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1.
Cell ; 175(2): 530-543.e24, 2018 10 04.
Article in English | MEDLINE | ID: mdl-30220458

ABSTRACT

The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.


Subject(s)
Nephritis, Interstitial/virology , Parvovirus/isolation & purification , Parvovirus/pathogenicity , Animals , Australia , Disease Progression , Female , Fibrosis/pathology , Fibrosis/virology , Humans , Kidney/metabolism , Kidney/physiology , Male , Mice , Mice, Inbred C57BL , Nephritis, Interstitial/physiopathology , North America , Parvoviridae Infections/metabolism
2.
Nat Immunol ; 14(6): 564-73, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23603794

ABSTRACT

Type 2 immunity is critical for defense against cutaneous infections but also underlies the development of allergic skin diseases. We report the identification in normal mouse dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depended on interleukin 7 (IL-7) and constitutively produced IL-13. Intravital multiphoton microscopy showed that dermal ILC2 cells specifically interacted with mast cells, whose function was suppressed by IL-13. Treatment of mice deficient in recombination-activating gene 1 (Rag1(-/-)) with IL-2 resulted in the population expansion of activated, IL-5-producing dermal ILC2 cells, which led to spontaneous dermatitis characterized by eosinophil infiltrates and activated mast cells. Our data show that ILC2 cells have both pro- and anti-inflammatory properties and identify a previously unknown interactive pathway between two innate populations of cells of the immune system linked to type 2 immunity and allergic diseases.


Subject(s)
Dermatitis/immunology , Immunity, Innate/immunology , Lymphocytes/immunology , Skin/immunology , Animals , Cell Communication/immunology , Cells, Cultured , Dermatitis/genetics , Dermatitis/metabolism , Dermis/cytology , Dermis/immunology , Dermis/metabolism , Eosinophils/immunology , Eosinophils/metabolism , Flow Cytometry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/immunology , Homeodomain Proteins/metabolism , Immunity, Innate/genetics , Interleukin-13/immunology , Interleukin-13/metabolism , Interleukin-17/immunology , Interleukin-17/metabolism , Interleukin-2/immunology , Interleukin-2/pharmacology , Lymphocytes/drug effects , Lymphocytes/metabolism , Mast Cells/immunology , Mast Cells/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton , Skin/metabolism , Videotape Recording
3.
Biophys J ; 123(15): 2328-2342, 2024 Aug 06.
Article in English | MEDLINE | ID: mdl-38327056

ABSTRACT

T cells migrate constitutively with a polarized morphology, underpinned by signaling compartmentalization and discrete cytoskeletal organizations, giving rise to a dynamic and expansive leading edge, distinct from the stable and constricted uropod at the rear. In vivo, the motion and function of T cells at various stages of differentiation is highly directed by chemokine gradients. When cognate ligands bind chemokine receptors on their surface, T cells respond by reorientating their polarity axis and migrating toward the source of the chemokine signal. Despite the significance of such chemotactic repolarization to the accurate navigation and function of T cells, the precise signaling mechanisms that underlie it remain elusive. Notably, it remained unclear whether the distribution of chemokine receptors on the T cell surface is altered during repolarization. Here, we developed parallel cell-secreted and microfluidics-based chemokine gradient delivery methods and employed both fixed imaging and live lattice light-sheet microscopy to investigate the dynamics of chemokine receptor CCR5 on the surface of primary murine CD8+ T cells. Our findings show that, during constitutive migration, chemokine receptor distribution is largely isotropic on the T cell surface. However, upon exposure to a CCL3 gradient, surface chemokine receptor distributions exhibit a transient bias toward the uropod. The chemokine receptors then progressively redistribute from the uropod to cover the T cell surface uniformly. This study sheds new light on the dynamics of surface chemokine receptor distribution during T cell repolarization, advancing our understanding of the signaling of immune cells in the complex chemokine landscapes they navigate.


