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BACKGROUND Ageing is a complex phenomenon that leads to decreased proliferative activity,loss of function of the cells,and cellular senescence. Senescence of the immune system exacerbates individual's immune response,both humoral and cellular but increases the frequency of infections. We hypothesized that physiological ageing of adaptive immune system occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at faster rate when compared to their respective donors since the small number of donor cells undergo immense proliferative stress restoring recipients hematopoiesis. We compared molecular characterizations of ageing between recipients and donors of allo-HCT: telomeric length and immunophenotypic changes in main lymphocyte subsets - CD4+,CD8+,CD19+,CD56+. RESULTS Median telomeric length (TL) of CD8+ lymphocytes was significantly longer in donors compared to recipients (on average 2,1 kb and 1,7 kb respectively,p??=??0,02). Similar trends were observed for CD4+ and CD19+ although the results did not reach statistical significance. We have also found trends in the immunophenotype between recipients and donors in the subpopulations of CD4+ (na{\{i}}ve and effector memory) CD8+ Eomes+ and B-lymphocytes (B1 and B2). Lower infection risk recipients had also a significantly greater percentage of NK cells (22 3%) than high-risk patients (9 3%) p??=??0 04. CONCLUSION Our data do not support the initial hypothesis of accelerated aging in the long term all-HCT recipients with the exception of the recipients lymphocytes (mainly CD8+) which present some molecular features characteristic for physiological ageing (telomeric shortening immunophenotype) when compared to their respective donors. However a history of lower infection numbers in HCT recipients seems to be associated with increased percentage of NK cells. The history of GVHD seems not to affect the rate of ageing. Therefore it is safe to conclude that the observed subtle differences between recipients' and donors' cells result mainly from the proliferative stress in the early period after allo-HCT and the difference between hosts' and recipients' microenvironments." View Publication

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) on multiple myeloma (MM) produces fast but not long-lasting responses. Reasons for treatment failure are poorly understood. CARs simultaneously targeting two antigens may represent an alternative. Here,we (1) designed and characterized novel A proliferation inducing ligand (APRIL) based dual-antigen targeting CARs,and (2) investigated mechanisms of resistance to CAR T cells with three different BCMA-binding moieties (APRIL,single-chain-variable-fragment,heavy-chain-only). METHODS Three new APRIL-CARs were designed and characterized. Human APRIL-CAR T cells were evaluated for their cytotoxic function in vitro and in vivo,for their polyfunctionality,immune synapse formation,memory,exhaustion phenotype and tonic signaling activity. To investigate resistance mechanisms,we analyzed BCMA levels and cellular localization and quantified CAR T cell-target cell interactions by live microscopy. Impact on pathway activation and tumor cell proliferation was assessed in vitro and in vivo. RESULTS APRIL-CAR T cells in a trimeric ligand binding conformation conferred fast but not sustained antitumor responses in vivo in mouse xenograft models. In vitro trimer-BB$\zeta$ CAR T cells were more polyfunctional and formed stronger immune synapses than monomer-BB$\zeta$ CAR T cells. After CAR T cell-myeloma cell contact,BCMA was rapidly downmodulated on target cells with all evaluated binding moieties. CAR T cells acquired BCMA by trogocytosis,and BCMA on MM cells was rapidly internalized. Since BCMA can be re-expressed during progression and persisting CAR T cells may not protect patients from relapse,we investigated whether non-functional CAR T cells play a role in tumor progression. While CAR T cell-MM cell interactions activated BCMA pathway,we did not find enhanced tumor growth in vitro or in vivo. CONCLUSION Antitumor responses with APRIL-CAR T cells were fast but not sustained. Rapid BCMA downmodulation occurred independently of whether an APRIL or antibody-based binding moiety was used. BCMA internalization mostly contributed to this effect,but trogocytosis by CAR T cells was also observed. Our study sheds light on the mechanisms underlying CAR T cell failure in MM when targeting BCMA and can inform the development of improved treatment strategies. View Publication

Immunotherapy has revolutionized cancer treatment,but preclinical models are required to understand immunotherapy resistance mechanisms underlying patient relapse. This protocol describes how to generate an acquired resistance humanized in vivo model to immunotherapies in patient-derived xenografts (PDX). We detail steps to inject human CD34+ cells into NSG mice,followed by generation of immunoresistant PDX in humanized mice. This approach recapitulates the human immune system,allowing investigators to generate preclinical resistance models to different immunotherapies for identifying the resistant phenotype. For complete details on the use and execution of this protocol,please refer to Mart{\'{i}}nez-Sabadell et al.,2022 and Arenas et al. (2021). View Publication

Rift Valley fever virus (RVFV) is a hemorrhagic fever virus with the potential for significant economic and public health impact. Vaccination with an attenuated strain,DelNSsRVFV,provides protection from an otherwise lethal RVFV challenge,but mechanistic determinants of protection are undefined. In this study,a murine model was used to assess the contributions of humoral and cellular immunity to DelNSsRVFV-mediated protection. Vaccinated mice depleted of T cells were protected against subsequent challenge,and passive transfer of immune serum from vaccinated animals to na{\{i}}ve animals was also protective demonstrating that T cells were dispensable in the presence of humoral immunity and that humoral immunity alone was sufficient. Animals depleted of B cells and then vaccinated were protected against challenge. Total splenocytes but not T cells alone B cells alone or B??+??T cells harvested from vaccinated animals and then transferred to na{\"{i}}ve animals were sufficient to confer protection suggesting that multiple cellular interactions were required for effective cellular immunity. Together these data indicate that humoral immunity is sufficient to confer vaccine-mediated protection and suggests that cellular immunity plays a role in protection that requires the interaction of various cellular components." View Publication

B cells orchestrate autoimmune responses in patients with systemic lupus erythematosus (SLE),but broad-based B cell-directed therapies show only modest efficacy while blunting humoral immune responses to vaccines and inducing immunosuppression. Development of more effective therapies targeting pathogenic clones is a currently unmet need. Here,we demonstrate enhanced activation of the ATR/Chk1 pathway of the DNA damage response (DDR) in B cells of patients with active SLE disease. Treatment of B cells with type I IFN,a key driver of immunity in SLE,induced expression of ATR via binding of interferon regulatory factor 1 to its gene promoter. Pharmacologic targeting of ATR in B cells,via a specific inhibitor (VE-822),attenuated their immunogenic profile,including proinflammatory cytokine secretion,plasmablast formation,and antibody production. Together,these findings identify the ATR-mediated DDR axis as the orchestrator of the type I IFN-mediated B cell responses in SLE and as a potential novel therapeutic target. View Publication

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