ABSTRACT
Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.
Subject(s)
Adenocarcinoma/immunology , Macrophages/immunology , Melanoma/immunology , Tumor Escape/immunology , Tumor Microenvironment/immunology , Acidosis/immunology , Adenocarcinoma/metabolism , Animals , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Glycolysis/immunology , Humans , Melanoma/metabolism , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the ß-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.
Subject(s)
Casein Kinase II/immunology , T-Lymphocytes, Regulatory/immunology , Th2 Cells/immunology , Animals , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Growth Processes/immunology , Cell Line , Dendritic Cells/enzymology , Dendritic Cells/immunology , Forkhead Transcription Factors/immunology , Humans , Hypersensitivity/blood , Hypersensitivity/immunology , Interferon Regulatory Factors/immunology , Leukocytes, Mononuclear/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Cell Surface/immunology , T-Lymphocytes, Regulatory/enzymology , Th2 Cells/enzymologyABSTRACT
IL-9 has lent its numerical designation to the Th9 subset of CD4+ Th cells, although it is also produced by additional cell types, including mast cells. It is a pleiotropic cytokine involved in allergic reactions, parasitic infections, autoimmune inflammation, and cancer immunity. In this article, we provide evidence that NFATc2 has contradictory functions in the expression of IL-9 in murine Th9 cells and bone marrow-derived mast cells (BMMC). The basis for this is our observation that the production of IL-9 in NFATc2-deficient Th9 cells is increased, whereas it is decreased in BMMC devoid of NFATc2. In addition, NFATc2 deficiency almost completely abrogates the expression of IL-3 in both cell types. However, selectively in BMMC, the production of IL-9 critically depends on autocrine IL-3 acting via the sustained activation of STAT5 on the expression of IL-9. Furthermore, we demonstrate that IL-3 acts independently and synergistically with IL-1ß on the production of IL-9. Taken together, we highlight NFATc2-driven production of autocrine IL-3 as a critical and cell type-specific component for IL-9 expression in BMMC.
Subject(s)
Interleukin-3/metabolism , Interleukin-9/metabolism , Mast Cells/immunology , NFATC Transcription Factors/metabolism , T-Lymphocytes, Helper-Inducer/immunology , Animals , Autocrine Communication , Cells, Cultured , Feedback, Physiological , Interleukin-9/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , NFATC Transcription Factors/genetics , STAT5 Transcription Factor/metabolism , Up-RegulationABSTRACT
T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance. Thus, the balance between Treg cells and TH17 cells determines the severity of a TH17 cell-driven disease and therefore is a promising target for treating autoimmune diseases. However, the molecular mechanisms controlling this balance are still unclear. Here, we report that pharmacological inhibition as well as genetic ablation of the protein kinase CK2 (CK2) ameliorates experimental autoimmune encephalomyelitis (EAE) severity and relapse incidence. Furthermore, CK2 inhibition or genetic ablation prevents TH17 cell development and promotes the generation of Treg cells. Molecularly, inhibition of CK2 leads to reduced STAT3 phosphorylation and strongly attenuated expression of the IL-23 receptor, IL-17, and GM-CSF. Thus, these results identify CK2 as a nodal point in TH17 cell development and suggest this kinase as a potential therapeutic target to treat TH17 cell-driven autoimmune responses.
Subject(s)
Casein Kinase II/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , T-Lymphocytes, Regulatory/immunology , Th17 Cells/immunology , Animals , Casein Kinase II/deficiency , Casein Kinase II/genetics , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Forkhead Transcription Factors , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor , Humans , Interleukin-17 , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multiple Sclerosis/genetics , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments , Phosphorylation , Receptors, Interleukin , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/immunology , Severity of Illness Index , Signal Transduction , T-Lymphocytes, Regulatory/cytology , Th17 Cells/pathologyABSTRACT
Efficient and reproducible sample preparation is a prerequisite for any robust and sensitive quantitative bottom-up proteomics workflow. Here, we performed an independent comparison between single-pot solid-phase-enhanced sample preparation (SP3), filter-aided sample preparation (FASP), and a commercial kit based on the in-StageTip (iST) method. We assessed their performance for the processing of proteomic samples in the low µg range using varying amounts of HeLa cell lysate (1-20 µg of total protein). All three workflows showed similar performances for 20 µg of starting material. When handling sample sizes below 10 µg, the number of identified proteins and peptides as well as the quantitative reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low µg range. Even when digesting 1 µg of starting material, both methods still enabled the identification of over 3000 proteins and between 25â¯000 and 30â¯000 peptides. On average, the quantitative reproducibility between experimental replicates was slightly higher in case of SP3 (R2 = 0.97 (SP3); R2 = 0.93 (iST)). Applying SP3 toward the characterization of the proteome of FACS-sorted tumor-associated macrophages in the B16 tumor model enabled the quantification of 2965 proteins and revealed a "mixed" M1/M2 phenotype.
