ABSTRACT
The surveillance of body barriers relies on resident T cells whose repertoires are biased toward particular γδ T cell antigen receptors (TCRs) according to location. These γδ TCRs can recognize ligands that emerge after stress. Through the use of intravital dynamics-immunosignal correlative microscopy, we found that γ-chain variable region 5 (V(γ)5) TCRs expressed by epidermal T cells were constitutively clustered and functionally activated in vivo at steady state, forming true immunological synapses that polarized and anchored T cell projections at squamous keratinocyte tight junctions. This synaptogenesis depended on TCR variable domains, the kinase Lck and the integrin α(E)ß(7) but not the γδ lineage or the receptor NKG2D. In response to tissue stress, TCR-proximal signals did not increase substantially but underwent stress mode-dependent relocalization toward the basal epidermis and Langerhans cells. Thus, the γδ TCR orchestrates barrier surveillance proactively, presumably by recognizing tissue ligands expressed in the steady state.
Subject(s)
Epidermis/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Animals , Keratinocytes/immunology , Ligands , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Mice , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Signal Transduction , T-Lymphocytes/immunologyABSTRACT
Assays based on Förster resonance energy transfer (FRET) can be used to study many processes in cell biology. Although this is most often done with microscopy for fluorescence detection, we report two ways to measure FRET in living cells by flow cytometry. Using a conventional flow cytometer and the "3-cube method" for intensity-based calculation of FRET efficiency, we measured the enzymatic activity of specific kinases in cells expressing a genetically-encoded reporter. For both AKT and protein kinase A, the method measured kinase activity in time-course, dose-response, and kinetic assays. Using the Cytek Aurora spectral flow cytometer, which applies linear unmixing to emission measured in multiple wavelength ranges, FRET from the same reporters was measured with greater single-cell precision, in real time and in the presence of other fluorophores. Results from gene-knockout studies suggested that spectral flow cytometry might enable the sorting of cells on the basis of FRET. The methods we present provide convenient and flexible options for using FRET with flow cytometry in studies of cell biology.
Subject(s)
Fluorescence Resonance Energy Transfer , Proto-Oncogene Proteins c-akt , Cyclic AMP-Dependent Protein Kinases/metabolism , Flow Cytometry/methods , Fluorescence Resonance Energy Transfer/methods , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.
Subject(s)
Lymphoma, Large B-Cell, Diffuse/metabolism , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Antigens/metabolism , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Cell Proliferation , Cluster Analysis , Gene Knockout Techniques , Germinal Center/pathology , Humans , Lymphoma, Large B-Cell, Diffuse/pathology , Mutation/genetics , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
The non-canonical NF-κB pathway forms a major arm of NF-κB signalling that mediates important biological functions, including lymphoid organogenesis, B-lymphocyte function, and cell growth and survival. Activation of the non-canonical NF-κB pathway involves degradation of an inhibitory protein, TNF receptor-associated factor 3 (TRAF3), but how this signalling event is controlled is still unknown. Here we have identified the deubiquitinase OTUD7B as a pivotal regulator of the non-canonical NF-κB pathway. OTUD7B deficiency in mice has no appreciable effect on canonical NF-κB activation but causes hyperactivation of non-canonical NF-κB. In response to non-canonical NF-κB stimuli, OTUD7B binds and deubiquitinates TRAF3, thereby inhibiting TRAF3 proteolysis and preventing aberrant non-canonical NF-κB activation. Consequently, the OTUD7B deficiency results in B-cell hyper-responsiveness to antigens, lymphoid follicular hyperplasia in the intestinal mucosa, and elevated host-defence ability against an intestinal bacterial pathogen, Citrobacter rodentium. These findings establish OTUD7B as a crucial regulator of signal-induced non-canonical NF-κB activation and indicate a mechanism of immune regulation that involves OTUD7B-mediated deubiquitination and stabilization of TRAF3.
