技术资料

FMS-related tyrosine kinase 3 ligand (FLT3L),encoded by FLT3LG,is a hematopoietic factor essential for the development of natural killer (NK),B cells,and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant,with a history of various recurrent infections,including severe cutaneous warts. The patients’ bone marrow was hypoplastic,with low levels of hematopoietic progenitors,particularly myeloid and B-cell precursors. Counts of B cells,monocytes,and DCs were low in the patients’ blood,whereas the other blood subsets,including NK cells,were affected only moderately,if at all. The patients had normal counts of Langerhans cells and dermal macrophages in the skin but lacked dermal DCs. Thus,FLT3L is required for B-cell and DC development in mice and humans. However,unlike its murine counterpart,human FLT3L is required for the development of monocytes but not NK cells. View Publication

The fine control of synaptic function requires robust trans-synaptic molecular interactions. However,it remains poorly understood how trans-synaptic bridges change to reflect the functional states of the synapse. Here,we develop optical tools to visualize in firing synapses the molecular behavior of two trans-synaptic proteins,LGI1 and ADAM23,and find that neuronal activity acutely rearranges their abundance at the synaptic cleft. Surprisingly,synaptic LGI1 is primarily not secreted,as described elsewhere,but exo- and endocytosed through its interaction with ADAM23. Activity-driven translocation of LGI1 facilitates the formation of trans-synaptic connections proportionally to the history of activity of the synapse,adjusting excitatory transmission to synaptic firing rates. Accordingly,we find that patient-derived autoantibodies against LGI1 reduce its surface fraction and cause increased glutamate release. Our findings suggest that LGI1 abundance at the synaptic cleft can be acutely remodeled and serves as a critical control point for synaptic function. View Publication

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC),which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover,immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids,which will advance the use of kidney organoids for transplantation research. View Publication

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines. View Publication

CRISPR-Cas9 genome editing often induces unintended,large genomic rearrangements,posing potential safety risks. However,there are no methods for mitigating these risks. Using long-read individual-molecule sequencing (IDMseq),we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs,while depleting or overexpressing RPA increases or reduces LD frequency,respectively. Interestingly,small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR,suggesting new strategies for safer and more precise genome editing. The online version contains supplementary material available at 10.1186/s12915-024-01896-z. View Publication

Allogeneic cell therapies,such as those involving macrophages or Natural Killer (NK) cells,are of increasing interest for cancer immunotherapy. However,the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges,such as required cell pre-activation and inefficiency in transduction,which hinder the assessment of preclinical efficacy and clinical translation. In our study,we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein,which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors,this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood,as well as freshly obtained monocytes,which were differentiated to M1 macrophages as well as B cells from multiple donors,achieving up to 80% reporter gene expression within three days post-transduction. Importantly,KoRV-based transduction does not compromise the expression of crucial immune cell receptors,nor does it impair immune cell functionality,including NK cell viability,proliferation,cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion,our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics,requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics,expediting their availability to patients in need. Subject terms: Genetic transduction,Tumour immunology,Immunotherapy,Genetic vectors,Innate immune cells View Publication

In preclinical studies on the T-cell engager MP0533,the authors show that targeting multiple tumor-associated antigens may lead to better selectivity and efficacy in eliminating leukemic stem cells and blasts,representing a promising therapeutic strategy for AML. View Publication

Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here,we report the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients – one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study. View Publication

The implementation of antiretroviral therapy (ART) has effectively restricted the transmission of Human Immunodeficiency Virus (HIV) and improved overall clinical outcomes. However,a complete cure for HIV remains out of reach,as the virus persists in a stable pool of infected cell reservoir that is resistant to therapy and thus a main barrier towards complete elimination of viral infection. While the mechanisms by which host proteins govern viral gene expression and latency are well-studied,the emerging regulatory functions of non-coding RNAs (ncRNA) in the context of T cell activation,HIV gene expression and viral latency have not yet been thoroughly explored. Here,we report the identification of the Cytoskeleton Regulator (CYTOR) long non-coding RNA (lncRNA) as an activator of HIV gene expression that is upregulated following T cell stimulation. Functional studies show that CYTOR suppresses viral latency by directly binding to the HIV promoter and associating with the cellular positive transcription elongation factor (P-TEFb) to activate viral gene expression. CYTOR also plays a global role in regulating cellular gene expression,including those involved in controlling actin dynamics. Depletion of CYTOR expression reduces cytoplasmic actin polymerization in response to T cell activation. In addition,treating HIV-infected cells with pharmacological inhibitors of actin polymerization reduces HIV gene expression. We conclude that both direct and indirect effects of CYTOR regulate HIV gene expression. View Publication

SARS-CoV-2 Omicron variant has evolved into sublineages. Here,we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However,there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population. Subject areas: Immunology,Immune response,Virology View Publication

N6‐methyladenosine (m6A) is an RNA modification involved in RNA processing and widely found in transcripts. In cancer cells,m6A is upregulated,contributing to their malignant transformation. In this study,we analyzed gene expression and m6A modification in cancer tissues,ducts,and acinar cells derived from pancreatic cancer patients using MeRIP‐seq. We found that dozens of RNAs highly modified by m6A were detected in cancer tissues compared with ducts and acinar cells. Among them,the m6A‐activated mRNA TCEAL8 was observed,for the first time,as a potential marker gene in pancreatic cancer. Spatially resolved transcriptomic analysis showed that TCEAL8 was highly expressed in specific cells,and activation of cancer‐related signaling pathways was observed relative to TCEAL8‐negative cells. Furthermore,among TCEAL8‐positive cells,the cells expressing the m6A‐modifying enzyme gene METTL3 showed co‐activation of Notch and mTOR signaling,also known to be involved in cancer metastasis. Overall,these results suggest that m6A‐activated TCEAL8 is a novel marker gene involved in the malignant transformation of pancreatic cancer. View Publication

Timothy syndrome (TS) is a severe,multisystem disorder characterized by autism,epilepsy,long-QT syndrome and other neuropsychiatric conditions 1 . TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A,as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1,including delayed channel inactivation,prolonged depolarization-induced calcium rise,impaired interneuron migration,activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A 2 – 6 . We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and,following transplantation,in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed 7,we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons,suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly,these experiments illustrate how a multilevel,in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. Subject terms: Autism spectrum disorders,Development of the nervous system View Publication