Subject(s)
Receptors, CCR5 , Animals , Mice , Receptors, CCR5/metabolism , Cell Membrane/metabolism , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , Mice, Inbred C57BL , Cell Movement , Receptors, Chemokine/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/cytology , Cell Polarity
4.
J Cell Sci ; 133(5)2020 03 05.
Article in English | MEDLINE | ID: mdl-32041902

ABSTRACT

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8+ T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activation-induced gains in fluorescence were also observed in naïve and CD4+ T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.


Subject(s)
Actin Cytoskeleton , CD8-Positive T-Lymphocytes , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Gene Expression Regulation , Mice
5.
Immunol Cell Biol ; 99(3): 299-308, 2021 03.
Article in English | MEDLINE | ID: mdl-33002241

ABSTRACT

Staphylococcus aureus is one of the most common causes of community- and hospital-acquired bacterial infection worldwide. While neutrophils play an important role in anti-S. aureus immune defense, the role of adaptive immunity is less clear. In this study, we generated a model antigen-expressing S. aureus strain to investigate the dynamics and magnitude of T cell immune responses against this pathogen. We demonstrate that S. aureus is delivered to the draining lymph nodes (LNs) by lymphatic flow immediately after intradermal inoculation. There, the bacterium initiates CD8+ cytotoxic T lymphocyte (CTL) proliferation via activating LN-resident dendritic cells. Large numbers of neutrophils are recruited to the draining LNs to engulf bacteria; however, neutrophil depletion did not impact on CTL proliferation, despite increasing bacterial burden. Tissue-resident memory T cells were formed in the skin at bacteria-inoculated sites. Yet, blood and tissue-resident memory T cells failed to prevent secondary cutaneous S. aureus infection. Our study defines the delivery kinetics of S. aureus from the skin and suggests that CTLs are dispensable for protection against skin infections.


Subject(s)
Staphylococcal Infections , Staphylococcus aureus , Animals , Antigens, Bacterial , CD8-Positive T-Lymphocytes , Humans , Lymph Nodes , Mice , Mice, Inbred C57BL , Skin
6.
Hepatology ; 70(6): 2047-2061, 2019 12.
Article in English | MEDLINE | ID: mdl-31099022

ABSTRACT

Recombinant adeno-associated viral (rAAV) vectors are highly promising vehicles for liver-targeted gene transfer, with therapeutic efficacy demonstrated in preclinical models and clinical trials. Progressive familial intrahepatic cholestasis type 3 (PFIC3), an inherited juvenile-onset, cholestatic liver disease caused by homozygous mutation of the ABCB4 gene, may be a promising candidate for rAAV-mediated liver-targeted gene therapy. The Abcb4-/- mice model of PFIC3, with juvenile mice developing progressive cholestatic liver injury due to impaired biliary phosphatidylcholine excretion, resulted in cirrhosis and liver malignancy. Using a conventional rAAV strategy, we observed markedly blunted rAAV transduction in adult Abcb4-/- mice with established liver disease, but not in disease-free, wild-type adults or in homozygous juveniles prior to liver disease onset. However, delivery of predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression due to hepatocyte proliferation in the growing liver. Conclusion: A hybrid vector system, combining the high transduction efficiency of rAAV with piggyBac transposase-mediated somatic integration, was developed to facilitate stable human ABCB4 expression in vivo and to correct juvenile-onset chronic liver disease in a murine model of PFIC3. A single dose of hybrid vector at birth led to life-long restoration of bile composition, prevention of biliary cirrhosis, and a substantial reduction in tumorigenesis. This powerful hybrid rAAV-piggyBac transposon vector strategy has the capacity to mediate lifelong phenotype correction and reduce the tumorigenicity of progressive familial intrahepatic cholestasis type 3 and, with further refinement, the potential for human clinical translation.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/deficiency , Cholestasis, Intrahepatic/prevention & control , DNA Transposable Elements/genetics , Dependovirus/genetics , Genetic Therapy , Liver Neoplasms, Experimental/prevention & control , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Humans , Male , Mice , Transduction, Genetic , ATP-Binding Cassette Sub-Family B Member 4
7.
J Immunol ; 193(5): 2087-95, 2014 Sep 01.
Article in English | MEDLINE | ID: mdl-25070847