Subject(s)
Endopeptidases/metabolism , NF-kappa B/metabolism , TNF Receptor-Associated Factor 3/metabolism , Ubiquitination , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Bacteria/immunology , Cells, Cultured , Endopeptidases/deficiency , Endopeptidases/genetics , Female , Fibroblasts , HEK293 Cells , Homeostasis , Humans , Intestines/immunology , Male , Mice , Proteolysis , Receptors, Cell Surface/metabolismABSTRACT
Recent work has demonstrated that following the clearance of infection a stable population of memory T cells remains present in peripheral organs and contributes to the control of secondary infections. However, little is known about how tissue-resident memory T cells behave in situ and how they encounter newly infected target cells. Here we demonstrate that antigen-specific CD8(+) T cells that remain in skin following herpes simplex virus infection show a steady-state crawling behavior in between keratinocytes. Spatially explicit simulations of the migration of these tissue-resident memory T cells indicate that the migratory dendritic behavior of these cells allows the detection of antigen-expressing target cells in physiologically relevant time frames of minutes to hours. Furthermore, we provide direct evidence for the identification of rare antigen-expressing epithelial cells by skin-patrolling memory T cells in vivo. These data demonstrate the existence of skin patrol by memory T cells and reveal the value of this patrol in the rapid detection of renewed infections at a previously infected site.
Subject(s)
Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory , Epithelium/immunology , HumansABSTRACT
Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.
Subject(s)
Autocrine Communication , Hematopoietic Stem Cells , Interferon Type I , STAT3 Transcription Factor , Animals , STAT3 Transcription Factor/metabolism , Mice , Hematopoietic Stem Cells/metabolism , Interferon Type I/metabolism , Signal Transduction , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Plasmacytoid dendritic cells (pDCs) sense viral and microbial DNA through endosomal Toll-like receptors to produce type 1 interferons. pDCs do not normally respond to self-DNA, but this restriction seems to break down in human autoimmune disease by an as yet poorly understood mechanism. Here we identify the antimicrobial peptide LL37 (also known as CAMP) as the key factor that mediates pDC activation in psoriasis, a common autoimmune disease of the skin. LL37 converts inert self-DNA into a potent trigger of interferon production by binding the DNA to form aggregated and condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger Toll-like receptor 9. Thus, our data uncover a fundamental role of an endogenous antimicrobial peptide in breaking innate tolerance to self-DNA and suggest that this pathway may drive autoimmunity in psoriasis.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , Autoantigens/metabolism , Autoimmune Diseases/metabolism , Autoimmunity , DNA/metabolism , Dendritic Cells/metabolism , Psoriasis/metabolism , Autoantigens/immunology , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Cathelicidins , DNA/immunology , Dendritic Cells/immunology , Endocytosis , Endosomes/immunology , Endosomes/metabolism , Humans , Psoriasis/immunology , Psoriasis/pathology , Toll-Like Receptor 9/immunology , Toll-Like Receptor 9/metabolismABSTRACT
STAT3 function in hematopoietic stem and progenitor cells (HSPCs) has been difficult to discern as Stat3 deficiency in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. To address this, we established mixed bone marrow (BM) chimeric mice with CreER-mediated Stat3 deletion in 20% of the hematopoietic compartment. Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM in contrast to Stat3-sufficient (CreER) controls. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells revealed altered transcriptional responses in Stat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, including intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation, Stat3-deficient Lin-ckit+Sca1+ cells accumulated γH2AX over time. Following secondary BM transplantation, Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs.
