14 research outputs found
Regulation of Syk activity by antiviral adaptor MAVS in FcεRI signaling pathway
BackgroundMast cells are the major effector cell type for IgE-mediated allergic reactions. Recent studies revealed a role for mast cells in orchestrating the host response to viral infections.ObjectiveWe studied the relationship between FcεRI (high-affinity IgE receptor) and RIG-I-like receptor (RLR)-mediated antiviral signaling pathways.MethodsMast cells (BMMCs) were cultured from bone marrow cells from mice deficient in MAVS or other RLR signaling molecules. MAVS expression was restored by retroviral transduction of MAVS-deficient BMMCs. These cells were stimulated with IgE and antigen and their activation (degranulation and cytokine production/secretion) was quantified. FcεRI-mediated signaling events such as protein phosphorylation and Ca2+ flux were analyzed by western blotting and enzyme assays. WT and mutant mice as well as mast cell-deficient KitW−sh/W−sh mice engrafted with BMMCs were subjected to passive cutaneous anaphylaxis.ResultsUnexpectedly, we found that mast cells devoid of the adaptor molecule MAVS exhibit dramatically increased cytokine production upon FcεRI stimulation, despite near-normal degranulation. Consistent with these observations, MAVS inhibited tyrosine phosphorylation, thus catalytic activity of Syk kinase, the key signaling molecule for FcεRI-mediated mast cell activation. By contrast, mast cells deficient in RIG-I, MDA5 or IRF3, which are antiviral receptor and signaling molecules upstream or downstream of MAVS, exhibited reduced or normal mast cell activation. MAVS-deficient mice showed enhanced late-phase responses in passive cutaneous anaphylaxis.ConclusionThis study demonstrates that the adaptor MAVS in the RLR innate immune pathway uniquely intersects with the adaptive immune FcεRI signaling pathway
Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition.
The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy
Regulation of CD8+ T Lymphocyte Fate Specification by Proteasome Activity and Cellular Metabolism
In an immune response, CD8+ T lymphocytes can differentiate into effector cells that acutely clear the infection as well as memory cells that provide lasting immunity against the same pathogen. Previous studies have shown that differentiation of CD8+ T cells in to effector and memory subsets occurs in a heterogeneous manner. Single-cell RNA-seq has revealed that transcriptional heterogeneity is present as early as the first cell division. Using single-cell transcriptional profiling to create supervised classifiers revealed that the outcome of intermediate-stage cells could be predicted. In ‘pre-effector’ and ‘pre-memory’ cells resulting from the first CD8+ T cell division in vivo, we observed distinctly low and high levels of proteasome activity, respectively. Pharmacological inhibition of proteasome activity early during differentiation resulted in acquisition of terminal effector cell characteristics, while enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulation of proteasome activity in CD8+ T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Overall, these findings demonstrate that proteasome activity is an important regulator of CD8+ T cell fate early during the differentiation process
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Regulation of CD8+ T Lymphocyte Fate Specification by Proteasome Activity and Cellular Metabolism
In an immune response, CD8+ T lymphocytes can differentiate into effector cells that acutely clear the infection as well as memory cells that provide lasting immunity against the same pathogen. Previous studies have shown that differentiation of CD8+ T cells in to effector and memory subsets occurs in a heterogeneous manner. Single-cell RNA-seq has revealed that transcriptional heterogeneity is present as early as the first cell division. Using single-cell transcriptional profiling to create supervised classifiers revealed that the outcome of intermediate-stage cells could be predicted. In ‘pre-effector’ and ‘pre-memory’ cells resulting from the first CD8+ T cell division in vivo, we observed distinctly low and high levels of proteasome activity, respectively. Pharmacological inhibition of proteasome activity early during differentiation resulted in acquisition of terminal effector cell characteristics, while enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulation of proteasome activity in CD8+ T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Overall, these findings demonstrate that proteasome activity is an important regulator of CD8+ T cell fate early during the differentiation process
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Heterogenous Populations of Tissue-Resident CD8+ T Cells Are Generated in Response to Infection and Malignancy
Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches
The long noncoding RNA Malat1 regulates CD8+ T cell differentiation by mediating epigenetic repression
During an immune response to microbial infection, CD8+ T cells give rise to short-lived effector cells and memory cells that provide sustained protection. Although the transcriptional programs regulating CD8+ T cell differentiation have been extensively characterized, the role of long noncoding RNAs (lncRNAs) in this process remains poorly understood. Using a functional genetic knockdown screen, we identified the lncRNA Malat1 as a regulator of terminal effector cells and the terminal effector memory (t-TEM) circulating memory subset. Evaluation of chromatin-enriched lncRNAs revealed that Malat1 grouped with trans lncRNAs that exhibit increased RNA interactions at gene promoters and gene bodies. Moreover, we observed that Malat1 was associated with increased H3K27me3 deposition at a number of memory cell-associated genes through a direct interaction with Ezh2, thereby promoting terminal effector and t-TEM cell differentiation. Our findings suggest an important functional role of Malat1 in regulating CD8+ T cell differentiation and broaden the knowledge base of lncRNAs in CD8+ T cell biology
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Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition.
The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy
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Early precursors and molecular determinants of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing
During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer
Proteasome activity regulates CD8+ T lymphocyte metabolism and fate specification
During an immune response, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt terminal effector and memory cell fates. Here we show that "pre-effector" and "pre-memory" cells resulting from the first CD8+ T cell division in vivo exhibited low and high rates of endogenous proteasome activity, respectively. Pharmacologic reduction of proteasome activity in CD8+ T cells early during differentiation resulted in acquisition of terminal effector cell characteristics, whereas enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in CD8+ T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Taken together, these results demonstrate that proteasome activity is an important regulator of CD8+ T cell fate and raise the possibility that increasing proteasome activity may be a useful therapeutic strategy to enhance the generation of memory lymphocytes
Immunodeficiency and Autoimmune Enterocolopathy Linked to NFAT5 Haploinsufficiency
The link between autoimmune diseases and primary immunodeficiency syndromes has been increasingly appreciated. Immunologic evaluation of a young man with autoimmune enterocolopathy and unexplained infections revealed evidence of immunodeficiency, including IgG subclass deficiency, impaired Ag-induced lymphocyte proliferation, reduced cytokine production by CD8(+) T lymphocytes, and decreased numbers of NK cells. Genetic evaluation identified haploinsufficiency of NFAT5, a transcription factor regulating immune cell function and cellular adaptation to hyperosmotic stress, as a possible cause of this syndrome. Inhibition or deletion of NFAT5 in normal human and murine cells recapitulated several of the immune deficits identified in the patient. These results provide evidence of a primary immunodeficiency disorder associated with organ-specific autoimmunity linked to NFAT5 deficiency