技术资料

Bone disease represents a major cause of morbidity and mortality in Multiple Myeloma (MM); primarily driven by osteoclasts whose differentiation is dependent on expression of RANKL by MM cells. Notably,costimulation by ITAM containing receptors (i.e.,Fc$\gamma$R) can also play a crucial role in osteoclast differentiation. Modeling the pathology of the bone marrow microenvironment with an ex vivo culture system of primary human multiple myeloma cells,we herein demonstrate that Fc$\gamma$R-mediated signaling,via staphylococcal protein A (SpA) IgG immune-complexes,can act as a critical negative regulator of MM-driven osteoclast differentiation. Interrogation of the mode-of-action revealed that Fc$\gamma$R-mediated signaling causes epigenetic modulation of chromosomal 3D architecture at the RANK promoter; with altered spatial orientation of a proximal super enhancer. Combined this leads to substantial down-regulation of RANK at a transcript,protein,and functional level. These observations shed light on a novel mechanism regulating RANK expression and provide a rationale for targeting Fc$\gamma$R-signaling for the amelioration of osteolytic bone pathology in disease. View Publication

The mechanisms that control the inflammatory-immune response play a key role in tissue remodelling in cardiovascular diseases. T cell activation receptor CD69 binds to oxidized low-density lipoprotein (oxLDL),inducing the expression of anti-inflammatory NR4A nuclear receptors and modulating inflammation in atherosclerosis. To understand the downstream T cell responses triggered by the CD69-oxLDL binding,we incubated CD69-expressing Jurkat T cells with oxLDL. RNA sequencing revealed a differential gene expression profile dependent on the presence of CD69 and the degree of LDL oxidation. CD69-oxLDL binding induced the expression of NR4A receptors (NR4A1 and NR4A3),but also of PD-1. These results were confirmed using oxLDL and a monoclonal antibody against CD69 in CD69-expressing Jurkat and primary CD4??+??lymphocytes. CD69-mediated induction of PD-1 and NR4A3 was dependent on NFAT activation. Silencing NR4A3 slightly increased PD-1 levels,suggesting a potential regulation of PD-1 by this receptor. Moreover,expression of PD-1,CD69 and NR4A3 was increased in human arteries with chronic inflammation compared to healthy controls,with a strong correlation between PD-1 and CD69 mRNA expression (r??=??0.655 P?? View Publication

Immune-mediated red blood cell (RBC) destruction due to antibodies is an ongoing problem in transfusion medicine for the selection of the safest blood. Serological testing often revealed incompatibility with donors' RBCs. When this incompatible blood was transfused,destruction was due mostly to extravascular-mediated phagocytosis of the antibody-opsonized RBCs; however,intravascular hemolysis was sometimes observed without explanation. Based on serology,antibodies with potential for clinical sequalae could not be ascertained; thus,antigen-negative blood was usually selected for transfusion to avoid problems. Antibodies to antigens having very high frequency in the general population (>95%),however,made selection of antigen-negative blood difficult and sometimes impossible. Some patients,who were sensitized by previous transfusions or by pregnancy,developed multiple antibodies,again creating a problem for finding compatible blood for transfusion,without the ability to discern which of the antibodies may be clinically irrelevant and ignored. Transfusion medicine scientists began searching for an in vitro means to determine the in vivo outcome of transfusion of blood that was serologically incompatible. Methods such as chemiluminescence,monocyte-macrophage phagocytosis,and antibody-dependent cellular cytotoxicity (ADCC) were described. Over the years,the monocyte monolayer assay (MMA) has emerged as the most reliable in vitro assay for the prediction of the clinical relevance of a given antibody. ADCC has not been fully studied but has the potential to be useful for predicting which antibodies may result in intravascular hemolysis. This article captures the protocols for the implementation and readout of the MMA and ADCC assays for use in predicting the clinical significance of antibodies in a transfusion setting. {\textcopyright} 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Monocyte monolayer assay (MMA) Basic Protocol 2: Antibody-dependent cellular cytotoxicity assay (ADCC). View Publication

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity,we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity,non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition,we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells,termed iDuo NK cells,displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models,iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore,iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19,features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies. View Publication

T cell antigen-receptor (TCR) signaling controls the development,activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing,translation and stability of transcripts. In the present study,we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes,control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut ROR?t+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells. View Publication

