Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7.

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Derivation of functional thymic epithelial organoid lines from adult murine thymus.

Thymic epithelial cells (TECs) orchestrate T cell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term ex vivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single and double FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire-expressing medullary-like TECs upon RANK ligand + retinoic acid treatment. TEC organoids support T cell development from immature thymocytes in vitro as well as in vivo upon transplantation into athymic nude mice. This organoid-based platform allows in vitro study of TEC biology and offers a potential strategy for ex vivo T cell development.

In Vivo Induction of Regulatory T Cells Via CTLA-4 Signaling Peptide to Control Autoimmune Encephalomyelitis and Prevent Disease Relapse.

Regulatory T cells play a key role in immune tolerance to self-antigens, thereby preventing autoimmune diseases. However, no drugs targeting Treg cells have been approved for clinical trials yet. Here, a chimeric peptide is generated by conjugation of the cytoplasmic domain of CTLA-4 (ctCTLA-4) with dNP2 for intracellular delivery, dNP2-ctCTLA-4, and evaluated Foxp3 expression during Th0, Th1, Treg, and Th17 differentiation dependent on TGF-ß. The lysine motif of ctCTLA-4, not tyrosine motif, is required for Foxp3 expression for Treg induction and amelioration of experimental autoimmune encephalomyelitis (EAE). Transcriptome analysis reveals that dNP2-ctCTLA-4-treated T cells express Treg transcriptomic patterns with properties of suppressive functions. In addition, the molecular interaction between the lysine motif of ctCTLA-4 and PKC-η is critical for Foxp3 expression. Although both CTLA-4-Ig and dNP2-ctCTLA-4 treatment in vivo ameliorated EAE progression, only dNP2-ctCTLA-4 requires Treg cells for inhibition of disease progression and prevention of relapse. Furthermore, the CTLA-4 signaling peptide is able to induce human Tregs in vitro and in vivo as well as from peripheral blood mononuclear cells (PBMCs) of multiple sclerosis patients. These results collectively suggest that the chimeric CTLA-4 signaling peptide can be used for successful induction of regulatory T cells in vivo to control autoimmune diseases, such as multiple sclerosis.

Pathogenic function of bystander-activated memory-like CD4+ T cells in autoimmune encephalomyelitis.

T cells generate antigen-specific immune responses to their cognate antigen as a hallmark of adaptive immunity. Despite the importance of antigen-specific T cells, here we show that antigen non-related, bystander memory-like CD4+ T cells also significantly contribute to autoimmune pathogenesis. Transcriptome analysis demonstrates that interleukin (IL)-1ß- and IL-23-prime T cells that express pathogenic TΗ17 signature genes such as RORγt, CCR6, and granulocyte macrophage colony-stimulating factor (GM-CSF). Importantly, when co-transferred with myelin-specific 2D2 TCR-transgenic naive T cells, unrelated OT-II TCR-transgenic memory-like TH17 cells infiltrate the spinal cord and produce IL-17A, interferon (IFN)-γ, and GM-CSF, increasing the susceptibility of the recipients to experimental autoimmune encephalomyelitis in an IL-1 receptor-dependent manner. In humans, IL-1R1high memory CD4+ T cells are major producers of IL-17A and IFN-γ in response to IL-1ß and IL-23. Collectively, our findings reveal the innate-like pathogenic function of antigen non-related memory CD4+ T cells, which contributes to the development of autoimmune diseases.

Regulation of human T cell responses by dNP2-ctCTLA-4 inhibits human skin and microvessel graft rejection.

Manipulation of human T cell functioning by delivery of macromolecules such as DNA, RNA, or protein is limited, unless the human T cells have been stimulated or electropermeabilized. To achieve successful adaptation and survival of a grafted organ, the alloreactive T cells that induce graft rejection must be regulated. Corticosteroids, calcineurin inhibitors, and mTOR inhibitors, which are systemic immunosuppressants, are currently used for transplantation, with significant side effects. In this study, we demonstrated that a cell-permeable peptide (CPP), dNP2, could efficiently deliver proteins into human CD4 and CD8 T cells. We confirmed regulatory functioning of the cytoplasmic domain of CTLA-4 conjugated with dNP2 (dNP2-ctCTLA-4) in human T cell activation, proliferation, and chemokine receptor expression. We utilized a human skin allograft system in SCID/beige mice to examine whether dNP2-ctCTLA-4 could inhibit allograft rejection by controlling T cell responses. The grafted skin tissue inflammation, allogeneic T cell infiltration, and blood cytokine level was markedly reduced by dNP2-ctCTLA-4, resulting in successful transplantation. In addition, it also inhibited T cell alloresponses against microvessels formed form Bcl-2-transduced human umbilical vein endothelial cells implanted into Balb/c Rag1-/-/IL-2Rγ-/- double knockout (DKO) mice, assessed as reduced T cell infiltration and granzyme B expression. These results collectively suggest that dNP2 peptide conjugation offers a valuable tool for delivering macromolecules like proteins into human T cells, and dNP2-ctCTLA-4 is a novel agent that shows potential in controlling human T cell responses to allow successful adaptation of grafted tissues.

