Search

Engineered natural killer (NK) cell-based therapies have been potentially broadly applicable and exhibited promising results in clinical trials, particularly in the fight against cancers. NK cell immunotherapy however always remains variable. One major obstacle is the inhibitory pathway including PD1/PDL1, providing tumor cells an escape mechanism from immunosurveillance. In this regard, we rationally designed a chimeric switch-receptor (CSR) PD1-DAP10-41BB, which comprising the ectodomain of PD1 fused to the co-stimulatory receptor DAP10 and 41BB. Therefore, by exchanging the transmembrane and cytoplasmic tail of PD1 with positive costimulatory molecules DAP10 and 41BB signaling domains, the negative PD1/PDL1 signal pathway was thus converted into a positive one. This CSR-expressing NK92 cells showed a typical parental NK92 phenotype and improved cytotoxicity against human lung cancer H1299 cells. Besides, the expression of CSR elicited a significant increase of effector molecules such as perforin and granzymes, which can induce apoptosis of H1299 cells. More importantly, in the solid tumor cell H1299-bearing mice model, the CSR-modified NK92 cells significantly inhibited tumor growth. Collectively, we demonstrated that expression of PD1-DAP10-41BB augmented NK92-cell activation and killing in vitro and in vivo , which provides a considerable avenue of using NK-tailored chimeric receptor engineered NK92 cells to treat a wide range of solid tumors. • A novel NK-tailored chimeric switch-receptor PD1-DAP10-41BB is rationally designed. • Modified NK92 cells mediate the clearance of H1299 cells by triggering apoptosis. • PD1-DAP10-41BB-expressing NK92 cells elicit in vivo tumor control of H1299 cells. [ABSTRACT FROM AUTHOR]

Preliminary results and emerging data have shown that lipid droplet high (LDhi) immunosuppressive cells accumulate in tumour tissues. By tracking and phenotypic profiling of LDhi cells, we find that LDhiCD19+, LDhiCD11b+, and LDhiLy6G+ immune cell populations appear in the spleen, thymus, and tumour tissues in a syngeneic tumour model. Using a contact‐dependent reporter system, we discover a LDhiCCR7hi immunosuppressive cell population that migrates from tumour tissues to the spleen and thymus. Hence, we engineered a family of chimeric antigen receptor‐modified macrophages (CAR‐Ms) that direct macrophages to CCR7‐positive cells and show that the cytosolic domain from Mer receptor tyrosine kinase (MerTK) triggers tumour cell cytotoxicity by the CAR‐Ms. In vivo, CCR7‐targeted CAR‐Ms suppressed tumour growth and prolonged survival by preventing metastasis and by inducing systemic anti‐tumour immunity through retarding the migration of LDhiCCR7hi immunosuppressive cells from tumour tissues to distal immune organs, indicating an important role for CCR7 in tumour cell‐induced immune tolerance. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Objectives: synNotch receptors can get different inputs about the outside environment into cells; however, synNotch system doesn't work for soluble ligands. This study aims to explore the activation efficiency of different ligand carriers on synNotch system. Methods: SynNotch-based contact-dependent activation system was constructed in 293T cells (293T-synNotch), then the synNotch receptor ligands eGFP proteins were provided by three different carriers: 400 nm Ni magnetic beads (Mag Ni-eGFP), 300 nm carboxyl modified Fe3O4 magnetic beads (Mag COOH-deGFP), and 4T1 cell (4T1-mGFP). Three eGFP carriers were incubated with 293T-synNotch, the activation efficiencies of these three eGFP carriers were evaluated by fluorescence microscopy. Results: The 293T-synNotch cell activated by Mag Ni-eGFP shows no observable red fluorescence; when Mag COOH-deGFP are incubated with 293T-synNotch, about 50‰ cells with red fluorescence appeared, also eGFP proteins have not dropped off from the Mag COOH-deGFP beads; 4T1-mGFP cells show the highest activation efficiency, about 21% synNotch cells are activated, when 4T1-mGFP and 293T-synNotch are incubated at high cell density. Conclusion: The 4T1-mGFP cells are the most effective ligands for synNotch receptor activation. [ABSTRACT FROM AUTHOR]