Myeloid-derived suppressor cells (MDSCs) are known to promote tumor growth in part by their immunosuppressive activities and their angiogenesis support. It has been shown that Bv8 blockade inhibits the recruitment of MDSCs to tumors,thereby delaying tumor relapse associated with resistance to antiangiogenic therapy. However,the impact of Bv8 blockade on tumors resistant to the new immunotherapy drugs based on the blockade of immune checkpoints has not been investigated. Here,we demonstrate that granulocytic-MDSCs (G-MDSCs) are enriched in anti-PD1 resistant tumors. Importantly,resistance to anti-PD1 monotherapy is reversed upon switching to a combined regimen comprised of anti-Bv8 and anti-PD1 antibodies. This effect is associated with a decreased level of G-MDSCs and enrichment of active cytotoxic T cells in tumors. The blockade of anti-Bv8 has shown efficacy also in hyperprogressive phenotype of anti-PD1-treated tumors. In vitro,anti-Bv8 antibodies directly inhibit MDSC-mediated immunosuppression,as evidenced by enhanced tumor cell killing activity of cytotoxic T cells. Lastly,we show that anti-Bv8-treated MDSCs secrete proteins associated with effector immune cell function and T cell activity. Overall,we demonstrate that Bv8 blockade inhibits the immunosuppressive function of MDSCs,thereby enhancing anti-tumor activity of cytotoxic T cells and sensitizing anti-PD1 resistant tumors. Our findings suggest that combining Bv8 blockade with anti-PD1 therapy can be used as a strategy for overcoming therapy resistance. View Publication

Recent studies suggest that highly activated,polyfunctional CD4+ T cells are incredibly effective in strengthening and sustaining overall host antitumor immunity,promoting tumor-specific CD4+ T-cell responses and effectively enhancing antitumor immunity by immunotherapy. Previously,we developed a novel cryo-thermal therapy for local tumor ablation and achieved long-term survival rates in several tumor models. It was discovered that cryo-thermal therapy remodeled the tumor microenvironment and induced an antigen-specific CD4+ T-cell response,which mediated stronger antitumor immunity in vivo. In this study,the phenotype of bulk T cells in spleen was analyzed by flow cytometry after cryo-thermal therapy and both CD4+ Th1 and CD8+ CTL were activated. In addition,by using T-cell depletion,isolation,and adoptive T-cell therapy,it was found that cryo-thermal therapy induced Th1-dominant CD4+ T cells that directly inhibited the growth of tumor cells,promoted the maturation of MDSCs via CD4+ T-cell-derived IFN-? and enhanced the cytotoxic effector function of NK cells and CD8+ T cells,and promoted the maturation of APCs via cell-cell contact and CD4+ T-cell-derived IFN-?. Considering the multiple roles of cryo-thermal-induced Th1-dominant CD4+ T cells in augmenting antitumor immune memory,we suggest that local cryo-thermal therapy is an attractive thermo-immunotherapy strategy to harness host antitumor immunity and has great potential for clinical application. View Publication

OBJECTIVES Rheumatoid arthritis (RA) is a chronic autoimmune disease,that involves both pro- and anti-inflammatory mechanisms. The purpose of the present study is to investigate T-cell immunoglobulin and mucin domain 3 (Tim-3) in RA. METHODS Plasma levels of soluble (s) Tim-3 in early RA (n=98),were followed,to evaluate association with treatment and disease activity,acquired from a prospective collected biobank (clinicaltrials.gov (NCT00660647)). We also investigate the influence of Tim-3 on spontaneous cytokine production in synovial fluid mononuclear cells (SFMC) from RA patients after addition of neutralizing anti-Tim-3's antibodies,either alone or in combination with neutralizing anti-Programmed Cell death protein 1 (PD-1) antibodies. RESULTS Long-time stimulated CD4 T-cells expressed high levels of Tim-3,but tended to decrease their PD-1 expression. Tim-3 expression was exclusively seen co-expressed with PD-1 by CD3,CD4,CD45RO positive cells in the inflamed RA joint. Addition of neutralizing Tim-3 antibodies increased the secretion of IFN$\gamma$ and MCP-1,in SFMC cultures from RA. Whereas neutralizing anti-PD-1 antibodies showed a broader impact on cytokine production. Finally,we observed that soluble Tim-3 is increased in plasma and is associated with disease activity in early RA. CONCLUSION Taken together,our findings indicate disease-suppressive functions of Tim-3 in RA. View Publication

