Cell Cytokine Production Research Articles

Abstract 15: Massively parallel base-editing screens to map variant effects on anti-tumor hallmarks of primary human T cells and improve cell-based cancer immunotherapies

Abstract Base editing enables precise generation of single nucleotide variants, but large-scale screening in primary human T cells has been limited by low editing efficiency. Here, we developed a high-throughput approach for highly efficient, massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale pooled screens editing 108 genes with central functions in T cells and whole-gene tiling mutagenesis of selected genes. All screens were functionally read out to capture the effect of variants on hallmarks of T cell anti-tumor immunity, including activation, proliferation, long-term persistence, and cytokine production. We discovered a broad landscape of gain-of-function and loss-of-function mutations and characterized their impact across multiple axes of T cell functions. Among others, we identified gain-of-function mutations in PIK3CD and its regulatory subunit PIK3R1, LCK, AKT1, and loss-of-function mutants of CTLA-4, driving increased T cell polyfunctionality. We validated several of these mutations by probing downstream signaling and functional outputs. To demonstrate the feasibility of using such screen results for improving cell-based cancer immunotherapies, we used human T cells with engineered T cell receptors recognizing specific epitopes in melanoma models. We base-edited these T cells with mutations identified in our screens and demonstrate that these mutations indeed significantly enhance cytotoxic cytokine production and tumor-lytic capacity with minimal off-target activity. Lastly, we also provide proof-of-feasibility for high-efficiency combinatorial base-editing in T cells. This study presents a framework for large-scale base editing screens with multi-dimensional functional readouts in cells that are typically difficult to molecularly engineer in a precise manner. This work also has important therapeutic implications: T cells are the substrate for clinically active cell-based therapies, such as adoptive transfer of tumor-infiltrating lymphocytes (TILs) or chimeric-receptor antigen T (CAR-T) cells. Our screen-informed base editing to achieve an improved T cell product against melanoma exemplifies the therapeutic power of generating synthetic protein variants that improve a wide range of existing and future T cell-based cancer immunotherapies. Citation Format: Zachary Hudson Walsh, Parin Shah, Neeha Kothapalli, Gergo Nikolenyi, Shivem Shah, Giuseppe Leuzzi, Neil Vasan, Mohammed AlQuarishi, Alberto Ciccia, Johannes Melms, Benjamin Izar. Massively parallel base-editing screens to map variant effects on anti-tumor hallmarks of primary human T cells and improve cell-based cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 15.

Abstract 1340: Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™

Abstract Natural Killer (NK) cells are highly responsive cytotoxic immune cells of the innate immune system with well characterized anti-tumor properties. Their ability to directly kill malignant cells and elicit an adaptive immune response makes them a promising candidate for a precision guided immunotherapy in oncology. Bicycle® peptides are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle® platform technology to discover and evaluate a new class of fully synthetic molecules termed NK tumor immune cell agonists (NK-TICA™). The NK-TICA™ consists of chemically coupled Bicycle® peptides that bind specifically to the key activating receptor, NKp46, and to tumor antigens, that results in highly potent, antigen-dependent receptor activation and NK cell function. We demonstrate potent, selective binding of our Bicycle® peptides to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner. In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle® NK-TICA™ as a multifunctional immune cell engager will promote the modulation and anti-tumor activity of peripheral and intra-tumoral NK cells to solid tumors. Citation Format: Fay Dufort, Tucker Ezell, Alexandra Rezvaya, Kathleen Ho, Christopher Leitheiser, Philip Brandish, Kevin McDonnell, Nicholas Keen. Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1340.

