Mature Cells Research Articles

New generalized metric based on branch length distance to compare B cell lineage trees

The B cell lineage tree encapsulates the successive phases of B cell differentiation and maturation, transitioning from hematopoietic stem cells to mature, antibody-secreting cells within the immune system. Mathematically, this lineage can be conceptualized as an evolutionary tree, where each node represents a distinct stage in B cell development, and the edges reflect the differentiation pathways. To compare these lineage trees, a rigorous mathematical metric is essential. Analyzing B cell lineage trees mathematically and quantifying changes in lineage attributes over time necessitates a comparison methodology capable of accurately assessing and measuring these changes. Addressing the intricacies of multiple B cell lineage tree comparisons, this study introduces a novel metric that enhances the precision of comparative analysis. This metric is formulated on principles of metric theory and evolutionary biology, quantifying the dissimilarities between lineage trees by measuring branch length distance and weight. By providing a framework for systematically classifying lineage trees, this metric facilitates the development of predictive models that are crucial for the creation of targeted immunotherapy and vaccines. To validate the effectiveness of this new metric, synthetic datasets that mimic the complexity and variability of real B cell lineage structures are employed. We demonstrated the ability of the new metric method to accurately capture the evolutionary nuances of B cell lineages.

6421 Imaging Phenotype May Predict Genotype In Human Pheochromocytoma: Relationships Between Genetic Mutation Clusters, Metabolite Transporter Expression, And Radiotracer Uptake

Abstract Disclosure: I. Sakuma: None. M. Fujimoto: None. D.F. Vatner: None. K. Yokote: None. T. Tanaka: None. Context [1]23I-metaiodobenzylguanidine scintigraphy (MIBG) and [1]8F-fluoro-2-doxy-D-glucose positron emission tomography (FDG-PET) are helpful for the localization of pheochromocytoma. The norepinephrine transporter (NET) and the glucose transporter 1 (GLUT1) are thought to be critical for the uptake of these radioisotopes. NET expression is activated by Paired Like Homeobox (PHOX2) and glucocorticoid receptor (GR) in neuroblastoma cells. Around 70% of pheochromocytomas carry well-known mutations. Pheochromocytoma-associated genes are divided into two clusters: the pseudohypoxia-related cluster 1 and the kinase signaling-related cluster 2. Cluster 1 mutation are associated with noradrenergic pheochromocytomas with an elevated metastatic risk. Cluster 2 tumors demonstrate an adrenergic phenotype with a less aggressive course. How mutations of cluster 1 and cluster 2 impact the expression of NET/GLUT1 and on radiotracer uptake remains unclear. Hypothesis Cluster 1 and cluster 2 will have different effects on the expression of transcription factors associated with chromaffin cell differentiation with resultant differential effects on NET/GLUT1 expression. Methods 42 patients with pheochromocytoma were evaluated by MIBG and FDG-PET. Tumor gene expression was evaluated by real-time qPCR, and mutation analyses were performed by Sanger or next-generation sequencing. Results 88% of tumors were MIBG-avid, and 67% were FDG-avid. The expression of NET in MIBG-negative tumors was 70% lower than in MIBG-positive tumors. The expression of GLUT1 in FDG-PET-negative tumors was 75% lower than in FDG-PET-positive tumors. The expression of NET positively correlated with transcription factors involved in chromaffin cell maturation, including PHOX2A, PHOX2B, GATA binding protein 3 (GATA3), GR, catecholamine synthases, and insulinoma associated 1 (INSM1). The JASPAR database for promoter analysis revealed putative responsive regions of the transcription factors PHOX2A, PHOX2B, GATA3, GR, and INSM1 on the NET gene. Seven patients (16.7%) had mutations in cluster 1, and fourteen patients (33.3%) had mutations in cluster 2. Cluster 2 exhibited elevated NET, PHOX2A, PHOX2B, GATA3, GR, and INSM1 expression levels compared to cluster 1. Cluster 1 showed a higher expression level of GLUT1 and BMP4 that is associated with early chromaffin cell differentiation. Conclusions In pheochromocytomas, MIBG and FDG uptake correlated with the expression levels of NET and GLUT1, respectively. NET expression is associated with chromaffin cell differentiation transcription factor expression. We propose that pheochromocytomas that carry Cluster 1 mutations are more likely to be missed in MIBG due to low NET expression, while pheochromocytomas that carry Cluster 2 mutations are more likely to be missed on FDG-PET due to low GLUT1 expression with a more mature chromaffin cell phenotype. Presentation: 6/2/2024

