ABSTRACT
Stimulator of interferon genes (STING) signaling has been extensively studied in inflammatory diseases and cancer, while its role in T cell responses to infection is unclear. Using Listeria monocytogenes strains engineered to induce different levels of c-di-AMP, we found that high STING signals impaired T cell memory upon infection via increased Bim levels and apoptosis. Unexpectedly, reduction of TCR signal strength or T cell-STING expression decreased Bim expression, T cell apoptosis, and recovered T cell memory. We found that TCR signal intensity coupled STING signal strength to the unfolded protein response (UPR) and T cell survival. Under strong STING signaling, Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition also reduced apoptosis and led to a recovery of T cell memory in STING sufficient CD8 T cells. Thus, STING signaling regulates CD8 T cell memory fitness through both cell-intrinsic and extrinsic mechanisms. These studies provide insight into how IDO and STING therapies could improve long-term T cell protective immunity.
Subject(s)
Receptors, Antigen, T-Cell , Signal Transduction , Receptors, Antigen, T-Cell/genetics , CD8-Positive T-Lymphocytes , Memory T Cells , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolismABSTRACT
CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained, and the molecular programmes defining the autoimmune T cell state, are unknown. In type 1 diabetes, ß-cell-specific CD8 T cells destroy insulin-producing ß-cells. Here we followed the fate of ß-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas, where they differentiate further and destroy ß-cells. Whereas transplantation of as few as 20 autoimmune progenitors induced type 1 diabetes, as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived, and stem-like autoimmune progenitors must continuously seed the pancreas to sustain ß-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/pathology , Insulin-Secreting Cells/immunology , Stem Cells/pathology , Animals , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/transplantation , Cell Self Renewal , Clone Cells/immunology , Clone Cells/metabolism , Clone Cells/pathology , Disease Models, Animal , Female , Glucose-6-Phosphatase/immunology , Hepatocyte Nuclear Factor 1-alpha/metabolism , Insulin-Secreting Cells/pathology , Lymph Nodes/immunology , Male , Mice , Receptors, Antigen, T-Cell/metabolism , Single-Cell Analysis , Stem Cell Transplantation , Stem Cells/immunology , Stem Cells/metabolism , TranscriptomeABSTRACT
BACKGROUND: The safety and immunogenicity of JNJ-64041809 (JNJ-809), a live-attenuated, double-deleted Listeria monocytogenes (LADD Lm)-based immunotherapy targeting 4 relevant prostate cancer antigens, was evaluated in a phase 1 study in patients with metastatic castration-resistant prostate cancer (mCRPC). METHODS: Men with progressive mCRPC who had received ≥2 prior approved therapies were enrolled. Primary study objectives were to determine the recommended phase 2 dose (RP2D) and to evaluate the safety and immunogenicity of JNJ-809. RESULTS: A total of 26 patients received JNJ-809 (1 × 108 CFU (n = 6); 1 × 109 CFU (n = 20)). No dose-limiting toxicities were reported, and 1 × 109 CFU was selected as the RP2D. The most common adverse events (AEs) reported were chills (92%), pyrexia (81%), and fatigue (62%). The most frequent grade ≥3 AEs were lymphopenia (27%) and hypertension (23%). Serious AEs were reported in 27% of patients including 1 patient with grade 3 intestinal obstruction. JNJ-809 transiently induced peripheral cytokines, including interferon-γ, interleukin-10, and tumor necrosis factor-α. Of the 7 patients evaluable for T cell responses at the 1 × 109 CFU dose, evidence of post-treatment antigenic responses were observed in 6 to the Listeria antigen listeriolysin O and in 5 to ≥1 of the 4 encoded tumor antigens. Best overall response was stable disease in 13/25 response-evaluable patients. The study was terminated early as data collected were considered sufficient to evaluate safety and immunogenicity. CONCLUSIONS: JNJ-809 has manageable safety consistent with other LADD Lm-based therapies. Limited antigen-specific immune responses were observed, which did not translate into objective clinical responses.
