技术资料

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However,it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics,particularly those with Latin American ancestry. Using dPCR,we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both,suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification,early detection of cancers,and reduction of associated cancer health disparities. Rates of Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) continue to rise in Hispanics and Latinos. Authors developed a digital PCR assay to quantify activation-induced cytidine deaminase activity at risk loci involved in cancer etiology that may contribute to this health disparity. View Publication

SummarySensing of extracellular ATP (eATP) controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules,such as the release channel Pannexin 1 (Panx1). Whether Panx1 controls CD8+ T cell immune responses in vivo,however,has not been previously addressed. Here,we report that T-cell-specific Panx1 is needed for CD8+ T cell responses to viral infections and cancer. We found that CD8-specific Panx1 promotes both effector and memory CD8+ T cell responses. Panx1 favors initial effector CD8+ T cell activation through extracellular ATP (eATP) export and subsequent P2RX4 activation,which helps promote full effector differentiation through extracellular lactate accumulation and its subsequent recycling. In contrast,Panx1 promotes memory CD8+ T cell survival primarily through ATP export and subsequent P2RX7 engagement,leading to improved mitochondrial metabolism. In summary,Panx1-mediated eATP export regulates effector and memory CD8+ T cells through distinct purinergic receptors and different metabolic and signaling pathways. Graphical abstract Highlights•The hemichannel Panx1 promotes in vivo effector and memory CD8+ T cell responses•Panx1 promotes effector CD8+ T cells via eATP and lactate extracellular accumulation•Panx1 promotes memory CD8+ T cell survival by eATP export and activation of AMPK Natural sciences; Biological sciences; Biochemistry; Immunology; Immune response; Immune system View Publication

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways. However,targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here,we have developed an mRNA therapeutic for the lower lung using one-component Ionizable Amphiphilic Janus Dendrimers (IAJDs) as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA,transforming growth factor-beta (TGF-β),to produce transient protein expression in the lower regions of the lung. This study highlights IAJD’s potential for precise,effective,and safe delivery of TGF-β mRNA to the lung. This delivery system offers a promising approach for targeting therapeutics to the specific tissues,a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease. View Publication

Trained immunity may play a role in vaccine-induced protection against infections. We showed that the highly efficacious recombinant VZV-gE zoster vaccine (RZV) generated trained immunity in monocytes,natural killer (NK) cells,and dendritic cells (DCs) and that the less efficacious live zoster vaccine did not. RZV stimulated ex vivo gE-specific monocyte,DC and NK cell responses that did not correlate with CD4 + T-cell responses. These responses were also elicited in purified monocyte and NK cell cocultures stimulated with VZV-gE and persisted above prevaccination levels for ≥ 4 years post-RZV administration. RZV administration also increased ex vivo heterologous monocyte and NK cell responses to herpes simplex and cytomegalovirus antigens. ATAC-seq analysis and ex vivo TGFβ1 supplementation and inhibition experiments demonstrated that decreased tgfβ1 transcription resulting from RZV-induced chromatin modifications may explain the development of monocyte trained immunity. The role of RZV-trained immunity in protection against herpes zoster and other infections should be further studied. View Publication

BackgroundDiffuse large B-cell lymphoma (DLBCL) represents a prevalent malignant tumor,with approximately 40% of patients encountering treatment challenges or relapse attributed to rituximab resistance,primarily due to diminished or absent CD20 expression. Our prior research identified PDK4 as a key driver of rituximab resistance through its negative regulation of CD20 expression. Further investigation into PDK4’s resistance mechanism and the development of advanced exosome nanoparticle complexes may unveil novel resistance targets and pave the way for innovative,effective treatment modalities for DLBCL.MethodsWe utilized a DLBCL-resistant cell line with high PDK4 expression (SU-DHL-2/R). We infected it with short hairpin RNA (shRNA) lentivirus for RNA sequencing,aiming to identify significantly downregulated mRNA in resistant cells. Techniques including immunofluorescence,immunohistochemistry,and Western blotting were employed to determine PDK4’s localization and expression in resistant cells and its regulatory role in phosphorylation of Histone deacetylase 8 (HDAC8). Furthermore,we engineered advanced exosome nanoparticle complexes,aCD20@ExoCTX/siPDK4,through cellular,genetic,and chemical engineering methods. These nanoparticles underwent characterization via Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM),and their cellular uptake was assessed through flow cytometry. We evaluated the nanoparticles’ effects on apoptosis in DLBCL-resistant cells and immune cells using CCK-8 assays and flow cytometry. Additionally,their capacity to counteract resistance and exert anti-tumor effects was tested in a resistant DLBCL mouse model.ResultsWe found that PDK4 initiates HDAC8 activation by phosphorylating the Ser-39 site,suppressing CD20 protein expression through deacetylation. The aCD20@ExoCTX/siPDK4 nanoparticles served as effective intracellular delivery mechanisms for gene therapy and monoclonal antibodies,simultaneously inducing apoptosis in resistant DLBCL cells and triggering immunogenic cell death in tumor cells. This dual action effectively reversed the immunosuppressive tumor microenvironment,showcasing a synergistic therapeutic effect in a subcutaneous mouse tumor resistance model.ConclusionsThis study demonstrates that PDK4 contributes to rituximab resistance in DLBCL by modulating CD20 expression via HDAC8 phosphorylation. The designed exosome nanoparticles effectively overcome this resistance by targeting the PDK4/HDAC8/CD20 pathway,representing a promising approach for drug delivery and treating patients with Rituximab-resistant DLBCL.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-024-02057-0. View Publication