8660 Bodipy Labeled Vitamin D3 Associates With Lipid Droplets In Adipocytes

Abstract Disclosure: N. Ucar: None. J.T. Deeney: None. R. Pickering, PhD: None. M.T. Kirber: None. T. Fan: None. R. Loo, PhD: Research Investigator; Self; Carbogen Amcis BV. M.F. Holick: Grant Recipient; Self; Carbogen Amcis BV. Research Investigator; Self; Carbogen Amcis BV. Obese patients are often found to be deficient in vitamin D, requiring higher levels of supplementation than normal weight subjects. While this has been attributed to vitamin D3‘s lipid solubility and accumulation in fat tissue, little is known about how vitamin D3 enters adipocytes and associates with the intracellular lipid droplet. The goal of this study was to investigate this association using a novel fluorescently labelled vitamin D3. Bodipy, 4,4-difluoro-1,3,5, 7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503), was covalently linked to vitamin D3 and its structure was confirmed by NMR and mass spectroscopy. Murine 3T3L1 and primary adipocytes were used for our study. Mouse 3T3L1 preadipocytes were cultured in multiwell plates or glass bottom dishes in high glucose DMEM media and differentiated for 7-14 days. Primary adipocytes were obtained by collagenase digestion after surgical extraction of adipose tissue from male/female C57BL/6J mice and attached to glass bottom dishes. The cultured adipocytes were exposed to 10 microM BODIPY labelled Vitamin D3 (vitD-B). BODIPY was added to cultured cells and served as the control. Cells were imaged at various times on a wide-field epifluorescence microscope and analysed with NIH ImageJ software. VitD-B adipocyte uptake and accumulation in lipid droplets was observed over 24 hrs. Isolated lipid droplets were also incubated with vitD-B and BODIPY. VitD-B in mature 3T3L1 cells was observed in lipid droplets after 1h incubation and increased fluorescent intensity was observed over the next 24 hrs. BODIPY was observed in the lipid droplets after 15-20 mins. As a negative control, non-differentiated 3T3L1 cells did not exhibit staining by BODIPY even after 24-hrs. When vitD-B was added to primary cultured adipocytes, it appeared in the adipocytes within 1-hr. Fluorescence intensity of primary adipocytes was increased 1.2-fold at 8 hrs and 5.7-fold at 24 hrs compared to 1 hr. For comparison the positive control treated with BODIPY exhibited rapid fluorescence uptake into primary adipocytes that was increased 29-fold higher than that of vitD-B at 1 hr. VitD-B and BODIPY both demonstrated rapid uptake into isolated lipid droplets (less than 2 min) based on fluorescence microscopy.These results hold promising implications for understanding of the interaction between vitamin D and intracellular lipid droplets. Keywords: Adipocyte, Vitamin D3, BODIPY, adipocytes, lipid droplet, fluorescent labelled vitamin D3 Presentation: 6/2/2024

7704 Generating pituitary cells from iPSCs of patients with child onset congenital hypopituitarism harboring PROP1 pathogenic allelic variants

