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Purpose: To investigate the therapeutic potential of inducible pluripotent stem cell (hiPSC)-based vascular repair,we evaluated two vascular reparative cell populations,CD34+ cells derived from hiPSC (hiPSC-CD34+) and endothelial colony forming cells (ECFCs) derived from hiPSC (iPS-ECFCs),alone and in combination,in a type 2 diabetic (db/db) mouse model of DR. Methods: hiPSC-CD34+ cells (1 × 104) or iPSC- ECFCs (1 × 105) alone or in combination (1.1 × 105) were injected into the vitreous of immunosuppressed db/db mice with six months of established diabetes. One month post-injection,mice underwent electroretinography (ERG) and optical coherence tomography (OCT) to evaluate functional and structural retinal recovery with iPSC administration. Immunohistochemistry (IHC) was used to assess recruitment and incorporation of cells into the retinal vasculature. Retinas from the experimental groups were analyzed using Functional Proteomics via Reverse Phase Protein Array (RPPA). Results: Functional assessment via ERG demonstrated significant improvements in retinal response in the diabetic cohorts treated with either hiPSC-derived CD34+ cells or hiPSC-ECFCs. Retinal thickness,assessed by OCT,was restored to near-nondiabetic levels in mice treated with hiPSC-CD34+ cells alone and the combination group,whereas hiPSC-ECFCs alone did not significantly affect retinal thickness. One month following intravitreal injection,hiPSC-CD34+ cells were localized to perivascular regions,whereas hiPSC-ECFCs were observed to integrate directly into the retinal vasculature. RPPA analysis revealed interaction-significant changes,and this was interpreted as a combination-specific,non-additive host responses (m6A,PI3K–AKT–mTOR,glycolysis,endothelial junction pathways). Conclusions: The studies support that injection of hiPSC-CD34+ cells and hiPSC-ECFCs,both individually and in combination,showed benefit; however,iPSC combination-specific effects were identified by measurement of retinal thickness and by RPPA. View Publication

Retinoic acid (RA) plays a key role in mucosal immune regulation and tolerance,with implications for inflammatory bowel disease (IBD). However,its effects have not been extensively studied in humanized in vitro models that recapitulate epithelial–immune interactions. We established a 3D in vitro small intestinal model composed of three epithelial cell types,naïve CD4+ T cells,and monocyte/dendritic cell (M/DC) precursors derived from CD34+ umbilical cord blood hematopoietic stem/progenitor cells. The epithelial microenvironment strongly suppressed monocyte/DC differentiation and T cell activation,indicating a regulatory role of epithelial-derived signals. Retinoic acid (RA) priming of M/DC precursors induced CD103+CD11b+Sirp1α− regulatory DCs and promoted a shift from naive to memory-type T cells. Upon addition of pro-inflammatory cytokines (TNF-α,IFN-γ,IL-1β),the model mimicked an inflamed intestinal state,resulting in CD14+CD16+ inflammatory monocytes and increased T cell activation (CD25+CD69+). RA-primed DCs modestly counterbalanced T cell activation and IBD-like responses,even under inflammatory conditions. Flow cytometry and clustering analysis revealed distinct immune cell phenotypes depending on RA exposure and cytokine context. This model provides a reproducible and physiologically relevant human system to study RA-mediated immune programming in the intestinal mucosa and may support the development of novel therapeutic strategies for IBD and related inflammatory conditions. Statistical differences were evaluated using ANOVA with Tukey’s post-hoc test (n = 4; p < 0.05). View Publication

Interleukin-6 (IL-6) is a multifunctional cytokine that plays important roles in inflammation. Several studies have shown that IL-6 regulates various aspects of T cell function,including the differentiation of CD4+ T cells into the pro-inflammatory Th17 subset. Given the tight link between T cell metabolism and function,and the role of IL-6 in regulating cellular metabolism across tissues,we investigated the role of IL-6 signaling in Th17 cell metabolism. Using T cell specific IL-6 receptor (IL-6R) conditional knockout mice and littermate controls,we found that IL-6R signaling regulates the proportions of CD4+ and CD8+ T cells and drives CD4+ T cell differentiation into Th17 cells. We also found that IL-6R signaling is required for Th17 cell glycolytic metabolism. In T cell-specific IL-6R knockout mice,Th17 cells had reduced glucose uptake and glycolysis,as well as decreased expression of key glycolytic enzymes,while showing increased basal oxygen consumption. However,we also found that IL-6R signaling enhanced oxidative capacity and mitochondrial coupling efficiency in Th17 T cells. Importantly,inhibition of lactate dehydrogenase using FX11 selectively impaired Th17 cell differentiation with minimal effects on Treg cells. These findings suggest that targeting metabolic pathways regulated by IL-6R signaling can selectively inhibit inflammatory Th17 responses,offering a potential strategy for controlling IL-6 mediated inflammation. View Publication

Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges,including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer,functional,and tunable alternative for gene silencing without the need for DNA editing. As a proof of concept for multiplex gene silencing,we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors,enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models. Silenced (S) M5CAR T cells retained antitumor functionality comparable to,and in some cases exceeding,that of KO cells. In vivo,S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs). Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells,with potential advantages in potency,persistence,metastasis prevention,and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks. View Publication

IntroductionAmid the persistent threat of future pandemics,the continuous evolution of SARS-CoV-2 exposed critical challenges for vaccine efficacy and therapeutic interventions,highlighting the need for rapid and adaptable approaches to respond to immune escape variants.MethodsHere,we report the use of immortalized B cell libraries from human peripheral blood mononuclear cells (PBMCs) and tonsil tissues to uncover B cell clones exhibiting cross-reactive neutralization against various SARS-CoV-2 variants and perform directed evolution of immortalized B cell clones to produce antibodies with improved binding and neutralization against emerging SARS-CoV-2 variants.ResultsImmortalization of PBMC and tonsil-derived human B cells was achieved through transduction with retroviral vectors encoding apoptosis inhibitors,yielding transduction efficiencies of 67.5% for PBMCs and 50.2% for tonsil-derived cells. Analysis revealed that immortalized B cell libraries produced with this method retain diverse immunoglobulin isotype representations. Through high-throughput functional screening of approximately 40,000 B cells per library,we identified 12 unique clones with neutralization activity for SARS-CoV-2,leading to selection of monoclonal antibodies with robust neutralization activity against Delta and BA.5 variants. We applied our directed evolution approach to libraries generated by ex vivo AID-induced somatic hypermutation (SHM) of immortalized B cell clones to enhance the affinity and cross-reactivity,resulting in improved binding and neutralization potency to escape variants such as EG.5.1 and JN.1. Furthermore,we engineered a bi-paratopic antibody combining KBA2401,a broadly neutralizing antibody binding to highly conserved epitope on Spike-RBD,and KBA2402,a broadly binding non-neutralizing antibody,resulting in enhanced potency against SARS-CoV-2 variant JN.1 and KP.3.DiscussionOur findings illustrate the use of immortalized B cell libraries for development of therapeutics that adapt to viral evolution and highlight the application of ex vivo directed evolution in refining antibody responses against emerging immune escape SARS-CoV-2 variants. The approach here described offers a promising pathway for rapid therapeutic development in the face of evolving viral threats. View Publication

BackgroundLung cancer has become one of the most fatal cancers at present. Traditional treatments showed limited therapeutic effects on lung cancer. The phototherapy has emerged as a powerful approach for lung cancer treatment. Aggregation-induced emission luminogens (AIEgens) exhibit excellent optical performance such as strong fluorescence,enhanced reactive oxygen species (ROS) generation,and effective thermal effect after aggregation,which show great potential in phototherapy. However,the disadvantages including hydrophobicity,low specificity,and short circulation lifetime limited their efficacy on cancer therapy.MethodsWe developed a biomimetic AIEgens constructed using CD8+ T cells membrane to camouflage the AIEgen C41H37N2O3S2 (named BITT) nanoparticles (termed TB). The prepared TB improved the tumor accumulation of AIEgen by PD-1/PD-L1 recognition on the CD8+ T and LLC cell membranes,respectively.ResultsThe prepared TB showed improved binding efficiency,photothermal effects,and ROS generation ability to kill the lung cancer cells. TB also showed improved circulation lifetime and excellent tumor targeting ability,leading to effective phototherapy and immunotherapy in vivo based on BITT and the CD8+ T cell-derived membranes. Based on the AIE and immune checkpoint blockade (ICB) strategies,TB enhanced the antitumor activities of lung cancer by phototherapy and immunotherapy.ConclusionThe present work developed a type of biomimetic AIEgens,which overcame the inherent limitations of conventional AIEgens and leveraged immune recognition for targeted tumor accumulation. Furthermore,the integration of AIE-driven phototherapy with immune checkpoint blockade demonstrated potent synergistic antitumor efficacy,establishing a promising combinatorial strategy against aggressive lung malignancies. View Publication

