技术资料

N6-methyladenosine (m6A) modification plays a pivotal role in cell fate determination. Previous studies show that eliminating m6A using Mb1-Cre dramatically impairs B cell development. However,whether disturbing m6A modification at later stages affects B cell development and function remains elusive. Here,we deleted m6A methyltransferase Mettl3 from the pro-B stage on using Cd19-Cre (Mettl3 cKO) and found that the frequency of total B cells in peripheral blood,peritoneal cavity,and liver is comparable between Mettl3 cKO mice and wild-type (WT) littermates,while the percentage of whole splenic B cells slightly increases in Mettl3 cKO individuals. The proportion of pre-pro-B,pro-B,pre-B,immature,and mature B cells in the bone marrow were minimally affected. Loss of Mettl3 resulted in increased apoptosis but barely affected B cells' proliferation and IgG production upon LPS,CD40L,anti-IgM,or TNF-$\alpha$ stimulation. Different stimuli had different effects on B cell activation. In addition,B cell-specific Mettl3 knockout had no influence on the pro-fibrogenic activity of B cells in liver fibrosis,evidenced by comparable fibrosis in carbon tetrachloride- (CCl4-) treated Mettl3 cKO mice and WT controls. In summary,our study demonstrated that deletion of Mettl3 from the pro-B stage on has minimal effects on B cell development and function,as well as profibrogenic activity of B cells in liver fibrosis,revealing a stage-specific dependence on Mettl3-mediated m6A of B cell development. View Publication

BACKGROUND Obesity has often been associated with severe allergic asthma (AA). Here,we analyzed the frequency of different circulating CD4+T-cell subsets from lean,overweight and obese AA patients. METHODS Mononuclear cells from peripheral blood were obtained from 60 AA patients and the frequency of different CD4+T-cell subsets and type 1 regulatory B cells (Br1) was determined by cytometry. The effect of obese-related leptin dose on cytokine production and Treg cell function in AA-derived CD4+ T cell cultures was evaluated by ELISA and 3H thymidine uptake,respectively. Leptin levels were quantified in the plasma by ELISA. According to the BMI,patients were stratified as lean,overweight and obese. RESULTS AA severity,mainly among obese patients,was associated with an expansion of hybrid Th2/Th17 and Th17-like cells rather than classic Th2-like cells. On the other hand,the frequencies of Th1-like,Br1 cells and regulatory CD4+ T-cell subsets were lower in patients with severe AA. While percentages of the hybrid Th2/Th17 phenotype and Th17-like cells positively correlated with leptin levels,the frequencies of regulatory CD4+ T-cell subsets and Br1 cells negatively correlated with this adipokine. Interestingly,the obesity-related leptin dose not only elevated Th2 and Th17 cytokine levels,but also directly reduced the Treg function in CD4+ T cell cultures from lean AA patients. CONCLUSION In summary,our results indicated that obesity might increase AA severity by favoring the expansion of Th17-like and Th2/Th17 cells and decreasing regulatory CD4+T cell subsets,being adverse effects probably mediated by leptin overproduction. View Publication