ABSTRACT

Naive T cell activation is normally restricted to the lymphoid organs, in part because of their limited ability to migrate into the parenchyma of peripheral tissues. The liver vasculature is unique, however, and circulating leukocytes within the hepatic sinusoids have direct access to liver-resident cells, which include an abundant population of Kupffer cells. It is well accepted that recognition of cognate Ag within the liver leads to naive CD8(+) T cell activation in situ, but it is unclear whether the liver also supports naive CD4(+) T cell activation. In this study, we show that naive CD4(+) T cells can be activated to proliferate in the liver when cognate Ag expression is induced in hepatocytes by recombinant adeno-associated viral vectors. Ag-specific retention and activation of naive CD4(+) T cells within the liver are independent of lymphoid tissues but dependent on a clodronate liposome-sensitive population of liver-resident phagocytic cells. To our knowledge, this study provides the first unequivocal evidence that naive CD4(+) T cells can be activated in a nonlymphoid organ. It also gives critical insight into how CD4(+) T cells specific for Ag expressed in the liver are recruited to participate in protective or pathological responses during hepatotropic infections and autoimmune liver disease.


Subject(s)
Autoimmune Diseases/immunology , CD4-Positive T-Lymphocytes/immunology , Kupffer Cells/immunology , Liver Diseases/immunology , Liver/immunology , Animals , Autoimmune Diseases/genetics , Autoimmune Diseases/pathology , Bone Density Conservation Agents/pharmacology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Clodronic Acid/pharmacology , Kupffer Cells/pathology , Liposomes , Liver/pathology , Liver Diseases/genetics , Liver Diseases/pathology , Lymphocyte Activation , Mice , Mice, Transgenic
8.
J Allergy Clin Immunol ; 136(6): 1653-1663.e7, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26025126

ABSTRACT

BACKGROUND: Group 2 innate lymphoid cells (ILC2) have been implicated in the pathogenesis of allergic lung diseases. However, the upstream signals that regulate ILC2 function during pulmonary inflammation remain poorly understood. ILC2s have been shown to respond to exogenous IL-2, but the importance of endogenous IL-2 in ILC2 function in vivo remains unclear. OBJECTIVE: We sought to understand the role of IL-2 in the regulation of ILC2 function in the lung. METHODS: We used histology, flow cytometry, immunohistochemistry, ELISA, and quantitative PCR with knockout and reporter mice to dissect pulmonary ILC2 function in vivo. We examined the role of ILC2s in eosinophilic crystalline pneumonia, an idiopathic type 2 inflammatory lung condition of mice, and the effect of IL-2 deficiency on this disease. We determined the effect of IL-2 administration on pulmonary ILC2 numbers and function in mice in the steady state and after challenge with IL-33. RESULTS: We discovered an unexpected role for innate cell-derived IL-2 as a major cofactor of ILC2 function during pulmonary inflammation. Specifically, we found that IL-2 was essential for the development of eosinophilic crystalline pneumonia, a type 2 disease characterized by increased numbers of activated ILC2s. We show that IL-2 signaling serves 2 distinct functions in lung ILC2s, namely promoting cell survival/proliferation and serving as a cofactor for the production of type 2 cytokines. We further demonstrate that group 3 innate lymphoid cells are an innate immune source of IL-2 in the lung. CONCLUSION: Innate cell-derived IL-2 is a critical cofactor in regulating ILC2 function in pulmonary type 2 pathology.