Abstract 4216: Indication agnostic evaluation of CAR T cell function using patient-derived immunocompetent tumoroid modelling platform

Abstract Introduction: Engineered T cells redirected towards tumor-specific cytotoxicity is a promising immunotherapeutic modality that has been well characterized for efficacy against B cell malignancies and evaluated in multiple tumor types. However, the development and optimization of CAR T cell therapies requires a rigorous preclinical model for the evaluation of CAR T cell function, tumor killing, cytokine production, and memory responses. Experimental Procedure: We are evaluating our model platform Custom Organoid Modelling Platform for Accurate and Speedy SolutionS (COMPASS 2) as a comprehensive platform for testing developmental and functional stages of CAR T cells including activation, memory, and exhaustion. We are performing qualitative and quantitative analyses of surface markers to recapitulate the complexity of human cancer. Additionally, since CAR T cells mediate crosstalk with other immune components in the TME, we also evaluate and monitor the presence of B cells, NK cells, macrophages, and endogenous T cells. Triple Negative Breast Cancer (TNBC); Immunophenotyping (IP); Immunofluorescence (IF); Fluorescence Activated Cell Sorting (FACS); T regulatory (Treg). Summary of Results: Our evaluation of COMPASS 2 for functional testing of CAR T cells demonstrates the feasibility of the platform in preclinical CAR T cell evaluation. We showed T cell infiltration and analyzed cytokine production that may alter tumor cells and TME. Crosstalk with other immune cells will be evaluated. We are currently evaluating the tumor volume changes using an EGFRvIII-targeted CART cell therapy on TNBC model systems, followed by apoptosis and EGFR analysis using FACS. Conclusion: Our COMPASS 2 model platform enables studies that require recapitulating hurdles to CAR therapy, including antigen escape, T cell dysfunction and trafficking; to reveal an essential role of the endogenous immune response mediating the antitumor effect following CAR therapy. Citation Format: Damieanus O. Ochola, Huckie Dell Mundo, Preethi Samuel, Natalia Korkorina, Bhuvanesh Dave. Indication agnostic evaluation of CAR T cell function using patient-derived immunocompetent tumoroid modelling platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4216.

Abstract 5302: CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors

Abstract INTRODUCTION: CD137 is a TNF receptor superfamily (TNFRSF9) costimulatory receptor expressed by T cells and NK cells. Clustering-mediated CD137 signaling enhances immune cell survival, proliferation, cytokine production and memory formation. CD40 (previously TNFRSF5) is a related superfamily member expressed primarily on B cells and antigen-presenting cells (APCs) which upon clustering is also a key modulator of T cell responses. Mesothelin (MSLN) is highly upregulated on tumor cells in ovarian and pancreatic cancer and select other solid tumour histologies. To avoid systemic toxicity, CB699 is designed to be immunologically active in the presence of any two of its three binding targets, thus enhancing both immune-immune and tumour-immune crosstalk. We present here the preclinical pharmacology of CB699. EXPERIMENTAL PROCEDURES: Binding of CB699 to CD40, CD137 or MSLN was measured by surface plasmon resonance and by flow cytometry. CD137 and/or CD40 agonism, immune cell activation and cytokine secretion were evaluated in vitro using reporter gene assays and primary cell coculture assays in the presence/absence of MSLN-expressing tumor cells. The ability of CB699 to enhance tumor cell killing was characterized in 3D spheroid cocultures. CB699 toxicology and toxicokinetics was assessed in a dose range-finding study in cynomolgus macaques. CB699 was also assessed in vivo with a human CD34+ cell engrafted mouse model. RESULTS: CB699 binds to primary and immortalized cells that express CD40, CD137 or MSLN, with low nanomolar affinity. CB699 also binds to cells from primary disaggregated human solid tumors. CB699 induces CD137 reporter cell activity in cocultures containing cells expressing either CD40 or MSLN. Additionally, CB699 induces CD40 reporter cell activity in cocultures containing cells expressing either CD137 or MSLN. CB699 induces IL-2 secretion and upregulation of CD86 on B cells in primary cocultures containing healthy human PBMCs and MSLN expressing cancer cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB699 and was coupled with immune cell expansion. CB699 showed significant pharmacology in vivo with immune cell engrafted mice. Finally, CB699 was well tolerated in a dose range finding non-human primate toxicology study. CONCLUSIONS: CB699 is a novel MSLN-binding CD137 and CD40 dual agonist that selectively enhances immune cell activity by enhancing both T cell and APC signaling axes. CB699 is positioned to enter clinical development. Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Holly Spencer, Minjung Song, Timothy Wong, Phillip D. Bartlett, Kieran Williams, Lukas Jasaitis, Philip Bland-Ward. CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5302.