ABSTRACT
BH3 mimetics are used as an efficient strategy to induce cell death in several blood malignancies, including acute myeloid leukemia (AML). Venetoclax, a potent BCL-2 antagonist, is used clinically in combination with hypomethylating agents for the treatment of AML. Moreover, MCL1 or dual BCL-2/BCL-xL antagonists are under investigation. Yet, resistance to single or combinatorial BH3-mimetic therapies eventually ensues. Integration of multiple genome-wide CRISPR/Cas9 screens revealed that loss of mitophagy modulators sensitizes AML cells to various BH3 mimetics targeting different BCL-2 family members. One such regulator is MFN2, whose protein levels positively correlate with drug resistance in patients with AML. MFN2 overexpression is sufficient to drive resistance to BH3 mimetics in AML. Insensitivity to BH3 mimetics is accompanied by enhanced mitochondria-endoplasmic reticulum interactions and augmented mitophagy flux, which acts as a prosurvival mechanism to eliminate mitochondrial damage. Genetic or pharmacologic MFN2 targeting synergizes with BH3 mimetics by impairing mitochondrial clearance and enhancing apoptosis in AML. SIGNIFICANCE: AML remains one of the most difficult-to-treat blood cancers. BH3 mimetics represent a promising therapeutic approach to eliminate AML blasts by activating the apoptotic pathway. Enhanced mitochondrial clearance drives resistance to BH3 mimetics and predicts poor prognosis. Reverting excessive mitophagy can halt BH3-mimetic resistance in AML. This article is highlighted in the In This Issue feature, p. 1501.
Subject(s)
Antineoplastic Agents , Leukemia, Myeloid, Acute , Humans , Myeloid Cell Leukemia Sequence 1 Protein , Mitophagy , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Apoptosis , Cell Death , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic useABSTRACT
The co-receptors CD4 and CD8 are important in the activation of T cells primarily because of their ability to interact with the proteins of the MHC enhancing recognition of the MHC-peptide complex by the T cell receptor (TCR). An antigen-presenting cell presents a small number of antigenic peptides on its MHC molecules, in the presence of a much larger number of endogenous, mostly nonstimulatory, peptides. Recent work has demonstrated that these endogenous MHC-peptide complexes have an important role in modulating the sensitivity of the TCR. But the role of the endogenous nonstimulatory MHC-peptide complexes differs in MHC class I and class II-restricted T cells. This chapter discusses the data on the role of CD4 or CD8 co-receptors in T cell activation at the immunological synapse, and the role of non stimulatory MHC-peptide complexes in aiding antigen recognition.
Subject(s)
CD4 Antigens/physiology , CD8 Antigens/physiology , Immunological Synapses/physiology , Animals , Antigen Presentation , Autoantigens/immunology , Fluorescence Resonance Energy Transfer , Histocompatibility Antigens/physiology , Humans , Lymphocyte Activation , Receptors, Antigen, T-Cell/physiologyABSTRACT
IL-15 is crucial for the development of intestinal intraepithelial lymphocytes (IEL) and delivery is mediated by a unique mechanism known as trans-presentation. Parenchymal cells have a major role in the trans-presentation of IL-15 to IELs, but the specific identity of this cell type is unknown. To investigate whether the intestinal epithelial cells (IEC) are the parenchymal cell type involved, a mouse model that expresses IL-15Ralpha exclusively by the IECs (Villin/IL-15Ralpha Tg) was generated. Exclusive expression of IL-15Ralpha by the IECs restored all the deficiencies in the CD8alphaalpha(+)TCRalphabeta(+)and CD8alphaalpha(+)TCRgammadelta(+) subsets that exist in the absence of IL-15Ralpha. Interestingly, most of the IEL recovery was due to the preferential increase in Thy1(low) IELs, which compose a majority of the IEL population. The differentiation of Thy1(high)CD4(-)CD8(-) thymocytes into Thy1(-)CD8alphaalpha IELs was found to require IL-15Ralpha expression specifically by IECs and thus, provides evidence that differentiation of Thy1(low) IELs is one function of trans-presentation of IL-15 in the intestines. In addition to effects in IEL differentiation, trans-presentation of IL-15 by IECs also resulted in an increase in IEL numbers that was accompanied by increases in Bcl-2, but not proliferation. Collectively, this study demonstrates that trans-presentation of IL-15 by IECs alone is completely sufficient to direct the IL-15-mediated development of CD8alphaalpha(+) T cell populations within the IEL compartment, which now includes a newly identified role of IL-15 in the differentiation of Thy1(low) IELs.
