ABSTRACT
The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.
Subject(s)
CD8-Positive T-Lymphocytes , Neoplasms , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Immune Checkpoint Inhibitors , Immune Evasion , Interferon-gamma/genetics , Interferon-gamma/metabolism , NK Cell Lectin-Like Receptor Subfamily CABSTRACT
T cell exhaustion is an induced state of dysfunction that arises in response to chronic infection and cancer. Exhausted CD8+ T cells acquire a distinct epigenetic state, but it is not known whether that chromatin landscape is fixed or plastic following the resolution of a chronic infection. Here we show that the epigenetic state of exhaustion is largely irreversible, even after curative therapy. Analysis of chromatin accessibility in HCV- and HIV-specific responses identifies a core epigenetic program of exhaustion in CD8+ T cells, which undergoes only limited remodeling before and after resolution of infection. Moreover, canonical features of exhaustion, including super-enhancers near the genes TOX and HIF1A, remain 'epigenetically scarred.' T cell exhaustion is therefore a conserved epigenetic state that becomes fixed and persists independent of chronic antigen stimulation and inflammation. Therapeutic efforts to reverse T cell exhaustion may require new approaches that increase the epigenetic plasticity of exhausted T cells.
Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Hepacivirus/immunology , Hepatitis C, Chronic/immunology , Immunologic Memory/immunology , 2-Naphthylamine/therapeutic use , Anilides/therapeutic use , Antiviral Agents/therapeutic use , Chromatin/metabolism , Cyclopropanes/therapeutic use , Epigenesis, Genetic/genetics , Hepacivirus/drug effects , Hepatitis C, Chronic/drug therapy , High Mobility Group Proteins/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Lactams, Macrocyclic/therapeutic use , Proline/analogs & derivatives , Proline/therapeutic use , Ribavirin/therapeutic use , Ritonavir/therapeutic use , Sulfonamides/therapeutic use , Uracil/analogs & derivatives , Uracil/therapeutic use , Valine/therapeutic useABSTRACT
CD8+ T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6+ progenitor exhausted and Tim-3+ terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8+ T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3+ cells without altering Slamf6+ numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8+ T cells enhanced Tim-3+ anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3+CD8+ T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target.
Subject(s)
Adenocarcinoma/immunology , CD8-Positive T-Lymphocytes/physiology , Colonic Neoplasms/immunology , Immunotherapy/methods , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/physiology , Lymphoid Progenitor Cells/physiology , Melanoma/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism , Skin Neoplasms/immunology , Animals , Cellular Senescence , Cytotoxicity, Immunologic , Female , Hepatitis A Virus Cellular Receptor 2/metabolism , Immune Tolerance , Interferon Type I/metabolism , Male , Melanoma, Experimental , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , Signal Transduction , Signaling Lymphocytic Activation Molecule Family/metabolismABSTRACT
Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion-they can also shape the fate and function of CD8+ T cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Fibroblasts/physiology , Lymph Nodes/immunology , Animals , Cell Differentiation , Cell Proliferation , Cell Survival , Cells, Cultured , Cellular Reprogramming , Chromatin Assembly and Disassembly , Cytotoxicity, Immunologic , Epigenesis, Genetic , Gene Expression Regulation , Immunologic Memory , Interleukin-6/genetics , Interleukin-6/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/metabolismABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8+ tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8+ TILs include a subpopulation of 'progenitor exhausted' cells that retain polyfunctionality, persist long term and differentiate into 'terminally exhausted' TILs. Consequently, progenitor exhausted CD8+ TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8+ T cells might be an important component of improving the response to checkpoint blockade.
Subject(s)
Antibodies, Blocking/pharmacology , CD8-Positive T-Lymphocytes/drug effects , Lymphocytes, Tumor-Infiltrating/drug effects , Melanoma, Experimental/prevention & control , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Animals , Antibodies, Blocking/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Cell Line, Tumor , Female , Humans , Lymphocyte Subsets/drug effects , Lymphocyte Subsets/immunology , Lymphocyte Subsets/virology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/virology , Lymphocytic Choriomeningitis/immunology , Lymphocytic Choriomeningitis/prevention & control , Lymphocytic Choriomeningitis/virology , Lymphocytic choriomeningitis virus/drug effects , Lymphocytic choriomeningitis virus/immunology , Lymphocytic choriomeningitis virus/physiology , Melanoma, Experimental/immunology , Melanoma, Experimental/virology , Mice, Congenic , Mice, Inbred C57BL , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolismABSTRACT
Foxo transcription factors play an essential role in regulating specialized lymphocyte functions and in maintaining T cell quiescence. Here, we used a system in which Foxo1 transcription-factor activity, which is normally terminated upon cell activation, cannot be silenced, and we show that enforcing Foxo1 activity disrupts homeostasis of CD4 conventional and regulatory T cells. Despite limiting cell metabolism, continued Foxo1 activity is associated with increased activation of the kinase Akt and a cell-intrinsic proliferative advantage; however, survival and cell division are decreased in a competitive setting or growth-factor-limiting conditions. Via control of expression of the transcription factor Myc and the IL-2 receptor ß-chain, termination of Foxo1 signaling couples the increase in cellular cholesterol to biomass accumulation after activation, thereby facilitating immunological synapse formation and mTORC1 activity. These data reveal that Foxo1 regulates the integration of metabolic and mitogenic signals essential for T cell competitive fitness and the coordination of cell growth with cell division.