Subject(s)
Interleukin-2/immunology , Lymphocytes/immunology , Pneumonia/immunology , Pulmonary Eosinophilia/immunology , Animals , Cytokines/blood , Cytokines/immunology , DNA-Binding Proteins/genetics , Female , Homeodomain Proteins/genetics , Immunity, Innate/immunology , Interleukin-2/genetics , Lung/cytology , Lung/immunology , Lung/pathology , Male , Mice , Mice, Knockout , Pneumonia/blood , Pulmonary Eosinophilia/blood , Spleen/immunology
9.
Liver Int ; 35(1): 240-53, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24576072

ABSTRACT

BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) has been implicated in the processes of embryogenesis, tissue fibrosis and carcinogenesis. Transforming growth factor-ß (TGF-ß) has been identified as a key driver of EMT and plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). The aim was to identify microRNA (miR) expression in TGF-ß-induced hepatocyte EMT. METHODS: We treated a human hepatocyte cell line PH5CH8 with TGF-ß to induce an EMT-like change in phenotype and then identified dysregulated miRs using TaqMan Low Density Arrays. MiR expression was altered using miR-181a mimic and inhibitor in the same system and gene changes were identified using TaqMan gene arrays. MiR-181a gene expression was measured in human and mouse cirrhotic or HCC liver tissue samples. Gene changes were identified in rAAV-miR-181a-expressing mouse livers using TaqMan gene arrays. RESULTS: We identified miR-181a as a miR that was significantly up-regulated in response to TGF-ß treatment. Over-expression of a miR-181a mimic induced an in vitro EMT-like change with a phenotype similar to that seen with TGF-ß treatment alone and was reversed using a miR-181a inhibitor. MiR-181a was shown to be up-regulated in experimental and human cirrhotic and HCC tissue. Mouse livers expressing rAAV-miR-181a showed genetic changes associated with TGF-ß signalling and EMT. CONCLUSIONS: MiR-181a had a direct effect in inducing hepatocyte EMT and was able to replace TGF-ß-induced effects in vitro. MiR-181a was over-expressed in cirrhosis and HCC and is likely to play a role in disease pathogenesis.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Epithelial-Mesenchymal Transition/physiology , Gene Expression Regulation/drug effects , Hepatocytes/physiology , Liver Cirrhosis/metabolism , Liver Neoplasms/metabolism , MicroRNAs/metabolism , Animals , Cell Line , Hepatocytes/cytology , Humans , In Vitro Techniques , Mice , MicroRNAs/genetics , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology
10.
Proc Natl Acad Sci U S A ; 108(40): 16735-40, 2011 Oct 04.
Article in English | MEDLINE | ID: mdl-21933957

ABSTRACT

Although most self-reactive T cells are eliminated in the thymus, mechanisms to inactivate or control T cells specific for extrathymic antigens are required and exist in the periphery. By investigating the site in which autoreactive T cells are tolerized, we identify a unique mechanism of peripheral deletion in which naïve autoreactive CD8 T cells are rapidly eliminated in the liver after intrahepatic activation. T cells actively invade hepatocytes, enter endosomal/lysosomal compartments, and are degraded. Blockade of this process leads to accumulation of autoreactive CD8 T cells in the liver and breach of tolerance, with the development of autoimmune hepatitis. Cell into cell invasion, or emperipolesis, is a long-observed phenomenon for which a physiological role has not been previously demonstrated. We propose that this "suicidal emperipolesis" is a unique mechanism of autoreactive T-cell deletion, a process critical for the maintenance of tolerance.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Death/immunology , Emperipolesis/immunology , Hepatocytes/immunology , Peripheral Tolerance/immunology , Animals , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Homeodomain Proteins/genetics , Membrane Proteins/genetics , Mice , Mice, Knockout , Microscopy, Confocal , Microscopy, Electron , Proto-Oncogene Proteins/genetics
11.
Liver Transpl ; 19(11): 1224-35, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23913831