BACKGROUND The use of intralesional Mycobacterium bovis BCG (intralesional live BCG) for the treatment of metastatic melanoma resulted in regression of directly injected,and occasionally of distal lesions. However,intralesional-BCG is less effective in patients with visceral metastases and did not significantly improve overall survival. METHODS We generated a novel BCG lysate and developed it into a thermosensitive PLGA-PEG-PLGA hydrogel (BCG hydrogel),which was injected adjacent to the tumor to assess its antitumor effect in syngeneic tumor models (B16F10,MC38). The effect of BCG hydrogel treatment on contralateral tumors,lung metastases,and survival was assessed to evaluate systemic long-term efficacy. Gene expression profiles of tumor-infiltrating immune cells and of tumor-draining lymph nodes from BCG hydrogel-treated mice were analyzed by single-cell RNA sequencing (scRNA-seq) and CD8+ T cell receptor (TCR) repertoire diversity was assessed by TCR-sequencing. To confirm the mechanistic findings,RNA-seq data of biopsies obtained from in-transit cutaneous metastases of patients with melanoma who had received intralesional-BCG therapy were analyzed. RESULTS Here,we show that BCG lysate exhibits enhanced antitumor efficacy compared to live mycobacteria and promotes a proinflammatory tumor microenvironment and M1 macrophage (M$\Phi$) polarization in vivo. The underlying mechanisms of BCG lysate-mediated tumor immunity are dependent on M$\Phi$ and dendritic cells (DCs). BCG hydrogel treatment induced systemic immunity in melanoma-bearing mice with suppression of lung metastases and improved survival. Furthermore,BCG hydrogel promoted cathepsin S (CTSS) activity in M$\Phi$ and DCs,resulting in enhanced antigen processing and presentation of tumor-associated antigens. Finally,BCG hydrogel treatment was associated with increased frequencies of melanoma-reactive CD8+ T cells. In human patients with melanoma,intralesional-BCG treatment was associated with enhanced M1 M$\Phi$,mature DC,antigen processing and presentation,as well as with increased CTSS expression which positively correlated with patient survival. CONCLUSIONS These findings provide mechanistic insights as well as rationale for the clinical translation of BCG hydrogel as cancer immunotherapy to overcome the current limitations of immunotherapies for the treatment of patients with melanoma. View Publication

The high mobility group (HMG) transcription factor TCF-1 is essential for early T cell development. Although in vitro biochemical assays suggest that HMG proteins can serve as architectural elements in the assembly of higher-order nuclear organization,the contribution of TCF-1 on the control of three-dimensional (3D) genome structures during T cell development remains unknown. Here,we investigated the role of TCF-1 in 3D genome reconfiguration. Using gain- and loss-of-function experiments,we discovered that the co-occupancy of TCF-1 and the architectural protein CTCF altered the structure of topologically associating domains in T cell progenitors,leading to interactions between previously insulated regulatory elements and target genes at late stages of T cell development. The TCF-1-dependent gain in long-range interactions was linked to deposition of active enhancer mark H3K27ac and recruitment of the cohesin-loading factor NIPBL at active enhancers. These data indicate that TCF-1 has a role in controlling global genome organization during T cell development. View Publication

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages BA.2.12.1,BA.4 and BA.5 exhibit higher transmissibility than the BA.2 lineage1. The receptor binding and immune-evasion capability of these recently emerged variants require immediate investigation. Here,coupled with structural comparisons of the spike proteins,we show that BA.2.12.1,BA.4 and BA.5 (BA.4 and BA.5 are hereafter referred collectively to as BA.4/BA.5) exhibit similar binding affinities to BA.2 for the angiotensin-converting enzyme 2 (ACE2) receptor. Of note,BA.2.12.1 and BA.4/BA.5 display increased evasion of neutralizing antibodies compared with BA.2 against plasma from triple-vaccinated individuals or from individuals who developed a BA.1 infection after vaccination. To delineate the underlying antibody-evasion mechanism,we determined the escape mutation profiles2,epitope distribution3 and Omicron-neutralization efficiency of 1,640 neutralizing antibodies directed against the receptor-binding domain of the viral spike protein,including 614 antibodies isolated from people who had recovered from BA.1 infection. BA.1 infection after vaccination predominantly recalls humoral immune memory directed against ancestral (hereafter referred to as wild-type (WT)) SARS-CoV-2 spike protein. The resulting elicited antibodies could neutralize both WT SARS-CoV-2 and BA.1 and are enriched on epitopes on spike that do not bind ACE2. However,most of these cross-reactive neutralizing antibodies are evaded by spike mutants L452Q,L452R and F486V. BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1. Nevertheless,these neutralizing antibodies are largely evaded by BA.2 and BA.4/BA.5 owing to D405N and F486V mutations,and react weakly to pre-Omicron variants,exhibiting narrow neutralization breadths. The therapeutic neutralizing antibodies bebtelovimab4 and cilgavimab5 can effectively neutralize BA.2.12.1 and BA.4/BA.5,whereas the S371F,D405N and R408S mutations undermine most broadly sarbecovirus-neutralizing antibodies. Together,our results indicate that Omicron may evolve mutations to evade the humoral immunity elicited by BA.1 infection,suggesting that BA.1-derived vaccine boosters may not achieve broad-spectrum protection against new Omicron variants. View Publication

