技术资料

The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway is a cytosolic sensor of microbial and host-derived DNA and plays a key role in innate immunity. Activation of STING by cyclic dinucleotide (CDN) ligands in human monocytes induces a type I interferon response and production of pro-inflammatory cytokines associated with the induction of massive cell death. In this study we have re-evaluated the effect of signal strength of STING activation on the cytokine plasticity of human monocytes. CDN (2'3'c-GAMP) and non-CDN (diABZI,MSA-2) STING ligands in the range of EC50 concentrations (15 $\mu$M 2'3'c-GAMP,100 nM diABZI,25 $\mu$M MSA-2) induced IFN-$\beta$,IP-10,and large amounts of IL-1$\beta$ and TNF-$\alpha$,but no IL-10 or IL-19. Interestingly,LPS-induced production of IL-10 and IL-19 was abolished in the presence of diABZI or MSA-2,whereas IL-1$\beta$ and TNF-$\alpha$ were not inhibited. Surprisingly,we observed that tenfold lower (MSA-2,i.e. 2.5 $\mu$M) or 100-fold lower (diABZI,i.e. 1 nM) concentrations strongly stimulated secretion of anti-inflammatory IL-10 and IL-19,but little of IL-1$\beta$ and TNF-$\alpha$. Induction of IL-10 was associated with up-regulation of PRDM1 (Blimp-1). While cytokine secretion stimulated by the higher concentrations was accompanied by apoptosis as shown by cleavage of caspase-3 and PARP-1,the low concentrations did not trigger overt cell death yet induced cleavage of gasdermin-D. Our results reveal a previously unrecognized plasticity of human monocytes in their signal strength-dependent production of pro- versus anti-inflammatory cytokines upon STING activation. View Publication

Glucagon‐like peptide‐1 (GLP‐1) regulates glycemic excursions by augmenting insulin production and inhibiting glucagon secretion. Liraglutide,a long‐acting GLP‐1 analog,can improve glycemic control for treating type 2 diabetes and prevent neutrophil extravasation in inflammation. Here,we explored the role of liraglutide in the development and therapy of murine lung and liver cancers. In this study,liraglutide substantially decreased circulating neutrophil extracellular trap (NET) markers myeloperoxidase,elastase,and dsDNA in Lewis lung cancer (LLC) and Hepa1‐6 tumor‐bearing mice. Furthermore,liraglutide downregulated NETs and reactive oxygen species (ROS) of neutrophils in the tumor microenvironment. Functionally,in vitro experiments showed that liraglutide reduced NET formation by inhibiting ROS. In addition,we showed that liraglutide enhanced the anti‐tumoral efficiency of programmed cell death‐1 (PD‐1) inhibition in LLC and Hepa1‐6 tumor‐bearing C57BL/6 mice. However,the removal of NETs significantly weakened the antitumor efficiency of liraglutide. We further demonstrated that the long‐term antitumor CD8+ T cell responses induced by the combination therapy rejected rechallenges by respective tumor cell lines. Taken together,our findings suggest that liraglutide may promote the anti‐tumoral efficiency of PD‐1 inhibition by reducing NETs in lung and liver cancers. Liraglutide substantially decreases circulating and tumor‐infiltrated neutrophil extracellular traps by inhibiting reactive oxygen species production. In addition,liraglutide enhances the anti‐tumoral efficiency of programmed cell death‐1 inhibition in tumor‐bearing mice. Furthermore,combination therapy improves IFN‐γ release by CD8+ T cells. Finally,the long‐term antitumor CD8+ T cell response induced by the combination therapy rejects tumor rechallenges. View Publication

Na{\{i}}ve CD8+ T cells can differentiate into effector (Teff) memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff Tmem and Tex populations remain poorly understood. Here we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2 as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator Btg1 in establishing the Tex population. Finally these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections." View Publication