N6-methyladenosine (m6A) modification is involved in diverse immunoregulation,while the relationship between m6A modification and immune tolerance post kidney transplantation remains unclear. Expression of Wilms tumor 1-associating protein (WTAP),an m6A writer,was firstly detected in tolerant kidney transplant recipients (TOL). Then the role of WTAP on regulatory T (Treg) cell differentiation and function in CD4+ T cells from kidney transplant recipients with immune rejection (IR) was investigated. The potential target of WTAP and effect of WTAP on immune tolerance in vivo were subsequently verified. WTAP was upregulated in CD4+ T cells of TOL and positively correlated with Treg cell proportion. In vitro,WTAP overexpression promoted Treg cell differentiation and enhanced Treg cell-mediated suppression toward na?ve T cells. Forkhead box other 1 (Foxo1) was identified as a target of WTAP. WTAP enhanced m6A modification of Foxo1 mRNA in coding sequence (CDS) region,leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression,while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice,as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function. leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function." View Publication

Immunogenicity following an additional dose of Coronavirus disease 2019 (COVID-19) vaccine was investigated in an extended primary series among kidney transplant (KT) recipients. Eighty-five KT participants were randomized to receive either an mRNA (M group; n =??43) or viral vector (V group; n =??42) vaccine. Among them,62% were male,with a median (IQR) age of 50 (43-59) years and post-transplantation duration of 46 (26-82) months. At 2??weeks post-additional dose,there was no difference in the seroconversion rate between the M and V groups (70% vs. 65%,p =??.63). A median (IQR) of anti-RBD antibody level was not statistically different between the M group compared with the V group (51.8 [5.1-591] vs. 28.5 [2.9-119.3] BAU/ml,p =??.18). Furthermore,the percentage of participants with positive SARS-CoV-2 surrogate virus neutralization test results was not statistically different between groups (20% vs. 15%,p =??.40). S1-specific T cell and RBD-specific B cell responses were also comparable between the M and V groups (230 [41-420] vs. 268 [118-510],p =??.65 and 2 [0-10] vs. 2 [0-13] spot-forming units/106 peripheral blood mononuclear cells,p =??.60). In conclusion,compared with an additional dose of viral vector COVID-19 vaccine,a dose of mRNA COVID-19 vaccine did not elicit significantly different responses in KT recipients,regarding either humoral or cell-mediated immunity. (TCTR20211102003). View Publication

Monoclonal antibodies are at the vanguard of the most promising cancer treatments. Whereas traditional therapeutic antibodies have been limited to extracellular antigens,T cell receptor mimic (TCRm) antibodies can target intracellular antigens presented by cell surface major histocompatibility complex (MHC) proteins. TCRm antibodies can therefore target a repertoire of otherwise undruggable cancer antigens. However,the consequences of off-target peptide/MHC recognition with engineered T cell therapies are severe,and thus there are significant safety concerns with TCRm antibodies. Here we explored the specificity and safety profile of a new TCRm-based T cell therapy for hepatocellular carcinoma (HCC),a solid tumor for which no effective treatment exists. We targeted an alpha-fetoprotein peptide presented by HLA-A*02 with a highly specific TCRm,which crystallographic structural analysis showed binds directly over the HLA protein and interfaces with the full length of the peptide. We fused the TCRm to the ? and ? subunits of a TCR,producing a signaling AbTCR construct. This was combined with an scFv/CD28 co-stimulatory molecule targeting glypican-3 for increased efficacy towards tumor cells. This AbTC + co-stimulatory T cell therapy showed potent activity against AFP-positive cancer cell lines in vitro and an in an in vivo model and undetectable activity against AFP-negative cells. In an in-human safety assessment,no significant adverse events or cytokine release syndrome were observed and evidence of efficacy was seen. Remarkably,one patient with metastatic HCC achieved a complete remission after nine months and ultimately qualified for a liver transplant. View Publication

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here,we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline,in drug resistance,and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide,including CD53,CD10,EVI2B,and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally,we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological,therapeutic,and diagnostic understanding of myeloma. View Publication