Robust Proliferation Research Articles

Endothelial TRIM35-Regulated MMP10 Release Exacerbates Calcification of Vascular Grafts.

Vascular calcification is a highly regulated process in cardiovascular disease (CVD) and is strongly correlated with morbidity and mortality, especially in the adverse stage of vascular remodeling after coronary artery bypass graft surgery (CABG). However, the pathogenesis of vascular graft calcification, particularly the role of endothelial-smooth muscle cell interaction, is still unclear. To test how ECs interact with SMCs in artery grafts, single-cell analysis of wild-type mice is first performed using an arterial isograft mouse model and found robust cytokine-mediated signaling pathway activation and SMC proliferation, together with upregulated endothelial tripartite motif 35 (TRIM35) expression. Unexpectedly, severe SMC calcification in artery grafts is found in TRIM35 conditional endothelial knockout (cKO) mice. Calcified medium (comprising calcium chloride and beta-glycerophosphate)-induced calcium deposition in vitro is also found in SMCs cocultured with TRIM35 knockout endothelium. This extraordinary phenomenon is further confirmed to be induced by increased MMP10 secretion. Mechanistically, endothelial TRIM35 inhibits MMP10 expression and secretion by promoting K63-linked ubiquitination of RelB and maintaining its nuclear localization, consequently inhibiting nuclear transcription of MMP10 through the noncanonical NF-κB signaling pathway. Targeting MMP10 in situ in arterial isografts can effectively alleviate vascular calcification caused by conditional endothelial TRIM35 knockout. These findings demonstrated that TRIM35 inhibited vascular calcification during arterial isograft remodeling, a process that is driven by the aberrant secretion of endothelial MMP10. Targeting MMP10 pathway may be a potential therapeutic strategy for vascular calcification in vessel grafts.

GITRL enhances cytotoxicity and persistence of CAR-T cells in cancer therapy.

CAR T-cell therapy has achieved remarkable clinical success in treating hematological malignancies. However, its clinical efficacy in solid tumors is less satisfactory, partially due to poor in vivo expansion and limited persistence of CAR-T cells. Here, we demonstrated that the overexpression of glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL) enhances the anti-tumor activity of CAR-T cells. Compared to PSMA-BB-Z CAR-T, PSMA-BB-Z-GITRL CAR-T cells have much more IFN-γ, TNF-α, and IL-9 secretion, a higher proportion of central memory T (TCM) cells and Th9 cells, less expression of exhaustion markers, and robust proliferation capacity. Consequently, PSMA-BB-Z-GITRL CAR-T cells exhibited more potent anti-tumor activity against established solid tumors in vivo than PSMA-BB-Z CAR-T cells. The results of in vivo persistence experiment also indicated that PSMA-BB-Z-GITRL CAR-T cells exhibited much more retention in mouse blood, spleen, and tumor tissue than PSMA-BB-Z CAR-T cells 15 days after CAR-T cell therapy. In addition, PSMA-BB-Z-GITRL CAR-T cells produce higher levels of IFN-γ, TNF-α and IL-9 in mouse blood, exhibiting a higher proportion of TCM cells and a lower proportion of Treg cells compared to PSMA-BB-Z CAR-T cells. Our results demonstrate that the overexpression of GITRL has important implications for improving CAR-T cell-based human solid tumor immunotherapy.

IL-21 shapes the B cell response in a context-dependent manner.

The T-cell-derived cytokine IL-21 is crucial for germinal center (GC) responses, but its precise role in B cell function has remained elusive. Using IL-21 receptor (Il21r) conditional knockout mice and exvivo culture systems, we demonstrate that IL-21 has dual effects on B cells. While IL-21 induced apoptosis in a STAT3-dependent manner in naive B cells, it promoted the robust proliferation of pre-activated B cells, particularly IgG1+ B cells. Invivo, B-cell-specific Il21r deletion impaired IgG1 responses post-immunization and disrupted progression from pre-GC to GC states. Although Il21r deficiency did not affect the proportion of IgG1+ cells among GC B cells, it greatly diminished the proportion of IgG1+ cells among the plasmablast/plasma cell population. Collectively, our findings suggest that IL-21 serves as a critical regulator of B cell fates, influencing B cell apoptosis and proliferation in a context-dependent manner.

Intrafibrillar calcium carbonate mineralization of electrospinning polyvinyl alcohol/collagen films with improved mechanical and bioactive properties.