Subject(s)
Listeria monocytogenes , Prostatic Neoplasms, Castration-Resistant , Humans , Immunotherapy/adverse effects , Listeria monocytogenes/genetics , Male , Prostatic Neoplasms, Castration-Resistant/pathologyABSTRACT
Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Differentiation/immunology , High Mobility Group Proteins/metabolism , Homeodomain Proteins/metabolism , Neoplasms/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , High Mobility Group Proteins/deficiency , High Mobility Group Proteins/genetics , Homeodomain Proteins/genetics , Humans , Immunologic Memory , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Mice , Neoplasms/pathology , Phenotype , Receptors, Antigen, T-Cell/immunology , Transcription, GeneticABSTRACT
Live-attenuated Listeria monocytogenes has shown encouraging potential as an immunotherapy platform in preclinical and clinical settings. However, additional safety measures will enable application across malignant and infectious diseases. Here, we describe a new vaccine platform, termed Lm-RIID (L. monocytogenes recombinase-induced intracellular death), that induces the deletion of genes required for bacterial viability yet maintains potent T cell responses to encoded antigens. Lm-RIID grows normally in broth but commits suicide inside host cells by inducing Cre recombinase and deleting essential genes flanked by loxP sites, resulting in a self-limiting infection even in immunocompromised mice. Lm-RIID vaccination of mice induces potent CD8+ T cells and protects against virulent challenges, similar to live L. monocytogenes vaccines. When combined with α-PD-1, Lm-RIID is as effective as live-attenuated L. monocytogenes in a therapeutic tumor model. This impressive efficacy, together with the increased clearance rate, makes Lm-RIID ideal for prophylactic immunization against diseases that require T cells for protection.
Subject(s)
Bacterial Vaccines/immunology , Listeria monocytogenes/immunology , Animals , Female , Immunotherapy , Listeria monocytogenes/pathogenicity , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , T-Lymphocytes/immunology , Vaccines, Attenuated/immunology , VirulenceABSTRACT
BACKGROUND: Immune checkpoint inhibitors are not effective for pancreatic ductal adenocarcinoma (PDAC) as single agents. Vaccine therapy may sensitize PDACs to checkpoint inhibitor treatments. Annexin A2 (ANXA2) is a pro-metastasis protein, previously identified as a relevant PDAC antigen that is expressed by a GM-CSF-secreting allogenic whole pancreatic tumor cell vaccine (GVAX) to induce an anti-ANXA2 antibody response in patients with PDAC. We hypothesized that an ANXA2-targeting vaccine approach not only provokes an immune response but also mounts anti-tumor effects. METHODS: We developed a Listeria-based, ANXA2-targeting cancer immunotherapy (Lm-ANXA2) and investigated its effectiveness within two murine models of PDAC. RESULTS: We show that Lm-ANXA2 prolonged the survival in a transplant model of mouse PDACs. More importantly, priming with the Lm-ANXA2 treatment prior to administration of anti-PD-1 antibodies increased cure rates in the implanted PDAC model and resulted in objective tumor responses and prolonged survival in the genetically engineered spontaneous PDAC model. In tumors treated with Lm-ANXA2 followed by anti-PD-1 antibody, the T cells specific to ANXA2 had significantly increased INFγ expression. CONCLUSIONS: For the first time, a listeria vaccine-based immunotherapy was shown to be able to induce a tumor antigen-specific T cell response within the tumor microenvironment of a "cold" tumor such as PDAC and sensitize the tumor to checkpoint inhibitor therapy. Moreover, this combination immunotherapy led to objective tumor responses and survival benefit in the mice with spontaneously developed PDAC tumors. Therefore, our study supports developing Lm-ANXA2 as a therapeutic agent in combination with anti-PD-1 antibody for PDAC treatment.
