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Capsular polysaccharides are common virulence factors of extracellular,but not intracellular bacterial pathogens,due to the antiphagocytic properties of these surface structures. It is therefore paradoxical that Salmonella enterica subspecies enterica serovar Typhi,an intracellular pathogen,synthesizes a virulence-associated (Vi) capsule,which exhibits antiphagocytic properties. Here,we show that the Vi capsular polysaccharide has different functions when S. Typhi interacts with distinct subsets of host phagocytes. The Vi capsular polysaccharide allowed S. Typhi to selectively evade phagocytosis by human neutrophils while promoting human macrophage phagocytosis. A screen of C-type lectin receptors identified human DC-SIGN as the receptor involved in macrophage binding and phagocytosis of capsulated S. Typhi. Consistent with the anti-inflammatory activity of DC-SIGN,purified Vi capsular polysaccharide reduced inflammatory responses in macrophages. These data suggest that binding of the human C-type lectin receptor DC-SIGN by the Vi capsular polysaccharide contributes to the pathogenesis of typhoid fever. IMPORTANCE Salmonella enterica subspecies enterica serovar Typhi is the causative agent of typhoid fever. The recent emergence of S. Typhi strains which are resistant to antibiotic therapy highlights the importance of vaccination in managing typhoid fever. The virulence-associated (Vi) capsular polysaccharide is an effective vaccine against typhoid fever,but the role the capsule plays during pathogenesis remains incompletely understood. Here,we identify the human C-type lectin receptor DC-SIGN as the receptor for the Vi capsular polysaccharide. Binding of capsulated S. Typhi to DC-SIGN resulted in phagocytosis of the pathogen by macrophages and induction of an anti-inflammatory cytokine response. Thus,the interaction of the Vi capsular polysaccharide with human DC-SIGN contributes to the pathogenesis of typhoid fever and should be further investigated in the context of vaccine development. View Publication

BACKGROUND Chronic kidney disease patients are at increased risk of mortality with cardiovascular diseases and infections as the two leading causes of death for end-stage kidney disease treated with hemodialysis (HD). Mortality from bacterial infections in HD patients is estimated to be 100-1000 times higher than in the healthy population. METHODS We comprehensively characterized highly pure circulating neutrophils from HD and healthy donors. RESULTS Protein levels and transcriptome of HD patients' neutrophils indicated massive neutrophil degranulation with a dramatic reduction in reactive oxygen species (ROS) production during an oxidative burst and defective oxidative cellular signaling. Moreover,HD neutrophils exhibit severely impaired ability to generate extracellular NET formation (NETosis) in NADPH oxidase-dependent or independent pathways,reflecting their loss of capacity to kill extracellular bacteria. Ectopic hydrogen peroxidase (H2O2) or recombinant human SOD-1 (rSOD-1) partly restores and improves the extent of HD dysfunctional neutrophil NET formation. CONCLUSIONS Our report is one of the first singular examples of severe and chronic impairment of NET formation leading to substantial clinical susceptibility to bacteremia that most likely results from the metabolic and environmental milieu typical to HD patients and not by common human genetic deficiencies. In this manner,aberrant gene expression and differential exocytosis of distinct granule populations could reflect the chronic defect in neutrophil functionality and their diminished ability to induce NETosis. Therefore,our findings suggest that targeting NETosis in HD patients may reduce infections,minimize their severity,and decrease the mortality rate from infections in this patient population. View Publication

The role of NK cells against HIV-1 infections remains to be elucidated in vivo. While humanized mouse models potentially could be used to directly evaluate human NK cell responses during HIV-1 infection,improved functional development of human NK cells in these hosts is needed. Here,we report the humanized MISTRG-6-15 mouse model,in which NK cells were quick to expand and exhibit degranulation,cytotoxicity,and proinflammatory cytokine production in nonlymphoid organs upon HIV-1 infection but had reduced functionality in lymphoid organs. Although HIV-1 infection induced functional impairment of NK cells,antiretroviral therapy reinvigorated NK cells in response to HIV-1 rebound after analytic treatment interruption. Moreover,a broadly neutralizing antibody,PGT121,enhanced NK cell function in vivo,consistent with antibody-dependent cellular cytotoxicity. Monoclonal antibody depletion of NK cells resulted in higher viral loads in multiple nonlymphoid organs. Overall,our results in humanized MISTRG-6-15 mice demonstrated that NK cells provided direct anti-HIV-1 responses in vivo but were limited in their responses in lymphoid organs. View Publication

OBJECTIVE Systemic sclerosis (SSc) is a complex autoimmune disease with a strong genetic component. However,most of the genes associated with the disease are still unknown because associated variants affect mostly noncoding intergenic elements of the genome. We used functional genomics to translate the genetic findings into a better understanding of the disease. METHODS Promoter capture Hi-C and RNA-sequencing experiments were performed in CD4+ T cells and CD14+ monocytes from 10 SSc patients and 5 healthy controls to link SSc-associated variants with their target genes,followed by differential expression and differential interaction analyses between cell types. RESULTS We linked SSc-associated loci to 39 new potential target genes and confirmed 7 previously known SSc-associated genes. We highlight novel causal genes,such as CXCR5,as the most probable candidate gene for the DDX6 locus. Some previously known SSc-associated genes,such as IRF8,STAT4,and CD247,showed cell type-specific interactions. We also identified 15 potential drug targets already in use in other similar immune-mediated diseases that could be repurposed for SSc treatment. Furthermore,we observed that interactions were directly correlated with the expression of important genes implicated in cell type-specific pathways and found evidence that chromatin conformation is associated with genotype. CONCLUSION Our study revealed potential causal genes for SSc-associated loci,some of them acting in a cell type-specific manner,suggesting novel biologic mechanisms that might mediate SSc pathogenesis. View Publication

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. 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