Collagen films play an essential role in guided bone-regeneration (GBR) techniques, which create space, promote cell adhesion, and induce osteogenic differentiation. It is therefore crucial to design appropriate GBR films to facilitate bone regeneration. However, current electrospun collagen scaffolds used as bioactive materials have limited clinical applications due to their poor mechanical properties. In this study, polyvinyl alcohol (PVA)/collagen (Col) films were electrospun by mixing PVA and type I collagen solution. For the first time, the intrafibrillar mineralization of aragonite nanocrystals within the PVA/Col fibrils was achieved, resulting in the formation of a hierarchical, bioactive film. The PVA/Col-CaCO3 film exhibited good mechanical properties, with hardness and Young's modulus values of 211.6 ± 0.1 MPa and 5.6 ± 1.7 GPa, respectively. Furthermore, bone marrow mesenchymal stem cells (BMSCs) inoculated onto the PVA/Col-CaCO3 film demonstrated robust adhesion and proliferation. The mineralized fibrils effectively stimulated the growth of BMSCs while suppressing cell apoptosis. Besides, the PVA/Col-CaCO3 film significantly induced the osteogenic differentiation of BMSCs, revealing its potential biomedical applications in hard tissue engineering.

Foxa1 disruption enhances human cell integration in human-mouse interspecies chimeras.

Blastocyst complementation can potentially generate a rodent model with humanized nasopharyngeal epithelium (NE) that supports sustained Epstein-Barr virus (EBV) infection, enabling comprehensive studies of EBV biology in nasopharyngeal carcinoma. However, during this process, the specific gene knockouts required to establish a developmental niche for NE remain unclear. We performed bioinformatics analyses and generated Foxa1 mutant mice to confirm that Foxa1 disruption could potentially create a developmental niche for NE. Subsequently, MYD88-inactivated human pluripotent stem cells (hPSCs) were constructed and complemented with Foxa1-deficient mouse blastocysts, withNosip-deficient mouse blastocysts as a control. The chimerism of human cells in mouse embryos was evaluated from E8.5 to E12.5 using genomic DNA PCR and immunohistochemistry. Our bioinformatics analysis indicated that the expression patterns of Foxa1 in E8.5 to E16.5 mouse embryos underscore its critical role in NE development. The generated mice with Foxa1 disordered region mutations displayed morphological abnormality in NE, suggesting Foxa1-knockouts could potentially establish a developmental niche for NE. In chimeric assays, human cells integrated into 80.00% of Foxa1-deficient embryos, compared with the 4.17% in controls. Immunohistochemistry results revealed robust proliferation of human cells in Foxa1-deficient mouse embryos. However, chimeras from Foxa1-deficient mouse embryos did not survive beyond E10.5, hindering the evaluation of human cell integration in mouse NE. Foxa1 disruption in mouse embryos significantly enhances the integration of human cells in human-mouse interspecies chimeras, thereby facilitating the generation of endoderm-derived organs through blastocyst complementation. Overcoming chimeras' embryonic lethality is crucial for successfully generating humanized NE in Foxa1-deficient mouse embryos.

Dental Resin for Periodontal and Tooth Root Regeneration via Metformin to Enhance Osteogenic and Cementogenic Differentiation of Human Periodontal Ligament Stem Cells

ObjectivesThis study developed a novel dental resin incorporating metformin to repair root caries and periodontitis defects. The objectives were to: (1) Develop a novel dental resin with metformin release to fulfill the clinical requirements for mechanical properties; and (2) investigate the metformin release pattern and the effects on osteogenic and cementogenic differentiation of human periodontal ligament stem cells (hPDLSCs). MethodsResin specimens with different concentrations of metformin were fabricated. Metformin release was measured using high-performance liquid chromatography. Cellular growth and proliferation on resin were assessed. Alizarin red S staining and an alkaline phosphatase (ALP) activity were determined. Quantitative real-time reverse transcription PCR was employed to determine osteogenic and cementogenic differentiation. ResultsThe resin with 7.5% metformin had mechanical properties comparable to those of a commercial control, thus satisfying the clinical requirements (mean ± SD; n = 10). hPDLSCs showed robust growth and proliferation on resin. At 14 days, the number of hPDLSCs increased by four-fold (p > 0.1). ALP activity in the metformin group was 1.8 times higher than that of control (p < 0.05). At 21 days, the metformin group showed a significant increase in mineral synthesis. Metformin group had cementogenic and osteogenic differentiation values that were 2.5 and 2.3 times higher, respectively, than control without metformin (p < 0.05). SignificanceA metformin-resin was formulated that greatly promoted both cementogenic and osteogenic differentiation for the first time. This resin was mechanically strong and supported hPDLSC adhesion, growth and proliferation. This resin substantially enhanced hPDLSC osteogenic and cementogenic differentiation, yielding mineral synthesis of hPDLSCs more than 2-fold that of control without metformin. This new metformin-resin shows promise for the restoration of tooth root caries and the regeneration of periodontal tissues including alveolar bone and cementum.