BACKGROUND Two opposing B cell subsets have been defined based on their cytokine profile: IL-6 producing effector B cells (B-effs) versus IL-10 producing regulatory B cells (B-regs) that respectively positively or negatively regulate immune responses. B-regs are decreased and/or impaired in many autoimmune diseases and inflammatory conditions. Since there is increasing evidence that links B cells and B cell-rich lymphoid follicles to the pathogenesis of COPD,the aim of this study was to investigate the presence and function of B-regs in COPD. METHODS First,presence of IL-10 producing regulatory B cells in human lung tissue was determined by immunohistochemistry. Secondly,quantification of IL-10??+??B-regs and IL-6??+??B-effs in peripheral blood mononuclear cells (PBMCs) from healthy controls,smokers without airflow limitation,and COPD patients (GOLD stage I-IV) was performed by flow cytometry. Thirdly,we exposed blood-derived B cells from COPD patients in vitro to cigarette smoke extract (CSE) and quantified IL-10??+??B-regs and IL-6??+??B-effs. Furthermore,we aimed at restoring the perturbed IL10 production by blocking BAFF. Fourthly,we determined mRNA expression of transcription factors involved in IL-10 production in FACS sorted memory- and naive B cells upon exposure to medium or CSE. RESULTS The presence of IL-10 producing regulatory B cells in parenchyma and lymphoid follicles in lungs was confirmed by immunohistochemistry. The percentage of IL-10??+??B-regs was significantly decreased in blood-derived memory B cell subsets from smokers without airflow limitation and patients with COPD,compared to never smokers. Furthermore,the capacity of B cells to produce IL-10 was reduced upon in vitro exposure to CSE and this could not be restored by BAFF-blockade. Finally,upon CSE exposure,mRNA levels of the transcription factors IRF4 and HIF-1$\alpha$,were decreased in memory B cells. CONCLUSION Decreased numbers and impaired function of B-regs in smokers and patients with COPD might contribute to the initiation and progression of the disease. View Publication

Chimeric antigen receptor (CAR)-T cells are engineered to identify and eliminate cells expressing a target antigen. Current manufacturing protocols vary between commercial CAR-T cell products warranting an assessment of these methods to determine which approach optimally balances successful manufacturing capacity and product efficacy. One difference between commercial product manufacturing methods is whether T cell engineering begins with fresh (unfrozen) patient cells or cells that have been cryopreserved prior to manufacture. Starting with frozen PBMC material allows for greater manufacturing flexibility,and the possibility of collecting and storing blood from patients prior to multiple lines of therapy. We prospectively analyzed if second generation anti-CD19 CAR-T cells with either CD28 or 4-1BB co-stimulatory domains have different phenotype or function when prepared side-by-side using fresh or cryopreserved PBMCs. We found that cryopreserved PBMC starting material is associated with slower CAR-T cell expansion during manufacture but does not affect phenotype. We also demonstrate that CAR-T cell activation,cytokine production and in vitro anti-tumor cytotoxicity were not different when CAR-T cells were manufactured from fresh or cryopreserved PBMC. As CAR-T cell therapy expands globally,the need for greater flexibility around the timing of manufacture will continue to grow. This study helps support the concept that cryopreservation of PBMCs could be the solution to these issues without compromising the quality of the final CAR-T product. View Publication

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders,hence they make suitable targets in proteomic studies. However,extraction of membrane proteins is challenging due to their low abundance,stability,and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction,the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols,internally identified as Protocol 1 and 2,were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation,while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer's recommendation. Using Protocol 2,protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150-200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships,particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states,such as ME/CFS. View Publication

While inhibitory Siglec receptors are known to regulate myeloid cells,less is known about their expression and function in lymphocytes subsets. Here we identified Siglec-7 as a glyco-immune checkpoint expressed on non-exhausted effector memory CD8+ T cells that exhibit high functional and metabolic capacities. Seahorse analysis revealed higher basal respiration and glycolysis levels of Siglec-7+ CD8+ T cells in steady state,and particularly upon activation. Siglec-7 polarization into the T cell immune synapse was dependent on sialoglycan interactions in trans and prevented actin polarization and effective T cell responses. Siglec-7 ligands were found to be expressed on both leukemic stem cells and acute myeloid leukemia (AML) cells suggesting the occurrence of glyco-immune checkpoints for Siglec-7+ CD8+ T cells,which were found in patients' peripheral blood and bone marrow. Our findings project Siglec-7 as a glyco-immune checkpoint and therapeutic target for T cell-driven disorders and cancer. View Publication