Type 1 diabetes is an autoimmune disease with no cure,where clinical translation of promising therapeutics has been hampered by the reproducibility crisis. Here,short-term administration of an antagonist to the receptor for advanced glycation end products (sRAGE) protected against murine diabetes at two independent research centers. Treatment with sRAGE increased regulatory T cells (Tregs) within the islets,pancreatic lymph nodes,and spleen,increasing islet insulin expression and function. Diabetes protection was abrogated by Treg depletion and shown to be dependent on antagonizing RAGE with use of knockout mice. Human Tregs treated with a RAGE ligand downregulated genes for suppression,migration,and Treg homeostasis (FOXP3,IL7R,TIGIT,JAK1,STAT3,STAT5b,CCR4). Loss of suppressive function was reversed by sRAGE,where Tregs increased proliferation and suppressed conventional T-cell division,confirming that sRAGE expands functional human Tregs. These results highlight sRAGE as an attractive treatment to prevent diabetes,showing efficacy and reproducibility at multiple research centers and in human T cells. View Publication

Allogeneic T cells are key immune therapeutic cells to fight cancer and other clinical indications. High T cell dose per patient and increasing patient numbers result in clinical demand for a large number of allogeneic T cells. This necessitates a manufacturing platform that can be scaled up while retaining cell quality. Here we present a closed and scalable platform for T cell manufacturing to meet clinical demand. Upstream manufacturing steps of T cell activation and expansion are done in-vessel,in a stirred-tank bioreactor. T cell selection,which is necessary for CAR-T-based therapy,is done in the bioreactor itself,thus maintaining optimal culture conditions through the selection step. Platform's attributes of automation and performing the steps of T cell activation,expansion,and selection in-vessel,greatly contribute to enhancing process control,cell quality,and to the reduction of manual labor and contamination risk. In addition,the viability of integrating a closed,automated,downstream process of cell concentration,is demonstrated. The presented T cell manufacturing platform has scale-up capabilities while preserving key factors of cell quality and process control. View Publication

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here,we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state,followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. View Publication

BACKGROUND & AIMS N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. METHODS Mettl3 knockout mice,CD34+ humanized mice,and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay,respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. RESULTS We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells,and eventually suppressed CRC in ApcMin/+Mettl3+/- mice,CD34+ humanized mice,and syngeneic mice models. Mechanistically,METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing,RNA sequencing,and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner,which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However,the effect was negligible on BHLHE41 depletion,CXCL1 protein or CXCR2 inhibitor SB265610 administration,inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently,depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly,targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. CONCLUSIONS Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC. View Publication