Glucose limitation protects cancer cells from apoptosis induced by pyrimidine restriction and replication inhibition.

Cancer cells often experience nutrient-limiting conditions because of their robust proliferation and inadequate tumour vasculature, which results in metabolic adaptation to sustain proliferation. Most cancer cells rapidly consume glucose, which is severely reduced in the nutrient-scarce tumour microenvironment. In CRISPR-based genetic screens to identify metabolic pathways influenced by glucose restriction, we find that tumour-relevant glucose concentrations (low glucose) protect cancer cells from inhibition of de novo pyrimidine biosynthesis, a pathway that is frequently targeted by chemotherapy. We identify two mechanisms to explain this result, which is observed broadly across cancer types. First, low glucose limits uridine-5-diphosphate-glucose synthesis, preserving pyrimidine nucleotide availability and thereby prolonging the time to replication fork stalling. Second, low glucose directly modulates apoptosis downstream of replication fork stalling by suppressing BAK activation and subsequent cytochrome c release, key events that activate caspase-9-dependent mitochondrial apoptosis. These results indicate that the low glucose levels frequently observed in tumours may limit the efficacy of specific chemotherapeutic agents, highlighting the importance of considering the effects of the tumour nutrient environment on cancer therapy.

Potassium tantalum niobate: Structural insights and biocompatibility for biomedical applications

AbstractThis study explores the biomedical applications of electrically active potassium tantalum niobate (KTN) ceramics (KTa1 − xNbxO3; where x = 0.0, 0.1, 0.3, 0.5, and 0.7), focusing on their dielectric and biological properties for bioelectrets and electroactive scaffolds. Varying Nb concentrations induced a structural shift from cubic to orthorhombic phases, with increased Nb inhibiting grain growth and maintaining stoichiometry. Dielectric analysis across temperatures and frequencies revealed two phase transitions (orthorhombic to tetragonal at 433–453 K and tetragonal to cubic at 653–703 K). Nyquist plots indicated reduced bulk resistance with increasing temperature. The AC conductivity (10−4 S/cm) suggests potential enhancement in bone regeneration scaffolds. Bioactivity assessments showed negative surface charges promoting dense apatite layer formation, while cytocompatibility studies demonstrated robust cell proliferation (&gt;100% by Day 3 for PSVK‐1 and Wi‐38 cell lines). This comprehensive investigation underscores KTN ceramics' promise for biomedical applications in enhancing electrical properties and fostering favorable biological interactions.

Enhanced therapeutic potential of a self-healing hyaluronic acid hydrogel for early intervention in osteoarthritis

Osteoarthritis (OA) is characterized by symptoms such as abnormal lubrication function of synovial fluid and heightened friction on the cartilage surface in its early stages, prior to evident cartilage damage. Current early intervention strategies employing lubricated hydrogels to shield cartilage from friction often overlook the significance of hydrogel-cartilage adhesion and enhancement of the cartilage extracellular matrix (ECM). Herein, we constructed a hydrogel based on dihydrazide-modified hyaluronic acid (HA) (AHA) and catechol-conjugated aldehyde-modified HA (CHA), which not only adheres to the cartilage surface as an effective lubricant but also improves the extracellular environment of chondrocytes in OA. Material characterization experiments on AHA/CHA hydrogels with varying concentrations validated their exceptional self-healing capabilities, superior injectability and viscoelasticity, sustained adhesion strength to cartilage, and a low friction coefficient. Chondrocytes exhibited robust adhesion and proliferation on the AHA/CHA hydrogel surface, with the upregulation of cartilage matrix protein expression. Intra-articular injection of AHA/CHA hydrogels was performed following destabilization of the medial meniscus (DMM) surgery in mice to assess its protective effect on cartilage. The AHA/CHA hydrogel effectively attenuated the degree of cartilage wear, facilitated chondrocytes' anabolic metabolism, and restored the ECM of cartilage. Therefore, the AHA/CHA hydrogel emerges as a promising therapeutic approach in clinical practices of OA treatment.