Abstract 6357: Establishing a reverse translational platform to dissect mode of action and mechanisms of resistance to GUCY2C-CD3 T cell engager

Abstract PF-07062119 (GUCY2C x CD3), a bispecific T cell engager (TCE) targeting Guanylyl Cyclase C (GUCY2C), a GI tumor associated antigen, is currently being evaluated in a Phase 1 clinical trial in colorectal cancer (CRC) and other GI malignancies. Improved pre-clinical understanding of the correlates of PF-07062119 resistance mechanisms and PD in a human disease relevant context is critical to better inform drug combination and patient selection strategy. Due to limited translatability of mouse syngeneic tumor models, establishing a more human disease-relevant platform leveraging tumor patient material to better inform PF-07062119 clinical strategy is needed. Here, we will discuss our efforts to perform immune profiling of T cell and myeloid tumor infiltrates, and to evaluate tumor cell depletion and cytokine production of colorectal carcinoma (CRC) samples treated ex vivo with PF-07062119. This platform may be leveraged to 1) provide clinically relevant disease insights to the effect of TCEs across a range of tumor indications; 2) determine impact of different combinatorial partners on tumor cell killing to inform clinical decisions; 3) address questions related to the minimal efficacy of TCEs in the solid tumor space; and 4) provide potential solutions for future combination partners to overcome immunosuppression in the tumor microenvironment. Citation Format: Joana Borlido, Jade Lolarga, Rachael Whaley, Michael A. Damore, Cen Guo, Keith Abayasiriwardana, Xiaojun Xu, Maggie Liu, Gosia Nocula-Lugowska, Sam Stoner, Christopher Kraser, Ioanna Cheronis, Maria Delioukina, Lee Clark, Raj Bhadra, Murali Gururajan. Establishing a reverse translational platform to dissect mode of action and mechanisms of resistance to GUCY2C-CD3 T cell engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6357.

Abstract 481: Proteomimetic polymers limit tumor growth in multiple models via potent antigen-specific T cell activation

Abstract Background: Patient-specific cancer vaccines derived from tumor antigen have been explored as a promising therapeutic strategy, however, challenges delivering vaccine components in a coordinated fashion to elicit antitumor responses remain. To overcome these, we utilize a novel nanoplatform called the Protein-Like Polymer (PLP), which allows for sustained and targeted delivery of tumor antigens in conjunction with adjuvants. Methods: PLPs containing peptide antigens were synthesized via ring-opening metathesis polymerization (ROMP) and characterized. A library of compounds with different sidechain linkage chemistries, degrees of polymerization (DP), and inclusion/exclusion of Oligo-ethylene glycol (OEG) were made to determine design rules for immune activation. Cell uptake and functional assays using payload-specific T Cells were conducted. Immunization in three independent tumor models was done to show generalizability. Ability of PLPs to co-deliver adjuvants was tested by electrostatically coupling small molecule STING agonist, 2’3’ cGAMP. Results: Conjugating peptide antigens to the polymer via a cleavable disulfide linkage, which reduces intracellularly in APCs, resulted in increased endosomal localization, higher levels of induced T cell proliferation, cytokine production, and expression of activation markers in CTLs and APCs. Incorporating a diluent amount of OEG side chains reduced enzymatic degradation while increasing immunogenicity and uptake. Additionally, increasing the DP, and therefore the density of antigen side chains, further improved vaccine efficacy and resistance to proteolysis. Antigen-PLP conjugates enhanced dendritic cell activation and T-cell response only when paired with cells from their cognate system, with no activity in immune cells not expressing receptors for the payload demonstrating antigen-specificity. Mice bearing established B16F10, MC38, or TC-1 tumors treated with PLPs containing gp100, adpgk, or E7 respectively all showed increased survival times, reduced tumor burden with corresponding changes in immune cell profiles, and immunological memory upon rechallenge. Impressively, mice treated with STING-PLP complexes had significantly smaller tumors vs control at day 14 (0.038g vs 0.76g; p <0.0001) and allowed for subcutaneous administration of 2’3’ cGAMP, which traditionally requires intratumoral injection. Studies on the effects of vaccinating with pools of neoantigens multiplexed onto one PLP are ongoing. Conclusion: This work validates the ability of PLPs to overcome major limitations in cancer vaccine development. The modularity of the platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling, highlighting its potential to revolutionize cancer vaccinology. Citation Format: Max Wang, Miran Choi, Bin Zhang, Nathan Gianneschi. Proteomimetic polymers limit tumor growth in multiple models via potent antigen-specific T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 481.

