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Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here,we investigated the role of arginase 1 (ARG1) in V$\kappa$*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in EY-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of V$\kappa$*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However,arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether,these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors. View Publication

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs,focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations,gene expression,and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma,including severe type 2 inflammation,airway fibrosis,and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF-CD11c-CD11b+ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages,especially M2a and M2c. Furthermore,MSCs downregulated the excessive accumulation of Ly6c- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c- monocytes promoted the infiltration of MoM and Th2 inflammation,that of MSC-exposed Ly6c- monocytes did not. Ex vivo Ly6c- MoMs upregulated M2-related genes,which were reduced by MSC treatment. Molecules secreted by Ly6c- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts,which were also suppressed by MSC treatment. In conclusion,intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c- monocytes could be a therapeutic target for asthma management. View Publication

BACKGROUND Systemic sclerosis (SSc) is a multiple-organ disease characterized by vascular damage,autoimmunity,and tissue fibrosis. Organ injuries such as interstitial lung diseases (ILD),resulting from inflammatory and fibrosis processes,lead to poor prognosis. Although autoantibodies are detected in the serum of patients with SSc,the mechanisms by which immune cells are involved in tissue inflammation and fibrosis is not fully understood. Recent studies have revealed carcinoembryonic antigen related cell adhesion molecule (CEACAM)-positive monocytes are involved in murine bleomycin-induced lung fibrosis. We investigated CEACAM-positive monocytes in patients with SSc to clarify the role of monocytes in the pathogenesis of SSc. METHODS The proportion of of CEACAM-positive classical monocytes in healthy controls (HCs) and patients with rheumatoid arthritis (RA) and SSc was evaluated using flow cytometry. The correlation between the proportion of CEACAM-positive monocytes and clinical parameters was analyzed in patients with SSc. Gene expression microarrays were performed in CEACAM-positive and negative monocytes in patients with SSc. Infiltration of CEACAM-positive monocytes into scleroderma skin was evaluated by immunohistochemical staining. RESULTS The proportion of CEACAM-positive classical monocytes was increased in patients with early SSc within 2 years after diagnosis,which positively correlated with ESR,serum IgG,and serum KL-6 and negatively correlated with %forced vital capacity. The percentage of CEACAM-positive monocytes decreased after immunosuppressive therapy. CEACAM6-positive cells among classical monocytes were significantly increased in patients with SSc compared with HCs and patients with rheumatoid arthritis. SSc serum induced CEACAM6 expression on monocytes from HCs. Functionally,CEACAM-positive monocytes produced higher levels of TNF-$\alpha$ and IL-1$\beta$ compared to CEACAM-negative cells and showed activation of the NF-$\kappa$B pathway. Furthermore,CEACAM6-positive monocytes infiltrated the dermis of SSc. CONCLUSIONS CEACAM-positive monocytes showed inflammatory phenotypes and may be involved in the tissue inflammation and fibrosis in early SSc. CEACAM-positive monocytes may be one of biomarkers to detect patients with progressive ILD,requiring therapeutic intervention. View Publication

INTRODUCTION Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently,cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping,which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously. OBJECTIVE B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors. METHODS The immunomagnetic negative selection was used for the enrichment of B and T cells fractions,and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis,principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells. RESULTS Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as $\beta$-carotene. In addition,the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability,the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells. CONCLUSIONS This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system. 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

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