Subject(s)
Antigen Presentation , Epithelial Cells/immunology , Interleukin-15/immunology , Intestinal Mucosa/cytology , Lymphocytes/cytology , Animals , CD8 Antigens , Cell Differentiation , Interleukin-15 Receptor alpha Subunit/deficiency , Interleukin-15 Receptor alpha Subunit/physiology , Intestinal Mucosa/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Thymus Gland/cytologyABSTRACT
Zygomycosis is an emerging frequently fatal opportunistic mycosis whose immunopathogenesis is poorly understood. We developed a zygomycosis model by injecting Drosophila melanogaster flies with a standardized amount of fungal spores from clinical Zygomycetes isolates to study virulence and host defense mechanisms. We found that, as opposed to most other fungi, which are nonpathogenic in D. melanogaster (e.g., Aspergillus fumigatus), Zygomycetes rapidly infect and kill wild-type flies. Toll-deficient flies exhibited increased susceptibility to Zygomycetes, whereas constitutive overexpression of the antifungal peptide Drosomycin in transgenic flies partially restored resistance to zygomycosis. D. melanogaster Schneider 2 phagocytic cells displayed decreased phagocytosis and caused less hyphal damage to Zygomycetes compared with that to A. fumigatus. Furthermore, phagocytosis-defective eater mutant flies displayed increased susceptibility to Zygomycetes infection. Classic enhancers of Zygomycetes virulence in humans, such as corticosteroids, increased iron supply, and iron availability through treatment with deferoxamine dramatically increased Zygomycetes pathogenicity in our model. In contrast, iron starvation induced by treatment with the iron chelator deferasirox significantly protected flies infected with Zygomycetes. Whole-genome expression profiling in wild-type flies after infection with Zygomycetes vs. A. fumigatus identified genes selectively down-regulated by Zygomycetes, which act in pathogen recognition, immune defense, stress response, detoxification, steroid metabolism, or tissue repair or have unknown functions. Our results provide insights into the factors that mediate host-pathogen interactions in zygomycosis and establish D. melanogaster as a promising model to study this important mycosis.
Subject(s)
Drosophila melanogaster/immunology , Drosophila melanogaster/microbiology , Zygomycosis/immunology , Zygomycosis/pathology , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/pathogenicity , Dexamethasone/pharmacology , Disease Models, Animal , Disease Susceptibility , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/drug effects , Drosophila melanogaster/genetics , Fungi/drug effects , Fungi/isolation & purification , Fungi/pathogenicity , Gene Expression Regulation/drug effects , Genes, Insect , Humans , Hyphae/drug effects , Hyphae/physiology , Phagocytes/drug effects , Phagocytes/immunology , Phagocytes/microbiology , Phagocytosis/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survival Analysis , Virulence/drug effectsABSTRACT
Nanoparticles with ultrasmall sizes (less than 10 nm) offer many advantages in biomedical applications compared to their bigger counterparts, including better intratumoral distribution, improved pharmacokinetics (PK), and efficient body clearance. When functionalized with a biocompatible coating and a target-specific antibody, ultrasmall nanoparticles represent an attractive clinical translation platform. Although there is a tremendous body of work dedicated to PK and the biological effects of various nanoparticles, little is known about the fate of different components of functionalized nanoparticles in a biological environment such as in live cells. Here, we used luminescence properties of 5 nm gold nanoparticles (AuNPs) to study the intracellular trafficking and fate of the AuNPs functionalized with an organic layer consisting of a polyethylene glycol (PEG) coating and epidermal growth factor receptor (EGFR)-targeting antibody. We showed that intracellular uptake of the targeted 5 nm AuNPs results in a strong two-photon luminescence (TPL) that is characterized by broad emission and very short lifetimes compared to the fluorescence of the nanoparticle-conjugated fluorophore-tagged antibody, thereby allowing selective imaging of these components using TPL and two-photon excited fluorescence lifetime microscopy (2P-FLIM). Our results indicate that the nanoparticle's coating is detached from the particle's surface inside cells, leading to formation of nanoparticle clusters with a strong TPL. Furthermore, we observed an optically resolved spatial separation of the gold core and the antibody coating of the particles inside cells. We used data from two-photon microscopy, 2P-FLIM, electron microscopy, and in vitro assays to propose a model of interactions of functionalized 5 nm AuNPs with live cells.