ABSTRACT

Donor passenger leukocytes (PLs) from transplanted livers migrate to recipient lymphoid tissues, where they are thought to induce the deletion of donor-specific T cells and tolerance. Difficulties in tracking alloreactive T cells and PLs in rats and in performing this complex surgery in mice have limited progress in identifying the contribution of PL subsets and sites and the kinetics of T cell deletion. Here we developed a mouse liver transplant model in which PLs, recipient cells, and a reporter population of transgenic CD8 T cells specific for the graft could be easily distinguished and quantified in allografts and recipient organs by flow cytometry. All PL subsets circulated rapidly via the blood as soon as 1.5 hours after transplantation. By 24 hours, PLs were distributed differently in the lymph nodes and spleen, whereas donor natural killer and natural killer T cells remained in the liver and blood. Reporter T cells were activated in both liver and lymphoid tissues, but their numbers dramatically decreased within the first 48 hours. These results provide the first unequivocal demonstration of the differential recirculation of liver PL subsets after transplantation, and show that alloreactive CD8 T cells are deleted more rapidly than initially reported. This model will be useful for dissecting early events leading to the spontaneous acceptance of liver transplants.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Movement , Leukocytes/physiology , Liver Transplantation , Allografts , Animals , Leukocytes/cytology , Lymphocyte Activation , Male , Mice, Inbred C57BL , Natural Killer T-Cells/physiology , Tissue Donors
12.
Cancer Res Commun ; 3(8): 1524-1537, 2023 08.
Article in English | MEDLINE | ID: mdl-37575281

ABSTRACT

Solid cancer cells escape the primary tumor mass by transitioning from an epithelial-like state to an invasive migratory state. As they escape, metastatic cancer cells employ interchangeable modes of invasion, transitioning between fibroblast-like mesenchymal movement to amoeboid migration, where cells display a rounded morphology and navigate the extracellular matrix in a protease-independent manner. However, the gene transcripts that orchestrate the switch between epithelial, mesenchymal, and amoeboid states remain incompletely mapped, mainly due to a lack of methodologies that allow the direct comparison of the transcriptomes of spontaneously invasive cancer cells in distinct migratory states. Here, we report a novel single-cell isolation technique that provides detailed three-dimensional data on melanoma growth and invasion, and enables the isolation of live, spontaneously invasive cancer cells with distinct morphologies and invasion parameters. Via the expression of a photoconvertible fluorescent protein, compact epithelial-like cells at the periphery of a melanoma mass, elongated cells in the process of leaving the mass, and rounded amoeboid cells invading away from the mass were tagged, isolated, and subjected to single-cell RNA sequencing. A total of 462 differentially expressed genes were identified, from which two candidate proteins were selected for further pharmacologic perturbation, yielding striking effects on tumor escape and invasion, in line with the predictions from the transcriptomics data. This work describes a novel, adaptable, and readily implementable method for the analysis of the earliest phases of tumor escape and metastasis, and its application to the identification of genes underpinning the invasiveness of malignant melanoma. Significance: This work describes a readily implementable method that allows for the isolation of individual live tumor cells of interest for downstream analyses, and provides the single-cell transcriptomes of melanoma cells at distinct invasive states, both of which open avenues for in-depth investigations into the transcriptional regulation of the earliest phases of metastasis.


Subject(s)
Melanoma , Transcriptome , Humans , Transcriptome/genetics , Neoplasm Invasiveness/genetics , Cell Movement/genetics , Melanoma/genetics , Cell Line, Tumor
13.
ACS Appl Mater Interfaces ; 13(14): 16142-16154, 2021 Apr 14.
Article in English | MEDLINE | ID: mdl-33787198