Regulation of chitinase-3-like-1 in T cell elicits Th1 and cytotoxic responses to inhibit lung metastasis.

Chitinase-3-like-1 (Chi3l1) is known to play a significant role in the pathogenesis of Type 2 inflammation and cancer. However, the function of Chi3l1 in T cell and its clinical implications are largely unknown. Here we show that Chi3l1 expression was increased in activated T cells, especially in Th2 cells. In addition, Chi3l1-deficient T cells are hyper-responsive to TcR stimulation and are prone to differentiating into Th1 cells. Chi3l1-deficient Th1 cells show increased expression of anti-tumor immunity genes and decreased Th1 negative regulators. Deletion of Chi3l1 in T cells in mice show reduced melanoma lung metastasis with increased IFNγ and TNFα-producing T cells in the lung. Furthermore, silencing of Chi3l1 expression in the lung using peptide-siRNA complex (dNP2-siChi3l1) efficiently inhibit lung metastasis with enhanced Th1 and CTL responses. Collectively, this study demonstrates Chi3l1 is a regulator of Th1 and CTL which could be a therapeutic target to enhance anti-tumor immunity.

dNP2-ctCTLA-4 inhibits German cockroach extract-induced allergic airway inflammation and hyper-responsiveness via inhibition of Th2 responses.

German cockroaches are major household allergens that can trigger allergic airway inflammatory diseases with sensitive T-cell responses. Although the use of immune modulatory biologics, such as antibodies, to mediate allergic responses has recently been examined, only systemic administration is available because of the size limitations on intranasal administration. Here we utilized a cell-permeable peptide, dNP2, to deliver the cytoplasmic domain of cytotoxic T-lymphocyte antigen-4 (ctCTLA-4) through the airway epithelium to modulate Th2 responses in a German cockroach extract (GCE)-induced allergic airway inflammation model. The intranasal delivery efficiency of the dNP2-dTomato protein to the lungs was higher in GCE-induced asthmatic lung parenchymal cells compared to the sham cells. Intranasal administration of the dNP2-ctCTLA-4 protein inhibited airway hyper-responsiveness and reduced airway inflammation and remodeling, including goblet cell metaplasia and collagen deposition around the bronchi. The number of infiltrated cells, including eosinophils, and the levels of IL-4, IL-5, IL-13 and IFN-γ in the lungs were significantly reduced, presumably owing to inhibition of Th2 differentiation. However, intranasal administration of CTLA4-Ig did not inhibit airway inflammation. These results collectively suggest that dNP2-ctCTLA-4 is an efficient intranasally applicable candidate biologic for treating allergic asthma.

Cell Type Preference of a Novel Human Derived Cell-Permeable Peptide dNP2 and TAT in Murine Splenic Immune Cells.

Cell-permeable peptides (CPPs) have been widely studied as an attractive drug delivery system to deliver therapeutic macromolecules such as DNA, RNA, and protein into cells. However, its clinical application is still limited and controversial due to the lack of a complete understanding of delivery efficiency in target cells. Previously we identified and characterized the novel and superior CPP, named dNP2, and here we comparatively analyzed intracellular delivery efficiency of dNP2 and TAT in various immune cells of mouse spleen to demonstrate their cell type preference. dNP2- or TAT-conjugated fluorescent proteins were most efficiently taken up by phagocytic cells such as dendritic cells and macrophages while little protein uptake was seen by lymphocytes including T cells, B cells, and NK cells. Interestingly CD8+ lymphoid dendritic cells and CD62LloCD44hi memory like T cell subsets showed significantly better uptake efficiency in vitro and in vivo relative to other dendritic cells or T cells, respectively. In addition, activated macrophages, T cells, and B cells took up the proteins more efficiently relative to when in the resting state. Importantly, only dNP2, not TAT, shows significant intracellular protein delivery efficiency in vivo. Collectively, this study provides important information regarding heterogeneous intracellular delivery efficiency of CPPs such as dNP2 and TAT with cell type preference in the spleen needed for its application in phagocytic cells or activated immune cells.

Use of Cell-Penetrating Peptides in Dendritic Cell-Based Vaccination.