High CD62L expression predicts the generation of chimeric antigen receptor T cells with potent effector functions.

Efficient generation of chimeric antigen receptor (CAR) T cells is highly influenced by the quality of apheresed T cells. Healthy donor-derived T cells usually proliferate better than patients-derived T cells and are precious resources to generate off-the-shelf CAR-T cells. However, relatively little is known about the determinants that affect the efficient generation of CAR-T cells from healthy donor-derived peripheral blood mononuclear cells (PBMC) compared with those from the patients' own PBMC. We here examined the efficiency of CAR-T cell generation from multiple healthy donor samples and analyzed its association with the phenotypic features of the starting peripheral blood T cells. We found that CD62L expression levels within CD8+ T cells were significantly correlated with CAR-T cell expansion. Moreover, high CD62L expression within naïve T cells was associated with the efficient expansion of T cells with a stem cell-like memory phenotype, an indicator of high-quality infusion products. Intriguingly, genetic disruption of CD62L significantly impaired CAR-T cell proliferation and cytokine production upon antigen stimulation. Conversely, ectopic expression of a shedding-resistant CD62L mutant augmented CAR-T cell effector functions compared to unmodified CAR-T cells, resulting in improved antitumor activity in vivo. Collectively, we identified surface expression of CD62L as a concise indicator of potent T cell proliferation. CD62L expression is also associated with functional properties of CAR-T cells. These findings are potentially applicable to selecting optimal donors to massively generate CAR-T cell products.

Abstract 6333: A high-parameter mass cytometry panel for the functional characterization of CAR T cells

Abstract High-parameter flow cytometry provides a powerful tool for in-depth analysis of CAR T cell therapy, from characterizing CAR T cell populations to understanding their interactions within the tumor microenvironment. This information is crucial for advancing CAR T cell therapies and improving their effectiveness in treating cancer. To address the need for deep immunoprofiling at all stages of CAR T cell therapy, we created a robust and comprehensive mass cytometry panel of antibodies against 44 cell surface and intracellular markers. This assay allows for comprehensive characterization of CAR T cell features including surface marker expression, activation state, cytokine production, and differentiation status. These data will provide researchers with a better understanding of the phenotype of the CAR T cells used in therapy and can guide optimization efforts to enhance expansion, persistence, and self-renewal. For example, newer-generation CAR T cells (T cells redirected for antigen-unrestricted cytokine-initiated killing, or TRUCKs) that are engineered to produce cytokines such as IL-2 to enhance their own survival can be evaluated in vitro to select CAR constructs with the most potent anti-tumor activity. The lyophilized and validated 30-marker Maxpar® Direct™ Immune Profiling Assay™ enables comprehensive characterization of immune cell populations in both whole blood and PBMC. The 7-marker Maxpar Direct T Cell Expansion Panel 3 (OX40, TIGIT, CD69, PD-1, Tim-3, ICOS, and 4-1BB) and the Maxpar Direct Basic Activation Expansion Panel (CD107a, IL-2, TNFα, IFNγ, perforin, and granzyme B) are add-on modules that can further characterize CAR T cell exhaustion, degranulation, cytokine production, and cytotoxicity against target cells. We also compared CAR T detection options for sensitivity and specificity using a CD19 CAR-transduced cell line spiked into healthy donor PBMC. Indirect staining with Miltenyi Biotec biotinylated CD19 CAR Detection Reagent followed by anti-biotin- or streptavidin-conjugated metal tags was compared against a directly conjugated or biotinylated anti-G4S linker antibody. We included sample multiplexing using 6 metal-tagged CD45 antibodies to address the need to minimize batch effects, crucial to multi-site and longitudinal studies. Samples were acquired on a CyTOF® XT™ instrument, and preliminary data analysis was performed using Maxpar Pathsetter™ for automated enumeration of immune cells. By combining the lyophilized single-tube Maxpar Immune Profiling Assay and Expansion Panels with specific detection of CAR T cells, this assay enables researchers to analyze multiple key parameters simultaneously at the single-cell level at all stages of CAR T therapy development. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Deeqa Mahamed, Geneve Awong, Thiru Selvanatham. A high-parameter mass cytometry panel for the functional characterization of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6333.

Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury

BackgroundAcute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton’s jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI.MethodsA cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-β)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI.ResultsThe CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-β-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function.ConclusionThis study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

Granular Insights: Neutrophil Predominance and Elastase Release in Severe Asthma Exacerbations in a Pediatric Cohort.

The chronic inflammatory component of asthma is propagated by granulocytes, including neutrophils and eosinophils, in the peripheral circulation and airway. Previous studies have suggested that these cells have an altered expression of adhesion-related molecules and a propensity for the release of granule contents that may contribute to tissue damage and enhance inflammatory complications in patients with status asthmaticus. The goal of this prospective cohort study at a tertiary care pediatric hospital with a large population of asthma patients was to assess the role of granulocyte-based inflammation in the development of asthma exacerbation. Subjects were enrolled from two patient populations: those with mild-to-moderate asthma exacerbations seen in the emergency department and those with severe asthma admitted to the intensive care unit (PICU). Clinical data were collected, and blood was drawn. Granulocytes were immediately purified, and the phenotype was assessed, including the expression of cell surface markers, elastase release, and cytokine production. Severe asthmatics admitted to the PICU displayed a significantly higher total neutrophil count when compared with healthy donors. Moreover, little to no eosinophils were found in granulocyte preparations from severe asthmatics. Circulating neutrophils from severe asthmatics admitted to the PICU displayed significantly increased elastase release ex vivo when compared with the PMN from healthy donors. These data suggest that the neutrophil-based activation and release of inflammatory products displayed by severe asthmatics may contribute to the propagation of asthma exacerbations.

CD44 expression on murine hepatic stellate cells promotes the induction of monocytic and polymorphonuclear myeloid-derived suppressor cells.

In chronic inflammation, regulatory immune cells, such as regulatory T cells and myeloid-derived suppressor cells, can develop. Local signals in the inflamed tissue, such as cytokines and eicosanoids, but also contact-dependent signals, can promote myeloid-derived suppressor cell development. In the liver, hepatic stellate cells may provide such signals via the expression of CD44. Myeloid-derived suppressor cells generated in the presence of hepatic stellate cells and anti-CD44 antibodies were functionally and phenotypically analyzed. We found that both monocytic and polymorphonuclear myeloid-derived suppressor cells generated in the presence of αCD44 antibodies were less suppressive toward T cells as measured by T-cell proliferation and cytokine production. Moreover, both monocytic and polymorphonuclear myeloid-derived suppressor cells were phenotypically altered. Monocytic myeloid-derived suppressor cells mainly changed their expression of CD80 and CD39, and polymorphonuclear myeloid-derived suppressor cells showed altered expression of CD80/86, PD-L1, and CCR2. Moreover, both polymorphonuclear and monocytic myeloid-derived suppressor cells lost expression of Nos2 messenger RNA, whereas monocytic myeloid-derived suppressor cells showed reduced expression of TGFb messenger RNA and polymorphonuclear myeloid-derived suppressor cells reduced expression of Il10 messenger RNA. In summary, the presence of CD44 in hepatic stellate cells promotes the induction of both monocytic and polymorphonuclear myeloid-derived suppressor cells, although the mechanisms by which these myeloid-derived suppressor cells may increase suppressive function due to interaction with CD44 are only partially overlapping.

Role of differentiated embryo-chondrocyte expressed gene 2 in immunity.

Differentiated embryo-chondrocyte expressed gene 2 (DEC2) is a member of the basic helix-loop-helix (bHLH) subfamily of transcription factors. DEC2 is implicated in tumor immunotherapy, immune system function regulation, and autoimmune diseases. DEC2 enhances Th2 cell differentiation by regulating the IL-2 and IL-4 signaling pathways and mediates the growth of B-1a cells, thereby promoting the occurrence and development of inflammatory responses. In this study, we review the reported roles of DEC2, including the regulation of immune cell differentiation and cytokine production in various cells in humans, and discuss its potential in treating autoimmune diseases and tumors.