Subject(s)
Antineoplastic Agents, Immunological/administration & dosage , Cancer Vaccines/administration & dosage , Carcinoma, Pancreatic Ductal/therapy , Listeria/immunology , Pancreatic Neoplasms/therapy , Animals , Annexin A2/antagonists & inhibitors , Annexin A2/genetics , Annexin A2/immunology , Antigens, Neoplasm/immunology , Cancer Vaccines/immunology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor/transplantation , Disease Models, Animal , Female , Humans , Mice , Mice, Transgenic , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Proto-Oncogene Proteins p21(ras)/genetics , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Tumor Suppressor Protein p53/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunologyABSTRACT
Dysregulated signaling via the epidermal growth factor receptor (EGFR)-family is believed to contribute to the progression of a diverse array of cancers. The most common variant of EGFR is EGFRvIII, which results from a consistent and tumor-specific in-frame deletion of exons 2-7 of the EGFR gene. This deletion generates a novel glycine at the junction and leads to constitutive ligand-independent activity. This junction forms a novel shared tumor neo-antigen with demonstrated immunogenicity in both mice and humans. A 21-amino acid peptide spanning the junctional region was selected, and then one or five copies of this 21-AA neo-peptide were incorporated into live-attenuated Listeria monocytogenes-based vaccine vector. These vaccine candidates demonstrated efficient secretion of the recombinant protein and potent induction of EGFRvIII-specific CD8+ T cells, which prevented growth of an EGFRvIII-expressing squamous cell carcinoma. These data demonstrate the potency of a novel cancer-specific vaccine candidate that can elicit EGFRvIII-specific cellular immunity, for the purpose of targeting EGFRvIII positive cancers that are resistant to conventional therapies.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/therapeutic use , Carcinoma, Squamous Cell/metabolism , ErbB Receptors/metabolism , Animals , Cancer Vaccines/immunology , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/therapy , Female , Immunotherapy , Mice , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
Background. Sipuleucel T, an autologous cell-based vaccine targeting prostatic acid phosphatase (PAP), has demonstrated efficacy for the treatment of advanced prostate cancer. DNA vaccines encoding PAP and live attenuated Listeria vaccines have entered clinical trials for patients with prostate cancer, and have advantages in terms of eliciting predominantly Th1-biased immunity. In this study, we investigated whether the immunogenicity and anti-tumor efficacy of a DNA and Listeria vaccine, each encoding PAP, could be enhanced by using them in a heterologous prime/boost approach. Methods. Transgenic mice expressing HLA-A2.01 and HLA-DRB1*0101 were immunized alone or with a heterologous prime/boost strategy. Splenocytes were evaluated for MHC class I and II-restricted, PAP-specific immune responses by IFNγ ELISPOTs. Anti-tumor activity to a syngeneic, PAP-expressing tumor line was evaluated. Results. PAP-specific cellular immunity and anti-tumor activity were elicited in mice after immunization with DNA- or listeria-based vaccines. Greater CD4+ and CD8+ responses, and anti-tumor responses, were elicited when mice were immunized first with DNA and boosted with Listeria, but not when administered in the opposite order. This was found to be dependent on CD4+ T cells elicited with DNA priming, and was not due to inflammatory signals by Listeria itself or due to B cells serving as antigen-presenting cells for DNA during priming. Conclusions. Heterologous prime/boost vaccination using DNA priming with Listeria boosting may provide better anti-tumor immunity, similar to many reports evaluating DNA priming with vaccines targeting foreign microbial antigens. These findings have implications for the design of future clinical trials.
ABSTRACT
Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L- CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206- M1 phenotype. Remarkably, these LADD-Ag-induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.
Subject(s)
Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Listeria monocytogenes/immunology , Neoplasms/therapy , Tumor Microenvironment/immunology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/therapeutic use , Cancer Vaccines/genetics , Cancer Vaccines/therapeutic use , Cell Line, Tumor , Drug Evaluation, Preclinical , Female , Humans , Listeria monocytogenes/genetics , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasms/immunology , Treatment Outcome , Vaccination/methods , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Attenuated/therapeutic use , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Vaccines, DNA/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
