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The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease,the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study,a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus,preventing it from infecting cells in an in vitro cell-based assay,including multiple viral variants that are currently circulating in the human population. In addition,we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease. View Publication

Cytokine storm refers to the dysregulated production of inflammatory mediators leading to hyperinflammation. They are often detrimental,and worsen the severity of COVID-19 and other infectious or inflammatory diseases. Cannabinoids are known to have anti-inflammatory effects but their possible therapeutic value on cytokine storms has not been fully elucidated. In vivo and ex vivo studies were carried out to investigate the effects of high-THC and high-CBD extracts on cytokine production in immune cells. Significant differences between the extracts were observed. Subsequent experiments focusing on a specific high CBD extract (CBD-X) showed significant reductions in pro-inflammatory cytokines in human-derived PBMCs,neutrophils and T cells. In vivo mouse studies,using a systemically inflamed mouse model,showed reductions in pro-inflammatory cytokines TNF$\alpha$ and IL-1$\beta$ and a concurrent increase in the anti-inflammatory cytokine IL-10 in response to CBD-X extract treatment. Lung inflammation,as in severe COVID-19 disease,is characterized by increased T-cell homing to the lungs. Our investigation revealed that CBD-X extract impaired T-cell migration induced by the chemoattractant SDF1. In addition,the phosphorylation levels of T cell receptor (TCR) signaling proteins Lck and Zap70 were significantly reduced,demonstrating an inhibitory effect on the early events downstream to TCR activation. In a lung inflamed mouse model,we observed a reduction in leukocytes including neutrophil migration to the lungs and decreased levels of IL-1$\beta$,MCP-1,IL-6 and TNF$\alpha$,in response to the administration of the high-CBD extract. The results presented in this work offer that certain high-CBD extract has a high potential in the management of pathological conditions,in which the secretion of cytokines is dysregulated,as it is in severe COVID-19 disease or other infectious or inflammatory diseases. View Publication

Conditioning regimens used for hematopoietic stem cell transplantation (HCT) can escalate the severity of acute T cell-mediated graft-versus-host disease (GVHD) by disrupting gastrointestinal integrity and initiating lipopolysaccharide (LPS)-dependent innate immune cell activation. Activation of the complement cascade has been associated with murine GVHD,and previous work has shown that alternative pathway complement activation can amplify T cell immunity. Whether and how mannan-binding lectin (MBL),a component of the complement system that binds mannose as well as oligosaccharide components of LPS and lipoteichoic acid,affects GVHD is unknown. In this study,we tested the hypothesis that MBL modulates murine GVHD and examined the mechanisms by which it does so. We adoptively transferred C3.SW bone marrow (BM) cells ± T cells into irradiated wild type (WT) or MBL-deficient C57Bl/6 (B6) recipients with or without inhibiting MBL-initiated complement activation using C1-esterase inhibitor (C1-INH). We analyzed the clinical severity of disease expression and analyzed intestinal gene and cell infiltration. In vitro studies assessed MBL expression on antigen-presenting cells (APCs) and compared LPS-induced responses of WT and MBL-deficient APCs. MBL-deficient recipients of donor BM ± T cells exhibited significantly less weight loss over the first 2 weeks post-transplantation weeks compared with B6 controls (P < .05),with similar donor engraftment in the 2 groups. In recipients of C3.SW BM + T cells,the clinical expression of GVHD was less severe (P < .05) and overall survival was better (P < .05) in MBL-deficient mice compared with WT mice. On day-7 post-transplantation,analyses showed that the MBL-deficient recipients exhibited less intestinal IL1b,IL17,and IL12 p40 gene expression (P < .05 for each) and fewer infiltrating intestinal CD11c+,CD11b+,and F4/80+ cells and TCR$\beta$+,CD4+,CD4+IL17+,and CD8+ T cells (P < .05 for each). Ovalbumin or allogeneic cell immunizations induced equivalent T cell responses in MBL-deficient and WT mice,demonstrating that MBL-deficiency does not directly impact T cell immunity in the absence of irradiation conditioning. Administration of C1-INH did not alter the clinical expression of GVHD in preconditioned WT B6 recipients,suggesting that MBL amplifies clinical expression of GVHD via a complement-independent mechanism. WT,but not MBL-deficient,APCs express MBL on their surfaces. LPS-stimulated APCs from MBL-deficient mice produced less proinflammatory cytokines (P < .05) and induced weaker alloreactive T cell responses (P < .05) compared with WT APCs. Together,our data show that MBL modulates murine GVHD,likely by amplifying complement-independent,LPS-initiated gastrointestinal inflammation. The results suggest that devising strategies to block LPS/MBL ligation on APCs has the potential to reduce the clinical expression of GVHD. View Publication

Ankylosing spondylitis (AS) is an immune-mediated inflammatory disorder that primarily affects the axial skeleton,especially the sacroiliac joints and spine. This results in chronic back pain and,in extreme cases,ankylosis of the spine. Despite its debilitating effects,the pathogenesis of AS remains to be further elucidated. This study used single cell CITE-seq technology to analyze peripheral blood mononuclear cells (PBMCs) in AS and in healthy controls. We identified a number of molecular features associated with AS. CD52 was found to be overexpressed in both RNA and surface protein expression across several cell types in patients with AS. CD16+ monocytes overexpressed TNFSF10 and IL-18R$\alpha$ in AS,while CD8+ TEM cells and natural killer cells overexpressed genes linked with cytotoxicity,including GZMH,GZMB,and NKG7. Tregs underexpressed CD39 in AS,suggesting reduced functionality. We identified an overrepresented NK cell subset in AS that overexpressed CD16,CD161,and CD38,as well as cytotoxic genes and pathways. Finally,we developed machine learning models derived from CITE-seq data for the classification of AS and achieved an Area Under the Receiver Operating Characteristic (AUROC) curve of > 0.95. In summary,CITE-seq identification of AS-associated genes and surface proteins in specific cell subsets informs our understanding of pathogenesis and potential new therapeutic targets,while providing new approaches for diagnosis via machine learning. View Publication