Cell-penetrating peptides (CPPs) are short amino acids that have been widely used to deliver macromolecules such as proteins, peptides, DNA, or RNA, to control cellular behavior for therapeutic purposes. CPPs have been used to treat immunological diseases through the delivery of immune modulatory molecules in vivo. Their intracellular delivery efficiency is highly synergistic with the cellular characteristics of the dendritic cells (DCs), which actively uptake foreign antigens. DC-based vaccines are primarily generated by pulsing DCs ex vivo with various immunomodulatory antigens. CPP conjugation to antigens would increase DC uptake as well as antigen processing and presentation on both MHC class II and MHC class I molecules, leading to antigen specific CD4(+) and CD8(+) T cell responses. CPP-antigen based DC vaccination is considered a promising tool for cancer immunotherapy due to the enhanced CTL response. In this review, we discuss the various applications of CPPs in immune modulation and DC vaccination, and highlight the advantages and limitations of the current CPP-based DC vaccination.

dNP2 is a blood-brain barrier-permeable peptide enabling ctCTLA-4 protein delivery to ameliorate experimental autoimmune encephalomyelitis.

Central nervous system (CNS)-infiltrating effector T cells play critical roles in the development and progression of multiple sclerosis (MS). However, current drugs for MS are very limited due to the difficulty of delivering drugs into the CNS. Here we identify a cell-permeable peptide, dNP2, which efficiently delivers proteins into mouse and human T cells, as well as various tissues. Moreover, it enters the brain tissue and resident cells through blood vessels by penetrating the tightly organized blood-brain barrier. The dNP2-conjugated cytoplasmic domain of cytotoxic T-lymphocyte antigen 4 (dNP2-ctCTLA-4) negatively regulates activated T cells and shows inhibitory effects on experimental autoimmune encephalomyelitis in both preventive and therapeutic mouse models, resulting in the reduction of demyelination and CNS-infiltrating T helper 1 and T helper 17 cells. Thus, this study demonstrates that dNP2 is a blood-brain barrier-permeable peptide and dNP2-ctCTLA-4 could be an effective agent for treating CNS inflammatory diseases such as MS.

Cell membrane penetrating function of the nuclear localization sequence in human cytokine IL-1α.

Cytokines are released from the cell, bind to their receptors, and affect cellular responses. The precursor form of interleukin 1 alpha (pIL-1α) has a nuclear localization sequence (NLS) that causes it to be localized to the nucleus and regulate specific gene expression. The amino acids of the NLS are basic amino acid-rich sequences, as is the cell penetrating peptide (CPP), which has been widely studied as a way to deliver macromolecules into cells. Here, we hypothesized that the NLS in pIL-1α (pIL-1αNLS) can penetrate the cell membrane and it could deliver macromolecules such as protein in vivo. We characterized cell membrane penetration ability of pIL-1αNLS or its tandem repeated form (2pIL-1αNLS) to enhance its intracellular delivery efficiency. 2pIL-1αNLS showed comparable protein delivery efficiency to TAT-CPP and it mediates endocytosis following heparan sulfate interaction. 2pIL-1αNLS conjugated enhanced green fluorescence protein was localized to the nucleus and the cytoplasm. Intra-peritoneal administration of 2pIL-1αNLS conjugated dTomato protein showed remarkable in vivo intracellular delivery efficiency in various tissues including spleen, liver, and intestine in mice. Moreover, cytotoxicity of 2pIL-1αNLS was not observed even at 100 µM. Our results demonstrate cell membrane-penetrating function of NLS in pIL-1α, which can be used as a safe therapeutic macromolecular delivery peptide.

The use of a rigid tritopic phosphonic ligand for the synthesis of a robust honeycomb-like layered zirconium phosphonate framework.

1,3,5-Tris(4-phosphonophenyl)benzene was synthesized via a microwave heating assisted route and was subsequently used for the preparation of a new zirconium phosphonate with honeycomb-like structure displaying remarkable thermal stability and hydrolysis resistance.

Insights into the role of follicular helper T cells in autoimmunity.

Follicular helper T (TFH) cells are recently highlighted as their crucial role for humoral immunity to infection as well as their abnormal control to induce autoimmune disease. During an infection, naïve T cells are differentiating into TFH cells which mediate memory B cells and long-lived plasma cells in germinal center (GC). TFH cells are characterized by their expression of master regulator, Bcl-6, and chemokine receptor, CXCR5, which are essential for the migration of T cells into the B cell follicle. Within the follicle, crosstalk occurs between B cells and TFH cells, leading to class switch recombination and affinity maturation. Various signaling molecules, including cytokines, surface molecules, and transcription factors are involved in TFH cell differentiation. IL-6 and IL-21 cytokine-mediated STAT signaling pathways, including STAT1 and STAT3, are crucial for inducing Bcl-6 expression and TFH cell differentiation. TFH cells express important surface molecules such as ICOS, PD-1, IL-21, BTLA, SAP and CD40L for mediating the interaction between T and B cells. Recently, two types of microRNA (miRNA) were found to be involved in the regulation of TFH cells. The miR-17-92 cluster induces Bcl-6 and TFH cell differentiation, whereas miR-10a negatively regulates Bcl-6 expression in T cells. In addition, follicular regulatory T (TFR) cells are studied as thymus-derived CXCR5(+)PD-1(+)Foxp3(+) Treg cells that play a significant role in limiting the GC response. Regulation of TFH cell differentiation and the GC reaction via miRNA and TFR cells could be important regulatory mechanisms for maintaining immune tolerance and preventing autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Here, we review recent studies on the various factors that affect TFH cell differentiation, and the role of TFH cells in autoimmune diseases.

