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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

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

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

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

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