参考文献

Large-scale cancer genetic/genomic studies demonstrated that papillary renal cell carcinoma (pRCC) is featured with a frequent shallow deletion of the upstream tumor suppressors of the Hippo/YAP signaling pathway,suggesting that this signaling pathway may play a role in pRCC development. Here we develop a transgenic mouse model with a renal epithelial cell-specific hyperactivation of YAP1 and find that hyperactivation of YAP1 can induce dedifferentiation and transformation of renal tubular epithelial cells leading to the development of pRCC. We analyze at the single-cell resolution the cellular landscape alterations during cancer initiation and progression. Our data indicate that the hyperactivated YAP1,via manipulating multiple signaling pathways,induces epithelial cell transformation,MDSC (Myeloid-derived suppressor cells) accumulation,and pRCC development. Interestingly,we find that depletion of MDSC blocks YAP1-induced kidney overgrowth and tumorigenesis. Inhibiting YAP1 activity with MGH-CP1,a recently developed TEAD inhibitor,impedes MDSC accumulation and suppresses tumor development. Our results identify the disrupted Hippo/YAP signaling as a major contributor to pRCC and suggest that targeting the disrupted Hippo pathway represents a plausible strategy to prevent and treat pRCC. Deletion of upstream tumor suppressors of the Hippo/YAP pathway is frequent in papillary renal cell carcinoma (pRCC). Here,the authors employ a transgenic mouse model,single-cell transcriptomics and public genomic datasets to show that targeting hyperactivated YAP1 prevents neoplastic renal epithelial cell immune evasion and impairs the development of pRCC. View Publication

AbstractThere is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats),coupled with light’s unparalleled spatiotemporal resolution deliverable from a controllable source,makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here,we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs,BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR,including indels,CRISPRa,and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing,achieving optogenetic gene editing in T lymphocytes in vivo. View Publication

Data of low-density neutrophils (LDN),the neutrophils in the peripheral blood mononuclear cells (PBMC) fraction,in sepsis is still less. As such,LDN (CD66b-positive cells in PBMC) was highest in intensive care unit (ICU) patients with sepsis (n=24) compared with non-sepsis (n=10) and healthy control (n=20),with a negative correlation with lymphocyte count and could predict secondary infection and mortality with the area under the curve (AUC) at 0.79 and 0.84,respectively. Compared with sepsis normal-density neutrophils (NDN),sepsis-LDN demonstrated higher expression of CD66b,CD63,CD11b,and CD184,but lower expression of CD62L and CD182 and defects of effector functions,including phagocytosis and apoptosis. The t-distributed stochastic neighbor embedding (t-SNEs) demonstrated high program cell death ligand-1 (PD-L1) in sepsis-LDN. In sepsis samples,the T cell proliferation in PBMC (T cells with LDNs) was lower than that in the isolated T cells (T cells alone) and incubation of anti-PD-L1 neutralizing antibody,but not a reactive oxygen species (ROS) scavenger (N-acetyl cysteine),improved the T cell suppression. Additionally,30 min lipopolysaccharide (LPS) activation altered healthy control NDN into LPS-LDN (reduced density) and LPS-NDN (maintain density) with similarly elevated CD66b,CD11B,and CD62L. However,LPS-LDN (in vitro LDN) showed lower expression of CD63,CD184,and PD-L1 compared with LDN from patients (sepsis-LDN),suggesting a partial LPS impact on LDN generation. From the microscopic-based method (Wright’s staining in PBMC),sepsis-LDN demonstrated a mixed population of mature and immature cells with a good correlation with the flow-based analysis (Bland–Altman analysis and AUC). In conclusion,LDN in sepsis,partly generated by LPS activation,was associated with secondary infection and T cell suppression,mainly through the expression of PD-L1,which might be an immune suppression biomarker,especially with a less expensive microscopic-based method. View Publication