Listeria monocytogenes is a Gram-positive intracellular pathogen that causes a severe invasive disease. Upon infecting a host cell, L. monocytogenes upregulates the transcription of numerous factors necessary for productive infection. VirR is the response regulator component of a two-component regulatory system in L. monocytogenes In this report, we have identified the putative ABC transporter encoded by genes lmo1746-lmo1747 as necessary for VirR function. We have designated lmo1746-lmo1747 virAB We constructed an in-frame deletion of virAB and determined that the ΔvirAB mutant exhibited reduced transcription of VirR-regulated genes. The ΔvirAB mutant also showed defects in in vitro plaque formation and in vivo virulence that were similar to those of a ΔvirR deletion mutant. Since VirR is important for innate resistance to antimicrobial agents, we determined the MICs of nisin and bacitracin for ΔvirAB bacteria. We found that VirAB expression was necessary for nisin resistance but was dispensable for resistance to bacitracin. This result suggested a VirAB-independent mechanism of VirR regulation in response to bacitracin. Lastly, we found that the ΔvirR and ΔvirAB mutants had no deficiency in growth in broth culture, intracellular replication, or production of the ActA surface protein, which facilitates actin-based motility and cell-to-cell spread. However, the ΔvirR and ΔvirAB mutants produced shorter actin tails during intracellular infection, which suggested that these mutants have a reduced ability to move and spread via actin-based motility. These findings have demonstrated that L. monocytogenes VirAB functions in a pathway with VirR to regulate the expression of genes necessary for virulence and resistance to antimicrobial agents.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Listeria monocytogenes/metabolism , Listeria monocytogenes/pathogenicity , Listeriosis/microbiology , Nisin/pharmacology , Transcription Factors/genetics , ATP-Binding Cassette Transporters/genetics , Animals , Bacterial Proteins/genetics , Drug Resistance, Bacterial , Female , Gene Expression Regulation, Bacterial , Humans , Listeria monocytogenes/drug effects , Listeria monocytogenes/genetics , Mice, Inbred BALB C , Microbial Sensitivity Tests , Regulon , Transcription Factors/metabolism , VirulenceABSTRACT
The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell-cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin-mediated coupling of the bacterium to F-actin is not required.
Subject(s)
Cadherins/metabolism , Listeria monocytogenes/metabolism , alpha Catenin/metabolism , Actins/immunology , Animals , Antigens, Surface/metabolism , Bacterial Proteins/metabolism , Cadherins/immunology , Cell Adhesion/physiology , Cell Culture Techniques , Cell Line, Tumor , Cell Membrane/metabolism , Dogs , Epithelial Cells/microbiology , Humans , Intercellular Junctions/metabolism , Madin Darby Canine Kidney CellsABSTRACT
Tumour-specific CD8 T cells in solid tumours are dysfunctional, allowing tumours to progress. The epigenetic regulation of T cell dysfunction and therapeutic reprogrammability (for example, to immune checkpoint blockade) is not well understood. Here we show that T cells in mouse tumours differentiate through two discrete chromatin states: a plastic dysfunctional state from which T cells can be rescued, and a fixed dysfunctional state in which the cells are resistant to reprogramming. We identified surface markers associated with each chromatin state that distinguished reprogrammable from non-reprogrammable PD1hi dysfunctional T cells within heterogeneous T cell populations from tumours in mice; these surface markers were also expressed on human PD1hi tumour-infiltrating CD8 T cells. Our study has important implications for cancer immunotherapy as we define key transcription factors and epigenetic programs underlying T cell dysfunction and surface markers that predict therapeutic reprogrammability.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Chromatin/genetics , Chromatin/metabolism , Neoplasms/genetics , Neoplasms/immunology , Animals , CD8-Positive T-Lymphocytes/metabolism , Epigenesis, Genetic , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Immunologic Memory , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Male , Mice , Neoplasms/metabolism , Neoplasms/therapy , Transcription Factors/metabolismABSTRACT
CD8(+) T cells recognizing tumor-specific antigens are detected in cancer patients but are dysfunctional. Here we developed a tamoxifen-inducible liver cancer mouse model with a defined oncogenic driver antigen (SV40 large T-antigen) to follow the activation and differentiation of naive tumor-specific CD8(+) T (TST) cells after tumor initiation. Early during the pre-malignant phase of tumorigenesis, TST cells became dysfunctional, exhibiting phenotypic, functional, and transcriptional features similar to dysfunctional T cells isolated from late-stage human tumors. Thus, T cell dysfunction seen in advanced human cancers may already be established early during tumorigenesis. Although the TST cell dysfunctional state was initially therapeutically reversible, it ultimately evolved into a fixed state. Persistent antigen exposure rather than factors associated with the tumor microenvironment drove dysfunction. Moreover, the TST cell differentiation and dysfunction program exhibited features distinct from T cell exhaustion in chronic infections. Strategies to overcome this antigen-driven, cell-intrinsic dysfunction may be required to improve cancer immunotherapy.