Abstract Disclosure: J.M. Marques: None. F. Fattahi: None. L.R. Carvalho: None. Introduction: Reduced or absent levels of pituitary hormones due to genetic or organic causes is known as combined pituitary hormone deficiency (CPHD) and PROP1 is the most frequent cause of CPHD, but more than 85% of the cases are without molecular diagnosis. Novel Techniques such as induced pluripotent stem cells (iPSC), can be used to generate patient’s specific pituitary cells for disease modeling. Aim: To generate iPSC from CO-CH patients’ peripheral blood mononuclear cells (PBMC) and differentiate them into pituitary cells. Methods: Peripheral blood was collected from 2 siblings (HC1 and HC2) harboring compound heterozygous PROP1 allelic variants and their parents (HC3 and HC4). PBMCs were isolated and expanded with MNC medium prior to nucleoporation and transfection with plasmids containing pluripotency genes (OCT4 and SOX2; cMYC and KLF). After 48h, transfected cells were transferred to Geltrex coated plates and fed with E8, NaB and βFGF until pluripotent colonies were formed. iPSCs were differentiated into cranial placode cells using pituitary placode conditioned medium (PPCM) until day 15. For pituitary cell maturation, pituitary conditioned medium (PPCM without SB431542) was used until day 30. qPCR and flow cytometry using the Kit BD Stemflow - Human Pluripotent Stem Cell Transcription Factor Analysis (BD Biosciences) for stem cells and the eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization (Thermo Fisher Scientific) for the placode and pituitary markers were used. Pituitary hormonal levels from cell supernatant were measured at the Laboratory of Hormones and Molecular Genetics - HCFMUSP. Results: we generated iPSCs from patient and control blood cells. iPSCs were more than 90% positive for the pluripotency markers SOX2, NANOG, OCT3/4. Pituitary cells were generated with positivity for the placode marker SIX1 (58%) and pituitary markers, as LHX3 (56%) and PROP1 (59%), at day 15. Control cell lineages expressed higher levels of pituitary hormones at days 15 and 30 at RNA and protein levels while patient cells failed to induce hormone-producing cells properly. Pituitary hormonal release was impaired in patients pituitary cells. Conclusion: CO-CH patients’ PBMC were dedifferentiated to a pluripotent state, being able to generate pituitary hormone producing cells. Patient cells were able to mimic human phenotype. These cells can be used, in the future, to study CO-CH basis in patients specific background, to develop pituitary disease modeling as well as test possible new treatments. Presentation: 6/3/2024

Pseudo-3D Topological Alignments Regulate Mechanotransduction and Maturation of Smooth Muscle Cells.

Smooth muscle cells (SMCs) sense and respond to mechanical stimuli in their extracellular microenvironments (ECMs), playing a crucial role in muscle tissue engineering. Increasing evidence from topological cues-mediated mechanotransduction of SMCs in ECMs has suggested some potential underlying mechanisms of how SMC functions and maturation are regulated by their mechanosensing leading to transduction. However, how the expression of yes-associated protein 1 (YAP) influences the phenotypic shift from synthetic to contractile is still controversial. Here, pseudo-3D topological alignments mimicking native muscle tissues are generated using laser-cutter engraving to explore the influence of topological cues on SMC mechanotransduction and maturation. The analysis of topological cue-mediated mechanotransduction and maturation marker expression revealed YAP is involved in mechanotransduction for SMCs cultured on cross-patterned substrates in the presence of cell-cell interactions. Moreover, these SMCs with YAP-linked mechanosensing showed higher expression of calponin, indicating a shift toward contractile phenotypes in vitro and in vivo. Furthermore, it showed skeletal muscle cells has different mechanosensing and maturation mechanisms compared to SMCs, revealing muscle type-dependent different sensing of topological cues, and converting into maturation-associated signaling cascades. This study provides insights into the regulation of SMC mechanotransduction and maturation by topological cues, suggesting the involvement of YAP-linked signaling pathways in this process.

