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The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies,including acute myeloid leukemia (AML). Here,we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this,we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78,CD123,or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus,we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations. View Publication

The peripheral immune system is important in neurodegenerative diseases,both in protecting and inflaming the brain,but the underlying mechanisms remain elusive. Alzheimer’s Disease is commonly preceded by a prodromal period. Here,we report the presence of large Aβ aggregates in plasma from patients with mild cognitive impairment ( n = 38). The aggregates are associated with low level Alzheimer’s Disease-like brain pathology as observed by 11 C-PiB PET and 18 F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aβ aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aβ. Hydrodynamic calculations suggest Aβ aggregates associate with vessel walls of the cortical capillaries. In turn,we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis. Subject terms: Protein aggregation,Neuroimmunology,Dementia View Publication

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with a complex and multifactorial etiology,making it challenging to treat. While recent advances in immunomodulatory biologics,such as antitumor necrosis factor-α (TNF-α) antibodies,have shown moderate success,systemic administration of antibody therapeutics may lead to several adverse effects,including the risk of autoimmune disorders due to systemic cytokine depletion. Transient RNA interference using exogenous short interfering RNA (siRNA) to regulate target gene expression at the transcript level offers an alternative to systemic immunomodulation. However,siRNAs are susceptible to premature degradation and have poor cellular uptake. Graphene oxide (GO) nanoparticles have been shown to be effective nanocarriers for biologics due to their reduced cytotoxicity and enhanced bioavailability. In this study,we evaluate the therapeutic efficacy of GO mediated TNF-α_siRNA using in vitro models of chronic inflammation generated by treating murine small intestines (enteroids) and large intestines (colonoids) with inflammatory agents IL-1β,TNF-α,and LPS. The organotypic mouse enteroids and colonoids developed an inflammatory phenotype similar to that of IBD,characterized by impaired epithelial homeostasis and an increased production of inflammatory cytokines such as TNF-α,IL-1β,and IL-6. We assessed siRNA delivery to these inflamed organoids using three different GO formulations. Out of the three,small-sized GO with polymer and dendrimer modifications (smGO) demonstrated the highest transfection efficiency,which led to the downregulation of inflammatory cytokines,indicating an attenuation of the inflammatory phenotype. Moreover,the transfection efficiency and inflammation-ameliorating effects could be further enhanced by increasing the TNF-α_siRNA/smGO ratio from 1:1 to 3:1. Overall,the results of this study demonstrate that ex vivo organoids with disease-specific phenotypes are invaluable models for assessing the therapeutic potential of nanocarrier-mediated drug and biologic delivery systems. View Publication

The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However,the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs,no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process,we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study,we used a sized-based,label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (ie,>3 million cells/minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining,flow cytometry analysis,and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the “downstream processing” of cell manufacturing workflow,ensuring better quality and safety of transplanted cells. View Publication

RAB7,encoded by RAB7A in humans and Rab7 in mice,is a small GTPase that catalyzes endosome maturation. It mediates NF-κB activation through the assembly of intracellular membrane signalosomes in stimulated normal B cells and plays a B cell-intrinsic role in the antibody response in mice. Here we show RAB7A transcripts are expressed in primary diffuse large B-cell lymphomas (DLBCLs),and that RAB7 protein expression is heightened in activated human tonsil B cells as well as in DLBCL and Burkitt lymphoma cell lines. Treating these cell lines with CID1067700,a selective small-molecule RAB7 inhibitor,results in a dose-dependent decrease in cell growth,associated with impaired proliferation and survival. CID1067700 also suppressed tumor development from Daudi cells,a Burkitt lymphoma cell line,in Foxn1nu/nu nude mice. The inhibitory effect of CID1067700 on Daudi cell growth in vitro is further enhanced by methyl-β-cyclodextrin,which disrupts plasma membrane lipid rafts,and by FX1,a BCL6 inhibitor. These findings,together with the unfavorable prognosis of DLBCL patients showing high RAB7A expression,suggest that targeting RAB7 is a promising therapeutic approach for mature B cell-derived lymphomas. View Publication

Solid tumor malignancy (STM) patients experience increased risk of breakthrough SARS-CoV-2 infection owing to reduced COVID-19 vaccine immunogenicity. However,the underlying immunological causes of impaired neutralization remain poorly characterized. Furthermore,non-neutralizing antibody functions can contribute to reduced disease severity but remain understudied within high-risk populations. We dissected polyfunctional antibody responses in STM patients and age-matched controls who received adenoviral vector- or mRNA-based COVID-19 vaccine regimens. Elevated inflammatory biomarkers,including agalactosylated IgG,interleukin (IL)-6,IL-18,and an expanded population of CD11c−CD21− double negative 3 (DN3) B cells were observed in STM patients and were associated with impaired neutralization. In contrast,mRNA vaccination induced Fc effector functions that were comparable in patients and controls and were cross-reactive against SARS-CoV-2 variants. These data highlight the resilience of Fc functional antibodies and identify systemic inflammatory biomarkers that may underpin impaired neutralizing antibody responses,suggesting potential avenues for immunomodulation via rational vaccine design. View Publication