BACKGROUND Hypersensitivity pneumonitis (HP) is an interstitial lung disease characterized by antigen-triggered neutrophilic exacerbations. Although CD4+ T cells are sufficient for HP pathogenesis,this never translated into efficient T cell-specific therapies. Increasing evidence shows that B cells also play decisive roles in HP. Here,we aimed to further define the respective contributions of B and T cells in subacute experimental HP. METHODS Mice were subjected to a protocol of subacute exposure to the archaeon Methanosphaera stadmanae to induce experimental HP. Using models of adoptive transfers of B cells and T cells in Rag1-deficient mice and of B cell-specific S1P1 deletion,we assessed the importance of B cells in the development of HP by evaluating inflammation in bronchoalveolar lavage fluid. We also aimed to determine if injected antibodies targeting B and/or T cells could alleviate HP exacerbations using a therapeutic course of intervention. RESULTS Even though B cells are not sufficient to induce HP,they strongly potentiate CD4+ T cell-induced HP?‘associated neutrophilic inflammation in the airways. However,the reduction of 85% of lung B cells in mice with a CD19-driven S1P1 deletion does not dampen HP inflammation,suggesting that lung B cells are not necessary in large numbers to sustain local inflammation. Finally,we found that injecting antibodies targeting B cells after experimental HP was induced does not dampen neutrophilic exacerbation. Yet,injection of antibodies directed against B cells and T cells yielded a potent 76% inhibition of neutrophilic accumulation in the lungs. This inhibition occurred despite partial,sometimes mild,depletion of B cells and T cells subsets. CONCLUSIONS Although B cells are required for maximal inflammation in subacute experimental HP,partial reduction of B cells fails to reduce HP-associated inflammation by itself. However,co-modulation of T cells and B cells yields enhanced inhibition of HP exacerbation caused by an antigenic rechallenge. View Publication

BACKGROUND Current immunotherapies still have limited successful rates among cancers. It is now recognized that T cell functional state in the tumor microenvironment (TME) is a key determinant for effective antitumor immunity and immunotherapy. In addition to exhaustion,cellular senescence in tumor-infiltrating T cells (TILs) has recently been identified as an important T cell dysfunctional state induced by various malignant tumors. Therefore,a better understanding of the molecular mechanism responsible for T cell senescence in the TME and development of novel strategies to prevent effector T cell senescence are urgently needed for cancer immunotherapy. METHODS Senescent T cell populations in the TMEs in mouse lung cancer,breast cancer,and melanoma tumor models were evaluated. Furthermore,T cell senescence induced by mouse tumor and regulatory T (Treg) cells in vitro was determined with multiple markers and assays,including real-time PCR,flow cytometry,and histochemistry staining. Loss-of-function strategies with pharmacological inhibitors and the knockout mouse model were used to identify the potential molecules and pathways involved in T cell senescence. In addition,melanoma mouse tumor immunotherapy models were performed to explore the synergistical efficacy of antitumor immunity via prevention of tumor-specific T cell senescence combined with anti-programmed death-ligand 1 (anti-PD-L1) checkpoint blockade therapy. RESULTS We report that both mouse malignant tumor cells and Treg cells can induce responder T cell senescence,similar as shown in human Treg and tumor cells. Accumulated senescent T cells also exist in the TME in tumor models of lung cancer,breast cancer and melanoma. Induction of ataxia-telangiectasia mutated protein (ATM)-associated DNA damage is the cause for T cell senescence induced by both mouse tumor cells and Treg cells,which is also regulated by mitogen-activated protein kinase (MAPK) signaling. Furthermore,blockages of ATM-associated DNA damage and/or MAPK signaling pathways in T cells can prevent T cell senescence mediated by tumor cells and Treg cells in vitro and enhance antitumor immunity and immunotherapy in vivo in adoptive transfer T cell therapy melanoma models. Importantly,prevention of tumor-specific T cell senescence via ATM and/or MAPK signaling inhibition combined with anti-PD-L1 checkpoint blockade can synergistically enhance antitumor immunity and immunotherapy in vivo. CONCLUSIONS These studies prove the novel concept that targeting both effector T cell senescence and exhaustion is an effective strategy and can synergistically enhance cancer immunotherapy. View Publication

Currently immunomodulatory compounds are under investigation for use in patients with cardiovascular disease,caused by atherosclerosis. These trials,using recurrent cardiovascular events as endpoint,require enrollment of large patient groups. We investigated the effect of key risk factors for atherosclerosis development,ageing and smoking,on the immune system,with the objective to identify biomarkers differentiating between human populations,and potentially serving as endpoints for future phase 1B trials with immunomodulatory compounds. Blood was collected from young healthy volunteers (aged 18-25 years,n=30),young smokers (18-25 years,n=20),elderly healthy volunteers (>60 years,n=20),heavy smokers (>45 years,15 packyears,n=11) and patients with stable coronary artery disease (CAD) (>60 years,n=27). Circulating immune cell subsets were characterized by flow cytometry,and collected plasma was evaluated by proteomics (Olink). Clear ageing effects were observed,mostly illustrated by a lower level in CD8+ and na{\{i}}ve CD4+ and CD8+ T cells with an increase in CD4+ and CD8+ effector memory T cells in elderly healthy volunteers compared to young healthy volunteers. Heavy smokers showed a more inflammatory cellular phenotype especially a shift in Th1/Th2 ratio: higher Th1 and lower Th2 percentages compared to young healthy volunteers. A significant decrease in circulating atheroprotective oxLDL-specific IgM was found in patients with CAD compared to young healthy volunteers. Elevated pro-inflammatory and chemotactic proteins TREM1 and CCL11 were observed in elderly volunteers compared to young volunteers. In addition heavy smokers had an increase in pro-inflammatory cytokine IL-6 and lysosomal protein LAMP3. These data show that ageing and smoking are associated with an inflammatory immunophenotype and that heavy smokers or aged individuals may serve as potential populations for future clinical trials investigating immunomodulatory drugs targeted for cardiovascular disease." View Publication

