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

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

Hypoxia Reduces Cell Attachment of SARS-CoV-2 Spike Protein by Modulating the Expression of ACE2, Neuropilin-1, Syndecan-1 and Cellular Heparan Sulfate

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1αdependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), the European Research Council (ERC) (grant numbers: ERC-2018-StG 804236-NEXTGEN-IO to A.P and ERC-2017-AdG 788143-RECGLYCANMR to J.J-B.), the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the FERO Foundation. Personal fellowships: E.P. (Juan de la Cierva-Formación, FJC2018-035449-I), L.V. (Juan de la Cierva-Formación, FJCI-2017-34099), A.B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), A.G. (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), A.A (La Caixa Inphinit, LCF/BQ/DR20/11790022), B.J. (Basque Government, PRE_2019_1_0320), L.M. (Juan de la Cierva-Formación, FJC2019-039983-I), E.B. (MINECO, BFU2016-76872-R; Excellence Networks, SAF2017-90794-REDT) and A.P. (Ramón y Cajal, RYC2018-024183-I; Proyectos I+D+I, PID2019-107956RA-I00; and Ikerbasque Research Associate)

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.Acknowledgements: We thank Petros Tyrakis and Iván Martínez-Forero for critical reading and editing of the manuscript. Support was provided by the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the SPRI I+D COVID-19 fund (Gobierno Vasco). Personal fellowships: A.A.-V. (La Caixa Inphinit LCF/BQ/DR20/11790022), A.B. (AECC Bizkaia), A.G.d.R (Bikaintek), A.P. (Ramón y Cajal), B.J.-L. (Gob. Vasco), and E.P.-F. (Juan de la Cierva-Formación). M.L.M.-C. acknowledges RTC2019-007125-1, DTS20/00138, SAF2017-87301-R, and BBVA UMBRELLA project. M.L.-H. acknowledges the ISCIII for grant COV20-0170 and the Government of Cantabria for grant 2020UIC22-PUB-0019. O.M., J.-M.M., and N.G.A.A. acknowledge the Agencia Estatal de Investigación (Spain) for grants CTQ2015-68756-R, RTI2018-101269-BI00, and RTI2018-095700-B-I00, respectively. A.P. has received grant funding from the European Research Council (ERC), grant agreement number 804236 (Horizon 2020), and the FERO Foundation

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b

Funding Information: This work was supported by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B; ERC-2018-StG 804236-NEXTGEN-IO to A.P.) and the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement No 956758 to K.S.; ITN BactiVax under grant agreement no. 860325 to U.A. and ITN DIRNANO grant agreement No 956544 to F.C.). X-ray diffraction experiments described in this paper were performed using beamlines XALOC synchrotron at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland). F.M., C.S. and H.C. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding projects: PTDC/BIA-MIB/31028/2017 and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy—i4HB project (LA/P/0140/2020), to the CEEC contracts 2020.00233.CEECIND and 2020.03261.CEECIND for F.M. and H.C., respectively, and to PhD grant 2022.11723.BD of C.S. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 22161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). F.M. and J.J.-B. acknowledge to the European funding for the GLYCOTwinning project (No. 101079417) and -COST Action GLYCONANOPROBES. A.P.’s research is funded by “La Caixa” Foundation (HR21-00925), AECC (LABAE211744PALA), Fundación FERO, Ikerbasque, and BIOEF EITB MARATOIA BIO19/CP/002. We thank Agencia Estatal de Investigación of Spain for grants PID2019-107956RA-I00 (A.P.), PID2019-107770RA-I00 (J.E.-O.), RTI2018-099592-B-C21 (F.C.), ID2020-114178GB (R.B. and J.D.S.), RYC2018-024183-I (A.P.), and the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain) A.A.-V. receives funding from “La Caixa” Foundation (ID 100010434, LCF/BQ/DR20/11790022). A. B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC). F.C. acknowledges the Mizutani Foundation for Glycoscience (Grant 220115). Funding Information: This work was supported by the European Research Council (ERC-2017-AdG, 788143-RECGLYCANMR to J.J.-B; ERC-2018-StG 804236-NEXTGEN-IO to A.P.) and the Marie-Skłodowska-Curie actions (ITN Glytunes grant agreement No 956758 to K.S.; ITN BactiVax under grant agreement no. 860325 to U.A. and ITN DIRNANO grant agreement No 956544 to F.C.). X-ray diffraction experiments described in this paper were performed using beamlines XALOC synchrotron at ALBA (Spain) and PXIII in Swiss Light Source (Switzerland). F.M., C.S. and H.C. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding projects: PTDC/BIA-MIB/31028/2017 and UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy—i4HB project (LA/P/0140/2020), to the CEEC contracts 2020.00233.CEECIND and 2020.03261.CEECIND for F.M. and H.C., respectively, and to PhD grant 2022.11723.BD of C.S. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 22161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). F.M. and J.J.-B. acknowledge to the European funding for the GLYCOTwinning project (No. 101079417) and -COST Action GLYCONANOPROBES. A.P.’s research is funded by “La Caixa” Foundation (HR21-00925), AECC (LABAE211744PALA), Fundación FERO, Ikerbasque, and BIOEF EITB MARATOIA BIO19/CP/002. We thank Agencia Estatal de Investigación of Spain for grants PID2019-107956RA-I00 (A.P.), PID2019-107770RA-I00 (J.E.-O.), RTI2018-099592-B-C21 (F.C.), ID2020-114178GB (R.B. and J.D.S.), RYC2018-024183-I (A.P.), and the Severo Ochoa Center of Excellence Accreditation CEX2021-001136-S, all funded by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro, as well as CIBERES, and initiative of Instituto de Salud Carlos III (ISCIII, Spain) A.A.-V. receives funding from “La Caixa” Foundation (ID 100010434, LCF/BQ/DR20/11790022). A. B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC). F.C. acknowledges the Mizutani Foundation for Glycoscience (Grant 220115). Publisher Copyright: © 2023, The Author(s).Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.publishersversionpublishe

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.Funding: This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), BIOEF EITB Maratoia (BIO21/COV/037 to AP), the European Research Council (ERC) (ERC-2018-StG 804236-NEXTGEN-IO to AP), the Instituto de Salud Carlos iii (ISCiii, DTS21/00094 to AP and DTS20/00138 to MM-C), Ministerio de Ciencia, Innovación y Universidades (MICINN, PID2019-107956RA-I00 and TED2021-129433B-C21 to AP; PID2020-117116RB-I00 and RTC2019-007125-1 to MM-C) and the FERO Foundation to AP. Personal fellowships: EP-F (Juan de la Cierva-Formación, FJC2018-035449-I), ABo (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), AG (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), AA-V (La Caixa Inphinit, LCF/BQ/DR20/11790022), BJ-L (Basque Government, PRE_2019_1_0320), ABl (AECC Bizkaia Scientific Foundation, PRDVZ21640DEBL), PV-B (Proyectos I +D+I, PRE2020-092342) and AP (Ramón y Cajal, RYC2018- 024183-I; and Ikerbasque Research Associate). Acknowledgments: The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). HEK293T-ACE2 cells were kindly provided by Dr. June Ereño-Orbea (CIC bioGUNE) and Dr. Jean-Philippe Julien (The Hospital for Sick Children Research Institute, Toronto)