Cytotoxicity and anti-inflammatory properties of a GLP-1 receptor agonist in a model of human peripheral blood mononuclear cell culture

<p><strong>Introduction. </strong>GLP-1R agonists (GLP-1RAs) are a type of anti-hyperglycemic medications used for the management of type 2 diabetes mellitus (T2DM). In addition to glucose-lowering effects, GLP-1R agonists (GLP-1RAs) also provide further advantages by promoting weight loss and controlling blood pressure. Furthermore, GLP-1RAs have been reported for their therapeutic benefits in neurological, cardiovascular, endocrine, and metabolic diseases. Emerging evidence from many clinical and experimental studies, as well as in vitro researches, suggests that GLP-1RA agonists may reduce inflamma tion and modify the immunological response, but the underlying molecu lar mechanisms remain unclear. In this research, we examined the effect of GLP-1RA on the cytotoxicity, proliferative activity, and cytokine production of human peripheral blood mononuclear cells (PBMCs) in vitro.</p> <p><strong> Methods. </strong>The peripheral blood mononuclear cells (PBMCs) cultures from healthy volunteers were used. GLP-1RA (DMB), in different concentrations, was added to the cell cultures. A flow cytometry assay was used to assess cell viability, while MTT and CFSE dye dilution assays were used to quantify proliferative activity. Cytokine levels were measured using a sandwich en zyme-linked immunosorbent assay (ELISA).</p> <p><strong>Results.</strong> The cytotoxicity data demonstrated that only the highest DMB concentration (1000 nM) decreased the metabolic activity (viability) of PBMCs (p<0.05) in comparison to untreated cultures, while concentra tions of 200 nM (p<0.05) and 1000 nM (p<0.001) of GLP-1RA increased the proportion of late apoptotic and necrotic cells (p<0.001). The prolif eration of PBMCs was significantly decreased in the presence of GLP-1RA at a 100 nM concentration when compared to the control (p<0.05). The same concentration of GLP-1RA significantly reduced the production of IL-6, IL-1β, and TNF-α.</p> <p><strong>Conclusion.</strong> Our results suggested that GLP-1RA (DMB), at a non-toxic concentration of 100 nM, inhibited the proliferation and production of pro-inflammatory cytokines by human PBMCs in vitro. These findings open the perspective to study the immune response in detail in future experiments.</p>

Alteration in gut microbiota is associated with immune imbalance in Graves' disease.

Graves' disease (GD), characterized by immune aberration, is associated with gut dysbiosis. Despite the growing interest, substantial evidence detailing the precise impact of gut microbiota on GD's autoimmune processes remains exceedingly rare. This study was designed to investigate the influence of gut microbiota on immune dysregulation in GD. It encompassed 52 GD patients and 45 healthy controls (HCs), employing flow cytometry and enzyme-linked immunosorbent assay to examine lymphocyte and cytokine profiles, alongside lipopolysaccharide (LPS) levels. Gut microbiota profiles and metabolic features were assessed using 16S rRNA gene sequencing and targeted metabolomics. Our observations revealed a disturbed B-cell distribution and elevated LPS and pro-inflammatory cytokines in GD patients compared to HCs. Significant differences in gut microbiota composition and a marked deficit in short-chain fatty acid (SCFA)-producing bacteria, including ASV263(Bacteroides), ASV1451(Dialister), and ASV503(Coprococcus), were observed in GD patients. These specific bacteria and SCFAs showed correlations with thyroid autoantibodies, B-cell subsets, and cytokine levels. In vitro studies further showed that LPS notably caused B-cell subsets imbalance, reducing conventional memory B cells while increasing naïve B cells. Additionally, acetate combined with propionate and butyrate showcased immunoregulatory functions, diminishing cytokine production in LPS-stimulated cells. Overall, our results highlight the role of gut dysbiosis in contributing to immune dysregulation in GD by affecting lymphocyte status and cytokine production.

Exploring the significance of microbiota metabolites in rheumatoid arthritis: uncovering their contribution from disease development to biomarker potential.