Prostate cancer (PCa) ranks as the second leading cause of cancer-related deaths among men in the United States. Prostate-specific membrane antigen (PSMA) represents a well-established biomarker of PCa,and its levels correlate positively with the disease progression,culminating at the stage of metastatic castration-resistant prostate cancer. Due to its tissue-specific expression and cell surface localization,PSMA shows superior potential for precise imaging and therapy of PCa. Antibody-based immunotherapy targeting PSMA offers the promise of selectively engaging the host immune system with minimal off-target effects. Here we report on the design,expression,purification,and characterization of a bispecific engager,termed 5D3-CP33,that efficiently recruits macrophages to the vicinity of PSMA-positive cancer cells mediating PCa death. The engager was engineered by fusing the anti-PSMA 5D3 antibody fragment to a cyclic peptide 33 (CP33),selectively binding the Fc gamma receptor I (FcγRI/CD64) on the surface of phagocytes. Functional parts of the 5D3-CP33 engager revealed a nanomolar affinity for PSMA and FcγRI/CD64 with dissociation constants of KD =  3 nM and KD =  140 nM,respectively. At a concentration as low as 0.3 nM,the engager was found to trigger the production of reactive oxygen species by U937 monocytic cells in the presence of PSMA-positive cells. Moreover,flow cytometry analysis demonstrated antibody-dependent cell-mediated phagocytosis of PSMA-positive cancer cells by U937 monocytes when exposed to 0.15 nM 5D3-CP33. Our findings illustrate that 5D3-CP33 effectively and specifically activates monocytes upon PSMA-positive target engagement,resulting in the elimination of tumor cells. The 5D3-CP33 engager can thus serve as a promising lead for developing new immunotherapy tools for the efficient treatment of PCa. View Publication

Tumor-associated macrophages (TAMs) significantly influence tumor progression and patient responses to conventional chemotherapy. However,the interplay between anti-cancer drugs,immune responses in the tumor microenvironment,and their implications for cancer treatment remains poorly understood. This study investigates the effects of vinorelbine on M2 macrophages in lung cancer and its capacity to modulate TAMs toward an M1 phenotype. Peripheral blood mononuclear cells (PBMCs) were polarized into M2 macrophages,and subsequent phenotype alterations upon vinorelbine treatment were assessed. Additionally,we evaluated vinorelbine’s impact on gene and protein expression associated with cancer progression and cell invasion in non-small-cell lung cancer (NSCLC) cells indirectly co-cultured with M2 macrophages. Notably,vinorelbine,particularly at low concentrations,reprogrammed M2 macrophages to exhibit M1-like characteristics. While M2 macrophages enhanced cancer cell invasion,vinorelbine significantly mitigated this effect. M2 macrophages led to the overexpression of numerous genes linked to tumor growth,angiogenesis,invasion,and immune suppression in NSCLC cells,increasing the BCL2/BAX ratio and promoting cellular resistance to apoptosis. The anti-tumor efficacy of vinorelbine appears to be partly attributed to the reprogramming of M2 macrophages to the M1 phenotype,suggesting that low-dose vinorelbine may optimize therapeutic outcomes while minimizing toxicity in cancer patients. View Publication

This work demonstrates that group 2 KLF family transcription factors are critical for specifying the identity of distinct tissue-resident macrophages. KLF2 directly controls expression of genes previously shown to be necessary in cavity macrophages,while KLF4 may play a similar role in alveolar macrophages. Tissue-resident macrophages adopt distinct gene expression profiles and exhibit functional specialization based on their tissue of residence. Recent studies have begun to define the signals and transcription factors that induce these identities. Here we describe an unexpected and specific role for the broadly expressed transcription factor Krüppel-like factor 2 (KLF2) in the development of embryonically derived large cavity macrophages (LCMs) in the serous cavities. KLF2 not only directly regulates the transcription of genes previously shown to specify LCM identity,such as retinoic acid receptors and GATA6,but also is required for induction of many other transcripts that define the identity of these cells. Our results suggest that KLF4 may similarly regulate the identity of alveolar macrophages in the lung. These data demonstrate that broadly expressed transcription factors,such as group 2 KLFs,can play important roles in the specification of distinct identities of tissue-resident macrophages. View Publication