Subject(s)
Antigens, Polyomavirus Transforming/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Immunotherapy, Adoptive/methods , Liver Neoplasms/immunology , Animals , Carcinogenesis , Cell Differentiation , Cells, Cultured , Cellular Senescence , Disease Models, Animal , Humans , Liver Neoplasms/chemically induced , Liver Neoplasms/therapy , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Tamoxifen , Tumor MicroenvironmentABSTRACT
PURPOSE: Immune responses to antigens originating in the central nervous system (CNS) are generally attenuated, as collateral damage can have devastating consequences. The significance of this finding for the efficacy of tumor-targeted immunotherapies is largely unknown. EXPERIMENTAL DESIGN: The B16 murine melanoma model was used to compare cytotoxic responses against established tumors in the CNS and in the periphery. Cytokine analysis of tissues from brain tumor-bearing mice detected elevated TGFß secretion from microglia and in the serum and TGFß signaling blockade reversed tolerance of tumor antigen-directed CD8 T cells. In addition, a treatment regimen using focal radiation therapy and recombinant Listeria monocytogenes was evaluated for immunologic activity and efficacy in this model. RESULTS: CNS melanomas were more tolerogenic than equivalently progressed tumors outside the CNS as antigen-specific CD8 T cells were deleted and exhibited impaired cytotoxicity. Tumor-bearing mice had elevated serum levels of TGFß; however, blocking TGFß signaling with a small-molecule inhibitor or a monoclonal antibody did not improve survival. Conversely, tumor antigen-specific vaccination in combination with focal radiation therapy reversed tolerance and improved survival. This treatment regimen was associated with increased polyfunctionality of CD8 T cells, elevated T effector to T regulatory cell ratios, and decreased TGFß secretion from microglia. CONCLUSIONS: These data suggest that CNS tumors may impair systemic antitumor immunity and consequently accelerate cancer progression locally as well as outside the CNS, whereas antitumor immunity may be restored by combining vaccination with radiation therapy. These findings are hypothesis-generating and warrant further study in contemporary melanoma models as well as human trials.
Subject(s)
Brain Neoplasms/therapy , Central Nervous System Neoplasms/therapy , Immune Tolerance , Melanoma, Experimental/therapy , Transforming Growth Factor beta/blood , Animals , Antigens, Neoplasm/administration & dosage , Antigens, Neoplasm/immunology , Brain Neoplasms/blood , Brain Neoplasms/immunology , Brain Neoplasms/radiotherapy , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/radiation effects , Central Nervous System Neoplasms/blood , Central Nervous System Neoplasms/immunology , Central Nervous System Neoplasms/radiotherapy , Female , Humans , Melanoma, Experimental/blood , Melanoma, Experimental/immunology , Melanoma, Experimental/radiotherapy , Mice , Microglia/immunology , Microglia/pathology , T-Lymphocytes, Cytotoxic/immunology , Transforming Growth Factor beta/antagonists & inhibitors , VaccinationABSTRACT
Stimulator of interferon genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs), activating TBK1/IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and proinflammatory cytokine responses. CDN ligands were formulated with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing cellular cancer vaccines--termed STINGVAX--that demonstrated potent in vivo antitumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and noncanonical c[A(2',5')pA(3',5')p] phosphate bridge structure, enhanced antitumor efficacy of STINGVAX in multiple aggressive therapeutic models of established cancer in mice. Antitumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen-specific CD8(+) T cells. Tumors from STINGVAX-treated mice demonstrated marked PD-L1 (programmed death ligand 1) up-regulation, which was associated with tumor-infiltrating CD8(+)IFNγ(+) T cells. When combined with PD-1 (programmed death 1) blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone.
Subject(s)
Antineoplastic Agents/chemistry , Cancer Vaccines/chemistry , Membrane Proteins/agonists , Programmed Cell Death 1 Receptor/metabolism , Animals , CD8-Positive T-Lymphocytes/cytology , Cell Line, Tumor , Cytosol/metabolism , Dendritic Cells/cytology , Female , Humans , Interferon-gamma/metabolism , Ligands , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Monocytes/cytology , NF-kappa B/metabolism , Neoplasm Transplantation , Phosphates/chemistry , Protein Serine-Threonine Kinases/metabolism , STAT6 Transcription Factor/metabolismABSTRACT
Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.