Static and dynamic cell culture of small caliber bilayer vascular grafts electrospun from polycaprolactone

Developing a suitable vascular graft to replace damaged small-caliber vessels and promote tissue regeneration is a critical challenge. This study focuses on bilayer polycaprolactone (PCL) grafts, fabricated by electrospinning, with both randomly distributed and radially oriented fibers. The analysis covers surface morphology, fiber diameter, pore size, cellular behavior, and biodegradability. Human umbilical vein endothelial cells (HUVECs) show an impressive viability of 142.2% after 7 days, while umbilical artery smooth muscle cells (UASMCs) exhibit 112.6%. Fluorescent microscopy indicates a higher viability of HUVECs over UASMCs. On the other hand, less than 10% of HUVECs and less than 5% of UASMCs undergo cell death within 7 days. Biodegradation analysis over 12 months shows a significant weight loss of 65–70% for both types of fiber arrangements. A custom-designed bioreactor enables dynamic cell culture, revealing comparable activity to static cultures. In the dynamic cell culture environment, HUVECs maintain high viability and display robust proliferation at both the 4th and 7th days, supporting the results observed in static cell cultures. These findings underscore the significant potential of bilayer PCL grafts in advancing the field of blood vessels, offering promising avenues for the development of functional vascular replacements with enhanced regenerative capabilities.

Nanoparticle containing recombinant excretory/secretory-24 protein of Haemonchus contortus enhanced the cellular immune responses in mice.

Haemonchus contortus poses a global challenge as a parasite affecting small ruminants, yet the problem of absence of an effective vaccine against H. contortus infection still exists. This investigation sought to appraise the immunological reaction induced by recombinant H. contortus excretory/secretory-24 (rHcES-24) in combination with complete Freund's adjuvant (CFA) and bio-polymeric nanoparticles (NPs) within a murine model. In this study, rHcES-24 was encapsulated in poly(d, l-lactide-co-glycolide) (PLGA) and chitosan (CS) NPs, administered subcutaneously to mice. Researchers analyzed the NPs using scanning electron microscope (SEM) and assessed lymphocyte proliferation, specific antibodies, cytokines, T cell proliferation (CD3e+CD4+, CD3e+CD8a+), and phenotypic alteration in splenocytes (CD11c+CD83+, CD11c+CD86+) through flow cytometry to understand the immune response. The results demonstrated that the administration of nanovaccines (NVs) prompted immune responses towards Th1 pathway. This was indicated by notable enhancements in the production of specific antibodies, heightened cytokine levels, and a robust proliferation of lymphocytes observed in mice that received the NVs compared to control groups. Remarkably, mice vaccinated with the antigen-loaded NPs formulations exhibited considerably higher proportions of splenic dendritic cells (DCs) and T cells in comparison to those receiving the traditional adjuvant or the control groups. Incorporating HcES-24 protein into NPs effectively conferred immunity against H. contortus, paving the way for developing a targeted and commercial vaccine.

Dual-Synergistic Nanomodulator Alleviates Exosomal PD-L1 Expression Enabling Exhausted Cytotoxic T Lymphocytes Rejuvenation for Potentiated iRFA-Treated Hepatocellular Carcinoma Immunotherapy.

The tumor immunosuppressive microenvironment (TME) induced by incomplete radiofrequency ablation (iRFA) in hepatocellular carcinoma (HCC) is a critical driver of tumor progression and metastasis. Herein, we proposed a therapeutic strategy aimed at remodeling the post-iRFA TME by targeting exosome biogenesis, secretion, and PD-L1 expression, thereby rejuvenating cytotoxic T lymphocyte function to mitigate the progression and metastasis of HCC. Leveraging the versatile properties of polydopamine nanomodulators, we have engineered a tailored delivery platform for GW4869 and amlodipine (AM), enabling precise and tumor-specific release of these therapeutic agents. Initially, GW4869, a neutral sphingomyelinase inhibitor, synergized with AM, an intracellular calcium modulator, to suppress exosome biogenesis and secretion. Subsequently, AM triggered the autophagic degradation of PD-L1. In vitro and in vivo experiments demonstrated that this synergistic approach significantly enhanced the robust activation and proliferation of various functional T-cell subsets following iRFA, particularly CD8+T cells, IFN-γ+ CD8+ cytotoxic T cells, natural killer cells, and innate lymphoid cells. Concurrently, it effectively reduced the infiltration of immunosuppressive cell types, including regulatory T cells and myeloid-derived suppressor cells. This favorable remodeling of the TME substantially inhibited the progression and metastasis of HCC post-iRFA. Collectively, our study presented a promising paradigm for enhancing HCC treatment efficacy by integrating radiofrequency ablation with advanced immune modulation strategies.

