技术资料

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

AbstractBackgroundHypoxia is associated with the evasion of triple-negative breast cancer (TNBC) from immune surveillance. Hypoxia increases the subpopulation of putative TNBC stem-like cells (TNBCSCs) through activating Wnt/β-Catenin signaling. The shedding of MHC class I-related chain A (MICA) is particularly noteworthy in cancer stem cells (CSCs),promoting the resistance of CSCs to natural killer (NK) cell cytotoxicity. To reestablish MICA/NKG2D-mediated immunosurveillance,we proposed the design of a fusion protein (SHH002-hu1-MICA) which consists of Frizzled-7 (Fzd7)-targeting antibody and MICA,serving as an engager retargeting NK cells against TNBCs,especially TNBCSCs.MethodsOpal multicolor immunohistochemistry staining was used to validate the expression of membrane MICA (mMICA) and existence of NK cells in TNBC tumors; flow cytometry (FCM) assay was used to detect the expression of Fzd7/mMICA on TNBCs. Biolayer interferometry (BLI) and surface plasmon resonance (SPR) assays were executed to assess the affinity of SHH002-hu1-MICA towards rhFzd7/rhNKG2D; near-infrared imaging assay was used to evaluate the targeting capability. A cytotoxicity assay was conducted to assess the effects of SHH002-hu1-MICA on NK cell-mediated killing of TNBCs,and FCM assay to analyze the effects of SHH002-hu1-MICA on the degranulation of NK cells. Finally,TNBC cell-line-derived xenografts were established to evaluate the anti-tumor activities of SHH002-hu1-MICA in vivo.ResultsThe expression of mMICA is significantly downregulated in hypoxic TNBCs and TNBCSCs,leading to the evasion of immune surveillance exerted by NK cells. The expression of Fzd7 is significantly upregulated in TNBCSCs and exhibits a negative correlation with the expression of mMICA and infiltration level of NK cells. On accurate assembly,SHH002-hu1-MICA shows a strong affinity for rhFzd7/rhNKG2D,specifically targets TNBC tumor tissues,and disrupts Wnt/β-Catenin signaling. SHH002-hu1-MICA significantly enhances the cytotoxicity of NK cells against hypoxic TNBCs and TNBCSCs by inducing the degranulation of NK cells and promotes the infiltration of NK cells in CD44high regions within TNBC xenograft tumors,exhibiting superior anti-tumor activities than SHH002-hu1.ConclusionsSHH002-hu1-MICA maintains the targeting property of SHH002-hu1,successfully activates and retargets NK cells against TNBCs,especially TNBCSCs,exhibiting superior antitumor activities than SHH002-hu1. SHH002-hu1-MICA represents a promising new engager for NK cell-based immunotherapy for TNBC. View Publication

BackgroundMacrophages are required for development and tissue repair and protect against microbial attacks. In response to external signals,monocytes differentiate into macrophages,but our knowledge of changes that promote this transition at the level of mRNA processing,in particular mRNA polyadenylation,needs advancement if it is to inform new disease treatments. Here,we identify CFIm25,a well-documented regulator of poly(A) site choice,as a novel mediator of macrophage differentiation.MethodsCFIm25 expression was analyzed in differentiating primary human monocytes and monocytic cell lines. Overexpression and depletion experiments were performed to assess CFIm25’s role in differentiation,NF-κB signaling,and alternative polyadenylation (APA). mRNA 3’ end-focused sequencing was conducted to identify changes in poly(A) site use of genes involved in macrophage differentiation and function. Cell cycle markers,NF-κB pathway components,and their targets were examined. The role of CFIm25 in NF-κB signaling was further evaluated through chemical inhibition and knockdown of pathway regulators.ResultsCFIm25 showed a striking increase upon macrophage differentiation,suggesting it promotes this process. Indeed,CFIm25 overexpression during differentiation amplified the acquisition of macrophage characteristics and caused an earlier slowing of the cell cycle,a hallmark of this transition,along with APA-mediated downregulation of cyclin D1. The NF-κB signaling pathway plays a major role in maturation of monocytes to macrophages,and the mRNAs of null,TBL1XR1,and NFKB1,all positive regulators of NF-κB signaling,underwent 3’UTR shortening,coupled with an increase in the corresponding proteins. CFIm25 overexpression also elevated phosphorylation of the NF-κB-p65 transcription activator,produced an earlier increase in the NF-κB targets p21,Bcl-XL,ICAM1 and TNF-α,and resulted in greater resistance to NF-κB chemical inhibition. Knockdown of Tables 2 and TBL1XR1 in CFIm25-overexpressing cells attenuated these effects,reinforcing the mechanistic link between CFIm25-regulated APA and NF-κB activation. Conversely,depletion of CFIm25 hindered differentiation and led to lengthening of NFKB1,TAB2,and TBL1XR1 3’ UTRs.ConclusionsOur study establishes CFIm25 as a key mediator of macrophage differentiation that operates through a coordinated control of cell cycle progression and NF-κB signaling. This linkage of mRNA processing and immune cell function also expands our understanding of the role of alternative polyadenylation in regulating cell signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02114-1. View Publication

ABSTRACTCell therapy based on chimeric antigen receptor (CAR) T cells has represented a revolutionary new approach for treating tumors,especially hematological diseases. Complete remission rates (CRR) > 80%–97% and 50%–90% overall response rates (ORR) have been achieved with a treatment based on CAR‐T cells in patients with malignant B‐cell tumors that have relapsed or are refractory to previous treatments. Toxicity remains the major problem. Most patients treated with CAR‐T cells develop high‐grade cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity syndrome (ICANS). However,the unprecedentedly high CRR and ORR have led to the approval of six CAR‐T cell therapeutics by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA),prompting researchers to improve existing products and develop new ones. By now,around 1000 clinical trials based on CAR‐T cells are registered at ClinicalTrials.gov: 82% are for hematological diseases,while the remaining 16% are for solid tumors. As a result of this increased research,an enormous amount of conflicting information has been accumulated in the literature,and each group follows its manufacturing protocols and performs specific in vitro testing. This review aimed to combine and compare clinical and preclinical information,highlighting the most used protocols to provide a comprehensive overview of the in vitro world of CAR‐T cells,from manufacturing to their characterization. The focus is on all steps of the CAR‐T cell manufacturing process,from the collection of patient or donor blood to the enrichment of T cells,their activation with anti‐CD3/CD28 beads,interleukin‐2 (IL‐2) or IL‐7 and IL‐15 (induction of a more functional memory phenotype),and their transfection (viral or non‐viral methods). Automation is crucial for ensuring a standardized final product. View Publication