ABSTRACT

The ability of upconversion nanoparticles (UCNPs) to convert low-energy near-infrared (NIR) light into high-energy visible-ultraviolet light has resulted in their development as novel contrast agents for biomedical imaging. However, UCNPs often succumb to poor colloidal stability in aqueous media, which can be conquered by decorating the nanoparticle surface with polymers. The polymer cloak, therefore, plays an instrumental role in ensuring good stability in biological media. This study aims to understand the relationship between the length and grafting density of the polymer shell on the physicochemical and biological properties of these core-shell UCNPs. Poly(ethylene glycol) methyl ether methacrylate block ethylene glycol methacrylate phosphate (PPEGMEMAn-b-PEGMP3) with different numbers of PEGMEMA repeating units (26, 38, and 80) was prepared and attached to the UCNPs via the phosphate ligand of the poly(ethylene glycol methacrylate phosphate) (PEGMP) block at different polymer densities. The in vitro and in vivo protein corona, cellular uptake in two-dimensional (2D) monolayer and three-dimensional (3D) multicellular tumor spheroid (MCTS) models, and in vivo biodistribution in mice were evaluated. Furthermore, the photoluminescence of single-polymer-coated UCNPs was compared in solid state and cancer cells using laser scanning confocal microscopy (LSCM). Our results showed that the bioactivity and luminescence properties are chain length and grafting density dependent. The UCNPs coated with the longest PPEGMEMA chain, grafted at low brush density, were able to reduce the formation of the protein corona in vitro and in vivo, while these UCNPs also showed the brightest upconversion luminescence in the solid state. Moreover, these particular polymer-coated UCNPs showed enhanced cellular uptake, extended in vivo blood circulation time, and more accumulation in the liver, brain, and heart.


Subject(s)
Nanoparticles/chemistry , Polymers/chemistry , Adsorption , Animals , Cell Line, Tumor , Humans , Ligands , Mice , Microscopy, Electron, Transmission , Molecular Weight , Proteins/chemistry , RAW 264.7 Cells , Spectroscopy, Fourier Transform Infrared , Thermogravimetry , Tissue Distribution
14.
JHEP Rep ; 2(1): 100065, 2020 Feb.
Article in English | MEDLINE | ID: mdl-32039406

ABSTRACT

BACKGROUND & AIMS: Genome editing technology has immense therapeutic potential and is likely to rapidly supplant contemporary gene addition approaches. Key advantages include the capacity to directly repair mutant loci with resultant recovery of physiological gene expression and maintenance of durable therapeutic effects in replicating cells. In this study, we aimed to repair a disease-causing point mutation in the ornithine transcarbamylase (OTC) locus in patient-derived primary human hepatocytes in vivo at therapeutically relevant levels. METHODS: Editing reagents for precise CRISPR/SaCas9-mediated cleavage and homology-directed repair (HDR) of the human OTC locus were first evaluated against an OTC minigene cassette transposed into the mouse liver. The editing efficacy of these reagents was then tested on the native OTC locus in patient-derived primary human hepatocytes xenografted into the FRG (Fah -/- Rag2 -/- Il2rg -/-) mouse liver. A highly human hepatotropic capsid (NP59) was used for adeno-associated virus (AAV)-mediated gene transfer. Editing events were characterised using next-generation sequencing and restoration of OTC expression was evaluated using immunofluorescence. RESULTS: Following AAV-mediated delivery of editing reagents to patient-derived primary human hepatocytes in vivo, OTC locus-specific cleavage was achieved at efficiencies of up to 72%. Importantly, successful editing was observed in up to 29% of OTC alleles at clinically relevant vector doses. No off-target editing events were observed at the top 10 in silico-predicted sites in the genome. CONCLUSIONS: We report efficient single-nucleotide correction of a disease-causing mutation in the OTC locus in patient-derived primary human hepatocytes in vivo at levels that, if recapitulated in the clinic, would provide benefit for even the most therapeutically challenging liver disorders. Key challenges for clinical translation include the cell cycle dependence of classical HDR and mitigation of unintended on- and off-target editing events. LAY SUMMARY: The ability to efficiently and safely correct disease-causing mutations remains the holy grail of gene therapy. Herein, we demonstrate, for the first time, efficient in vivo correction of a patient-specific disease-causing mutation in the OTC gene in primary human hepatocytes, using therapeutically relevant vector doses. We also highlight the challenges that need to be overcome for this technology to be translated into clinical practice.

15.
Elife ; 92020 10 13.
Article in English | MEDLINE | ID: mdl-33046212

ABSTRACT

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.


Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.