Subject(s)
Gene Expression Regulation, Bacterial/genetics , Glutathione/metabolism , Intracellular Space/metabolism , Intracellular Space/microbiology , Listeria monocytogenes/genetics , Listeria monocytogenes/pathogenicity , Allosteric Regulation/drug effects , Bacterial Proteins/metabolism , DNA/metabolism , Gene Expression Regulation, Bacterial/drug effects , Glutathione/pharmacology , Intracellular Space/drug effects , Listeria monocytogenes/drug effects , Macrophages/metabolism , Mutation/genetics , Peptide Termination Factors/metabolism , Protein Binding , Selection, Genetic/genetics , Suppression, Genetic/genetics , Virulence/geneticsABSTRACT
HIV-1 infection is characterized by myeloid dendritic cell (DC) dysfunction, which blunts the responsiveness to vaccine adjuvants. We previously showed that nonviral factors in HIV-seropositive plasma are partially responsible for mediating this immune suppression. In this study we investigated recombinant Listeria monocytogenes (Lm) vectors, which naturally infect and potently activate DCs from seronegative donors, as a means to overcome DC dysfunction associated with HIV infection. Monocyte-derived DCs were cocultured with plasma from HIV-infected donors (HIV-moDCs) to induce a dysregulated state and infected with an attenuated, nonreplicative vaccine strain of Lm expressing full length clade B consensus gag (KBMA Lm-gag). Lm infection stimulated cytokine secretion [interleukin (IL)-12p70, tumor necrosis factor (TNF)-α, and IL-6] and Th-1 skewing of allogeneic naive CD4 T cells by HIV-moDCs, in contrast to the suppressive effects observed by HIV plasma on moDCs on toll-like receptor ligand stimulation. Upon coculture of "killed" but metabolically active (KBMA) Lm-gag-infected moDCs from HIV-infected donors with autologous cells, expansion of polyfunctional, gag-specific CD8(+) T cells was observed. Reactivation of latent proviruses by moDCs following Lm infection was also observed in models of HIV latency in a TNF-α-dependent manner. These findings reveal the unique ability of Lm vectors to contend with dysregulation of HIV-moDCs, while simultaneously possessing the capacity to activate latent virus. Concurrent stimulation of innate and adaptive immunity and disruption of latency may be an approach to reduce the pool of latently infected cells during HIV infection. Further study of Lm vectors as part of therapeutic vaccination and eradication strategies may advance this evolving field.
Subject(s)
Dendritic Cells/immunology , HIV Infections/immunology , HIV-1/physiology , Listeria monocytogenes/immunology , Virus Activation , Adaptive Immunity/immunology , CD8-Positive T-Lymphocytes/immunology , Cells, Cultured , Coinfection/immunology , HIV Infections/virology , HIV-1/immunology , Humans , Interleukin-12/metabolism , Interleukin-6/metabolism , Myeloid Cells/immunology , Th1 Cells/immunology , Tumor Necrosis Factor-alpha/metabolism , Virus Latency , gag Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
A principal mechanism by which tumors evade immune-mediated elimination is through immunosuppression. Previous approaches to tumor immunotherapy have focused on modifying the immunosuppressive environment with immune checkpoint inhibitors, cytokine therapy, and other modalities with the intent to generate T-cell based anti-tumor immunity. We hypothesized that transformation of the suppressive ovarian cancer microenvironment could be achieved by introduction of the attenuated ΔactA/ΔinlB strain of Listeria monocytogenes. ΔactA/ΔinlB introduced into the microenvironment of the aggressive ID8-Defb29/Vegf-A murine ovarian carcinoma is preferentially phagocytosed by tumor-associated macrophages (TAMs) and reprograms that population from one of suppression to immunostimulation. TAMs in the peritoneum upregulated their co-stimulatory molecules CD80 and CD86, increased transcription of inflammatory cytokines, and downregulated transcription of suppressive effector molecules. Surprisingly, therapeutic benefit was not mediated by T- or NK-cell activity. ΔactA/ΔinlB-induced repolarization of TAMs activated direct tumor cell lysis via Nos2 production of nitric oxide. Modulation of the immunosuppressive nature of the ID8-Defb29/Vegf-A microenvironment, specifically by reprogramming of the TAM suppressive population from M2 to M1 polarization, is critical for our observed immune-mediated survival benefit.
