolished the im- Of note, heparin also reduced cluster formation between pact of histones in cell cluster formation and in CB-NK T-cells and K562 cells, without changes in cytotoxicity. J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 13 of 16 Protected by copyright. Fig. 7 Released NK cell histones also promote T cell/MM cell clustering increasing T cell anti-MM activity: a. CB-NK and MM cells were co-cultured and the SIPs were collected and added into 3 h cytotoxicity assays against MM cells adding as effectors T cells (b). SIPs were added in parallel to U266 cells alone as control, and CB-NK were also included as effectors to compare the efficacy. c-f: Impact of histones in the SIPs capacity to increase T cell anti-tumor activity. CB-NK ctrl (siRNA ctrl) and CB-NK with knockdown of histones (siRNA Hist) were co-cultured with MM cells to obtain both SIPs siRNA ctrl and SIPs siRNA Hist (c). These SIPs were added into T-cell/tumor cell co-cultures, and the impact on cell clustering formation after 4 h (d-e) and on T cell cytotoxicity after 48 h was analyzed (f). g-k: Impact of recombinant H2AZ (g-i) and H4 (j and k), in the formation of cell clustering after 4 h, and in the T cell cytotoxicity vs MM and non-MM K562 cells after 48 h. Heparin was added in parallel. *p < 0.05. ** p < 0.001 J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 14 of 16 Histone impact is not a common mechanism for other phenomenon performed by neutrophils in Neutrophil tumor types Extracelullar Traps (NETs) which consist of fibril matrix Since NK-histones did not increase the anti-tumor activ- containing histones to immobilize and eliminate micro- ity of NK and T cells against K562 non-MM cells, we bial pathogens [36, 37]. We confirmed that NK cells also aimed to assess whether this histone impact was specific release histones in vesicles and in similar structures to against MM. Thus, cytotoxicity assays of either NK or T NETs, and that in addition to their well-known anti- cells revealed a very low impact of exogenous H2AZ and microbial activity [36, 37], H2AZ and H4 per se exerted H4 in the NK and T cell anti-tumor activity against a growth inhibitory effect in MM cells in vitro and also lymphoid B cells (Ramos) (Additional file 1: Figure S6D in vivo for H2AZ, being in agreement with previously and E), and no impact was detected towards Jurkat T described anti-tumor properties of histones [15, 38]. In cells (Additional file 1: Figure S6D and E) indicating a addition to this growth inhibitory effect, we found that specific histone activity against MM. Interestingly, histones are involved in CB-NK anti-MM activity as part heparin reduced CB-NK and T cell cytotoxicity for both of the cytotoxic arsenal of NK cells to perform anti-MM Ramos and Jurkat cells (Additional file 1: Figure S6D activity. and E), suggesting that other proteins different to The existence of an immunoregulatory NK cell sub- histones and related to inflammation/immunothrombo- population [39] suggests a relevant role for this NK pro- sis could be involved in the anti-tumor activity against inflammatory activity [19]. In this regard, NK cells re- Ramos and Jurkat cells. To support this hypothesis, ei- lease pro-inflammatory granulysin and granzymes, ther Ramos or Jurkat T cells were co-cultured with CB- whose role is not completely understood [40], and NK to collect SIPs (Additional file 1: Figure S6F) that cytokines which coordinate the immune response by were added in cytotoxicity assays with T cells and tumor recruiting DCs [25–28] and promoting T-cell activity cells. As previously shown for MM, SIPs, containing a [41]. However, effector cytokines secreted by NK cells Protected by copyright. high number of inflammatory proteins, increased both are detected after 2–3 h of immune cell-tumor cell con- cell cluster formation and T cell anti-tumor activity tact, suggesting the existence of other molecules released against Ramos and Jurkat cells (Additional file 1: Figure earlier and responsible for initiating these processes. S6G and H). Taking together, our results demonstrate a Here, we provide evidence of the pro-inflammatory novel mechanism of histone-mediated NK cytotoxicity nature of NK histones [18, 36] by activating pyroptosis through binding to CD138 on MM cell surface. [13, 18, 42] in MM. Histones in collaboration with other pro-inflammatory molecules form the first line of Discussion defense to eliminate pathogens by initiating immuno- The field of immunotherapy to treat cancer has grown thrombosis, a complex process where neutrophil over recent years [29, 30]. While some strategies show histones contribute to generate an intravascular scaffold remarkable success [31, 32], others, including NK-based for containment, exposure and destruction of pathogens therapies, have still yet to realize their full therapeutic [12, 24]. This process, illustrating the relevance of multi- potential [1]. Access to banked CB has provided