Subject(s)
CD4-Positive T-Lymphocytes/physiology , Forkhead Box Protein O1/metabolism , T-Lymphocytes, Regulatory/physiology , Animals , Cell Proliferation , Cells, Cultured , Cholesterol/metabolism , Forkhead Box Protein O1/genetics , Gene Expression Profiling , Homeostasis , Immunological Synapses/metabolism , Interleukin-2 Receptor beta Subunit/genetics , Interleukin-2 Receptor beta Subunit/metabolism , Lymphocyte Activation , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Knockout , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Signal TransductionABSTRACT
CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.
Subject(s)
Carcinoma, Renal Cell/immunology , Genetic Testing/methods , Genetic Vectors/genetics , Immunotherapy/methods , Kidney Neoplasms/immunology , Killer Cells, Natural/immunology , Lentivirus/genetics , Animals , Antigen Presentation , Autophagy , Carcinoma, Renal Cell/therapy , Cells, Cultured , Clustered Regularly Interspaced Short Palindromic Repeats , Genetic Engineering , Histocompatibility Antigens Class I/metabolism , Kidney Neoplasms/therapy , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Targeted TherapyABSTRACT
Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.
Subject(s)
Drug Resistance, Neoplasm , Immune Evasion , Immunotherapy , Protein Serine-Threonine Kinases , Humans , Immune Evasion/genetics , Immune Evasion/immunology , Immunotherapy/methods , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Organoids , Tumor Necrosis Factors/immunology , Interferon-gamma/immunology , Spheroids, Cellular , Caspases , Janus Kinases , STAT Transcription FactorsABSTRACT
Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.
Subject(s)
Gene Silencing , Histone-Lysine N-Methyltransferase/metabolism , Neoplasms/genetics , Neoplasms/immunology , Animals , Antigens, Viral/immunology , CRISPR-Cas Systems/genetics , Chromatin/genetics , Chromatin/metabolism , DNA Transposable Elements/genetics , Disease Models, Animal , Female , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Mice , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
The transcription factor BATF is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and follicular helper T cells (TFH cells). Here we identified a fundamental role for BATF in regulating the differentiation of effector of CD8(+) T cells. BATF-deficient CD8(+) T cells showed profound defects in effector population expansion and underwent proliferative and metabolic catastrophe early after encountering antigen. BATF, together with the transcription factors IRF4 and Jun proteins, bound to and promoted early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors while paradoxically repressing genes encoding effector molecules (IFN-γ and granzyme B). Thus, BATF amplifies T cell antigen receptor (TCR)-dependent expression of transcription factors and augments the propagation of inflammatory signals but restrains the expression of genes encoding effector molecules. This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved.
Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , CD8-Positive T-Lymphocytes/immunology , T-Box Domain Proteins/metabolism , Th17 Cells/immunology , Transcription Factors/metabolism , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/immunology , Cell Differentiation/genetics , Cell Growth Processes/genetics , Cells, Cultured , Down-Regulation , Granzymes/genetics , Granzymes/metabolism , Interferon Regulatory Factors/metabolism , Interferon-gamma/genetics , Interferon-gamma/metabolism , Lymphocyte Activation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Positive Regulatory Domain I-Binding Factor 1 , Proto-Oncogene Proteins c-jun/metabolism , T-Box Domain Proteins/genetics , Transcription Factors/genetics , Transcriptional Activation/geneticsABSTRACT
Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8+ T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.