Affinity maturation and differentiation of B cells in the germinal center (GC) are tightly controlled by epigenetically regulated transcription programs,but the underlying mechanisms are only partially understood. Here we show that Cfp1,an integral component of the histone methyltransferase complex Setd1A/B,is critically required for GC responses. Cfp1 deficiency in activated B cells greatly impairs GC formation with diminished proliferation,somatic hypermutation and affinity maturation. Mechanistically,Cfp1 deletion reduces H3K4me3 marks at a subset of cell cycle and GC-related genes and impairs their transcription. Importantly,Cfp1 promotes the expression of transcription factors MEF2B and OCA-B and the Bcl6 enhancer-promoter looping for its efficient induction. Accordingly,Cfp1-deficient GCB cells upregulate IRF4 and preferentially differentiate into plasmablasts. Furthermore,Cfp1 ablation upregulates a panel of pre-memory genes with elevated H3K4me3 and leads to markedly expanded memory B populations. In summary,our study reveals that Cfp1-safeguarded epigenetic regulation ensures proper dynamics of GCB cells for affinity maturation and prevents the pre-mature exit from GC as memory cells. Cellular differentiation decisions,such as fates of B cells following entry into the germinal centres,are governed by epigenetically and transcriptionally regulated paths for bifurcating cell fates. Here the authors show that CFP1 is a master epigenetic regulator of activated B cells and controls their hypermutation and affinity maturation via the histone methyltransferase complex Setd1A/B. View Publication

Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However,the large size of CRISPR-based epigenome editors poses a challenge to their broad use in biomedical research and as future therapies. Here,we present Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi,DNMT3A-3L-dCas9,CRISPRoff) and activator (TET1-dCas9) as ribonucleoprotein complexes into human cells to modulate gene expression. After rational engineering,we show that RENDER induces durable epigenetic silencing of endogenous genes across various human cell types,including primary T cells. Additionally,we apply RENDER to epigenetically repress endogenous genes in human stem cell-derived neurons,including the reduction of the neurodegenerative disease associated V337M-mutated Tau protein. Together,our RENDER platform advances the delivery of CRISPR-based epigenome editors into human cells,broadening the use of epigenome editing in fundamental research and therapeutic applications. Epigenome editing programs gene silencing without inducing DNA breaks but challenges in delivery into human cells limit its broader use. Here,the authors present the RENDER platform,which uses virus-like particles to enable CRISPR-based epigenome editing for durable gene silencing in human cells. View Publication

Metastatic breast cancer remains largely incurable,and the mechanisms driving the transition from primary to metastatic breast cancer remain elusive. We analyzed the complex landscape of estrogen receptor (ER)-positive breast cancer primary and metastatic tumors using scRNA-seq data from twenty-three female patients with either primary or metastatic disease. By employing single-cell transcriptional profiling of unpaired patient samples,we sought to elucidate the genetic and molecular mechanisms underlying changes in the metastatic tumor ecosystem. We identified specific subtypes of stromal and immune cells critical to forming a pro-tumor microenvironment in metastatic lesions,including CCL2+ macrophages,exhausted cytotoxic T cells,and FOXP3+ regulatory T cells. Analysis of cell-cell communication highlights a marked decrease in tumor-immune cell interactions in metastatic tissues,likely contributing to an immunosuppressive microenvironment. In contrast,primary breast cancer samples displayed increased activation of the TNF-α signaling pathway via NF-kB,indicating a potential therapeutic target. Our study comprehensively characterizes the transcriptional landscape encompassing primary and metastatic breast cancer. View Publication

Natural killer (NK) cells,which are key components of the innate immune response,are crucial for ensuring the efficacy of vaccines as they rapidly eliminate infected cells and enhance the adaptive immune response,ensuring robust and lasting protection. In this report,we investigated the effect of Cladophora wrightiana var. minor (CW) extract,a marine alga,in activating NK cells,as an adjuvant to inactivated A/Puerto Rico/8/34 H1N1 influenza vaccine (iPR8). In vitro,CW extract significantly enhanced the level of activation markers CD69 and CD107a on NK cells and triggered intracellular secretion of interferon gamma (IFN‐γ) and granzyme B (GrB),indicating effective NK cell stimulation and cytotoxic function. In vivo,CW extract promoted substantial NK cell recruitment and activation,resulting in higher NK cell populations and elevated post‐immunization levels of activation markers. Additionally,CW extract increased IFN‐γ and GrB production in CD8+ T cells,highlighting its broader impact on the immune response. We also found direct evidence that CW‐activated NK cells and dendritic cells (DCs) interacted with and induced the activation of immature DCs and resting NK cells,respectively. These findings suggest that CW extract is a promising adjuvant for nasal vaccines,enhancing cellular immunity by activating NK cells and supporting interactions with DCs and CD8+ T cells. Cladophora wrightiana var. minor (CW) extract,administered as an adjuvant with inactivated influenza virus (iPR8),stimulates both innate and adaptive immune responses. CW enhances NK cell activation,cytotoxic function,and reciprocal crosstalk with dendritic cells,while also promoting CD8+ T cell responses and antigen‐specific IgG production. These findings support CW as a potent nasal vaccine adjuvant capable of boosting protective immunity. https://app.biorender.com/illustrations/6893f7d09e3fc89e9d953f76?slideId=2a6a42b6‐a3d9‐400a‐b839‐fa297b3108c5. View Publication