Tumor draining lymph nodes connected to cold triple-negative breast cancers are characterized by Th2-associated microenvironment

Tumor draining lymph nodes (TDLN) represent a key component of the tumor-immunity cycle. There are few studies describing how TDLNs impact lymphocyte infiltration into tumors. Here we directly compare tumor-free TDLNs draining “cold” and “hot” human triple negative breast cancers (TDLNCold and TDLNHot). Using machine-learning-based self-correlation analysis of immune gene expression, we find unbalanced intranodal regulations within TDLNCold. Two gene pairs (TBX21/GATA3-CXCR1) with opposite correlations suggest preferential priming of T helper 2 (Th2) cells by mature dendritic cells (DC) within TDLNCold. This is validated by multiplex immunofluorescent staining, identifying more mature-DC-Th2 spatial clusters within TDLNCold versus TDLNHot. Associated with this Th2 priming preference, more IL4 producing mast cells (MC) are found within sinus regions of TDLNCold. Downstream, Th2-associated fibrotic TME is found in paired cold tumors with increased Th2/T-helper-1-cell (Th1) ratio, upregulated fibrosis growth factors, and stromal enrichment of cancer associated fibroblasts. These findings are further confirmed in a validation cohort and public genomic data. Our results reveal a potential role of IL4+ MCs within TDLNs, associated with Th2 polarization and reduced immune infiltration into tumors.

CD28 Costimulation Augments CAR Signaling in NK Cells via the LCK/CD3ζ/ZAP70 Signaling Axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity.

Chimeric antigen receptor-T-cell therapies going viral: latent and incidental viral infections.

Infections are the leading cause of non-relapse mortality following chimeric antigen receptor (CAR)-T-cell therapy, with viral infections being frequent both in the early and late phases post-infusion. We review the epidemiology of viral infections and discuss critical approaches to prevention and management strategies in this setting. Herpesviruses dominate the early period. herpes simplex virus and varicella zoster virus infections are rare due to widespread antiviral prophylaxis, but cytomegalovirus (CMV) reactivation is increasingly observed, particularly in high-risk groups including B cell maturation antigen (BCMA)-CAR-T-cell therapy recipients and patients receiving corticosteroids. While CMV end-organ disease is rare, CMV is associated with increased mortality, emphasizing the need to evaluate the broader impact of CMV on long-term hematological, infection, and survival outcomes. Human herpesvirus-6 (HHV-6) has also emerged as a concern, with its diagnosis complicated by overlapping symptoms with neurotoxicity, underscoring the importance of considering viral encephalitis in differential diagnoses. Respiratory viruses are the most common late infections with a higher incidence after BCMA CAR-T-cell therapy. Vaccination remains a critical preventive measure against respiratory viruses but may be less immunogenic following CAR-T-cell therapy. The optimal timing, type of vaccine, and dosing schedule require further investigation. A better understanding of viral epidemiology and preventive trials are needed to improve infection prevention practices and outcomes following CAR-T-cell therapies.

Prolonged Omicron-specific B cell maturation alleviates immune imprinting induced by SARS-CoV-2 inactivated vaccine

ABSTRACT SARS-CoV-2 ancestral strain-induced immune imprinting poses great challenges to updating vaccines for new variants. Studies showed that repeated Omicron exposures could override immune imprinting induced by inactivated vaccines but not mRNA vaccines, a disparity yet to be understood. Here, we analyzed the immune imprinting alleviation in inactivated vaccine (CoronaVac) cohorts after a long-term period following breakthrough infections (BTI). We observed in CoronaVac-vaccinated individuals who experienced BA.5/BF.7 BTI, the proportion of Omicron-specific memory B cells (MBCs) substantially increased after an extended period post-Omicron BTI, with their antibodies displaying enhanced somatic hypermutation and neutralizing potency. Consequently, the neutralizing antibody epitope distribution encoded by MBCs post-BA.5/BF.7 BTI after prolonged maturation closely mirrors that in BA.5/BF.7-infected unvaccinated individuals. Together, these results indicate the activation and expansion of Omicron-specific naïve B cells generated by first-time Omicron exposure helped to alleviate CoronaVac-induced immune imprinting, and the absence of this process should have caused the persistent immune imprinting seen in mRNA vaccine recipients.

