ABSTRACT: The phenotype and proteome of these cells was compared to identify linage-dependent relationships and cancer-specific interactome expression modules.
Project description:In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, its fasting regimen-dependent regulation is unknown. Here, we show that cyclic intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, we provide evidence that this increase in LAM abundance is almost entirely dependent on p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation in AT during IF and increases the catabolic state of adipocytes, ameliorates metabolic flexibility, and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to long-term efficacy of a fasting-mimicking diet and that the expression of LAM markers and p53 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signaling in adipocytes dictates LAM accumulation in AT under IF and that adipocyte p53 modulates fasting effectiveness in mice and humans.
Project description:To improve our understanding of the relationships between methylation and expression we profiled mRNA expression and single-base resolution methylation levels for two breast cancer cell lines, MCF7 and T47D. Expression was profiled using RNA-seq. Methylation was assayed using Methyl-MAPS, which uses methylation-sensitive and -dependent restriction enzyme digests followed by high-throughput sequencing to identify methylation levels at individual CpGs (Edwards et al. 2010, Genome Research). DNA Methylation was assayed for two breast cancer cell lines using Methyl-MAPS.
Project description:T cell activation leads to dramatic changes in cellular phenotype. We used CD3/CD28-activated human CD4 T cells to study how RNA binding proteins define the post-transcriptional landscape. Using RIPseq, we identified the RNA interactome of U2AF2 and show at the global level that U2AF2 binds the majority of transcripts that are differentially expressed and/or alternatively spliced during CD4 T cell activation. A unique protein interactome centered on U2AF2 is assembled in response to activation. Knocking down specific U2AF2 interacting partners (U2AF1, SYNCRIP, SRRM2, ILF2) selectively affects cytokine secretion and expression of activation markers. Furthermore, the expression and/or alternative splicing of transcripts important for immune cell function are also affected by knocking down these U2AF2 interacting proteins. U2AF1 and SYNCRIP knockdowns affect the proteins and transcripts bound to U2AF2, altering the transcriptome. Our work highlights the importance of RNA binding protein complexes in regulating the differential expression and alternative splicing that defines T cell activation. RNAseq of total RNA from a primary CD4 T cell culture at rest and 48 hours after anti-CD3/CD28 bead activation
Project description:T cell activation leads to dramatic changes in cellular phenotype. We used CD3/CD28-activated human CD4 T cells to study how RNA binding proteins define the post-transcriptional landscape. Using RIPseq, we identified the RNA interactome of U2AF2 and show at the global level that U2AF2 binds the majority of transcripts that are differentially expressed and/or alternatively spliced during CD4 T cell activation. A unique protein interactome centered on U2AF2 is assembled in response to activation. Knocking down specific U2AF2 interacting partners (U2AF1, SYNCRIP, SRRM2, ILF2) selectively affects cytokine secretion and expression of activation markers. Furthermore, the expression and/or alternative splicing of transcripts important for immune cell function are also affected by knocking down these U2AF2 interacting proteins. U2AF1 and SYNCRIP knockdowns affect the proteins and transcripts bound to U2AF2, altering the transcriptome. Our work highlights the importance of RNA binding protein complexes in regulating the differential expression and alternative splicing that defines T cell activation. U2AF2 RIPseq on a primary CD4 T cell culture at rest and 48 hours after anti-CD3/CD28 bead activation
Project description:The identification of tyrosine phosphorylation-dependent interactome of immune coreceptors is crucial for the understanding of signal pathways involved in immunotherapy. However, identifying motif-specific interactome for each tyrosine phosphorylation site commonly found on these multi-phosphorylated membrane proteins remains challenging. Here we describe a photoaffinity-based chemical proteomic approach with synthetic full-length CD28 cytoplasmic tails (CD28cyto) to dissect the motif-specific cytoplasmic interactome of the critical immune coreceptor CD28 by covalent capturing and label-free quantification. Our chemical proteomic analysis well recapitulated reported CD28cyto interacting proteins to pY191 and pY209 motifs. We defined the stand-alone interaction of phospholipase PLCG1 to Y191 motif with enhanced affinity to the sequence neighboring the transmembrane domain. Importantly, we explored the interactome of previously undefined pY218 motif and verified that a critical kinase PKC? strongly and directly associated with pY218 through its C2 domain. This synthetic CD28cyto-based photoaffinity proteomic approach is generically applicable to study other immune coreceptors with multiple pY sites on their linear cytoplasmic tails.
Project description:Small cell lung cancer (SCLC) represents ~15% of all lung cancers and is characterized by its highly aggressive phenotype and its dismal outcome. Though the addition of immune checkpoint blockade to carboplatin and etoposide treatment has improved outcome in SCLC patients, SCLC cells finally acquire the ability to evade immunosurveillance and resistance against immune checkpoint blockade. To elaborate molecular mechanisms that mediate SCLC immune evasion, we performed high dimensional profiling of human and murine SCLC specimens. We herein comprehensively analyzed matched human samples of primary and metastatic SCLC and found a loss of MHC-I in SCLC metastases indicating that MHC-I loss mediates SCLC immune evasion. Silencing MHC-I in SCLC cells drastically diminished immune cell infiltration and facilitated the formation of metastasis in mice by circumventing immune surveillance. Using mass spectrometry and phospho-tyrosine kinase analysis, we discovered that ERBB2 signaling suppresses MHC-I expression in SCLC cells and stimulates immune modulating transcripts. Mechanistically, genetic and/or pharmacologic blockade of the ERBB2 signaling axis was sufficient to induce MHC-I expression and to prevent immune evasion in autochthonous murine SCLC in a STING-dependent manner. Finally, we demonstrate that the ERBB2 signaling axis regulates MHC-I expression on SCLC cells and is critical in maintaining immune evasion in SCLC. Most strikingly, we here uncover synergistic treatment efficacy by combining ERBB2 inhibition with PD-1 blockade eliciting profound responses in preclinical SCLC models, suggesting this combination for future clinical trials in patients with SCLC.