Reactivation of the latent viral reservoirs is crucial for a cure of HIV/AIDS. However,current latency reversing agents are inefficient,and the endogenous factors that have the potential to reactivate HIV in vivo remain poorly understood. To identify natural activators of latent HIV-1,we screened a comprehensive peptide/protein library derived from human hemofiltrate,representing the entire blood peptidome,using J-Lat cell lines harboring transcriptionally silent HIV-1 GFP reporter viruses. Fractions potently reactivating HIV-1 from latency contained human Retinol Binding Protein 4 (RBP4),the carrier of retinol (Vitamin A). We found that retinol-bound holo-RBP4 but not retinol-free apo-RBP4 strongly reactivates HIV-1 in a variety of latently infected T cell lines. Functional analyses indicate that this reactivation involves activation of the canonical NF-κB pathway and is strengthened by JAK/STAT5 and JNK signalling but does not require retinoic acid production. High levels of RBP4 were detected in plasma from both healthy individuals and people living with HIV-1. Physiological concentrations of RBP4 induced significant viral reactivation in latently infected cells from individuals on long-term antiretroviral therapy with undetectable viral loads. As a potent natural HIV-1 latency-reversing agent,RBP4 offers a novel approach to activating the latent reservoirs and bringing us closer to a cure. Subject terms: Preclinical research,Infectious diseases View Publication

BackgroundAutoantibodies against envelope (Env) protein encoded by human endogenous retrovirus group K (HERV-K) are prevalent in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE),but it remains unclear which proviruses are responsible for this autoantigen. It also remains poorly understood how the transcription of HERV-K loci is regulated in cells that can produce Env.ResultsWe aligned our neutrophil RNA sequencing data to the new telomere-to-telomere reference genome and found uniquely mapping transcripts from HERV-K101,K102,K104,K108,K109,K117 and ERVK5,of which only K102,K108,and K109 encode an intact Env. Expression of K102 and K108 were higher in SLE than in healthy donors or RA (padj < 0.05). Transcripts from these proviruses increased in response to interferon-α in monocytes and neutrophils from RA patients and healthy donors,but not in SLE,presumably because they have chronically elevated type I interferons in vivo. Indeed,HERV-K expression was significantly higher in SLE patients with high type I interferon gene signature. Tumor necrosis factor-α and other cytokines and TLR ligands also induced HERV-K102 and K108 transcripts. Interferon-α also increased detectable Env protein in monocytes,macrophages,and neutrophils from RA patients. Among the genes for epigenetic silencers of HERV-K,only TRIM28 was significantly decreased in SLE patients with high interferons (padj = 0.00024).ConclusionsOur data establish a role for interferons in maintaining increased HERV-K expression in SLE and suggest that interferons or other cytokines can upregulate HERV-K to similar levels in RA. A transient increase may also accompany normal immune responses,suggesting that endogenous retroviruses may have been co-opted for efficient immune responses.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00371-y. Key points Expression of HERV-K provirus is elevated in neutrophils from IFN-positive SLETNFα,IFN,and other cytokines induce similar HERV-K expression also in RAHealthy donor myeloid cells respond only transiently with HERV-K transcription Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00371-y. View Publication

T cell dysfunction is a pivotal driving factor in autoimmune diseases,yet its underlying regulatory mechanisms remain incompletely understood. The role of long non-coding RNAs (lncRNAs) in immune regulation has gradually been recognized,although their functional mechanisms in T cells remain elusive. This study focuses on lncBADR (LncRNA Branched-chain Amino acids Degradation Regulator),elucidating its mechanism by which it regulates branched-chain amino acids (BCAAs) metabolism to influence T cell effector functions. Mice with specific knockout of lncBADR (T celllncBADR−/−) exhibited markedly ameliorated experimental autoimmune encephalomyelitis (EAE) symptoms. Mechanistic investigations revealed that lncBADR inhibits BCAAs degradation by binding to the enzymes Mccc1 and Pcca,leading to the accumulation of BCAAs within T-cells. This,in turn,activates the mTOR-Stat1 signaling pathway,promoting IFN-γ secretion and exacerbating EAE pathology. In contrast,knockout of lncBADR restored BCAAs degradation,significantly reducing IFN-γ secretion in T cells and suppressing their pathogenic functions. Further studies demonstrated that high-BCAAs feeding partially reversed the protective effects of lncBADR knockout,indicating that lncBADR plays a crucial role in autoimmune inflammation by regulating BCAAs metabolism. This study offers new insights into targeting lncBADR or modulating BCAAs metabolism as potential therapeutic strategies for autoimmune diseases.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03538-9. View Publication

Primary human endothelial cells represent an essential tool to model endothelial dysfunction and to screen interventions for its treatment. Here,we developed a protocol for the synchronous isolation of primary human saphenous vein endothelial cells (HSaVEC),human internal thoracic artery endothelial cells (HITAEC),and human microvascular endothelial cells (HMVEC) from SV and ITA utilized as conduits during coronary artery bypass graft surgery and from subcutaneous adipose tissue excised while providing an access to the heart. Treatment by collagenase type IV and magnetic separation with anti-CD31-antibody-coated beads ensured relatively high efficiency of the isolation (≈60% for HSaVEC,≈50% for HITAEC,and ≈20% for HMVEC) and high purity (≥99%) of isolated ECs within ≈2 weeks (HSaVEC),≈2–3 weeks (HITAEC),and ≈3–4 weeks (HMVEC). A colorimetric assay of cell viability and proliferation,as well as real-time bioimpedance monitoring using the xCELLigence instrument,demonstrated high proliferative activity in HSaVEC,HITAEC,and HMVEC,whilst the in vitro tube formation assay indicated their angiogenic potential. The isolation of HSaVEC,HITAEC,and HMVEC from patients undergoing coronary artery bypass graft surgery is a promising option to investigate endothelial heterogeneity,to interrogate endothelial responses to various stresses,and to pinpoint the optimal approaches for restoring endothelial homeostasis,thereby reproducing them within the bedside-to-bench-to-bedside concept. View Publication