Transcutaneous Immunization of 1D Rod-Like Tobacco-Mosaic-Virus-Based Peptide Vaccine via Tip-Loaded Dissolving Microneedles.

Peptide vaccines induce specific neutralizing antibodies and are effective in disease prevention and treatment. However, peptide antigens have a low immunogenicity and are unstable, requiring efficient vaccine carriers to enhance their immunogenicity. Here, we develop a tobacco mosaic virus (TMV)-based peptide vaccine for transdermal immunization using a tip-loaded dissolving microneedle (MN) patch. TMV is decorated with the model peptide antigen PEP3. The prepared TMV-PEP3 promotes dendritic cell maturation and induces dendritic cells to overexpress MHC II, costimulatory factors, and pro-inflammatory factors. By encapsulation of TMV-PEP3 in the tips of a trehalose MN, TMV-PEP3 can be delivered by MN and significantly promote local immune cell infiltration. In vivo studies show that both subcutaneous injection and MN administration of TMV-PEP3 increase the production of anti-PEP3 IgG antibodies and the harvested serum can induce complement-dependent cytotoxicity. This work provides a promising strategy for constructing efficient and health-care-friendly peptide vaccines.

Polymeric nanocarrier via metabolism regulation mediates immunogenic cell death with spatiotemporal orchestration for cancer immunotherapy

The limited efficacy of cancer immunotherapy occurs due to the lack of spatiotemporal orchestration of adaptive immune response stimulation and immunosuppressive tumor microenvironment modulation. Herein, we report a nanoplatform fabricated using a pH-sensitive triblock copolymer synthesized by reversible addition-fragmentation chain transfer polymerization enabling in situ tumor vaccination and tumor-associated macrophages (TAMs) polarization. The nanocarrier itself can induce melanoma immunogenic cell death (ICD) via tertiary amines and thioethers concentrating on mitochondria to regulate metabolism in triggering endoplasmic reticulum stress and upregulating gasdermin D for pyroptosis as well as some features of ferroptosis and apoptosis. After the addition of ligand cyclic arginine-glycine-aspartic acid (cRGD) and mannose, the mixed nanocarrier with immune adjuvant resiquimod encapsulation can target B16F10 cells for in situ tumor vaccination and TAMs for M1 phenotype polarization. In vivo studies indicate that the mixed targeting nanoplatform elicits tumor ICD, dendritic cell maturation, TAM polarization, and cytotoxic T lymphocyte infiltration and inhibits melanoma volume growth. In combination with immune checkpoint blockade, the survival time of mice is markedly prolonged. This study provides a strategy for utilizing immunoactive materials in the innate and adaptive immune responses to augment cancer therapy.

Mechanism of modified mRNA structure in COVID-19 vaccines for inducing neutralizing antibodies

The development of SARS-CoV-2 mRNA vaccines is closely linked to advancements in mRNA manufacturing technology. Structural modifications, such as replacing uridine with 1-methylpseudouridine (1mψ), enhance translation efficiency and help the mRNA evade immune detection. Lipid nanoparticles (LNPs) serve as an effective delivery system. Vaccines like BNT162b2 and mRNA-1273 target the receptor-binding domain (RBD) of the spike (S) protein, prompting B cells to produce neutralizing antibodies that block the RBD from binding to the Angiotensin-Converting Enzyme 2 (ACE2) receptor, preventing infection. These vaccines also stimulate adaptive immune responses by activating CD4+ and CD8+ T cells, with mRNA functioning as an endogenous antigen. Antigen-presenting cells (APCs) present the vaccine antigens via major histocompatibility complex (MHC) class I and II pathways, with CD8+ T cells recognizing MHC class I and destroying infected cells, while CD4+ T cells recognize MHC class II and assist in B cell maturation and antibody production. While mRNA vaccines have proven effective in neutralizing SARS-CoV-2, challenges remain, including the decline in neutralizing antibody titers over time and the emergence of new viral variants.

