ABSTRACT
Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.
Subject(s)
Dendritic Cells, Follicular/immunology , Fibroblasts/immunology , Lymph Nodes/immunology , Stromal Cells/immunology , Aged , Animals , Apoptosis/genetics , Apoptosis/immunology , Cell Proliferation/genetics , Cell Survival/genetics , Cell Survival/immunology , Dendritic Cells, Follicular/metabolism , Female , Fibroblasts/metabolism , Gene Expression Regulation/immunology , Gene Knock-In Techniques , Humans , Immunity, Cellular/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Lymph Nodes/cytology , Male , Mice , Mice, Transgenic , RNA-Seq , Single-Cell Analysis , Stromal Cells/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/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
In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.
Subject(s)
Collagen/metabolism , Fibroblasts/cytology , Lectins, C-Type/metabolism , Lymph Nodes/cytology , Membrane Glycoproteins/metabolism , Amides/pharmacology , Animals , Cell Survival/immunology , Collagen/immunology , Cytoskeleton/immunology , Cytoskeleton/ultrastructure , Enzyme Inhibitors/pharmacology , Female , Fibroblasts/immunology , Fibroblasts/ultrastructure , Lectins, C-Type/immunology , Lymph Nodes/immunology , Lymph Nodes/ultrastructure , Male , Membrane Glycoproteins/immunology , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Phosphorylation , Pyridines/pharmacology , Specific Pathogen-Free OrganismsABSTRACT
Fibroblastic reticular cells (FRCs) are known to inhabit T cell-rich areas of lymphoid organs, where they function to facilitate interactions between T cells and dendritic cells. However, in vivo manipulation of FRCs has been limited by a dearth of genetic tools that target this lineage. Here, using a mouse model to conditionally ablate FRCs, we demonstrated their indispensable role in antiviral T cell responses. Unexpectedly, loss of FRCs also attenuated humoral immunity due to impaired B cell viability and follicular organization. Follicle-resident FRCs established a favorable niche for B lymphocytes via production of the cytokine BAFF. Thus, our study indicates that adaptive immunity requires an intact FRC network and identifies a subset of FRCs that control B cell homeostasis and follicle identity.
Subject(s)
B-Lymphocytes/immunology , Fibroblasts/immunology , Homeostasis/immunology , T-Lymphocytes/immunology , Animals , B-Cell Activating Factor/immunology , B-Cell Activating Factor/metabolism , B-Lymphocytes/metabolism , Cell Movement/immunology , Cell Survival/immunology , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/metabolism , Fibroblasts/metabolism , Flow Cytometry , Immunity, Humoral/immunology , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lymph Nodes/immunology , Lymph Nodes/metabolism , Male , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , T-Lymphocytes/metabolismABSTRACT
Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.
Subject(s)
ADAM Proteins/immunology , Colitis/immunology , Extracellular Matrix/metabolism , Fibroblasts/immunology , Intestinal Mucosa/immunology , Mesenchymal Stem Cells/immunology , ADAM Proteins/deficiency , ADAM Proteins/genetics , Animals , Cell Differentiation , Colitis/chemically induced , Colitis/genetics , Colitis/pathology , Colon/immunology , Colon/pathology , Extracellular Matrix/immunology , Fibroblasts/pathology , Fibrosis , Gene Expression Regulation , Humans , Inflammation , Interleukin-11/genetics , Interleukin-11/immunology , Intestinal Mucosa/pathology , Male , Mesenchymal Stem Cells/pathology , Mice , Mice, Inbred C57BL , Sequence Analysis, RNA , Single-Cell Analysis , Sodium Dodecyl Sulfate/administration & dosage , Transcription, Genetic , Transcriptome , Wound Healing/genetics , Wound Healing/immunologyABSTRACT
BACKGROUND & AIMS: Transforming growth factor-b (TGFb) plays pleiotropic roles in pancreatic cancer, including promoting metastasis, attenuating CD8 T-cell activation, and enhancing myofibroblast differentiation and deposition of extracellular matrix. However, single-agent TGFb inhibition has shown limited efficacy against pancreatic cancer in mice or humans. METHODS: We evaluated the TGFß-blocking antibody NIS793 in combination with gemcitabine/nanoparticle (albumin-bound)-paclitaxel or FOLFIRINOX (folinic acid [FOL], 5-fluorouracil [F], irinotecan [IRI] and oxaliplatin [OX]) in orthotopic pancreatic cancer models. Single-cell RNA sequencing and immunofluorescence were used to evaluate changes in tumor cell state and the tumor microenvironment. RESULTS: Blockade of TGFß with chemotherapy reduced tumor burden in poorly immunogenic pancreatic cancer, without affecting the metastatic rate of cancer cells. Efficacy of combination therapy was not dependent on CD8 T cells, because response to TGFß blockade was preserved in CD8-depleted or recombination activating gene 2 (RAG2-/-) mice. TGFß blockade decreased total α-smooth muscle actin-positive fibroblasts but had minimal effect on fibroblast heterogeneity. Bulk RNA sequencing on tumor cells sorted ex vivo revealed that tumor cells treated with TGFß blockade adopted a classical lineage consistent with enhanced chemosensitivity, and immunofluorescence for cleaved caspase 3 confirmed that TGFß blockade increased chemotherapy-induced cell death in vivo. CONCLUSIONS: TGFß regulates pancreatic cancer cell plasticity between classical and basal cell states. TGFß blockade in orthotropic models of pancreatic cancer enhances sensitivity to chemotherapy by promoting a classical malignant cell state. This study provides scientific rationale for evaluation of NIS793 with FOLFIRINOX or gemcitabine/nanoparticle (albumin-bound) paclitaxel chemotherapy backbone in the clinical setting and supports the concept of manipulating cancer cell plasticity to increase the efficacy of combination therapy regimens.