Identification of a novel cell-penetrating peptide from human phosphatidate phosphatase LPIN3.

Biomolecules such as proteins, DNA, and RNA are macromolecules and can not cross the cell membrane. However, cell-penetrating peptide (CPP) has been shown to deliver therapeutic biomolecules successfully into cells. The various and widely used CPPs including TAT, VP22, and Antp are mostly non-human originated CPPs, and are limited by their potential toxicity and immunogenicity. We report here on a newly identified novel cell-penetrating sequence (LPIN; RRKRRRRRK) from the nuclear localization sequence (NLS) of human nuclear phosphatase, LPIN3. LPIN-EGFP recombinant protein was concentration- and time-dependently delivered into cells and localized to the nucleus as well as the cytoplasm. It penetrated the cell membrane by lipid raft-mediated endocytosis by binding to heparan sulfate proteoglycan. LPIN-EGFP was successfully delivered into primary mouse splenocytes in vitro and it could be delivered into various tissues including liver, kidney, and intestine in mice after intra-peritoneal injection. This research suggests that LPIN-CPP could be used in a drug delivery system to deliver therapeutic biomolecules including peptides, proteins, DNA, and RNA and without the limitations of non-human originated CPPs such as TAT-CPP.

Crystalline Ge quantum dots embedded in SiO2 matrix synthesized by plasma immersion ion implantation.

A new plasma process, i.e. a combination of plasma immersion ion implantation and deposition (PIII&D) and high power impulse magnetron sputtering (HiPIMS), was developed to implant non-gaseous ions into material surfaces. The new process has the great advantage that thin film deposition and non-gaseous ion implantation can be achieved in a single plasma chamber. In this study, Ge ions were successfully implanted into SiO(2) thin film, which resulted in uniformly and homogeneously distributed crystalline Ge quantum dots (Ge-QDs) embedded in a SiO(2) matrix even without a further annealing process. Broader areas of application of PIII&D technology are envisaged with this newly developed process.

Successful removal of indigested shell using a rigid esophagoscope and a pneumatic lithotriptor.

Foreign body ingestion is a commonly encountered clinical problem in pediatric emergency cases. The authors report a case of an esophageal foreign body caused by the accidental ingestion of a shell in an 8-month-old girl. Endoscopic removal was attempted but failed because of the sharp margin of the shell and caused it to be deeply impacted into the esophageal wall. Accordingly, a pneumatic lithotriptor was inserted through a rigid esophagoscope and used to fragment the shell.

No enhancement of sensory and motor blockade by ketamine added to ropivacaine interscalene brachial plexus blockade.

BACKGROUND: Ketamine can enhance anesthetic and analgesic actions of a local anesthetic via a peripheral mechanism. The authors' goal was to determine whether or not ketamine added to ropivacaine in interscalene brachial plexus blockade prolongs postoperative analgesia. In addition, we wanted to determine the incidence of adverse-effects in patients undergoing hand surgery. METHODS: Sixty adults scheduled for forearm or hand surgery under the interscalene brachial plexus block were prospectively randomized to receive one of the solutions of the study. Group P received 0.5% ropivacaine 30 ml, group K received 0.5% ropivacaine 30 ml with 30 mg ketamine, and group C received 0.5% ropivacaine with 30 mg ketamine i.v. Loss of shoulder abduction, elbow flexion, wrist flexion and loss of pinprick in the C4-7 sensory dermatomes were assessed at 1-min intervals. Adverse-effects were assessed every 5 min. The duration of the sensory and motor blocks was assessed after operation. Adverse-effects were also recorded. RESULTS: The onset time of sensory or motor blockade and the duration of sensory or motor blockade were similar in all groups. Adverse-effects occurred in 44% of patients in group K and 94% of group C. CONCLUSION: This study suggests that 30 mg ketamine added to ropivacaine in the brachial plexus block does not improve the onset or duration of sensory block, but it does cause a relatively high incidence of adverse-effects. These two findings do not encourage the use of ketamine with local anesthetics for brachial plexus blockade.