Project description:HOTAIR was found to be overepressed in a subset of urothelial cancer tissues and cell lines compared to normal controls. Ectopic HOTAIR expression in urothelial cancer cells in vitro demonstrated cell type dependent changes in phenotype. While some cell lines showed increased proloferation activity and induction of an aggressive phenotypes (e.g. stable transfected VM-CUB1 cells), others displayed rather a reduction of proliferation and migration. Stable transfection of 5637 cells resulted in induction of an immune response. Results of microarray analysis of stable transfected VM-CUB1 and 5637 cells concurred well with observed phenotypical cell type-specific changes. For differential gene expression analyses three independent high quality RNA preparations from VM-CUB1 cells, stably transfected with HOTAIR (clone 20), and 5637 cells, stably transfected with HOTAIR (clone 4), were compared to the respective vector control cells.
Project description:Small cell lung cancer (SCLC) represents ~15% of all lung cancers and is characterized by its highly aggressive phenotype and its dismal outcome. Though the addition of immune checkpoint blockade to carboplatin and etoposide treatment has improved outcome in SCLC patients, SCLC cells finally acquire the ability to evade immunosurveillance and resistance against immune checkpoint blockade. To elaborate molecular mechanisms that mediate SCLC immune evasion, we performed high dimensional profiling of human and murine SCLC specimens. We herein comprehensively analyzed matched human samples of primary and metastatic SCLC and found a loss of MHC-I in SCLC metastases indicating that MHC-I loss mediates SCLC immune evasion. Silencing MHC-I in SCLC cells drastically diminished immune cell infiltration and facilitated the formation of metastasis in mice by circumventing immune surveillance. Using mass spectrometry and phospho-tyrosine kinase analysis, we discovered that ERBB2 signaling suppresses MHC-I expression in SCLC cells and stimulates immune modulating transcripts. Mechanistically, genetic and/or pharmacologic blockade of the ERBB2 signaling axis was sufficient to induce MHC-I expression and to prevent immune evasion in autochthonous murine SCLC in a STING-dependent manner. Finally, we demonstrate that the ERBB2 signaling axis regulates MHC-I expression on SCLC cells and is critical in maintaining immune evasion in SCLC. Most strikingly, we here uncover synergistic treatment efficacy by combining ERBB2 inhibition with PD-1 blockade eliciting profound responses in preclinical SCLC models and circumventing MHC-I loss under immunotherapy, suggesting this combination for future clinical trials in patients with SCLC.
Project description:Sepsis is a highly heterogeneous syndrome that impacts immune function and response to infection. To develop targeted therapeutics, immunophenotyping is needed to identify distinct immune cell functional phenotypes. Employing organ-on-chip for neutrophil functional analysis, we identified three distinct sepsis neutrophil phenotypes based on adhesion and migration patterns across human lung endothelial cells in response to cytokine activation (cytomix, TNF/IL-1β/IFNγ). The phenotypes were categorized as: a Hyperimmune phenotype characterized by enhanced cytomix-induced neutrophil adherence and migration, a Hypoimmune phenotype that was unresponsive to cytomix treatment, and a Hybrid phenotype with increased adherence but blunted migration in response to stimulation. Proteomic analysis identified both unique and shared proteins between the three phenotypes as compared to healthy controls. Proteins associated with neutrophil adherence were significantly upregulated in the Hyperimmune and Hybrid neutrophils, while the Hypoimmune group showed significant downregulation of these proteins. Clinically, the Hypoimmune group had significantly fewer patients requiring mechanical ventilation (29%) compared to the Hyperimmune group (70%). The Hypoimmune group and Hybrid group had significantly shorter ICU length of stay (LOS) than the Hyperimmune group. The Hypoimmune group also showed a trend for a lower mortality rate (35.7%) compared to the Hyperimmune group (50%). Thus, we identified associations between neutrophil phenotypes and important clinical outcomes, such as mechanical ventilation requirements, ICU LOS, and possibly mortality. Classification of sepsis patient phenotypes with diverse functional neutrophil responses and proteomic signatures can help distinguish patients who would benefit from specific treatments, such as immunosuppressive therapies and those who may be negatively impacted.
Project description:Metastasis to the cerebrospinal fluid (CSF)-filled leptomeninges, or leptomeningeal metastasis (LM), represents a fatal complication of cancer. Proteomic and transcriptomic analyses of human CSF reveal a substantial inflammatory infiltrate in LM. We find the solute and immune composition of CSF in the setting of LM changes dramatically, with notable enrichment in IFN-gamma signaling. To investigate the mechanistic relationships between immune cell signaling and cancer cells within the leptomeninges, we developed syngeneic lung, breast, and melanoma LM mouse models. We find that transgenic host mice, lacking IFN-gamma or its receptor, fail to control LM growth. Overexpression of Ifng through a targeted AAV system controls cancer cell growth independent of adaptive immunity. Instead, leptomeningeal IFN-gamma actively recruits and activates peripheral myeloid cells, generating a diverse spectrum of dendritic cell subsets. These migratory, CCR7+ dendritic cells orchestrate the influx, proliferation, and cytotoxic action of natural killer cells to control cancer cell growth in the leptomeninges. This work uncovers leptomeningeal-specific IFN-gamma signaling and suggests a novel immune-therapeutic approach against tumors within this space.