Subject(s)
Metal Nanoparticles , Neoplasms , Gold , Kinetics , Luminescence , Polyethylene GlycolsABSTRACT
Amyloid-induced neurodegeneration plays a central role in Alzheimer's disease (AD) pathogenesis. Here, we show that telomerase reverse transcriptase (TERT) haploinsufficiency decreases BDNF and increases amyloid-ß (Aß) precursor in murine brain. Moreover, prior to disease onset, the TERT locus sustains accumulation of repressive epigenetic marks in murine and human AD neurons, implicating TERT repression in amyloid-induced neurodegeneration. To test the impact of sustained TERT expression on AD pathobiology, AD mouse models were engineered to maintain physiological levels of TERT in adult neurons, resulting in reduced Aß accumulation, improved spine morphology, and preserved cognitive function. Mechanistically, integrated profiling revealed that TERT interacts with ß-catenin and RNA polymerase II at gene promoters and upregulates gene networks governing synaptic signaling and learning processes. These TERT-directed transcriptional activities do not require its catalytic activity nor telomerase RNA. These findings provide genetic proof-of-concept for somatic TERT gene activation therapy in attenuating AD progression including cognitive decline.
Subject(s)
Alzheimer Disease , Telomerase , Mice , Humans , Animals , Alzheimer Disease/genetics , Telomerase/genetics , Amyloid beta-Peptides/metabolism , Cognition , Neurons/metabolismABSTRACT
The use of appropriate fluorescent proteins has allowed the use of FRET microscopy for investigation of intermolecular interactions in living cells. This method has the advantage of both being dynamic and of working at the subcellular level, so that the time and place where proteins interact can be visualized. We have used FRET microscopy to analyze the interactions between the T cell antigen receptor and the coreceptors CD4 and CD8. This chapter reviews data on how these coreceptors are recruited to the immunological synapse, and how they interact when the T cell is stimulated by different ligands.
Subject(s)
CD4 Antigens/metabolism , CD8 Antigens/metabolism , Fluorescence Resonance Energy Transfer/methods , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , Animals , Antigen Presentation , Humans , Nanotubes , T-Lymphocytes/metabolismABSTRACT
Abnormally low level of interstitial oxygen, or hypoxia, is a hallmark of tumor microenvironment and a known promoter of cancer chemoresistance. Inside a solid tumor mass, the hypoxia stems largely from inadequate supply of oxygenated blood through sparse or misshapen tumor vasculature whilst oxygen utilization rates are low in typical tumor's glycolytic metabolism. In acute leukemias, however, markers of intracellular hypoxia such as increased pimonidazole adduct staining and HIF-1α stabilization are observed in advanced leukemic bone marrows (BM) despite an increase in BM vasculogenesis. We utilized intravital fast scanning two-photon phosphorescence lifetime imaging microscopy (FaST-PLIM) in a BCR-ABL B-ALL mouse model to image the extracellular oxygen concentrations (pO2) in leukemic BM, and we related the extracellular oxygen levels to intracellular hypoxia, vascular markers and local leukemia burden. We observed a transient increase in BM pO2 in initial disease stages with intermediate leukemia BM burden, which correlated with an expansion of blood-carrying vascular network in the BM. Yet, we also observed increased formation of intracellular pimonidazole adducts in leukemic BM at the same time. This intermediate stage was followed by a significant decrease of extracellular pO2 and further increase of intracellular hypoxia as leukemia cellularity overwhelmed BM in disease end-stage. Remarkably, treatment of leukemic mice with IACS-010759, a pharmacological inhibitor of mitochondrial Complex I, substantially increased pO2 in the BM with advanced B-ALL, and it alleviated intracellular hypoxia reported by pimonidazole staining. High rates of oxygen consumption by B-ALL cells were confirmed by Seahorse assay including in ex vivo cells. Our results suggest that B-ALL expansion in BM is associated with intense oxidative phosphorylation (OxPhos) leading to the onset of metabolic BM hypoxia despite increased BM vascularization. Targeting mitochondrial respiration may be a novel approach to counteract BM hypoxia in B-ALL and, possibly, tumor hypoxia in other OxPhos-reliant malignancies.