Synthetic Notch (synNotch) receptors are genetically encoded,modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control,applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus,we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally,we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues. Synthetic Notch (synNotch) receptors are genetically encoded,modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Here the authors apply synNotch receptors to spatially control differentiation of endothelial and skeletal muscle cells in a multicellular construct on assorted biomaterials. View Publication

Immune evasion is one of the critical hallmarks of malignant tumors,especially non-small cell lung cancer (NSCLC). Emerging findings have illustrated the roles of N6-methyladenosine (m6A) on NSCLC immune evasion. Here,this study investigated the function and underlying mechanism of m6A reader YTH domain family protein 3 (YTHDF3) on NSCLC immune evasion. YTHDF3 was found to be highly expressed in NSCLC tissue and act as an independent prognostic factor for overall survival. Functionally,up-regulation of YTHDF3 impaired the CD8+ T antitumor activity to deteriorate NSCLC immune evasion,while YTHDF3 silencing recovered the CD8+ T antitumor activity to inhibit immune evasion. Besides,YTHDF3 up-regulation reduced the apoptosis of NSCLC cells. Mechanistically,PD-L1 acted as the downstream target for YTHDF3,and YTHDF3 could upregulate the transcription stability of PD-L1 mRNA. Overall,YTHDF3 targeted PD-L1 to promote NSCLC immune evasion partially through escaping effector cell cytotoxicity CD8+ T mediated killing and antitumor immunity. In summary,this study provides an essential insight for m6A modification on CD8+ T cell-mediated antitumor immunity in NSCLC,which might inspire an innovation for lung cancer tumor immunotherapy. View Publication

Streptococcus pneumoniae (Sp),a leading cause of community-acquired pneumonia,can spread from the lung into the bloodstream to cause septicemia and meningitis,with a concomitant three-fold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor,pneumolysin (PLY),triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids,including hepoxilin A3 (HXA3),a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination,we utilized bronchial stem cell-derived air-liquid interface (ALI) cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration,monolayer disruption,and concomitant Sp barrier breach. In contrast,PMN transmigration in response to the non-eicosanoid chemoattractant fMLP did not lead to epithelial disruption or bacterial translocation. Correspondingly,HXA3-ME but not fMLP increased release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus,HXA3 promotes barrier disrupting PMN transmigration and NE release,pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia. View Publication

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients,we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence,we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition,we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays,CD69,CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155,one important TIGIT-ligand,is reliably expressed on AMLs,we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally,our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype,whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively,our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-024-03766-7. View Publication

AbstractBackgroundCancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating cancers such as hematological malignancies and breast cancers. However,approaches to obtain cancer‐reactive TCR‐T cells have been unsuccessful.MethodsHere,we developed a novel strategy to screen for cancer‐targeted TCR‐T cells using a special humanized mouse model with person‐specific immune fingerprints. Rare steady‐state circulating hematopoietic stem and progenitor cells were expanded via three‐dimensional culture of steady‐state peripheral blood mononuclear cells,and then the expanded cells were applied to establish humanized mice. The human immune system was evaluated according to the kinetics of dendritic cells,monocytes,T‐cell subsets,and cytokines. To fully stimulate the immune response and to obtain B‐cell precursor NAML‐6‐ and triple‐negative breast cancer MDA‐MB‐231‐targeted TCR‐T cells,we used the inactivated cells above to treat humanized mice twice a day every 7 days. Then,human T cells were processed for TCR β‐chain (TRB) sequencing analysis. After the repertoires had been constructed,features such as the fraction,diversity,and immune signature were investigated.ResultsThe results demonstrated an increase in diversity and clonality of T cells after treatment. The preferential usage and features of TRBV,TRBJ,and the V–J combination were also changed. The stress also induced highly clonal expansion. Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice.ConclusionsWe constructed a personalized humanized mouse model to screen cancer‐targeted TCR‐T pools. Our platform provides an effective source of cancer‐targeted TCR‐T cells and allows for the design of patient‐specific engineered T cells. It therefore has the potential to greatly benefit cancer treatment. Cancer‐targeted T‐cell receptor T (TCR‐T) cells hold promise in treating malignancies but with limited source. We applied steady‐state peripheral blood mononuclear cells via three‐dimensional culture to construct humanized mouse model for cancer‐targeted TCR‐T repertoire screening. View Publication

IntroductionBispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets,bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody,which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time.MethodsThis study comprised antibodies purification,Elisa assay for antigen binding,cytotoxicity assays,T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo.ResultsWe present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin β2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially,BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo.DiscussionMoreover,PHE1 domain was further shortened with D14G and R41S mutations,named PHE-S,and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo,bringing more possibilities for the development and optimization of different bsAbs. To sum up,PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy. View Publication

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However,it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here,Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells,type 1 conventional dendritic cells were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8+ T cell responses to Cryptosporidium. View Publication