Subject(s)
Chemokine CCL3/immunology , Chemokine CCL4/immunology , Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Cell Movement , Chemokine CCL3/genetics , Chemokine CCL4/genetics , Humans , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Neoplasms/genetics , Neoplasms/physiopathology , Signal Transduction , T-Lymphocytes, Cytotoxic/cytology
16.
Sci Rep ; 9(1): 6504, 2019 04 24.
Article in English | MEDLINE | ID: mdl-31019238

ABSTRACT

The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down; depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.


Subject(s)
Actin Cytoskeleton/metabolism , Formins/metabolism , Actin Cytoskeleton/drug effects , Actins/metabolism , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line , Cells, Cultured , Formins/genetics , HEK293 Cells , Humans , Marine Toxins/pharmacology , Microscopy, Confocal , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Interference , Thiazolidines/pharmacology , Tropomyosin/genetics , Tropomyosin/metabolism
17.
Mol Ther Nucleic Acids ; 6: 1-14, 2017 Mar 17.
Article in English | MEDLINE | ID: mdl-28325276

ABSTRACT

In early gene therapy trials for SCID-X1, using γ-retroviral vectors, T cell leukemias developed in a subset of patients secondary to insertional proto-oncogene activation. In contrast, we have reported development of T cell leukemias in SCID-X1 mice following lentivirus-mediated gene therapy independent of insertional mutagenesis. A distinguishing feature in our study was that only a proportion of transplanted γc-deficient progenitors were transduced and therefore competent for reconstitution. We hypothesized that reconstitution of SCID-X1 mice with limiting numbers of hematopoietic progenitors might be a risk factor for lymphoid malignancy. To test this hypothesis, in the absence of transduction, SCID-X1 mice were reconstituted with serially fewer wild-type hematopoietic progenitors. A robust inverse correlation between hematopoietic progenitor cell dose and T-lymphoid malignancy was observed, with earlier disease onset at lower cell doses. Malignancies were of donor origin and carried activating Notch1 mutations. These findings align with emerging evidence that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Although insertional proto-oncogene activation is required for the development of malignancy in humans, failure of γc-deficient thymocytes to effectively compete with this at-risk cell population may have also contributed to oncogenesis observed in early SCID-X1 trials.

18.
J Invest Dermatol ; 135(1): 84-93, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25007044

ABSTRACT

Site-specific differences in skin response to pathogens and in the course of cutaneous inflammatory diseases are well appreciated. The composition and localization of cutaneous leukocytes has been studied extensively using histology and flow cytometry. However, the precise three-dimensional (3D) distribution of distinct immune cell subsets within skin at different body sites requires visualization of intact living skin. We used intravital multiphoton microscopy in transgenic reporter mice in combination with quantitative flow cytometry to generate a 3D immune cell atlas of mouse skin. The 3D location of innate and adaptive immune cells and site-specific differences in the densities of macrophages, T cells, and mast cells at four defined sites (ear, back, footpad, and tail) is presented. The combinatorial approach further demonstrates an as yet unreported age-dependent expansion of dermal gamma-delta T cells. Localization of dermal immune cells relative to anatomical structures was also determined. Although dendritic cells were dispersed homogeneously within the dermis, mast cells preferentially localized to the perivascular space. Finally, we show the functional relevance of site-specific mast cell disparities using the passive cutaneous anaphylaxis model. These approaches are applicable to assessing immune cell variations and potential functional consequences in the setting of infection, as well as the pathogenesis of inflammatory skin conditions.


Subject(s)
Dermatitis/pathology , Dermoscopy/methods , Imaging, Three-Dimensional/methods , Leukocytes/pathology , Microscopy, Fluorescence, Multiphoton/methods , Skin/pathology , Animals , Bacterial Proteins/genetics , Dendritic Cells/pathology , Dermis/blood supply , Dermis/pathology , Ear, External , Epidermis/pathology , Female , Flow Cytometry , Green Fluorescent Proteins/genetics , Luminescent Proteins/genetics , Lymphocyte Subsets/pathology , Macrophages/pathology , Male , Mast Cells/pathology , Mice, Inbred C57BL , Mice, Transgenic , Skin/blood supply
19.
J Gerontol A Biol Sci Med Sci ; 70(12): 1499-507, 2015 Dec.
Article in English | MEDLINE | ID: mdl-25335766