Subject(s)
Antineoplastic Agents , Pancreatic Neoplasms , Humans , Mice , Animals , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Transforming Growth Factor beta/metabolism , Antineoplastic Agents/therapeutic use , Gemcitabine , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Albumins , Transforming Growth Factors/therapeutic use , Tumor Microenvironment , Pancreatic NeoplasmsABSTRACT
Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.
Subject(s)
Immunotherapy , Molecular Targeted Therapy , Protein Phosphatase 2/metabolism , Tumor Microenvironment/immunology , Animals , Antigens, Neoplasm/immunology , Apoptosis/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , Cytokines/immunology , Cytokines/metabolism , Female , Gene Knockdown Techniques , High-Throughput Nucleotide Sequencing , Immunotherapy/methods , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , Protein Phosphatase 2/deficiency , Protein Phosphatase 2/genetics , RNA, Small Interfering/genetics , Reproducibility of ResultsABSTRACT
Fibroblastic reticular cells (FRCs) form the cellular scaffold of lymph nodes (LNs) and establish distinct microenvironmental niches to provide key molecules that drive innate and adaptive immune responses and control immune regulatory processes. Here, we have used a graph theory-based systems biology approach to determine topological properties and robustness of the LN FRC network in mice. We found that the FRC network exhibits an imprinted small-world topology that is fully regenerated within 4 wk after complete FRC ablation. Moreover, in silico perturbation analysis and in vivo validation revealed that LNs can tolerate a loss of approximately 50% of their FRCs without substantial impairment of immune cell recruitment, intranodal T cell migration, and dendritic cell-mediated activation of antiviral CD8+ T cells. Overall, our study reveals the high topological robustness of the FRC network and the critical role of the network integrity for the activation of adaptive immune responses.
Subject(s)
Cell Communication/immunology , Cell Movement/immunology , Fibroblasts/immunology , Lymph Nodes/immunology , T-Lymphocytes/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Count , Cell Movement/genetics , Chemokine CCL19/genetics , Chemokine CCL19/immunology , Chemokine CCL19/metabolism , Dendritic Cells/cytology , Dendritic Cells/immunology , Fibroblasts/cytology , Fibroblasts/metabolism , Lymph Nodes/cytology , Lymph Nodes/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Models, Immunological , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolismABSTRACT
Multiple sclerosis (MS) is a presumed autoimmune disease directed against central nervous system (CNS) myelin, in which diet and obesity are implicated as risk factors. Immune responses can be influenced by molecules produced by fat cells, called adipokines. Adiponectin is an adipokine with anti-inflammatory effects. We tested the hypothesis that adiponectin has a protective role in the EAE model for MS, that can be induced by immunization with myelin antigens or transfer of myelin-specific T lymphocytes. Adiponectin deficient (ADPKO) mice developed worse EAE with greater CNS inflammation, demyelination, and axon injury. Lymphocytes from myelin-immunized ADPKO mice proliferated more, produced higher amounts of IFN-γ, IL-17, TNF-α, IL-6, and transferred more severe EAE than wild type (WT) lymphocytes. At EAE peak, the spleen and CNS of ADPKO had fewer regulatory T (Treg) cells than WT mice and during EAE recovery, Foxp3, IL-10 and TGF-ß expression levels in the CNS were reduced in ADPKO compared with WT mice. Treatment with globular adiponectin in vivo ameliorated EAE, and was associated with an increase in Treg cells. These data indicate that adiponectin is an important regulator of T-cell functions during EAE, suggesting a new avenue of investigation for MS treatment.
