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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

Innate lymphoid cells 2 (ILC2) play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). An important aspect of ILC2-mediated tumorigenesis is the expansion of the resident ILC2 and simultaneous recruitment of the peripheral ILC2. Here,we describe a protocol for isolation,enrichment,and DiD labeling of ILC2 for in vivo tracking of ILC2s in the mouse. Further,we describe steps for the adoptive transfer of ILC2 to a recipient mouse model of PDAC. For complete details on the use and execution of this protocol,please refer to Alam et al. (2022). View Publication

Due to viral envelope glycoprotein D binding to cellular membrane HVEM receptor,HSV-1 can infect certain dendritic cells,which becomes an event in the viral strategy to interfere with the host's immune system. We previously generated the HSV-1 mutant strain M6,which produced an attenuated phenotype in mice and rhesus monkeys. The attenuated M6 strain was used to investigate how HSV-1 infection of dendritic cells interferes with both innate and adaptive immunity. Our study showed that dendritic cells membrane HVEM receptors could mediate infection of the wild-type strain and attenuated M6 strain and that dendritic cells infected by both viruses in local tissues of animals exhibited changes in transcriptional profiles associated with innate immune and inflammatory responses. The infection of pDCs and cDCs by the two strains promoted cell differentiation to the CD103+ phenotype,but varied transcriptional profiles were observed,implying a strategy that the HSV-1 wild-type strain interferes with antiviral immunity,probably due to viral modification of the immunological phenotype of dendritic cells during processing and presentation of antigen to T cells,leading to a series of deviations in immune responses,ultimately generating the deficient immune phenotype observed in infected individuals in the clinical. 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

The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans,with high mortality in untreated patients. Opportunistic properties of the disease have been established based on the increased incidence in immunocompromised patients and mouse models,indicating that an appropriate adaptive immune response is required for the control of the disease. However,cellular interactions and the kinetics of the local hepatic immune responses during the different stages of infection with E. multilocularis remain unknown. In a mouse model of oral infection that mimics the normal infection route in human patients,the networks of the hepatic immune response were assessed using single-cell RNA sequencing (scRNA-seq) of isolated hepatic CD3+ T cells at different infection stages. We observed an early and sustained significant increase in natural killer T (NKT) cells and regulatory T cells (Tregs). Early tumor necrosis factor (TNF)- and integrin-dependent interactions between these two cell types promote the formation of hepatic lesions. At late time points,downregulation of programmed cell death protein 1 (PD-1) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)-dependent signaling suppress the resolution of parasite-induced pathology. The obtained data provide fresh insight into the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice. View Publication

BACKGROUND This study aimed to investigate the role of circulating tumor cells (CTCs) and circulating cancer stem-like cells (cCSCs) before and after one cycle of chemotherapy and assessed the effects of early changes in CTCs and cCSCs on the outcomes of patients with metastatic breast cancer. METHODS Patients with stage IV invasive ductal carcinoma of the breast who received first-line chemotherapy between April 2014 and January 2016 were enrolled. CTCs and cCSCs were measured before the first cycle of chemotherapy (baseline) and on day 21,before the second cycle of chemotherapy commenced; a negative selection strategy and flow cytometry protocol were employed. RESULTS CTC and cCSC counts declined in 68.8 and 45.5% of patients,respectively. Declines in CTCs and cCSCs following the first chemotherapy cycle were associated with superior chemotherapy responses,longer progression-free survival (PFS),and longer overall survival (OS). An early decline in cCSCs remained an independent prognostic indicator for OS and PFS in multivariate analysis. CONCLUSIONS A cCSC decline after one cycle of chemotherapy for metastatic breast cancer is predictive of a superior chemotherapy response and longer PFS and OS,implying that cCSC dynamic monitoring may be helpful in early prediction of treatment response and prognosis. View Publication

This study describes the characterization of one induced pluripotent stem cell line (iPSC) from a healthy female. It is crucial to use iPSCs derived from healthy individuals as controls in genetic disease studies. Thus,we established a human iPSC cell line derived from healthy people. The iPSC cell line was generated in our lab from the peripheral blood mononuclear cells (PBMCs) of a 28-year-old girl. The generated hiPSC line is free of episomal vectors,has a normal karyotype,expresses pluripotency markers and can differentiate into three germ layers in vivo. 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

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

BACKGROUND Airway remodeling is a significant contributor to impaired lung function in chronic allergic airway disease. Currently,no therapy exists that is capable of targeting these structural changes and the consequent loss of function. In the context of chronic allergic inflammation,pericytes have been shown to uncouple from the pulmonary microvasculature,migrate to areas of inflammation,and significantly contribute to airway wall remodeling and lung dysfunction. This study aimed to elucidate the mechanism by which pulmonary pericytes accumulate in the airway wall in a model of chronic allergic airway inflammation. METHODS Mice were subjected to a protocol of chronic airway inflammation driven by the common environmental aeroallergen house dust mite. Phenotypic changes to lung pericytes were assessed by flow cytometry and immunostaining,and the functional capacity of these cells was evaluated using in vitro migration assays. The molecular mechanisms driving these processes were targeted pharmacologically in vivo and in vitro. RESULTS Pericytes demonstrated increased CXCR4 expression in response to chronic allergic inflammation and migrated more readily to its cognate chemokine,CXCL12. This increase in migratory capacity was accompanied by pericyte accumulation in the airway wall,increased smooth muscle thickness,and symptoms of respiratory distress. Pericyte uncoupling from pulmonary vessels and subsequent migration to the airway wall were abrogated following topical treatment with the CXCL12 neutraligand LIT-927. CONCLUSION These results provide new insight into the role of the CXCL12/CXCR4 signaling axis in promoting pulmonary pericyte accumulation and airway remodeling and validate a novel target to address tissue remodeling associated with chronic inflammation. View Publication

Fucoidan has sparked considerable interest in biomedical applications because of its inherent (bio)physicochemical characteristics,particularly immunomodulatory effects on macrophages,neutrophils,and natural killer cells. However,the effect of fucoidan on T cells and the following regulatory interaction on cellular function has not been reported. In this work,the effect of sterile fucoidan on the T-cell response and the subsequent modulation of osteogenesis is investigated. The physicochemical features of fucoidan treated by high-temperature autoclave sterilization are characterized by UV-visible spectroscopy,X-ray diffraction,Fourier transform infrared and nuclear magnetic resonance analysis. It is demonstrated that high-temperature autoclave treatment resulted in fucoidan depolymerization,with no change in its key bioactive groups. Further,sterile fucoidan promotes T cells proliferation and the proportion of differentiated T cells decreases with increasing concentration of fucoidan. In addition,the supernatant of T cells co-cultured with fucoidan greatly suppresses the osteogenic differentiation of MC3T3-E1 by downregulating the formation of alkaline phosphatase and calcium nodule compared with fucoidan. Therefore,our work offers new insight into the fucoidan-mediated T cell and osteoblast interplay. View Publication