Transient proliferation by reversible YAP and mitogen-control of the cyclin D1/p27 ratio.

Hippo-YAP signaling orchestrates epithelial tissue repair and is therefore an attractive target in regenerative medicine. Yet it is unresolved how YAP integrates with mitogen signaling and contact inhibition to control the underlying transient proliferative response. Here we show that reduced contact inhibition, increased mitogen signaling, and YAP-TEAD activation converge on increasing the nuclear cyclin D1/p27 protein ratio during G1 phase, towards a threshold ratio that dictates whether individual cells enter or exit the cell cycle. YAP increases this ratio indirectly, in concert with mitogen signaling, by increasing EGFR and other receptors that signal primarily through ERK. After a delay, contact inhibition suppresses YAP activity which gradually downregulates mitogen signaling and the cyclin D1/p27 ratio. Increasing YAP activity by ablating the suppressor Merlin/NF2 reveals a balancing mechanism in which YAP suppression and contact inhibition of proliferation can be recovered but only at higher local cell density. Thus, critical for tissue repair, robust proliferation responses result from the YAP-induced and receptor-mediated prolonged increase in the cyclin D1/p27 ratio, which is only reversed by delayed suppression of receptor signaling after contact inhibition of YAP.

Nitric oxide-producing monocyte-myeloid suppressor cells expand and accumulate in the spleen and mesenteric lymph nodes of Yersinia enterocolitica-infected mice.

Yersinia enterocolitica (Ye) is a Gram-negative bacterium that causes gastrointestinal infections. The myeloid-derived suppressor cells (MDSCs) constitute a cellular population with the capacity of inducing the specific suppression of T cells. Although there is evidence supporting the role of MDSCs in controlling the immune responses in several bacterial infections, its role during Ye infection has not yet been reported. Therefore, the purpose of the present work was to analyze MDSCs after oral Ye infection. C57BL/6 wild-type mice were infected with Ye WAP-314 serotype O:8. The proliferation of splenocytes and mesenteric lymph nodes (MLN) cells was measured as well as the levels of cytokines and nitric oxide (NO) in culture supernatants. The frequency and subsets of MDSCs were analyzed in the intestinal mucosa and spleen by flow cytometry. Furthermore, monocytic-MDSCs (Mo-MDSCs) and polymorphonuclear-MDSCs (PMN-MDSCs) were purified from the spleen of infected mice and their suppressor activity was evaluated in co-cultures with purified T cells. we observed a marked expansion of CD11b+Gr-1+ cells, a phenotype consistent with MDSCs, in the spleen and intestinal mucosa of Ye-infected mice. Interestingly, a robust proliferation of splenocytes and MLN cells was observed only when the MDSCs were depleted or the NO production was blocked. In addition, we determined that only Mo-MDSCs had the ability to suppress T-cell proliferation. Our results highlight a mechanism by which Ye may induce suppression of the immune responses. We suggest that NO-producing Mo-MDSCs expand and accumulate in MLN and spleen of Ye-infected mice. These cells can then suppress the T-cell function without interfering with the anti-bacterial effector response. Instead, these immature myeloid cells may perform an important function in regulating the inflammatory response and protecting affected tissues.

Phosphorylation-driven epichaperome assembly is a regulator of cellular adaptability and proliferation

The intricate network of protein-chaperone interactions is crucial for maintaining cellular function. Recent discoveries have unveiled the existence of specialized chaperone assemblies, known as epichaperomes, which serve as scaffolding platforms that orchestrate the reconfiguration of protein-protein interaction networks, thereby enhancing cellular adaptability and proliferation. This study explores the structural and regulatory aspects of epichaperomes, with a particular focus on the role of post-translational modifications (PTMs) in their formation and function. A key finding is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 within an intrinsically disordered region, as critical determinants of epichaperome assembly. Our data demonstrate that phosphorylation of these serine residues enhances HSP90’s interactions with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Moreover, we establish a direct link between epichaperome function and cellular physiology, particularly in contexts where robust proliferation and adaptive behavior are essential, such as in cancer and pluripotent stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone assemblies in diseases characterized by epichaperome dysregulation, thereby bridging the gap between fundamental research and precision medicine.