Physalia gonodendra are not yet sexually mature when released

The blue bottle genus Physalia is one of the well-known siphonophore belonging to the Cnidaria, Hydrozoa. Physalia is also known as a ferocious predator, occasionally stinging and fatally wounding humans, but key details of its life cycle and reproductive biology are unclear. Physalia have separate sexes, and sexual reproduction occurs through the release of complex structures called gonodendra that contain many gonophores that will release either eggs or sperm. It is not known how mature the gonophores are when the gonodendra are released. In this study, we aim to characterize germ cell maturation by conducting histological, cytological, and gene expression analyses of the gonodendron of Physalia utriculus from Japan. We found a layered structure of the gonophore, consistent with other studies; however, gametes were not found even in gonophores that were within the released gonodendra. Moreover, haploid cells were not detected by flow cytometry. Analysis of the expression of putative germ cell marker and meiosis related genes showed high expression in the gonophore. These results strongly suggest that germ cells do not mature until after gonodendra are released. These findings provide valuable insights into the reproductive ecology and life cycle of Physalia.

Decoupling immunomodulatory properties from lipid binding in the α-pore-forming toxin Sticholysin II

Sticholysin II (StII), a pore-forming toxin from the marine anemone Stichodactyla helianthus, enhances an antigen-specific cytotoxic T lymphocyte (CTL) response when co-encapsulated in liposomes with a model antigen. This capacity does not depend exclusively on its pore-forming activity and is partially supported by its ability to activate Toll-like receptor 4 (TLR4) in dendritic cells, presumably by interacting with this receptor or by triggering signaling cascades upon binding to lipid membrane. In order to investigate whether the lipid binding capacity of StII is required for immunomodulation, we designed a mutant in which the aromatic amino acids from the interfacial binding site Trp110, Tyr111 and Trp114 were substituted by Ala. In the present work, we demonstrated that StII3A keeps the secondary structure composition and global folding of StII, while it loses its lipid binding and permeabilization abilities. Despite this, StII3A upregulates dendritic cells maturation markers, enhances an antigen-specific effector CD8+ T cells response and confers antitumor protection in a preventive scenario in C57BL/6 mice. Our results indicate that a mechanism independent of its lipid binding ability is involved in the immunomodulatory capacity of StII, pointing to StII3A as a promising candidate to improve the reliability of the Sts-based vaccine platform.

Reprogramming the Tumor Immune Microenvironment Through Activatable Photothermal Therapy and GSH depletion Using Liposomal Gold Nanocages to Potentiate Anti-Metastatic Immunotherapy.

Cancer immunotherapy offers significant clinical benefits for patients with advanced or metastatic tumors. However, immunotherapeutic efficacy is often hindered by the tumor microenvironment's high redox levels, leading to variable patient outcomes. Herein, a therapeutic liposomal gold nanocage (MGL) is innovatively developed based on photo-triggered hyperthermia and a releasable strategy by combining a glutathione (GSH) depletion to remodel the tumor immune microenvironment, fostering a more robust anti-tumor immune response. MGL comprises a thermosensitive liposome shell and a gold nanocage core loaded with maleimide. The flexible shell promotes efficient uptake by cancer cells, enabling targeted destruction through photothermal therapy while triggering immunogenic cell death and the maturation of antigen-presenting cells. The photoactivated release of maleimide depletes intracellular GSH, increasing tumor cell sensitivity to oxidative stress and thermal damage. Conversely, GSH reduction also diminishes immunosuppressive cell activity, enhances antigen presentation, and activates T cells. Moreover, photothermal immunotherapy decreases elevated levels of heat shock proteins in tumor cells, further increasing their sensitivity to hyperthermia. In summary, MGL elicited a robust systemic antitumor immune response through GSH depletion, facilitating an effective photothermal immunotherapeutic strategy that reprograms the tumor microenvironment and significantly inhibits primary and metastatic tumors. This approach demonstrates considerable translational potential and clinical applicability.