ABSTRACTBacteriophages (phages) are emerging as a viable adjunct to antibiotics for the treatment of multidrug‐resistant (MDR) bacterial infections. While intravenous phage therapy has proven successful in many cases,clinical outcomes remain uncertain due to a limited understanding of host response to phages. In this study,we conducted a comprehensive examination of the interaction between clinical‐grade phages used to treat MDR Escherichia coli and Klebsiella pneumoniae infections,and human peripheral blood immune cells. Using whole transcriptome as well as proteomic approaches,we identified a strong inflammatory response to E. coli phage vB_EcoM‐JIPh_Ec70 (herein,JIPh_Ec70) that was absent upon exposure to K. pneumoniae phage JIPh_Kp127. We confirmed that JIPh_Ec70's DNA recognition by the STING pathway was principally responsible for the activation of NF‐kB and the subsequent inflammatory response. We further show that monocytes and neutrophils play a dominant role in phage uptake,primarily through complement‐mediated phagocytosis. Significant differences in complement‐mediated phagocytosis of JIPh_Kp127 and JIPh_Ec70 were observed,suggesting that reduced recognition,phagocytosis,and immunogenicity all contribute to the significantly decreased response to JIPh_Kp127. Our findings contribute to the progress of our understanding of the innate immune response to therapeutic phages and offer potential insights into how to improve the safety and effectiveness of phage therapy. Clinical grade JIPh_Ec70 phages but not JIPh_Kp127 phages elicit a potent inflammatory response in peripheral immune cells. JIPh_Ec70 phagocytic engulfment is facilitated by complement opsonization,resulting in STING activation by phage DNA,driving an inflammatory signaling cascade. Understanding phage immunogenicity will be a key factor in developing effective phage therapies in the coming years. View Publication

Uveal melanoma (UM) is the most common intraocular cancer in adults,with metastatic disease (mUM) occurring in approximately half of the patients. Tebentafusp,an immune-mobilizing monoclonal T cell receptor against cancer (ImmTAC),is a therapeutic shown to improve overall survival (OS) in HLA-A*02:01+ adult patients with mUM. Here we investigate the impact of tumor-associated macrophages (TAM) on ImmTAC activity. In vitro,M2 macrophages inhibit ImmTAC-mediated tumor-killing in a dose-dependent and contact-dependent manner. Accordingly,high baseline intratumoral TAM-to-T cell ratios correlate with shorter OS (HR = 2.09,95% CI,1.31–3.33,p = 0.002) in tebentafusp-treated mUM patients from a phase 2 trial. By contrast,IL-2 conditioning of T cells overcomes M2 macrophage-mediated suppression in vitro,while ImmTAC treatment leads to M2-to-M1 macrophage reprogramming both in vitro and in tebentafusp-treated mUM patients. Overall,we show that tebentafusp reshapes the tumor microenvironment to enhance anti-tumor T cell activity,whilst combining tebentafusp with IL-2 may enhance benefit in patients with high levels of TAM. ‘T cell engagers promote antitumor immunity,but how macrophage modulates this activity in tumor is still unclear. Here the authors show,using biopsies from patients with uveal melanoma and single cell analyses,that a T cell engager,tebentafusp,reprograms tumor-associated macrophages and ameliorates,in synergy with IL-2,immunosuppression to cancer. View Publication

The levels of transcription factor Ets1 are high in resting B and T cells,but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells,Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response,stringent control of Ets1 gene expression levels is required for homeostasis. However,the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100 kb upstream and into the gene body. Additionally,we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs,we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells,but variegated expression in T cells,suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells,we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly,functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of Ets1. View Publication

Immune evasion by cancer cells involves reshaping the tumor microenvironment (TME) via communication with non-malignant cells. However,resistance-promoting interactions during treatment remain lesser known. Here we examine the composition,communication,and phenotypes of tumor-associated cells in serial biopsies from stage II and III high-risk estrogen receptor positive (ER+ ) breast cancers of patients receiving endocrine therapy (letrozole) as single agent or in combination with ribociclib,a CDK4/6-targeting cell cycle inhibitor. Single-cell RNA sequencing analyses on longitudinally collected samples show that in tumors overcoming the growth suppressive effects of ribociclib,first cancer cells upregulate cytokines and growth factors that stimulate immune-suppressive myeloid differentiation,resulting in reduced myeloid cell- CD8 + T-cell crosstalk via IL-15/18 signaling. Subsequently,tumors growing during treatment show diminished T-cell activation and recruitment. In vitro,ribociclib does not only inhibit cancer cell growth but also T cell proliferation and activation upon co-culturing. Exogenous IL-15 improves CDK4/6 inhibitor efficacy by augmenting T-cell proliferation and cancer cell killing by T cells. In summary,response to ribociclib in stage II and III high-risk ER +  breast cancer depends on the composition,activation phenotypes and communication network of immune cells. The CDK4/6 inhibitor ribociclib holds promise in cancer therapy but how cell cycle inhibitory drugs affect the anti-tumor immune response remains a question. Here authors show that poor response of early-stage estrogen receptor positive breast cancers to ribociclib is caused by changes in the immune cell composition and cancer-cell-immune-cell communication in the tumors rather than intrinsic cancer cell resistance to cell cycle inhibition. View Publication