Subject(s)
Proteomics/methods , Specimen Handling/methods , HeLa Cells , Humans , Proteomics/standards , Reproducibility of Results , Sample Size , Specimen Handling/standards , WorkflowABSTRACT
Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1ß and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.
Subject(s)
Cystatins/pharmacology , Immunity, Innate/drug effects , Immunosuppressive Agents/pharmacology , Interferon Regulatory Factors/immunology , Interleukin-9/immunology , Mast Cells/drug effects , Animals , Asthma/genetics , Asthma/immunology , Asthma/pathology , Binding Sites , Cell Degranulation/immunology , Cystatins/immunology , Gene Expression Regulation , Host-Parasite Interactions/immunology , Interferon Regulatory Factors/deficiency , Interferon Regulatory Factors/genetics , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Interleukin-9/antagonists & inhibitors , Interleukin-9/genetics , Mast Cells/immunology , Mast Cells/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , Protein Binding , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/immunology , Signal Transduction , Transcription, GeneticABSTRACT
CD8 T lymphocytes are classically viewed as cytotoxic T cells. Whether human CD8 T cells can, in parallel, induce a tissue regeneration program is poorly understood. Here, antigen-specific assay systems revealed that human CD8 T cells not only mediated cytotoxicity but also promoted tissue remodeling. Activated CD8 T cells could produce the epidermal growth factor receptor (EGFR)-ligand amphiregulin (AREG) and sensitize epithelial cells for enhanced regeneration potential. Blocking the EGFR or the effector cytokines IFN-γ and TNF could inhibit tissue remodeling. This regenerative program enhanced tumor spheroid and stem cell-mediated organoid growth. Using single-cell gene expression analysis, we identified an AREG+, tissue-resident CD8 T cell population in skin and adipose tissue from patients undergoing abdominal wall or abdominoplasty surgery. These tissue-resident CD8 T cells showed a strong TCR clonal relation to blood PD1+TIGIT+ CD8 T cells with tissue remodeling abilities. These findings may help to understand the complex CD8 biology in tumors and could become relevant for the design of therapeutic T cell products.
Subject(s)
CD8-Positive T-Lymphocytes , T-Lymphocytes, Cytotoxic , Humans , ErbB Receptors , Adipose Tissue , Cell CycleABSTRACT
The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.
Subject(s)
Adenylyl Cyclase Inhibitors/pharmacology , Adenylyl Cyclases/genetics , Antineoplastic Agents/pharmacology , Cyclic AMP/antagonists & inhibitors , Melanoma, Experimental/drug therapy , Skin Neoplasms/drug therapy , Adenylyl Cyclase Inhibitors/chemical synthesis , Adenylyl Cyclases/immunology , Animals , Antineoplastic Agents/chemical synthesis , Benzyl Compounds/chemistry , Cyclic AMP/immunology , Cyclic AMP/metabolism , Esters , Female , Gene Expression , Humans , Immunity, Innate/drug effects , Injections, Intralesional , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin 1 Receptor Antagonist Protein/immunology , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Micelles , Myeloid Cells/drug effects , Myeloid Cells/immunology , Myeloid Cells/pathology , Peptides/chemistry , Polyglutamic Acid/chemistry , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Sarcosine/analogs & derivatives , Sarcosine/chemistry , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Skin Neoplasms/pathology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Tumor Burden/drug effects , Tumor Escape/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.