Environmental salinity differentiates responses to acute hypothermal stress in milkfish

Global warming has led to an increase in the frequency of cold extremes, causing significant economic losses in aquaculture, particularly in subtropical regions. Milkfish (Chanos chanos) holds significant importance in aquaculture within subtropical Asian regions. Despite milkfish's ability to tolerate varying salinity levels, frequent cold snaps associated with extreme weather events have caused substantial mortality. Understanding the molecular and cellular mechanisms underlying cold stress-induced cell death is crucial for developing effective strategies to mitigate such losses. Given the pivotal role of the liver in fish physiology, we established a primary milkfish hepatocyte culture demonstrating robust proliferation and expressing a unique marker leptin A. The molecular characterization of cold-treated hepatocytes at 18 °C showed that the mRNA levels of superoxide dismutase (sod1) and catalase (cat), responsible for neutralizing reactive oxygen species (ROS), were downregulated in freshwater (FW) conditions, while cat expression was upregulated in seawater (SW) conditions. This differential modulation of ROS signaling implies distinct responses in FW and SW, leading to higher ROS levels and increased cell death in FW condition compared to those in SW. Transcriptomic analysis of liver tissues from milkfish reared in FW or SW, with or without cold stress, revealed distinct gene expression patterns. Although cold stress affected a common set of genes in both FW and SW conditions, SW-specific cold responsive genes are associated with metabolic pathways while FW-specific genes were linked to cell proliferation pathways. Notably, gene set enrichment analysis highlighted the downregulation of cytochrome-related genes, a major source of ROS production, in response to cold stress in SW. In summary, our study unveils an insightful mechanism whereby the salinity of SW counteracts cold stress-induced ROS signaling, emphasizing the feasibility and practicality of preconditioning fish in SW as a preventive measure against cold stress-induced mortality.

Evaluating Nanoparticulate Vaccine Formulations for Effective Antigen Presentation and T-Cell Proliferation Using an In Vitro Overlay Assay.

Inducing T lymphocyte (T-cell) activation and proliferation with specificity against a pathogen is crucial in vaccine formulation. Assessing vaccine candidates' ability to induce T-cell proliferation helps optimize formulation for its safety, immunogenicity, and efficacy. Our in-house vaccine candidates use microparticles (MPs) and nanoparticles (NPs) to enhance antigen stability and target delivery to antigen-presenting cells (APCs), providing improved immunogenicity. Typically, vaccine formulations are screened for safety and immunostimulatory effects using in vitro methods, but extensive animal testing is often required to assess immunogenic responses. We identified the need for a rapid, intermediate screening process to select promising candidates before advancing to expensive and time-consuming in vivo evaluations. In this study, an in vitro overlay assay system was demonstrated as an effective high-throughput preclinical testing method to evaluate the immunogenic properties of early-stage vaccine formulations. The overlay assay's effectiveness in testing particulate vaccine candidates for immunogenic responses has been evaluated by optimizing the carboxyfluorescein succinimidyl ester (CFSE) T-cell proliferation assay. DCs were overlaid with T-cells, allowing vaccine-stimulated DCs to present antigens to CFSE-stained T-cells. T-cell proliferation was quantified using flow cytometry on days 0, 1, 2, 4, and 6 upon successful antigen presentation. The assay was tested with nanoparticulate vaccine formulations targeting Neisseria gonorrhoeae (CDC F62, FA19, FA1090), measles, H1N1 flu prototype, canine coronavirus, and Zika, with adjuvants including Alhydrogel® (Alum) and AddaVax™. The assay revealed robust T-cell proliferation in the vaccine treatment groups, with variations between bacterial and viral vaccine candidates. A dose-dependent study indicated immune stimulation varied with antigen dose. These findings highlight the assay's potential to differentiate and quantify effective antigen presentation, providing valuable insights for developing and optimizing vaccine formulations.