Subject(s)
Adiponectin/physiology , Encephalomyelitis, Autoimmune, Experimental/immunology , Lymphocyte Activation , T-Lymphocytes, Regulatory/immunology , Adiponectin/administration & dosage , Adiponectin/deficiency , Adiponectin/genetics , Adoptive Transfer , Animals , Autoimmunity , Cell Proliferation , Cells, Cultured , Central Nervous System/immunology , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Forkhead Transcription Factors/biosynthesis , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Interleukin-17/biosynthesis , Interleukin-6/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Multiple Sclerosis/immunology , Myelin Sheath/immunology , Risk Factors , Spleen/immunology , Th1 Cells/immunology , Th1 Cells/transplantation , Transforming Growth Factor beta/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
BACKGROUND: NIS793 is a human IgG2 monoclonal antibody that binds to transforming growth factor beta (TGF-ß). This first-in-human study investigated NIS793 plus spartalizumab treatment in patients with advanced solid tumors. METHODS: Patients received NIS793 (0.3-1 mg/kg every 3 weeks (Q3W)) monotherapy; following evaluation of two dose levels, dose escalation continued with NIS793 plus spartalizumab (NIS793 0.3-30 mg/kg Q3W and spartalizumab 300 mg Q3W or NIS793 20-30 mg/kg every 2 weeks [Q2W] and spartalizumab 400 mg every 4 weeks (Q4W)). In dose expansion, patients with non-small cell lung cancer (NSCLC) resistant to prior anti-programmed death ligand 1 or patients with microsatellite stable colorectal cancer (MSS-CRC) were treated at the recommended dose for expansion (RDE). RESULTS: Sixty patients were treated in dose escalation, 11 with NIS793 monotherapy and 49 with NIS793 plus spartalizumab, and 60 patients were treated in dose expansion (MSS-CRC: n=40; NSCLC: n=20). No dose-limiting toxicities were observed. The RDE was established as NIS793 30 mg/kg (2100 mg) and spartalizumab 300 mg Q3W. Overall 54 (49.5%) patients experienced ≥1 treatment-related adverse event, most commonly rash (n=16; 13.3%), pruritus (n=10; 8.3%), and fatigue (n=9; 7.5%). Three partial responses were reported: one in renal cell carcinoma (NIS793 30 mg/kg Q2W plus spartalizumab 400 mg Q4W), and two in the MSS-CRC expansion cohort. Biomarker data showed evidence of target engagement through increased TGF-ß/NIS793 complexes and depleted active TGF-ß in peripheral blood. Gene expression analyses in tumor biopsies demonstrated decreased TGF-ß target genes and signatures and increased immune signatures. CONCLUSIONS: In patients with advanced solid tumors, proof of mechanism of NIS793 is supported by evidence of target engagement and TGF-ß pathway inhibition. TRIAL REGISTRATION NUMBER: NCT02947165.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Kidney Neoplasms , Lung Neoplasms , Adult , Humans , Antibodies, Monoclonal/adverse effects , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Kidney Neoplasms/drug therapy , Lung Neoplasms/drug therapy , Transforming Growth Factor betaABSTRACT
High levels of IL1ß can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1ß could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1ß blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti-programmed cell death protein 1 (anti-PD-1), anti-VEGFα, and anti-TGFß treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1ß blockade enhanced the effectiveness of docetaxel and anti-PD-1. Accompanying these effects, blockade of IL1ß alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1ß inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1ß blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1ß inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.
Subject(s)
Interleukin-1beta , Neoplasms , Tumor Microenvironment , Animals , Mice , Cell Line, Tumor , Docetaxel/pharmacology , Immunity , Immunotherapy , Neoplasms/drug therapy , Interleukin-1beta/antagonists & inhibitorsABSTRACT
Objectives: Sabatolimab is a humanized monoclonal antibody (hIgG4, S228P) directed against human T-cell immunoglobulin domain and mucin domain-3 (TIM-3). Herein, we describe the development and characterization of sabatolimab. Methods: Sabatolimab was tested for binding to its target TIM-3 and blocking properties. The functional effects of sabatolimab were tested in T-cell killing and myeloid cell cytokine assays. Antibody-mediated cell phagocytosis (ADCP) by sabatolimab was also assessed. Results: Sabatolimab was shown to (i) enhance T-cell killing and inflammatory cytokine production by dendritic cells (DCs); (ii) facilitate the phagocytic uptake of TIM-3-expressing target cells; and (iii) block the interaction between TIM-3 and its ligands PtdSer/galectin-9. Conclusion: Taken together, our results support both direct anti-leukemic effects and immune-mediated modulation by sabatolimab, reinforcing the notion that sabatolimab represents a novel immunotherapy with immuno-myeloid activity, holding promise for the treatment of myeloid cell neoplasms.