Rheumatoid arthritis (RA) is a multifaceted autoimmune disorder characterized by chronic inflammation and joint destruction. Recent research has elucidated the intricate interplay between gut microbiota and RA pathogenesis, underscoring the role of microbiota-derived metabolites as pivotal contributors to disease development and progression. The human gut microbiota, comprising a vast array of microorganisms and their metabolic byproducts, plays a crucial role in maintaining immune homeostasis. Dysbiosis of this microbial community has been linked to numerous autoimmune disorders, including RA. Microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), tryptophan derivatives, Trimethylamine-N-oxide (TMAO), bile acids, peptidoglycan, and lipopolysaccharide (LPS), exhibit immunomodulatory properties that can either exacerbate or ameliorate inflammation in RA. Mechanistically, these metabolites influence immune cell differentiation, cytokine production, and gut barrier integrity, collectively shaping the autoimmune milieu. This review highlights recent advances in understanding the intricate crosstalk between microbiota metabolites and RA pathogenesis and also discusses the potential of specific metabolites to trigger or suppress autoimmunity, shedding light on their molecular interactions with immune cells and signaling pathways. Additionally, this review explores the translational aspects of microbiota metabolites as diagnostic and prognostic tools in RA. Furthermore, the challenges and prospects of translating these findings into clinical practice are critically examined.

Pulmonary Effects of Traumatic Brain Injury in Mice: A Gene Set Enrichment Analysis.

Acute lung injury occurs in 20-25% of cases following traumatic brain injury (TBI). We investigated changes in lung transcriptome expression post-TBI using animal models and bioinformatics. Employing unilateral controlled cortical impact for TBI, we conducted microarray analysis after lung acquisition, followed by gene set enrichment analysis of differentially expressed genes. Our findings indicate significant upregulation of inflammation-related genes and downregulation of nervous system genes. There was enhanced infiltration of adaptive immune cells, evidenced by positive enrichment in Lung-Th1, CD4, and CD8 T cells. Analysis using the Tabula Sapiens database revealed enrichment in lung-adventitial cells, pericytes, myofibroblasts, and fibroblasts, indicating potential effects on lung vasculature and fibrosis. Gene set enrichment analysis linked TBI to lung diseases, notably idiopathic pulmonary hypertension. A Venn diagram overlap analysis identified a common set of 20 genes, with FOSL2 showing the most significant fold change. Additionally, we observed a significant increase in ADRA1A→IL6 production post-TBI using the L1000 library. Our study highlights the impact of brain trauma on lung injury, revealing crucial gene expression changes related to immune cell infiltration, cytokine production, and potential alterations in lung vasculature and fibrosis, along with a specific spectrum of disease influence.

Restriction of Glycolysis Increases Serial Killing Capacity of Natural Killer Cells.

Tumor cells rely heavily on glycolysis to meet their high metabolic demands. While this results in nutrient deprivation within the tumor microenvironment and has negative effects on infiltrating immune cells such as natural killer (NK) cells, it also creates a potential target for cancer therapies. Here we use Glupin, an inhibitor of glucose transporters, to study the effect of limited glucose uptake on NK cells and their anti-tumor functions. Glupin treatment effectively inhibited glucose uptake and restricted glycolysis in NK cells. However, acute treatment had no negative effect on NK cell cytotoxicity or cytokine production. Long-term restriction of glucose uptake via Glupin treatment only delayed NK cell proliferation, as they could switch to glutaminolysis as an alternative energy source. While IFN-γ production was partially impaired, long-term Glupin treatment had no negative effect on degranulation. Interestingly, the serial killing activity of NK cells was even slightly enhanced, possibly due to changes in NAD metabolism. This demonstrates that NK cell cytotoxicity is remarkably robust and insensitive to metabolic disturbances, which makes cellular metabolism an attractive target for immune-mediated tumor therapies.