BACKGROUND While lipid emulsions in modern formulations for total parenteral nutrition (TPN) provide essential fatty acids and dense calories,they also promote inflammation and immunometabolic disruptions. OBJECTIVES We aimed to develop a novel lipid emulsion for TPN use with superior immunometabolic actions compared with available standard lipid emulsions. METHODS A novel lipid emulsion [Vegaven (VV)] containing 30% of 18-carbon n-3 fatty acids ($\alpha$-linolenic acid and stearidonic acid) was developed for TPN (VV-TPN) and compared with TPN containing soybean oil-based lipid emulsion (IL-TPN) and fish-oil-based lipid emulsion (OV-TPN). In vivo studies were performed in instrumented male C57BL/6 mice subjected to 7-d TPN prior to analysis of cytokines,indices of whole-body and hepatic glucose metabolism,immune cells,lipid mediators,and mucosal bowel microbiome. RESULTS IL-6 to IL-10 ratios were significantly lower in liver and skeletal muscle of VV-TPN mice when compared with IL-TPN or OV-TPN mice. VV-TPN and OV-TPN each increased hepatic insulin receptor abundance and resulted in similar HOMA-IR values,whereas only VV-TPN increased hepatic insulin receptor substrate 2 and maintained normal hepatic glycogen content,effects that were IL-10-dependent and mediated by glucokinase activation. The percentages of IFN-$\gamma$- and IL-17-expressing CD4+ T cells were increased in livers of VV-TPN mice,and liver macrophages exhibited primed phenotypes when compared with IL-TPN. This immunomodulation was associated with successful elimination of the microinvasive bacterium Akkermansia muciniphila from the bowel mucosa by VV-TPN as opposed to standard lipid emulsions. Assay of hepatic lipid mediators revealed a distinct profile with VV-TPN,including increases in 9(S)-hydroxy-octadecatrienoic acid. When co-administered with IL-TPN,hydroxy-octadecatrienoic acids mimicked the VV-TPN immunometabolic phenotype. CONCLUSIONS We here report the unique anti-inflammatory,insulin-sensitizing,and immunity-enhancing properties of a newly developed lipid emulsion designed for TPN use based on 18-carbon n-3 fatty acids. View Publication

Gas gangrene caused by Clostridium perfringens type A infection is a highly lethal infection of soft tissue characterized by rapid spread of tissue necrosis. This tissue destruction is related to profound attenuation of blood flow accompanied by formation of platelet-leukocyte aggregates in the blood vessels. Several studies have identified $\alpha$-toxin,which has both sphingomyelinase and phospholipase C activities,as a major virulence factor in the aggregate formation via activation of the platelet gpIIbIIIa. Here,we show that $\alpha$-toxin greatly and rapidly increases plasma membrane localization of CD11b,which binds to the platelet gpIIbIIIa via fibrinogen,in mouse neutrophils. Interestingly,short-term treatment of $\alpha$-toxin has little effect on gene expression profiles in neutrophils,and the toxin does not change the total protein expression levels of CD11b in whole cell lysates. The following analysis demonstrated that CD11b localizes to intracellular vesicles in intact cells,but the localization changed to the cytoplasmic membrane in $\alpha$-toxin-treated cells. These results suggest that CD11b is recruited to the cytoplasmic membrane by $\alpha$-toxin. Previously,we reported that $\alpha$-toxin promotes the formation of ceramide by its sphingomyelinase activity in mouse neutrophils. Interestingly,a synthetic cell-permeable ceramide analog,C2-ceramide,increases plasma membrane localization of CD11b,suggesting that ceramide production by $\alpha$-toxin recruits CD11b to the cytoplasmic membrane to promote platelet-leukocyte aggregation. Together,our results illustrate that the increase of cell membrane CD11b expression by $\alpha$-toxin might be crucial for the pathogenesis of C. perfringens to promote formation of platelet-leukocyte aggregates,leading to rapid tissue necrosis due to ischemia. View Publication