技术资料

BACKGROUND With the rapid development of immune checkpoint inhibitors and neoantigen (NeoV)-based personalized tumor vaccines,tumor immunotherapy has shown promising therapeutic results. However,the limited efficacy of available tumor vaccines impedes the development of personalized tumor immunotherapy. In this study,we developed a novel tumor vaccine system and proposed combined therapeutic strategies for improving treatment effects. METHODS We developed a novel tumor vaccine system comprising a newly synthesized peptidic microarchitecture (PMA) with high assembly efficacy. The PMA-trapped neoantigen vaccine was developed to codeliver tumor neoantigen and the Toll-like receptor 9 agonist CpG (NeoV),abbreviated as PMA-NeoV. A microfluidic chip was used to produce PMA particles in a uniform and precise manner. Vaccine effectiveness was investigated both in vitro and in vivo. The combined immunotherapeutic effect of PMA-NeoV with anti-programmed cell death ligand 1 antibody (aPD-L1) or with the phosphatidylinositol 3?‘kinase $\gamma$ (PI3K$\gamma$) inhibitor IPI-549 was further tested in MC38 mouse tumor model. RESULTS PMA-NeoV not only promoted codelivery of the tumor vaccine but also potentiated vaccine immunogenicity. Moreover,compared with free NeoV,PMA-NeoV significantly increased the number of tumor-infiltrating lymphocytes,promoted the neoantigen-specific systemic immune response,and suppressed murine colon MC38 tumor growth. Furthermore,PMA-NeoV increased the expression of programmed cell death receptor-1 on T lymphocytes,and in combination with aPD-L1 eradicated seven of eight MC38 tumors by rescuing exhausted T lymphocytes. Moreover,we combined the PMA-NeoV with the IPI-549,a molecular switch that controls immune suppression,and found that this combination significantly suppressed tumor growth and eradicated five of eight inoculated tumors,by switching suppressive macrophages to their active state and activating T cells to prime a robust tumor immune microenvironment. CONCLUSIONS We developed a tumor vaccine delivery system and presented a promising personalized tumor vaccine-based therapeutic regimen in which a tumor vaccine delivery system is combined with an aPD-L1 or PI3K$\gamma$ inhibitor to improve tumor immunotherapy outcomes. View Publication

Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage-inducible C-type lectin (Mincle,Clec4e) expression is up-regulated in B cells from heart allograft recipients treated with murine anti-thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro-inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely,treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non-transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell-depleted,but not in Mincle-deficient,recipients indicating that CD40 signaling induces T cell proliferation via a Mincle-dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage-associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation. View Publication

BACKGROUND AKI is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here,we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine. METHODS We performed single-cell RNA sequencing (scRNAseq) on cells recovered from the urine of eight hospitalized patients with COVID-19 with (n=5) or without AKI (n=3) as well as four patients with non-COVID-19 AKI (n=4) to assess differences in cellular composition and gene expression during AKI. RESULTS Analysis of 30,076 cells revealed a diverse array of cell types,most of which were kidney,urothelial,and immune cells. Pathway analysis of tubular cells from patients with AKI showed enrichment of transcripts associated with damage-related pathways compared with those without AKI. ACE2 and TMPRSS2 expression was highest in urothelial cells among cell types recovered. Notably,in one patient,we detected SARS-CoV-2 viral RNA in urothelial cells. These same cells were enriched for transcripts associated with antiviral and anti-inflammatory pathways. CONCLUSIONS We successfully performed scRNAseq on urinary sediment from hospitalized patients with COVID-19 to noninvasively study cellular alterations associated with AKI and established a dataset that includes both injured and uninjured kidney cells. Additionally,we provide preliminary evidence of direct infection of urinary bladder cells by SARS-CoV-2. The urinary sediment contains a wealth of information and is a useful resource for studying the pathophysiology and cellular alterations that occur in kidney diseases. View Publication

Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis,with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN,NK cells and ILC3 were the prominent subpopulations. Among the ILC3s,we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients,NK cells were prominent,and proportions of ILC3 subsets were low. In tumors and TD-LN,NK cells display reduced levels of NCR (Natural cytotoxicity receptors),despite high transcript levels and included a small subset CD127- CD56- NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN,all cytokine activated ILC populations produced TNF$\alpha$ in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR,but cytokine stimulation restored their functionality. View Publication

Monocytes undergo phenotypic and functional changes in response to inflammatory cues,but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes,which are preferentially recruited,dwell within glomerular capillaries,and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically,the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells,findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2-activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients,and their frequency correlates with clinical disease. In conclusion,CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2-activated vasculature and may establish a CCR2-based autocrine,feed-forward loop that amplifies renal inflammation. View Publication