Most cancer vaccines target peptide antigens,necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore,tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells,but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding,enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably,this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary,highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations. View Publication

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses,and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1,attenuate differentiation,and enhance proliferation and antitumor efficacy in vivo. Here,we used a cell-free culture system to provide an agonistic NOTCH1 signal during na{\{i}}ve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation transcription factor expression cytokine production and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from na{\"{i}}ve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF driving heightened production of interleukin-22 interleukin-10 and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors." View Publication

To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250??$\mu$g/ip day 0) and CTLA4-Ig (200??$\mu$g/ip day 2),we administered low-dose IL-2 (2000??IU/day) starting on posttransplant day 14 for 3??weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin,vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens,leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100??days),prevented chronic rejection,and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142,associated with the TGF$\beta$ pathway,was significantly downregulated in exosomes from IL-2-treated mice. In conclusion,low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules. View Publication

Metabolic reprogramming is associated with myeloid-derived suppressor cell (MDSC) immunosuppressive function. Here,we outline the process for acquiring MDSCs from human and murine sources for subsequent analysis of fatty acid oxidation,oxidative phosphorylation,and glycolysis using the Seahorse XFe 96 Analyzer. Murine MDSCs can be isolated directly from tumor-bearing mice or derived through IL-6 and GM-CSF culture of bone marrow cells from non-tumor-bearing mice. To generate human MDSCs,peripheral blood mononuclear cells (PBMCs) can be cultured with IL-6 and GM-CSF. For complete details on the use and execution of this protocol,please refer to Mohammadpour et al. (2021). View Publication

BACKGROUND The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here,we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. METHODS To further understand CISH functions in NK cells,we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression,signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line,metastasis evaluation was performed. Then,orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally,we targeted CISH in human NK-92 or primary NK cells,using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. RESULTS In Cishfl/flNcr1Ki/+ mice,we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways,triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor,optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression,limiting NK cell exhaustion. Finally,using CRISPRi,we then targeted CISH in human NK-92 or primary NK cells. In human NK cells,CISH deletion also favors NCR signaling and antitumor functions. CONCLUSION This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy. View Publication

Fibroblasts are the most abundant stromal constituents of the tumour microenvironment in primary as well as metastatic colorectal cancer (CRC). Their supportive effect on tumour cells is well established. There is growing evidence that stromal fibroblasts also modulate the immune microenvironment in tumours. Here,we demonstrate a difference in fibroblast-mediated immune modulation between primary CRC and peritoneal metastasis. Cancer-associated fibroblasts (CAFs) were isolated from primary cancer and from peritoneal metastases (MAFs) from a total of 17 patients. The ectoenzyme CD38 was consistently expressed on the surface of all MAFs,while it was absent from CAFs. Furthermore,MAFs secreted higher levels of IGFBP2,CXCL2,CXCL6,CXCL12,PDGF-AA,FGFb,and IL-6. This was associated with a decreased activation of macrophages and a suppression of CD25 expression and proliferation of co-cultivated T-cells. Downregulation of IGFBP2 abolished these immunosuppressive effects of MAFs. Taken together,these results show that MAFs contribute to an immunosuppressive tumour microenvironment in CRC metastases by modulating the phenotype of immune cells through an IGFBP2-dependent mechanism. View Publication

BACKGROUND Hypoxia and inflammation tumor microenvironment (TME) play a crucial role in tumor development and progression. Although increased understanding of TME contributed to gastric cancer (GC) progression and prognosis,the direct interaction between macrophage and GC cells was not fully understood. METHODS Hypoxia and normoxia macrophage microarrays of GEO database was analyzed. The peripheral blood mononuclear cell acquired from the healthy volunteers. The expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) in GC tissues and cell lines was detected by quantitative reverse transcription PCR (qRT-PCR),western-blot,Elisa and immunofluorescence. Cell proliferation,migration,and invasion were evaluated by cell counting kit 8 (CCK8),colony formation,real-time imaging of cell migration and transwell. Flow Cytometers was applied to identify the source of cytokines. Luciferase reporter assays and chromatin immunoprecipitation were used to identify the interaction between transcription factor and target gene. Especially,a series of truncated and mutation reporter genes were applied to identify precise binding sites. The corresponding functions were verified in the complementation test and in vivo animal experiment. RESULTS Our results revealed that hypoxia triggered macrophage secreted CXCL8,which induced GC invasion and proliferation. This macrophage-induced GC progression was CXCL8 activated C-X-C Motif Chemokine Receptor 1/2 (CXCR1/2) on the GC cell membrane subsequently hyperactivated Janus kinase 1/ Signal transducer and activator of transcription 1 (JAK/STAT1) signaling pathway. Then,the transcription factor STAT1 directly led to the overexpression and secretion of Interleukin 10 (IL-10). Correspondingly,IL-10 induced the M2-type polarization of macrophages and continued to increase the expression and secretion of CXCL8. It suggested a positive feedback loop between macrophage and GC. In clinical GC samples,increased CXCL8 predicted a patient's pessimistic outcome. CONCLUSION Our work identified a positive feedback loop governing cancer cells and macrophage in GC that contributed to tumor progression and patient outcome. View Publication

The family of hexokinases (HKs) catalyzes the first step of glycolysis,the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed,the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system,we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead,loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death,oxidative stress,and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing,showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns,we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM,which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival,possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation. View Publication