PAD4 Inhibitor-Loaded Layered Double Hydroxide Nanosheets as a Multifunctional Nanoplatform for Photodynamic Therapy-Mediated Tumor Metastasis Treatment.

Photodynamic therapy (PDT) is demonstrated to be effective in inducing antitumor immune responses for tumor metastasis treatment. However, tumor hypoxia, inferior tissue penetration of light, and low singlet oxygen (1O2) quantum yield significantly hamper the efficacy of PDT, thus weakening its immune function. Moreover, PDT-mediated neutrophil extracellular traps (NETs) formation can further reduce the therapeutic effectiveness. Herein, the use of defect-rich CoMo-layered double hydroxide (DR-CoMo-LDH) nanosheets as a carrier to load a typical peptidyl arginine deiminase 4 inhibitor, i.e., YW4-03, to construct a multifunctional nanoagent (403@DR-LDH) for PDT/immunotherapy, is reported. Specifically, 403@DR-LDH inherits excellent 1O2 generation activity under 1550nm laser irradiation and improves the half-life of YW4-03. Meanwhile, 403@DR-LDH plus 1550nm laser irradiation can stimulate immunogenic cell death to promote the maturation of dendric cells and activation/infiltration of T cells and significantly downregulate H3cit protein expression to inhibit NETs formation, synergistically promoting the antitumor metastasis effect. Taken together, 403@DR-LDH can kill cancer cells and inhibit tumor growth/metastasis under 1550nm laser irradiation. Single-cell analysis indicates that 403@DR-LDH can regulate the ratio of immune cells and immune-related proteins to improve the tumor immune microenvironment, showing strong efficacy to inhibit the tumor growth, metastasis, and recurrence.

B-343 Development of CD40 and HER2 Bispecific Antibodies for Enhanced Efficacy against HER2-Positive Tumors

Abstract Background CD40, a member of the tumor necrosis factor superfamily, is primarily expressed on antigen-presenting cells. This receptor plays a crucial role in facilitating the interaction between dendritic cells, activating antigen-specific T cells. Agonist antibodies targeting the CD40 receptor have the potential to mimic the natural CD40 ligand, fostering dendritic cell maturation and stimulating immunity against “cold” tumors. Although this approach has shown promise in preclinical studies, translating it to clinical success has been challenging. Only modest anti-tumor activity was observed in cancer patients, and none of the developed CD40 monoclonal antibodies (mAbs) progressed beyond early clinical trials, highlighting a need for improved anti-tumor efficacy. Methods In this study, we successfully developed an anti-human CD40 agonist rabbit monoclonal antibody using Yurogen's proprietary SMAB(TM) platform. The most potent CD40 mAb clone was combined with trastuzumab (an anti-HER2 mAb) to create a therapeutic bispecific antibody. Our goal is to leverage immune cell activation through HER2-targeted cancer therapy to achieve enhanced anti-tumor activity. Dozens of diverse CD40xHER2 bispecific antibody designs (both symmetry and asymmetry) were explored, with careful evaluation of expression yield, purity, and binding profiles for each target respectively. Results A few bispecific antibody designs demonstrated robust binding to both HER2 and CD40 antigen proteins, as well as receptor-expressing cell lines. Notably, two developed bispecific antibodies exhibited potent killing of HER2-positive cancer cells with the assistance of innate immune cells. Conclusions In sum, this study provides novel insights into designing bispecific antibodies against HER2 and CD40, offering a promising avenue for advancing cancer immunotherapy.