A novel T cell-redirecting anti-GPRC5D × CD3 bispecific antibody with potent antitumor activity in multiple myeloma preclinical models

G-protein-coupled receptor class 5 member D (GPRC5D) is detected in malignant plasma cells in approximately 90% of patients diagnosed with multiple myeloma (MM). Here, we constructed BsAb5003, a novel humanized bispecific monoclonal antibody targeting CD3 and GPRC5D, and evaluated its therapeutic impact on MM. BsAb5003 induced specific cytotoxicity of GPRC5D-positive MM cells with concomitant T cell activation and cytokine release. The efficacy of BsAb5003 was associated with GPRC5D expression levels in MM cell lines. Flow cytometry analysis of bone marrow mononuclear cells (BMMNCs) from 49 MM patients revealed that GPRC5D was expressed in a wide population of MM patients, including heavily treated and high-risk patients. In ex vivo assays using BMMNCs, BsAb5003 induced potent efficacy against CD138 + MM cells in both newly diagnosed and relapsed/refractory patient samples in a GPRC5D expression-dependent manner. BsAb5003 significantly enhanced T cell activation and cytokine production in combination with immunomodulatory drugs (IMiDs) against MM cell lines. BsAb5003 also demonstrated significant inhibition of in vivo tumor growth by recruiting T cells. Taken together, these results suggest that T cell-redirecting bispecific antibody targeting GPRC5D as monotherapy and combination therapy with IMiDs could be a highly potent and effective treatment approach for a wide population of MM patients.

Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

BackgroundPrevious studies have revealed dexmedetomidine have potential protective effects on vital organs by inhibiting the release of inflammatory cytokines. To investigate the effects of dexmedetomidine on sepsis, especially in the initial inflammatory stage of sepsis. RAW264.7 cells were used as the cell model in this study to elucidate the underlying mechanisms. MethodsIn this study, we conducted several assays to investigate the mechanisms of dexmedetomidine and HOTAIR in sepsis. Cell viability was assessed using the CCK-8 kit, while inflammation responses were measured using ELISA for IL-1β, IL-6, and TNF-α. Additionally, we employed qPCR, MeRIP, and RIP to further explore the underlying mechanisms. ResultsOur findings indicate that dexmedetomidine treatment enhanced cell viability and reduced the production of inflammatory cytokines in LPS-treated RAW264.7 cells. Furthermore, we observed that the expression of HOTAIR was increased in LPS-treated RAW264.7 cells, which was then decreased upon dexmedetomidine pre-treatment. Further investigation demonstrated that HOTAIR could counteract the beneficial effects of dexmedetomidine on cell viability and cytokine production. Interestingly, we discovered that YTHDF1 targeted HOTAIR and was upregulated in LPS-treated RAW264.7 cells, but reduced in dexmedetomidine treatment. We also found that YTHDF1 increased HOTAIR and HOTAIR m6A levels. ConclusionsCollectively, our results suggest that dexmedetomidine downregulates HOTAIR and YTHDF1 expression, which in turn inhibits the biological behavior of LPS-treated RAW264.7 cells. This finding has potential implications for the prevention and treatment of sepsis-induced kidney injury.

Microenvironment with NIR-Controlled ROS and Mechanical Tensions for Manipulating Cell Activities in Wound Healing.

The extracellular matrix (ECM) orchestrates cell behavior and tissue regeneration by modulating biochemical and mechanical signals. Manipulating cell-material interactions is crucial for leveraging biomaterials to regulate cell functions. Yet, integrating multiple cues in a single material remains a challenge. Here, near-infrared (NIR)-controlled multifunctional hydrogel platforms, named PIC/CM@NPs, are introduced to dictate fibroblast behavior during wound healing by tuning the matrix oxidative stress and mechanical tensions. PIC/CM@NPs are prepared through cell adhesion-medicated assembly of collagen-like polyisocyanide (PIC) polymers and cell-membrane-coated conjugated polymer nanoparticles (CM@NPs), which closely mimic the fibrous structure and nonlinear mechanics of ECM. Upon NIR stimulation, PIC/CM@NPs composites enhance fibroblast cell proliferation, migration, cytokine production, and myofibroblast activation, crucial for wound closure. Moreover, they exhibit effective and toxin removal antibacterial properties, reducing inflammation. This multifunctional approach accelerates healing by 95%, highlighting the importance of integrating biochemical and biophysical cues in the biomaterial design for advanced tissue regeneration.