Cell-based immunotherapy continues to be a promising avenue for cancers that standard therapy has failed. Although the specificity, avidity, and efficacy of infused cells have improved, immunocytotherapy still faces substantial hurdles. To this end, we developed a structure-based rational design approach and constructed a novel Dual Targeting Chimeric Receptor (DTCR) PD1-DAP10/NKG2D comprising the truncated ectodomain of PD1 fused to a key co-stimulatory receptor DAP10, and subsequently harnessed the activating receptor NKG2D, which evaluated the capacity of solid tumor cell killing. Retroviral transduction of DTCR dramatically increased NK92 cell surface expression of PD1 and NKG2D, which boosted robust cytotoxicity against human gastric cell SGC-7901. Chimeric receptor DTCR stimulation elicited a significant increase of TNF-α and TRAIL, which can trigger apoptosis of SGC-7901 cells. More importantly, DTCR-NK92 cells had considerable antitumor activity in the solid tumor cell SGC-7901-bearing mice model. Collectively, we demonstrated that expression of DTCR markedly augmented the cytotoxic potential of NK92 cells against solid tumor cells, and this potentially promising treatment modality will facilitate clinical translation of potent NK-tailored chimeric receptor strategy for a generalized cellular therapy that may be conducive to treat a wide range of solid tumors. • A novel NK-tailored bispecific chimeric receptor PD1-DAP10/NKG2D is rationally designed with a structure-based approach. • Engineered NK92 cells can not only trigger apoptosis, but seemingly induce pyroptosis of SGC-7901 solid tumor cells. • Expressing PD1-DAP10/NKG2D mediates robust in vitro cytotoxicity and in vivo tumor control of NK92 cells. [ABSTRACT FROM AUTHOR]

The potential adverse effects of graphene oxide (GO) on organisms have received great attention. However, the underlying molecular mechanisms for response to GO exposure are still largely unclear. Canonical Wnt/β-catenin signaling is an evolutionarily conserved signaling, and plays a key role in the development. Using assay system of Caenorhabditis elegans , we investigated the potential involvement of canonical Wnt/β-catenin signaling pathway in the regulation of response to GO. GO exposure caused the damage on the functions of both primary and secondary targeted organs by dysregulating the expression of β-catenin BAR-1, APR-1 and GSK-3 in APC complex, Dishevelled proteins of DSH-1, and DSH-2, and Frizzled receptors of MOM-5 and CFZ-2 in the Wnt/β-catenin signaling pathway. Based on genetic evidence, we further raised a signaling cascade of MOM-5/CFZ-2-DSH-1/DSH-2-APR-1/GSK-3-BAR-1 in the Wnt/β-catenin signaling pathway in the regulation of GO toxicity. The homeobox protein EGL-5, an important downstream target of Wnt/β-catenin signaling, regulated GO toxicity by modulating expression of the genes required for the control of oxidative stress. Moreover, β-catenin BAR-1 acted in parallel with insulin or p38 MAPK signaling to regulate the GO toxicity. Therefore, our results suggest the crucial function of Wnt/β-catenin signaling pathway in the regulation of GO toxicity in organisms. [ABSTRACT FROM AUTHOR]

The microRNA (miRNA) let-7 is an important miRNA identified in Caenorhabditis elegans and has been shown to be involved in the control of innate immunity. The underlying molecular mechanisms for let-7 regulation of innate immunity remain largely unclear. In this study, we investigated the molecular basis for intestinal let-7 in the regulation of innate immunity. Infection with Pseudomonas aeruginosa PA14 decreased let-7::GFP expression. Intestine- or neuron-specific activity of let-7 was required for its function in the regulation of innate immunity. During the control of innate immune response to P. aeruginosa PA14 infection, SDZ-24 was identified as a direct target for intestinal let-7. SDZ-24 was found to be predominantly expressed in the intestine, and P. aeruginosa PA14 infection increased SDZ-24::GFP expression. Intestinal let-7 regulated innate immune response to P. aeruginosa PA14 infection by suppressing both the expression and the function of SDZ-24. Knockout or RNA interference knockdown of sdz-24 dampened the resistance of let-7 mutant to P. aeruginosa PA14 infection. Intestinal overexpression of sdz-24 lacking 3’-UTR inhibited the susceptibility of nematodes overexpressing intestinal let-7 to P. aeruginosa PA14 infection. In contrast, we could observed the effects of intestinal let-7 on innate immunity in P. aeruginosa PA14 infected transgenic strain overexpressing sdz-24 containing 3’-UTR. In the intestine, certain SDZ-24-mediated signaling cascades were formed for nematodes against the P. aeruginosa PA14 infection. Our results highlight the crucial role of intestinal miRNAs in the regulation of the innate immune response to pathogenic infection. [ABSTRACT FROM AUTHOR]