ABSTRACT

There is a strong association between aging, diet, and immunity. The effects of macronutrients and energy intake on splanchnic and hepatic lymphocytes were studied in 15 month old mice. The mice were ad-libitum fed 1 of 25 diets varying in the ratios and amounts of protein, carbohydrate, and fat over their lifetime. Lymphocytes in liver, spleen, Peyers patches, mesenteric lymph nodes, and inguinal lymph nodes were evaluated using flow cytometry. Low protein intake reversed aging changes in splenic CD4 and CD8 T cells, CD4:CD8 T cell ratio, memory/effector CD4 T cells and naïve CD4 T cells. A similar influence of total caloric intake in these ad-libitum fed mice was not apparent. Protein intake also influenced hepatic NK cells and B cells, while protein to carbohydrate ratio influenced hepatic NKT cells. Hepatosteatosis was associated with increased energy and fat intake and changes in hepatic Tregs, effector/memory T, and NK cells. Hepatic NK cells were also associated with body fat, glucose tolerance, and leptin levels while hepatic Tregs were associated with hydrogen peroxide production by hepatic mitochondria. Dietary macronutrients, particularly protein, influence splanchnic lymphocytes in old age, with downstream associations with mitochondrial function, liver pathology, and obesity-related phenotype.


Subject(s)
Dietary Carbohydrates/pharmacology , Dietary Fats/pharmacology , Dietary Proteins/pharmacology , Liver/immunology , Lymphocytes/drug effects , Viscera/immunology , Age Factors , Animals , Female , Male , Mice , Mice, Inbred C57BL
20.
Transplantation ; 78(7): 987-94, 2004 Oct 15.
Article in English | MEDLINE | ID: mdl-15480163

ABSTRACT

BACKGROUND: In vitro studies have shown that cognate recognition of antigen presented by endothelial cells (EC) causes T cell activation, proliferation, and cytokine release and alters the transmigration of T cells. Here we have investigated chemokine induction caused by cognate interactions between human CD4+ T cells and MHC class II-expressing EC. METHODS: HLA-DR-restricted CD4+ T cells were cocultured with HLA-DR-expressing allogeneic Eahy.926, aortic, or heart microvascular EC. Chemokine mRNA expression was measured by RTPCR, and chemokine protein secreted was measured by a cytokine array system and ELISA. Molecules involved in chemokine secretion were identified using blocking monoclonal antibodies, and cellular sources of chemokines determined by intracellular chemokine staining. Coculture supernatants were also used in chemotaxis assays. RESULTS: Nine different chemokine mRNA and proteins were expressed because of noncognate interactions between T cells and EC. Cognate interactions induced de novo expression of four chemokines and upregulation of seven chemokines. Levels of CCL3, CCL8, and CXCL10 secreted into supernatants were in the nanomolar range and were chemotactic for T cells and monocytes. Blocking antibodies to HLA-DR and LFA-3 abrogated production of CCL3, CCL8, and CXCL10. Blocking antibodies to interferon-gamma and tumor necrosis factor-alpha inhibited CCL8 and CXCL10 but not CCL3 production. CCL3 and CXCL10 were produced by both T cells and EC. CONCLUSIONS: Cognate interactions between alloreactive CD4+ T cells and MHC class II-expressing EC results in a specific pattern of chemokine production. These chemokines could play important roles in recruitment of leukocytes into vascularised allografts.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Cell Communication , Chemokines, CC/biosynthesis , Chemokines, CXC/biosynthesis , Endothelial Cells/metabolism , CD4-Positive T-Lymphocytes/cytology , Chemokine CXCL10 , Chemokines, CC/genetics , Chemokines, CXC/analysis , Coculture Techniques , Endothelial Cells/cytology , Enzyme-Linked Immunosorbent Assay , Humans , RNA, Messenger/analysis
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