ABSTRACT
High-grade serous ovarian cancers (HGSOC) represent the most common subtype of ovarian malignancies. Due to the frequency of late-stage diagnosis and high rates of recurrence following standard of care treatments, novel therapies are needed to promote durable responses. We investigated the anti-tumor activity of CD3 T cell engaging bispecific antibodies (TCBs) directed against the PAX8 lineage-driven HGSOC tumor antigen LYPD1 and demonstrated that anti-LYPD1 TCBs induce T cell activation and promote in vivo tumor growth inhibition in LYPD1-expressing HGSOC. To selectively target LYPD1-expressing tumor cells with high expression while sparing cells with low expression, we coupled bivalent low-affinity anti-LYPD1 antigen-binding fragments (Fabs) with the anti-CD3 scFv. In contrast to the monovalent anti-LYPD1 high-affinity TCB (VHP354), the bivalent low-affinity anti-LYPD1 TCB (QZC131) demonstrated antigen density-dependent selectivity and showed tolerability in cynomolgus monkeys at the maximum dose tested of 3 mg/kg. Collectively, these data demonstrate that bivalent TCBs directed against LYPD1 have compelling efficacy and safety profiles to support its use as a treatment for high-grade serous ovarian cancers.
Subject(s)
Antibodies, Bispecific/therapeutic use , Immunotherapy/methods , Ovarian Neoplasms/pathology , Ovarian Neoplasms/therapy , PAX8 Transcription Factor/immunology , T-Lymphocytes/immunology , Tumor Suppressor Proteins/immunology , Animals , CD3 Complex/immunology , Female , GPI-Linked Proteins/immunology , Macaca fascicularis , Mice , Neoplasm Grading , Xenograft Model Antitumor AssaysABSTRACT
SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.
Subject(s)
Immunity, Cellular , Neoplasm Proteins/immunology , Neoplasms, Experimental/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Animals , Cell Line, Tumor , Gene Knockout Techniques , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Neoplasm Proteins/genetics , Neoplasms, Experimental/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Signal Transduction/geneticsABSTRACT
Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogen-associated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C-gamma2 (PLC-gamma2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-gamma2 activation, release of intracellular Ca2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav.
Subject(s)
Cell Adhesion/physiology , Neutrophils/immunology , Phospholipase C gamma/metabolism , Proto-Oncogene Proteins c-vav/metabolism , Respiratory Burst , Signal Transduction/physiology , Animals , Calcium/metabolism , Diglycerides/metabolism , Granulomatous Disease, Chronic/metabolism , Humans , Mice , Mice, Knockout , NADPH Oxidases/metabolism , Phospholipase C gamma/genetics , Pneumonia, Bacterial/metabolism , Pneumonia, Bacterial/microbiology , Protein Subunits/metabolism , Proto-Oncogene Proteins c-vav/genetics , Pseudomonas Infections/metabolism , Reactive Oxygen Species/metabolism , Sepsis/metabolism , Sepsis/microbiology , Staphylococcal Infections/metabolismABSTRACT
Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFß in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFß and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cancer-Associated Fibroblasts/immunology , Carcinoma/drug therapy , Interferon-beta/immunology , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cancer-Associated Fibroblasts/drug effects , Carcinoma/immunology , Carcinoma/pathology , Cell Line, Tumor/transplantation , Cell Plasticity/drug effects , Cell Plasticity/immunology , Disease Models, Animal , Drug Synergism , Female , Humans , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Mice , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Stromal Cells/drug effects , Stromal Cells/immunology , Transforming Growth Factor beta/metabolism , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
OBJECTIVE: Accumulating evidence indicates an important role of neutrophils in the development of rheumatoid arthritis (RA). Recruitment of neutrophils to the joint space and release of proteolytic enzymes can exacerbate tissue damage and the inflammatory response related to RA. Engagement of beta2 integrin and subsequent activation of downstream signaling have been shown to be fundamental for activation of neutrophil effector functions. The aim of this study was to test the hypothesis that Vav and phospholipase Cgamma2 (PLCgamma2), two molecules involved in integrin signaling, are required for arthritis generation and neutrophil activation in a mouse model of arthritis. METHODS: Arthritis was induced in wild-type (WT), Vav(null), and PLCgamma2(-/-) mice using the K/BxN serum-transfer model. Neutrophil function was assessed by analyses of adhesion, spreading, and degranulation on integrin-dependent substrates. Regulation of integrin signaling was determined by analyzing the phosphorylation of Pyk-2, Src, and ERK. RESULTS: Vav(null) and PLCgamma2(-/-) mice were protected from inflammation and bone erosion in the K/BxN serum-transfer model of arthritis. Mechanistically, Vav and PLCgamma2 control neutrophils mediated spreading and degranulation on integrin-dependent substrates. Consequently, the Vav/PLCgamma2 axis, acting downstream of the integrin receptor, modulated the activation of Pyk-2, Src, and ERK. CONCLUSION: Our findings show that Vav cooperates with PLCgamma2 in modulating neutrophil activation downstream of the integrin receptor. This study identifies a Vav/PLCgamma2-dependent signaling pathway as a possible therapeutic target for the treatment of inflammation and bone disruption in arthritis.