ABSTRACT
BACKGROUND & AIMS: Premalignant lesions and early stage tumors contain immunosuppressive microenvironments that create barriers for cancer vaccines. Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which express an activated form of Kras in pancreatic tissues, develop pancreatic intraepithelial neoplasms (PanIN) that progress to pancreatic ductal adenocarcinoma (PDA). We used these mice to study immune suppression in PDA. METHODS: We immunized KPC and Kras(G12D/+);Pdx-1-Cre mice with attenuated intracellular Listeria monocytogenes (which induces CD4(+) and CD8(+) T-cell immunity) engineered to express Kras(G12D) (LM-Kras). The vaccine was given alone or in sequence with an anti-CD25 antibody (PC61) and cyclophosphamide to deplete T-regulatory (Treg) cells. Survival times were measured; pancreatic and spleen tissues were collected and analyzed by histologic, flow cytometry, and immunohistochemical analyses. RESULTS: Interferon γ-mediated, CD8(+) T-cell responses were observed in KPC and Kras(G12D/+);Pdx-1-Cre mice given LM-Kras, but not in unvaccinated mice. Administration of LM-Kras to KPC mice 4-6 weeks old (with early stage PanINs), depleted of Treg cells, significantly prolonged survival and reduced PanIN progression (median survival, 265 days), compared with unvaccinated mice (median survival, 150 days; P = .002), mice given only LM-Kras (median survival, 150 days; P = .050), and unvaccinated mice depleted of Treg cells (median survival, 170 days; P = .048). In 8- to 12-week-old mice (with late-stage PanINs), LM-Kras, alone or in combination with Treg cell depletion, did not increase survival time or slow PanIN progression. The combination of LM-Kras and Treg cell depletion reduced numbers of Foxp3(+)CD4(+) T cells in pancreatic lymph nodes, increased numbers of CD4(+) T cells that secrete interleukin 17 and interferon γ, and caused CD11b(+)Gr1(+) cells in the pancreas to acquire an immunostimulatory phenotype. CONCLUSIONS: Immunization of KPC mice with Listeria monocytogenes engineered to express Kras(G12D), along with depletion of Treg cells, reduces progression of early stage, but not late-stage, PanINs. This approach increases infiltration of the lesion with inflammatory cells. It might be possible to design immunotherapies against premalignant pancreatic lesions to slow or prevent progression to PDA.
Subject(s)
Cancer Vaccines/therapeutic use , Carcinoma in Situ/drug therapy , Carcinoma, Pancreatic Ductal/drug therapy , Listeria monocytogenes/immunology , Pancreatic Neoplasms/drug therapy , T-Lymphocytes, Regulatory/immunology , Animals , Antibodies, Monoclonal/pharmacology , CD11b Antigen/metabolism , Cancer Vaccines/immunology , Carcinoma in Situ/genetics , Carcinoma in Situ/immunology , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cyclophosphamide/pharmacology , Disease Models, Animal , Disease Progression , Forkhead Transcription Factors/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Inflammation Mediators/metabolism , Integrases/genetics , Integrases/metabolism , Interferon-gamma/metabolism , Interleukin-17/metabolism , Listeria monocytogenes/genetics , Listeria monocytogenes/metabolism , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Receptors, Chemokine/metabolism , T-Lymphocytes, Regulatory/metabolism , Time Factors , Trans-Activators/genetics , Trans-Activators/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
In this study, we engineered Listeria monocytogens (Lm) by deleting the LmΔactA/ΔinlB virulence determinants and inserting HCV-NS5B consensus antigens to develop a therapeutic vaccine against hepatitis C virus (HCV) infection. We tested this recombinant Lm-HCV vaccine in triggering of innate and adaptive immune responses in vitro using immune cells from HCV-infected and uninfected individuals. This live-attenuated Lm-HCV vaccine could naturally infect human dendritic cells (DC), thereby driving DC maturation and antigen presentation, producing Th1 cytokines, and triggering CTL responses in uninfected individuals. However, vaccine responses were diminished when using DC and T cells derived from chronically HCV-infected individuals, who express higher levels of inhibitory molecule Tim-3 on immune cells. Notably, blocking Tim-3 signaling significantly improved the innate and adaptive immune responses in chronically HCV-infected patients, indicating that novel strategies to enhance the potential of antigen presentation and cellular responses are essential for developing an effective therapeutic vaccine against HCV infection.