BACKGROUND Acute respiratory distress syndrome (ARDS) is a neutrophil-associated disease. Delayed neutrophil apoptosis and increased levels of neutrophil extracellular traps (NETs) have been described in ARDS. We aimed to investigate the relationship between these phenomena and their potential as inflammation drivers. We hypothesized that delayed neutrophil apoptosis might enhance NET formation in ARDS. METHOD Our research was carried out in three aspects: clinical research,animal experiments,and in vitro experiments. First,we compared the difference between neutrophil apoptosis and NET levels in healthy controls and patients with ARDS and analyzed the correlation between neutrophil apoptosis and NET levels in ARDS. Then,we conducted animal experiments to verify the effect of neutrophil apoptosis on NET formation in Lipopolysaccharide-induced acute lung injury (LPS-ALI) mice. Furthermore,this study explored the relationship between neutrophil apoptosis and NETs at the cellular level. Apoptosis was assessed using morphological analysis,flow cytometry,and western blotting. NET formation was determined using immunofluorescence,PicoGreen assay,SYTOX Green staining,and western blotting. RESULTS ARDS neutrophils lived longer because of delayed apoptosis,and the cyclin-dependent kinase inhibitor,AT7519,reversed this phenomenon both in ARDS neutrophils and neutrophils in bronchoalveolar lavage fluid (BALF) of LPS-ALI mice. Neutrophils in a medium containing pro-survival factors (LPS or GM-CSF) form more NETs,which can also be reversed by AT7519. Tissue damage can be reduced by promoting neutrophil apoptosis. CONCLUSIONS Neutrophils with extended lifespan in ARDS usually enhance NET formation,which aggravates inflammation. Enhancing neutrophil apoptosis in ARDS can reduce the formation of NETs,inhibit inflammation,and consequently alleviate ARDS. View Publication

OBJECTIVE To identify immune cells,cytokines,and immune cell transcriptome in the menstrual effluent (ME) of women with endometriosis compared with that of healthy donors. DESIGN Live immune cells were isolated from human ME samples and were analyzed by flow cytometry to identify various immune cell populations. Selected cytokines from the same patients were evaluated using multiplex cytokine analyses. The transcriptome of the immune cell population was subsequently profiled using NanoString nCounter's PanCancer Immune panel. SETTING Academic institution. PATIENT(S) Surgically confirmed endometriosis patients (n = 14) and healthy fertile donors (n = 19). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) In-depth immune cell profiling of ME obtained from women with endometriosis compared with that of healthy donors. RESULT(S) ME analysis revealed that the number of T helper 17 (TH17) cells was significantly lower in patients with endometriosis compared with that of healthy donors; the number of macrophages was also lower (P=.06) in the former. Multiplex cytokine analysis revealed significantly lower transforming growth factor $\alpha$ in the ME serum" of patients with endometriosis. Transcriptomic analysis of CD45+ cells revealed 47 differentially expressed genes mainly associated with the TH17 axis (IL10 IL23A and IL6) as well as genes associated with macrophage signaling/activation (CD74 CD83 CXCL16 and CCL3). CONCLUSION(S) We demonstrate for the first time that the levels of TH17 axis macrophages and transforming growth factor $\alpha$ were altered in the ME of women with endometriosis compared with that of healthy donors. These findings shed light on the potential immune pathways that could partly explain the pathogenesis and progression of endometriosis. Future large-scale studies on ME samples are warranted to exploit the use of these markers to study the pathogenesis of endometriosis." View Publication

Aging plays a critical role in the incidence and severity of infection,with age emerging as an independent predictor of mortality in sepsis. Trained immunity reprograms immunocytes to respond more rapidly and effectively to pathogens and serves as a potential approach to improve immune function in aging and/or sepsis. However,there is very little data on trained immunity in the aging immune system or in the presence of sepsis. We examined the impact of ?-glucan induced innate immune training on monocytes from aging healthy humans (>60 years old) as well as sepsis patients. We observed increased metabolic capacity,upregulated cytokine secretion,increased H3K27 acetylation,and upregulation of crucial intracellular signaling pathways in trained monocytes from healthy aging subjects. The response to trained immunity in healthy aging monocytes was equivalent to the response of monocytes from younger,i.e.,18 - 59 years,individuals. Additionally,we found that trained immunity induced a unique expression pattern of cell surface markers in monocytes that was consistent across age groups. Trained monocytes from sepsis patients also displayed enhanced metabolic capacity and increased cytokine production. These results indicate that immune training can be induced in aging monocytes as well as monocytes from critically ill sepsis patients. View Publication