a source cellular clusters to enable cross-talk between immune of NK to expand NK cells to clinically-relevant doses cells to eliminate microbial pathogens [7, 8], appears making NK-based cellular immunotherapies a real possi- analogous to the capacity of histones to create both NK/ bility [33–35]. The mechanisms by which NK kill tumor tumor and T-cell/tumor clusters to improve NK and T cells are becoming better understood. Previous evidence cell anti-MM activity. This additional immunoregulatory showing the relevance of NK-tumor cell communication ability of NK cells should be considered in immunother- events in mechanisms leading to a transmissible anti- apy strategies, especially given the fact that NK cells tumor activity [9], suggested us to perform TRANS- constitute 1–6% of leukocytes in PB. Importantly, DCs SILAC proteomics to unravel novel transmitted cyto- promote clustering and NK cell activation after bacterial toxic NK molecules. Surprisingly, we identified a high infection [7] leading to T cell activation, a process which number of CB-NK histones actively and early transferred is detected after a few hours [7]. Here, we demonstrate to MM and participating in the killing of MM cells. NK- that histones secreted by NK cells at early time points histones, through binding to CD138 on MM cells, contribute to this effect in the absence of DCs, suggest- promoted the formation of immune-tumor cell clusters, ing a role for other proteins as initiators of the whole ef- facilitating the immune attack of not only NK cells but fector immune response. also T lymphocytes. In addition, our proteomic approach provided with a Here, TRANS-SILAC proteomic analysis and further method to detect NK proteins with anti-MM activity, assays showed that histones were dynamically trans- suggesting the use of this technique to detect specific ferred from CB-NK through different MM cells, and also anti-tumor proteins. The observed anti-MM activity of released to the extracellular milieu. Histones release is a NK-histones was proposed to be mediated through J Immunother Cancer: first published as 10.1186/s40425-019-0739-1 on 16 October 2019. Downloaded from http://jitc.bmj.com/ on February 25, 2021 at Univesity Autonomous of Barcelona. Martín-Antonio et al. Journal for ImmunoTherapy of Cancer (2019) 7:259 Page 15 of 16 binding to CD138, a HSPG present on the surface of Ethics approval and consent to participate MM cells [43]. HSPGs are composed of proteins bound This study was approved by the Ethical Committee of Hospital Clinic, Barcelona. Cord blood units and peripheral blood from healthy individuals to polyanionic heparan sulfate chains that provide add- were obtained from healthy donors who gave informed consent. itional negative charges to the already anionic nature of lipid membranes [44]. These negative charges enable Consent for publication HSPG-mediated endocytosis of macromolecules. Specif- Donors and patients provided informed consent for publication of the data. ically, HSPG can uptake arginine and lysine rich peptides [45, 46], which interestingly are highly enriched Competing interests All authors declare no competing interests. in histones [47], and could explain the observed histone affinity for CD138. The disappearance of histone impact Author details 1 after CD138 knock-down, and after heparin treatment, a Department of Hematology, Hospital Clinic, IDIBAPS, Carrer Rosselló 149-153, 08036 Barcelona, Spain. 2Josep Carreras Leukaemia Research competitor of HSPG for their binding to cationic ligands Institute, Carrer Rosselló 149-153, 08036 Barcelona, Spain. 3Department of [11], and the co-localization of histones with CD138 Pediatrics – Research, The University of Texas M. D. Anderson Cancer Center, confirmed that histone activity requires binding to Houston, TX, USA. 4Immunoreceptors of the Innate and Adaptive System Group, IDIBAPS, Barcelona, Spain. 5Josep Carreras Leukemia Research Institute CD138 on MM cells. Even though we only analyzed the and Cell Therapy Program of the School of Medicine, University of Barcelona, effect of histones over CD138, NK histones might bind Barcelona, Spain. 6Department of Immunology, Hospital Clinic of Barcelona, to other types of HSPG and should require further Barcelona, Spain. 7Department of Biomedical Sciences, School of Medicine, University of Barcelona, Barcelona, Spain. 8Proteomic department, University investigation. of Barcelona, Barcelona, Spain. 9Cell Death Regulation Group, Oncobell In conclusion, this study demonstrates by first time a Program, Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain. 10 new anti-MM mechanism of CB-NK mediated by early Department of Stem Cell Transplantation & Cellular Therapy, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA. 11Department of histone-transfer and release, where histones bind to Hematology, University of Barcelona, Barcelona, Spain. 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