Subject(s)
Adenosine Deaminase/deficiency , Adenosine Deaminase/metabolism , Cell Cycle Checkpoints/drug effects , Drug Resistance, Neoplasm/drug effects , Melanoma, Experimental/drug therapy , Melanoma, Experimental/genetics , Programmed Cell Death 1 Receptor/antagonists & inhibitors , RNA-Binding Proteins/metabolism , Adenosine Deaminase/genetics , Animals , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Female , Histocompatibility Antigens Class I/immunology , Immunotherapy , Inflammation/genetics , Inflammation/immunology , Interferon-Induced Helicase, IFIH1/metabolism , Interferons/immunology , Melanoma, Experimental/immunology , Melanoma, Experimental/radiotherapy , Mice , Mice, Inbred C57BL , Phenotype , RNA Editing , RNA, Double-Stranded/genetics , RNA-Binding Proteins/genetics , Receptors, G-Protein-Coupled/metabolismABSTRACT
Syngeneic mouse models are tumors derived from murine cancer cells engrafted on genetically identical mouse strains. They are widely used tools for studying tumor immunity and immunotherapy response in the context of a fully functional murine immune system. Large volumes of syngeneic mouse tumor expression profiles under different immunotherapy treatments have been generated, although a lack of systematic collection and analysis makes data reuse challenging. We present Tumor Immune Syngeneic MOuse (TISMO), a database with an extensive collection of syngeneic mouse model profiles with interactive visualization features. TISMO contains 605 in vitro RNA-seq samples from 49 syngeneic cancer cell lines across 23 cancer types, of which 195 underwent cytokine treatment. TISMO also includes 1518 in vivo RNA-seq samples from 68 syngeneic mouse tumor models across 19 cancer types, of which 832 were from immune checkpoint blockade (ICB) studies. We manually annotated the sample metadata, such as cell line, mouse strain, transplantation site, treatment, and response status, and uniformly processed and quality-controlled the RNA-seq data. Besides data download, TISMO provides interactive web interfaces to investigate whether specific gene expression, pathway enrichment, or immune infiltration level is associated with differential immunotherapy response. TISMO is available at http://tismo.cistrome.org.
Subject(s)
Biomarkers, Pharmacological , Neoplasms/genetics , Software , Tumor Microenvironment/immunology , Animals , Databases, Genetic , Disease Models, Animal , Humans , Immunotherapy/trends , Mice , Neoplasms/immunology , Neoplasms/therapy , Tumor Microenvironment/geneticsABSTRACT
Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing , Immunotherapy/methods , Melanoma, Experimental/immunology , Melanoma, Experimental/therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism , Tumor Escape/drug effects , Tumor Escape/immunology , Animals , Antigen Presentation/genetics , Antigen Presentation/immunology , Genomics , Humans , Interferons/immunology , Loss of Function Mutation , Melanoma, Experimental/genetics , Melanoma, Experimental/pathology , Mice , NF-kappa B/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 2/deficiency , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Escape/genetics , Unfolded Protein Response , Xenograft Model Antitumor AssaysABSTRACT
Adenoviruses are the major cause of ocular viral infections worldwide. Currently, there is no approved antiviral treatment for these eye infections. Cyclopentenylcytosine (CPE-C) is an antiviral that has demonstrated activity against more than 20 viruses. The goals of the current study were to determine the in vitro and in vivo antiviral activity of CPE-C as well as its ocular toxicity. Antiviral activity was evaluated in vitro using standard plaque reduction assays to determine the 50% effective concentrations (EC50s) and in vivo in the Ad5/NZW rabbit ocular replication model. Ocular toxicity was determined in uninfected rabbit eyes following topical ocular application. The in vitro EC50s for CPE-C ranged from 0.03 to 0.059 µg/mL for nine adenovirus types that commonly infect the eye. Ocular toxicity testing determined CPE-C to be non-irritating or practically non-irritating by Draize scoring. In vivo, 3% CPE-C topically administered 4X or 2X daily for 7 days to adenovirus-infected eyes demonstrated effective antiviral activity compared with the negative control and comparable antiviral activity to the positive control, 0.5% cidofovir, topically administered twice daily for 7 days. We conclude CPE-C was relatively non-toxic to rabbit eyes and demonstrated potent anti-adenoviral activity in vitro and in vivo.
Subject(s)
Adenoviridae Infections , Adenoviruses, Human , Eye Infections , Organophosphonates , Animals , Rabbits , Antiviral Agents/therapeutic use , Organophosphonates/pharmacology , Toxic Optic Neuropathy/drug therapy , Cytosine/pharmacology , Adenoviridae Infections/drug therapy , Adenoviridae , Eye Infections/drug therapy , Virus ReplicationABSTRACT
In this study, we tested the hypothesis that the conserved bacterial IgaA-family protein, GumB, mediates microbial pathogenesis associated with Serratia marcescens ocular infections through regulation of the Rcs stress response system. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known, and the role of IgaA proteins in mammalian pathogenesis models has only been tested with partial-function allele variants of Salmonella. Here, we observed that an Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h and demonstrated a notable reduction in inflammation based on inflammatory signs, including the absence of hypopyons, and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid. We observed that bacteria with an inactivated Rcs stress response system induced high levels of ocular inflammation and restored corneal virulence to the gumB mutant. The high virulence of the ΔrcsB mutant was dependent upon the ShlA cytolysin transporter ShlB. Similar results were found for testing the cytotoxic effects of wild-type and mutant bacteria on a human corneal epithelial cell line in vitro. Together, these data indicate that GumB regulates virulence factor production through the Rcs system, and this overall stress response system is a key mediator of a bacterium's ability to induce vision-threatening keratitis.