Functional Activity of Cytokine-Induced Killer Cells Enhanced by CAR-CD19 Modification or by Soluble Bispecific Antibody Blinatumomab.

Strategies to increase the anti-tumor efficacy of cytokine-induced killer cells (CIKs) include genetic modification with chimeric antigen receptors (CARs) or the addition of soluble T-cell engaging bispecific antibodies (BsAbs). Here, CIKs were modified using a transposon system integrating two distinct anti-CD19 CARs (CAR-MNZ and CAR-BG2) or combined with soluble CD3xCD19 BsAb blinatumomab (CIK + Blina). CAR-MNZ bearing the CD28-OX40-CD3ζ signaling modules, and CAR-BG2, designed on the Tisagenlecleucel CAR sequence (Kymriah®), carrying the 4-1BB and CD3ζ signaling elements, were employed. After transfection and CIK expansion, cells expressed CAR-CD19 to a similar extent (35.9% CAR-MNZ and 17.7% CAR-BG2). In vitro evaluations demonstrated robust proliferation and cytotoxicity (~50% cytotoxicity) of CARCIK-MNZ, CARCIK-BG2, and CIK + Blina against CD19+ target cells, suggesting similar efficacy. All effectors formed an increased number of synapses, activated NFAT and NFkB, and secreted IL-2 and IFN-ɣ upon encountering targets. CIK + Blina displayed strongest NFAT and IFN-ɣ induction, whereas CARCIK-BG2 demonstrated superior synapse formation. All the effectors have shown therapeutic activity in vivo against the CD19+ Daudi tumor model, with CARCIK cells showing a more durable response compared to CIK + Blina, likely due to the short half-life of Blina in this model.

IL-18 primes T cells with an antigen-inexperienced memory phenotype for proliferation and differentiation into effector cells through Notch signaling.

Recent studies have revealed that a subset of CD8+ T cells exhibit innate features and can be activated by cytokines. However, the precise mechanisms underlying the proliferation and differentiation of these cells remain unclear. Here, we demonstrated that CD44highCD8+ T cells in the mouse spleen express functional interleukin-18 (IL-18) receptors, whereas CD44lowCD8+ T cells do not. In response to IL-18 stimulation, these cells activated various metabolic pathways, upregulated the expression of surface molecules, such as c-Kit (CD117), CD25, and PD-1, and induced progression through the G1/S phase in the cell cycle. IL-18-primed cells, expressing a high-affinity receptor for IL-2, exhibited robust proliferation in response to IL-2 and underwent differentiation into effector cells. The splenic CD44highCD8+ T cells exhibited high expression levels of CD122, CD62L, CCR7, and CXCR3, along with CD5, indicating their potential for migration to the lymph nodes, where they could undergo expansion and terminal differentiation into effector cells. Additionally, in a tumor model, administration of IL-18 increased the accumulation of CD8+ T cells in both the lymph nodes and tumors. It is noteworthy that stimulation of CD44highCD8+ T cells with IL-18 upregulated the Notch-1 receptor and c-Myc. Moreover, inclusion of γ-secretase inhibitors attenuated the effect of IL-18 on both proliferation and interferon-γ production in the cells. These results demonstrate that IL-18 primes CD44highCD122highCXCR3highCD62LhighCD8+ T cells for expansion and differentiation into effector cells in a Notch signaling-dependent manner.

Combination anti-PD-1 and anti-CTLA-4 therapy generates waves of clonal responses that include progenitor-exhausted CD8+ T cells

Combination checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies has shown promising efficacy in melanoma. However, the underlying mechanism in humans remains unclear. Here, we perform paired single-cell RNA and Tcell receptor (TCR) sequencing across time in 36 patients with stage IV melanoma treated with anti-PD-1, anti-CTLA-4, or combination therapy. We develop the algorithm Cyclone to track temporal clonal dynamics and underlying cell states. Checkpoint blockade induces waves of clonal Tcell responses that peak at distinct time points. Combination therapy results in greater magnitude of clonal responses at 6 and 9weeks compared to single-agent therapies, including melanoma-specific CD8+ Tcells and exhausted CD8+ Tcell (TEX) clones. Focused analyses of TEX identify that anti-CTLA-4 induces robust expansion and proliferation of progenitor TEX, which synergizes with anti-PD-1 to reinvigorate TEX during combination therapy. These next generation immune profiling approaches can guide the selection of drugs, schedule, and dosing for novel combination strategies.