Subject(s)
Arthritis, Rheumatoid/immunology , Neutrophil Activation/immunology , Neutrophils/immunology , Phospholipase C gamma/immunology , Proto-Oncogene Proteins c-vav/immunology , Animals , Arthritis, Rheumatoid/chemically induced , Arthritis, Rheumatoid/metabolism , Blotting, Western , Cell Adhesion/immunology , Cell Movement/immunology , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Inflammation/immunology , Integrins/immunology , Integrins/metabolism , Mice , Mice, Knockout , Neutrophils/metabolism , Phospholipase C gamma/genetics , Phosphorylation/immunology , Proto-Oncogene Proteins c-vav/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/immunologyABSTRACT
The chemokine receptor CXCR3 is involved in cell trafficking dysregulation associated with several inflammatory conditions, including autoimmune and viral diseases. Downregulation of CXCR3, through binding with its ligand CXCL11 (I-TAC), represents a key mechanism in lymphocyte recruitment. Determination of circulating I-TAC can provide useful information in the investigation of inflammatory/infectious conditions. The existing commercial kit does not measure CXCL11/I-TAC in complex matrices, such as human plasma and serum, as reliably as in in vitro-generated cell culture supernatants. We here describe means which lead to an improvement of CXCL11/I-TAC measurement in human plasma and serum.
Subject(s)
Chemokine CXCL11/blood , Enzyme-Linked Immunosorbent Assay , Ligands , Reagent Kits, Diagnostic , Receptors, CXCR3/metabolism , Anticoagulants , Chemokine CXCL11/metabolism , Edetic Acid , Heparin , HumansABSTRACT
Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN- population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFß signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide-dependent manner, whereas FAP+PDPN- pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.
Subject(s)
Breast Neoplasms/pathology , Gelatinases/metabolism , Membrane Proteins/metabolism , Serine Endopeptidases/metabolism , Stromal Cells/metabolism , Tumor Microenvironment/immunology , Animals , Breast Neoplasms/immunology , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Cell Proliferation , Endopeptidases , Female , Gene Expression Regulation , Humans , Membrane Glycoproteins/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Nitric Oxide/metabolism , Pericytes/metabolism , Pericytes/pathology , Stromal Cells/pathology , T-Lymphocytes/pathologyABSTRACT
Over the past 25 years, research in cancer therapeutics has largely focused on two distinct lines of enquiry. In one approach, efforts to understand the underlying cell-autonomous, genetic drivers of tumorigenesis have led to the development of clinically important targeted agents that result in profound, but often not durable, tumour responses in genetically defined patient populations. In the second parallel approach, exploration of the mechanisms of protective tumour immunity has provided several therapeutic strategies - most notably the 'immune checkpoint' antibodies that reverse the negative regulators of T cell function - that accomplish durable clinical responses in subsets of patients with various tumour types. The integration of these potentially complementary research fields provides new opportunities to improve cancer treatments. Targeted and immune-based therapies have already transformed the standard-of-care for several malignancies. However, additional insights into the effects of targeted therapies, along with conventional chemotherapy and radiation therapy, on the induction of antitumour immunity will help to advance the design of combination strategies that increase the rate of complete and durable clinical response in patients.