Biological barrier plays a crucial role for organisms against the possible toxicity from engineered nanomaterials (ENMs). Graphene oxide (GO) has been proven to cause potential toxicity on organisms. However, the molecular mechanisms for intestinal barrier of animals against GO toxicity are largely unclear. Usingin vivoassay system ofCaenorhabditis elegans, we found that mutation of genes encoding core p38 mitogen-activated protein kinase (MAPK) signaling pathway caused susceptible property to GO toxicity and enhanced translocation of GO into the body of nematodes. Genetic assays indicated that SKN-1/Nrf functioned downstream of p38 MAPK signaling pathway to regulate GO toxicity and translocation. Transcription factor of SKN-1 could regulate GO toxicity and translocation at least through function of its targeted gene ofgst-4encoding one of phase II detoxification proteins. Moreover, intestine-specific RNA interference (RNAi) assay demonstrated that the p38 MAPK-SKN-1/Nrf signaling cascade could function in intestine to regulate GO toxicity and intestinal permeability in GO exposed nematodes. Therefore, p38 MAPK-SKN-1/Nrf signaling cascade may act as an important molecular basis for intestinal barrier against GO toxicity in organisms. Exposure to GO induced significantly increased expression of genes encoding p38 MAPK-SKN-1/Nrf signaling cascade, which further implies that the identified p38 MAPK-SKN-1/Nrf signaling cascade may encode a protection mechanism for nematodes in intestine to be against GO toxicity. [ABSTRACT FROM AUTHOR]

• With the versatility, CRISPR/Cas9 has dominated the realm of genome editing. • Various challenges of CRISPR/Cas9 remain to be overcome for patients with cancer. • CRISPR/Cas9 is revolutionizing and opening a new era for cellular immunotherapies. Immunotherapy has become one of the most promising strategies in cancer therapies. Among the therapeutic alternatives, genetically engineered NK/T cell therapies have emerged as powerful and innovative therapeutic modalities for cancer patients with precise targeting and impressive efficacy. Nonetheless, this approach still faces multiple challenges, such as immunosuppressive tumor microenvironment, exhaustion of immune effector cells in tumors, off-target effects manufacturing complexity, and poor infiltration of effector cells, all of which need to be overcome for further utilization to cancers. Recently, CRISPR/Cas9 genome editing technology, with the goal of enhancing the efficacy and increasing the availability of engineered effector cell therapies, has shown considerable potential in the novel strategies and options to overcome these limitations. Here we review the current progress of the applications of CRISPR in cancer immunotherapy. Furthermore, we discuss issues related to the NK/T cell applications, gene delivery methods, efficiency, challenges, and implications of CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

In this study, we investigated the possible involvement of Wnt signals in the control of graphene oxide (GO) toxicity using the in vivo assay system of Caenorhabditis elegans. In nematodes, the Wnt ligands, CWN-1, CWN-2, and LIN-44, were found to be involved in the control of GO toxicity. Mutation of cwn-1 or lin-44 gene induced a resistant property to GO toxicity and resulted in the decreased accumulation of GO in the body of nematodes, whereas mutation of cwn-2 gene induces a susceptible property to GO toxicity and an enhanced accumulation of GO in the body of nematodes. Genetic interaction assays demonstrated that mutation of cwn-1 or lin-44 was able to suppress the susceptibility to GO toxicity shown in the cwn-2 mutants. Loss-of-function mutations in all three of these Wnt ligand genes resulted in the resistance of nematodes to GO toxicity. Moreover, the Wnt ligands might differentially regulate the toxicity and translocation of GO through different mechanisms. These findings could be important in understanding the function of Wnt signals in the regulation of toxicity from environmental nanomaterials. [ABSTRACT FROM AUTHOR]

Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies in vitro . Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies in vitro ., Competing Interests: The authors report no conflicts of interest regarding the publication of this article., (Copyright © 2022 Zhiyuan Niu et al.)

We performed a systematic identification of microRNAs (miRNAs) involved in the control of innate immunity. We identified 7 novel miRNA mutants with altered survival, colony forming in the body, and expression pattern of putative antimicrobial genes after Pseudomonas aeruginosa infection. Loss-of-function mutation of mir-45, mir-75, mir-246, mir-256, or mir-355 induced resistance to P. aeruginosa infection, whereas loss-of-function mutation of mir-63 or mir-360 induced susceptibility to P. aeruginosa infection. DAF-2 in the insulin signaling pathway acted as a target for intestinal mir-355 to regulate innate immunity. mir-355 functioned as an important link between p38 MAPK signaling pathway and insulin signaling pathway in the regulation of innate immunity. Our results provide an important molecular basis for further elucidation of the functions of various miRNAs in the regulation of innate immunity.