Enhancing CAR-T cell therapy against solid tumor by drug-free triboelectric immunotherapy

Chimeric antigen receptor (CAR) T cell therapy is a highly effective immunotherapy for hematological tumors, but its efficacy against most solid tumors remains challenging. Herein, a novel synergistic combination therapy of drug-free triboelectric immunotherapy and CAR-T cell therapy against solid tumor was proposed. A triboelectric nanogenerator (TENG) that can generate pulsed direct-current by coupling triboelectrification effect and electrostatic breakdown effect was fabricated. The TENG can generate up to 30 pulse direct-current peaks with peak current output ≈35 μA in a single sliding to power the triboelectric immunotherapy. The pulsed direct-current stimulation induced immunogenic cell death of tumor cells (survival rate of 35.9 %), which promoted dendritic cells maturation, accelerated the process of antigen presentation to CAR-T cells and enhanced the systemic adaptive immune response. Furthermore, triboelectric immunotherapy promoted M1-like macrophage polarization, reduced regulatory T cells differentiation and reprogrammed the tumor immunosuppressive microenvironment, which ultimately enhanced the efficacy of CAR-T cells to eradicate nearly 60 % of NALM6 solid tumor mass. Notably, considering that triboelectric immunotherapy is a safe and effective drug-free antitumor strategy, the combined therapy did not increase the burden of double-medication on patients.

Single-cell transcriptome analysis reveals evolving tumour microenvironment induced by immunochemotherapy in nasopharyngeal carcinoma.

Combinatory therapeutic strategy containing immunochemotherapy as part of induction therapy components is one of the current trends in the treatment of high-risk metastatic locally advanced nasopharyngeal carcinoma (NPC). However, the mechanism underlying the heterogeneity of response at the single-cell level has not been underexplored. 18 bulks and 11 single-cell RNA sequencing from paired before-treatment and on-treatment samples in patients with treatment-naive high-risk metastatic locally advanced NPCs were obtained. Following quality control, a total of 87191 cells were included in the subsequence bioinformatics analysis. Immunochemotherapy was associated with on-treatment tumour microenvironment (TME) remodelling, including upregulation of anti-TMEs signatures, downregulation of pro-TMEs signatures, reversing CD8+ T exhaustion, and repolarizing proinflammatory TAMs. For the patients achieving a complete response, the cytotoxic activity of CD8+ T cells was stimulated and more interferon-gamma was provided, which would be the key for TAMs proinflammatory repolarization and eventually promote the CD8+ T cells maturation in turn. Among patients who did not reach complete response, differentiation and hypoxia signatures for endothelial cells were elevated after therapy. These patients exhibited higher levels of immune checkpoint genes in malignant cells at the baseline (before treatment), and decreased tumour antigen presentation activity, which may underlie the resistance mechanism to therapy. This study pictures a map of TME modulation following immunochemotherapy-based combination induction therapy and provides potential future approaches. Immunochemotherapy remodeled T cell phenotypes. For the patients achieving complete response, more interferon gamma was provided by CD8+ T cells after therapy, which would be the key for TAMs pro-inflammatory repolarization and eventually promote the CD8+ T cells maturation in turns. Among patients who did not reach complete response, malignant cells exhibited higher level of immune checkpoint genes before therapy, and decreased tumor antigen presentation activity, which may underlie the resistance mechanism to therapy.

Helios-Illuminating the way for lymphocyte self-control.

Transcription factor Helios, encoded by the IKZF2 gene, has an important role in regulatory T cells by stabilizing their suppressive phenotype. While Helios is prominently expressed in regulatory T cells, its expression extends beyond to include effector T cells, follicular regulatory T cells, B cells, and innate-like lymphocyte populations. Recent characterizations of patients with inborn error of immunity due to damaging IKZF2 variants coupled with translational research on lymphocytes from healthy individuals, have increased our understanding on Helios' multifaceted role in controlling the human adaptive immune system. A less studied role for Helios beyond the stabilizing of regulatory T cells has emerged in directing effector T cell maturation. In the absence of functional Helios, effector T cells acquire more inflammatory phenotype and are prone to senescence. Loss of Helios expression disrupts the regulation of the germinal centre reaction, often resulting in either hypogammaglobulinemia or B cell autoimmunity. This review summarizes findings from studies in both mice and men offering a comprehensive understanding of the impact of the transcription factor Helios on the adaptive immune system.