Subject(s)
Bacterial Proteins/genetics , Keratitis/microbiology , Serratia Infections/microbiology , Serratia marcescens/physiology , Stress, Physiological , Animals , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Host-Pathogen Interactions , Mutation , Rabbits , Stress, Physiological/genetics , Virulence , Virulence Factors/geneticsABSTRACT
Regulatory T cells (Tregs) are key modulators of immune tolerance, capable of suppressing inflammatory immune responses and promoting nonlymphoid tissue homeostasis. Helios, a transcription factor (TF) that is selectively expressed by Tregs, has been shown to be essential for the maintenance of Treg lineage stability in the face of inflammatory conditions that include autoimmune disease and cancer. Helios-deficient Tregs within tumors acquire effector T cell function and contribute to immune responses against cancer. However, the underlying genetic basis of this Treg reprogramming is not well understood. Here, we report that Helios-deficient Tregs within the chronic inflammatory tumor microenvironment (TME) derepress genetic programs associated with T helper (Th) cell differentiation by up-regulating Th cell-associated TFs and effector cytokines. These genetic changes of Helios-deficient Tregs are most apparent in a Treg subpopulation with high affinity for self-antigens, as detected by both increased GITR/PD-1 expression and increased responsiveness to self-antigens. Their combined effects may promote a phenotype conversion of Tregs into effector T cells within the TME, where TCR engagement and costimulatory receptor expression by Tregs are increased. These data provide a genetic basis for the unstable phenotype of Helios-deficient Tregs within the inflammatory environment of tumors and suggest that immune milieu-dependent alterations in gene expression are a central feature of Treg conversion.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/physiology , Melanoma/metabolism , Neoplasms, Experimental/metabolism , T-Lymphocytes, Regulatory/physiology , Transcription Factors/metabolism , Animals , Bacterial Proteins/metabolism , DNA-Binding Proteins/genetics , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Profiling , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Transcription Factors/geneticsABSTRACT
There is no approved antiviral therapy for adenovirus (HAdV) ocular infections. Astodrimer sodium (SPL7013) is a polyanionic dendrimer with antiviral activity. The current study evaluated the ocular tolerability and anti-adenoviral efficacy of topical SPL7013 in rabbit ocular models. In a tolerability study, rabbits were treated with 3% SPL7013, vehicle, or 0.5% cidofovir. Their eyes were graded using the Draize scale. In antiviral efficacy studies, HAdV5 inoculated eyes were treated with 3% SPL7013, vehicle, or 0.5% cidofovir. Eyes were cultured for the virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Viral titers were determined. There were no differences in Draize scores between 3% SPL7013 and vehicle on any day. Cidofovir produced significantly higher Draize scores on day 12 than SPL7013 and vehicle. The 3% SPL7013 and 0.5% cidofovir significantly reduced daily viral titers and positive cultures per total compared with vehicle on several different days. The 3% SPL7013 and 0.5% cidofovir significantly reduced the duration of HAdV5 shedding compared to vehicle. The 3% SPL7013 demonstrated significantly more antiviral activity compared with vehicle in the Ad5/NZW rabbit ocular model. The 3% SPL7013 induced "minimal" to "practically non-irritating" Draize scores in the ocular tolerability study. Further development of astodrimer sodium as a topical antiviral therapy for adenoviral ocular infections is indicated.
Subject(s)
Adenoviridae Infections/drug therapy , Cidofovir/administration & dosage , Dendrimers/administration & dosage , Eye Infections, Viral/drug therapy , Polylysine/administration & dosage , A549 Cells , Adenoviruses, Human/drug effects , Adenoviruses, Human/physiology , Administration, Topical , Animals , Cidofovir/pharmacology , Dendrimers/pharmacology , Disease Models, Animal , Female , Humans , Polylysine/pharmacology , Rabbits , Treatment Outcome , Viral Load/drug effectsABSTRACT
ABSTRACT: With the increased attention to patient safety and quality care in health care, it is imperative that prelicensure health care provider students are taught to collaborate effectively to decrease medical errors. For this project, simulated participants were utilized as health care providers for a simulation in a stand-alone nursing school without affiliation to a medical or allied health school. Both simulated participants and students reacted positively to the experience. This project demonstrated that utilizing simulated participants to portray health care providers in simulation scenarios is a feasible and well-received method of providing learning experiences that emphasize the importance of collaboration.
Subject(s)
Students, Nursing , Delivery of Health Care , Health Personnel , Humans , Interprofessional Relations , LearningABSTRACT
The origins of plasmacytoid dendritic cells (pDCs) have long been controversial and progenitors exclusively committed to this lineage have not been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-pDCs were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-pDCs. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the pDC lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.