ABSTRACT
The strong cetyltrimethylammonium bromide (CTAB) surfactant responsible for the synthesis and stability of gold nanorod solutions complicates their biomedical applications. The critical parameter to maintain nanorod stability is the ratio of CTAB to nanorod concentration. The ratio is approximately 740,000 as determined by chloroform extraction of the CTAB from a nanorod solution. A comparison of nanorod stabilization by thiol-terminal PEG and by anionic polymers reveals that PEGylation results in higher yields and less aggregation upon removal of CTAB. A heterobifunctional PEG yields nanorods with exposed carboxyl groups for covalent conjugation to antibodies with the zero-length carbodiimide linker EDC. This conjugation strategy leads to approximately two functional antibodies per nanorod according to fluorimetry and ELISA assays. The nanorods specifically targeted cells in vitro and were visible with both two-photon and confocal reflectance microscopies. This covalent strategy should be generally applicable to other biomedical applications of gold nanorods as well as other gold nanoparticles synthesized with CTAB.
Subject(s)
Cetrimonium Compounds/chemistry , Gold/chemistry , Nanotubes/chemistry , Polyethylene Glycols/chemistry , Surface-Active Agents/chemistry , Cell Line , Cell Line, Tumor , Cetrimonium , Epithelial Cells/cytology , Epithelial Cells/ultrastructure , Humans , Luminescence , Microscopy, Confocal , Nanotubes/ultrastructureABSTRACT
BACKGROUND: Low availability of oxygen in tumors contributes to the hostility of the tumor microenvironment toward the immune system. However, the dynamic relationship between local oxygen levels and the immune surveillance of tumors by tumor infiltrating T-lymphocytes (TIL) remains unclear. This situation reflects a methodological difficulty in visualizing oxygen gradients in living tissue in a manner that is suitable for spatiotemporal quantification and contextual correlation with individual cell dynamics tracked by typical fluorescence reporter systems. METHODS: Here, we devise a regimen for intravital oxygen and cell dynamics co-imaging, termed 'Fast' Scanning Two-photon Phosphorescence Lifetime Imaging Microscopy (FaST-PLIM). Using FaST-PLIM, we image the cellular motility of T-lymphocytes in relation to the microscopic distribution of oxygen in mouse models of hematological and solid tumors, namely in bone marrow with or without B-cell acute lymphocytic leukemia (ALL), and in lungs with sarcoma tumors. RESULTS: Both in bone marrow leukemia and solid tumor models, TILs encountered regions of varying oxygen concentrations, including regions of hypoxia (defined as pO2 below 5 mmHg), especially in advanced-stage ALL and within solid tumor cores. T cell motility was sustained and weakly correlated with local pO2 above 5 mmHg but it was very slow in pO2 below this level. In solid tumors, this relationship was reflected in slow migration of TIL in tumor cores compared to that in tumor margins. Remarkably, breathing 100% oxygen alleviated tumor core hypoxia and rapidly invigorated the motility of otherwise stalled tumor core TILs. CONCLUSIONS: This study demonstrates a versatile and highly contextual FaST-PLIM method for phosphorescence lifetime-based oxygen imaging in living animal tumor immunology models. The initial results of this method application to ALL and solid lung tumor models highlight the importance of oxygen supply for the maintenance of intratumoral T cell migration, define a 5 mmHg local oxygen concentration threshold for TIL motility, and demonstrate efficacy of supplementary oxygen breathing in TIL motility enhancement coincident with reduction of tumor hypoxia.