Mutations causing loss of the NF-$\kappa$B regulator I$\kappa$BNS,result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming,we sought to investigate processes important for this transition using the bumble mouse strain,deficient for I$\kappa$BNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels,mitochondrial accumulation,increased OXPHOS and mROS production,along with a reduced capacity for autophagy,compared to wildtype B cells. Overall,our results demonstrate that PC differentiation in the absence of I$\kappa$BNS is characterized by excessive activation during early rounds of B cell division,increased mitochondrial metabolism and decreased autophagic capacity,thus improving our understanding of the role of I$\kappa$BNS in PC differentiation. View Publication

Altered energy metabolism and changes in glycolytic and oxidative phosphorylation pathways are hallmarks of all cancer cells. The expression of select genes associated with the production of various enzymes and proteins involved in glycolysis and oxidative phosphorylation were assessed in the clonal plasma cells derived from patients with newly diagnosed multiple myeloma (NDMM) enrolled in the Multiple Myeloma Research Foundation (MMRF) CoMMpass data set. A scoring system consisting of assigning a point for every gene where their fragments per kilobase of transcript per million (FPKM) was above the median yielded a minimum of 0 and a maximum of 12 for the set of genes in the glycolytic and oxidative phosphorylation pathways to create a total energy metabolism molecular signature (EMMS) score. This EMMS score was independently associated with worse progression free survival (PFS) and overall survival (OS) outcomes of patients with NDMM. A higher EMMS score was more likely to be present in clonal plasma cells derived from Multiple myeloma (MM) patients than those from patients with monoclonal gammopathy of undetermined significance (MGUS). This was functionally confirmed by the clonal plasma cells from MM patients having a higher rate of mitochondrial and glycolysis-derived ATP formation than clonal plasma cells from MGUS patients. Thus,this study provides evidence for the effect of energy metabolism within clonal plasma cells on pathogenesis and outcomes of patients with MM. Exploiting the energy-producing metabolic pathways within clonal plasma cells for diagnostic and therapeutic purposes in MM should be explored in the future. View Publication

BACKGROUND miR-155-5p is associated with autoimmune diseases. T helper 17 (Th17) cells,interleukin (IL)-17,and suppressor of cytokines signaling 1 (SOCS1) have important roles in the pathogenesis of systemic sclerosis (SSc). The purpose of this study was to explore the role of miR-155-5p in the regulation of IL-17 and SOCS1 expression in Th17 cells and the subsequent effect on SSc disease progression. METHODS Th17 cells were isolated from peripheral blood mononuclear cells of SSc patients and healthy controls (HCs). RT-qPCR and western blotting were used to examine the expression patterns of miR-155-5p,IL-17,and SOCS1. Luciferase reporter assays were performed to confirm SOCS1 as a target of miR-155-5p. RNA pull-down assays were performed to detect the interaction of IL-17 and SOCS1 with miR-155-5p. In situ hybridization was performed to analyze the co-expression pattern of miR-155-5p and IL17A in Th17 cells. RESULTS The levels of Th17 cell-derived miR-155-5p were significantly up-regulated in SSc patients compared with HCs,and its levels were negatively correlated with SOCS1 levels. Meanwhile,miR-155-5p positively regulated IL-17 expression levels in Th17 cells isolated from SSc patients as the disease progressed. Using pmirGLO vectors,SOCS1 was confirmed as a target of miR-155-5p. The binding status of IL-17 and SOCS1 to miR-155-5p was related to SSc progression. An increase in the co-localization of miR-155-5p and IL-17 was associated with greater SSc progression. CONCLUSIONS IL-17 and SOCS1 expression modulated by Th17 cell-derived miR-155-5p are critical for SSc progression,which may provide novel insights into the pathogenesis of SSc. View Publication

MALT1 forms part of a central signaling node downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors,across a broad range of immune cell subsets,and regulates NF-$\kappa$B driven transcriptional responses via dual scaffolding-protease activity. Allosteric inhibition of MALT1 activity has demonstrated benefit in animal models of inflammation. However,development of MALT1 inhibitors to treat autoimmune and inflammatory diseases (A&ID) has been hindered by reports linking MALT1 inhibition and genetic loss-of-function to reductions in regulatory T-cell (Treg) numbers and development of auto-inflammatory syndromes. Using an allosteric MALT1 inhibitor,we investigated the consequence of pharmacological inhibition of MALT1 on proinflammatory cells compared to regulatory T-cells. Consistent with its known role in ITAM-driven responses,MALT1 inhibition suppressed proinflammatory cytokine production from activated human T-cells and monocyte-derived macrophages,and attenuated B-cell proliferation. Oral administration of a MALT1 inhibitor reduced disease severity and synovial cytokine production in a rat collagen-induced arthritis model. Interestingly,reduction in splenic Treg numbers was less pronounced in the context of inflammation compared with na{\{i}}ve animals. Additionally in the context of the disease model we observed an uncoupling of anti-inflammatory effects of MALT1 inhibition from Treg reduction with lower systemic concentrations of inhibitor needed to reduce disease severity compared to that required to reduce Treg numbers. MALT1 inhibition did not affect suppressive function of human Tregs in vitro. These data indicate that anti-inflammatory efficacy can be achieved with MALT1 inhibition without impacting the number or function of Tregs further supporting the potential of MALT1 inhibition in the treatment of autoimmune disease." 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

Notoginsenoside R1 (NGR1) is the main monomeric component extracted from the dried roots and rhizomes of Panax notoginseng,and exerts pharmacological action against myocardial infarction (MI). Owing to the differences in compound distribution,absorption,and metabolism in vivo,exploring a more effective drug delivery system with a high therapeutic targeting effect is crucial. In the early stages of MI,CD11b-expressing monocytes and neutrophils accumulate at infarct sites. Thus,we designed a mesoporous silica nanoparticle-conjugated CD11b antibody with loaded NGR1 (MSN-NGR1-CD11b antibody),which allowed NGR1 precise targeted delivery to the heart in a noninvasively manner. By increasing targeting to the injured myocardium,intravenous injection of MSN-NGR1-CD11b antibody nanoparticle in MI mice improved cardiac function and angiogenesis,reduced cell apoptosis,and regulate macrophage phenotype and inflammatory factors and chemokines. In order to further explore the mechanism of NGR1 protecting myocardium,cell oxidative stress model and oxygen-glucose deprivation (OGD) model were established. NGR1 protected H9C2 cells and primary cardiomyocytes against oxidative injury induced by H2O2 and OGD treatment. Further network pharmacology and molecular docking analyses suggested that the AKT,MAPK and Hippo signaling pathways were involved in the regulation of NGR1 in myocardial protection. Indeed,NGR1 could elevate the levels of p-Akt and p-ERK,and promote the nuclear translocation of YAP. Furthermore,LY294002 (AKT inhibitor),U0126 (ERK1/2 inhibitor) and Verteporfin (YAP inhibitor) administration in H9C2 cells indicated the involvement of AKT,MAPK and Hippo signaling pathways in NGR1 effects. Meanwhile,MSN-NGR1-CD11b antibody nanoparticles enhanced the activation of AKT and MAPK signaling pathways and the nuclear translocation of YAP at the infarcted site. Our research demonstrated that MSN-NGR1-CD11b antibody nanoparticle injection after MI enhanced the targeting of NGR1 to the infarcted myocardium and improved cardiac function. More importantly,our pioneering research provides a new strategy for targeting drug delivery systems to the ischemic niche. View Publication

To date,driver genes for pancreatic cancer treatment are difficult to pursue therapeutically. Targeting mutated KRAS,the most renowned driver gene in pancreatic cancer,is an active area of study. We discovered a gene named SEMA3C was highly expressed in pancreatic cancer cell lines and patients with a G12D mutation in KRAS. High expression of SEMA3C in patients was significantly associated with the decreased survival of pancreatic cancer patients based on the TCGA database. In pancreatic cancer cells,SEMA3C knockdown or inhibition exhibited growth/colony inhibition and cell cycle arrest. In addition,SEMA3C inhibition sensitized KRAS or MEK1/2 inhibition in pancreatic cancer cells. Overexpression of SEMA3C resulted in the induction of autophagy,whereas depletion of SEMA3C compromised induction of autophagy. SEMA3C modified the PD-L1 expression in tumor and immune cells and is correlated with the M2-like macrophage marker ARG1/CD163 expression,which could reshape the tumor microenvironment. Inhibition of SEMA3C decreased tumor formation in the xenograft model in vivo. Taken together,our data suggest that SEMA3C plays a substantial role in promoting cancer cell survival by regulating the autophagy process and impacting the tumor environment immune response. SEMA3C can be used as a novel target or marker with therapeutic or diagnostic potential in pancreatic cancer especially in tumors harboring the specific KRAS G12D mutation. View Publication