Subject(s)
Lung Neoplasms/diagnostic imaging , Lung Neoplasms/secondary , Lymphocytes, Tumor-Infiltrating/metabolism , Microscopy, Fluorescence, Multiphoton/methods , Oxygen/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnostic imaging , Sarcoma/diagnostic imaging , Animals , Cell Tracking , Female , Humans , Image Interpretation, Computer-Assisted , Lung Neoplasms/metabolism , Male , Mice , Neoplasms, Experimental , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Sarcoma/metabolism , T-Lymphocytes/metabolism , Tumor MicroenvironmentABSTRACT
Ligand binding to cell membrane receptors sets off a series of protein interactions that convey the nuances ofligand identity to the cell interior. The information may be encoded in conformational changes, the interaction kinetics and, in the case of multichain immunoreceptors, by chain rearrangements. The signals may be modulated by dynamic compartmentalization of the cell membrane, cellular architecture, motility, and activation--all of which are difficult to reconstitute for studies of receptor signaling in vitro. In this chapter, we will discuss how protein interactions in general and receptor signaling in particular can be studied in living cells by different fluorescence imaging techniques. Particularly versatile are methods that exploit Förster resonance energy transfer (FRET), which is exquisitely sensitive to the nanometer-range proximity and orientation between fluorophores. Fluorescence correlation microscopy (FCM) can provide complementary information about the stoichiometry and diffusion kinetics of large complexes, while bimolecular fluorescence complementation (BiFC) and other complementation techniques can capture transient interactions. A continuing challenge is extracting from the imaging data the quantitative information that is necessary to verify different models of signal transduction.
Subject(s)
Luminescent Measurements/methods , Animals , Cell Survival , Fluorescence Resonance Energy Transfer , Humans , Immunological Synapses/immunology , Protein Binding , Receptors, Antigen, T-Cell/immunologyABSTRACT
Dendritic epidermal T cells (DETCs) represent a prototypical lineage of intraepithelial γδ T cells that participate in the maintenance of body barrier homeostasis. Unlike classical T cells, DETCs do not recirculate and they remain persistently activated through their T cell receptors (TCR) at steady state, i.e., in absence of infection or tissue wounding. The steady state TCR signals sustain the formation of immunological synapse-like phosphotyrosine-rich aggregates located on projections (PALPs) which act to anchor and polarize DETC's long cellular projections toward the apical epidermis while the cell bodies reside in the basal layers. The PALPs are known to contain pre-synaptic accumulations of TCR-containing and lysosomal granules, but how this cargo accumulates there remains unclear. Here, we combined anti-Vγ5 TCR, cholera toxin subunit B (CTB), and LysoTracker (LT)-based intravital labeling of intracellular granules, with high resolution dynamic microscopy and fluorescence recovery after photobleaching (FRAP) to characterize the steady state composition and transport of DETC granules in steady state epidermis. Intradermal fluorescent Vγ5 antibody decorated DETCs without causing cellular depletion, dendrite mobilization or rounding up and became slowly internalized over 48 h into intracellular granules that, after 6 days, colocalized with LAMP-1 and less so with LT or early endosomal antigen-1. Intradermal CTB was likewise internalized predominantly by DETCs in epidermis, labeling a partly overlapping set of largely LAMP-1+ intracellular granules. These as well as LT-labeled granules readily moved into newly forming dendrites and accumulated at the apical endings. FRAP and spatiotemporal tracking showed that the inside tubular lengths of DETC cellular projections supported dynamic trafficking of lysosomal cargo toward and away from the PALPs, including internalized TCR and lipid raft component ganglioside GM1 (labeled with CTB). By contrast, the rate of GM1 granules transport through comparable dendrites of non-DETCs was twice slower. Our observations suggest that DETCs use chronic TCR activation to establish a polarized conduit system for long-range trans-epithelial transport aimed to accumulate mature lysosomes at the barrier-forming apical epidermis. The biological strategy behind the steady state lysosome polarization by DETCs remains to be uncovered.