ABSTRACT: During the human B cell (Bc) recall response, rapid cell division results in multiple Bc subpopulations. The TLR-9 agonist CpG oligodeoxynucleotide, combined with cytokines, causes Bc activation and division in vitro and increased CD27 surface expression in a sub-population of Bc. We hypothesized that the proliferating CD27lo subpopulation, which has a lower frequency of antibody-secreting cells (ASC) than CD27hi plasmablasts, provides alternative functions such as cytokine secretion, costimulation, or antigen presentation. We performed genome-wide transcriptional analysis of CpG activated Bc sorted into undivided, proliferating CD27lo and proliferating CD27hi subpopulations. Our data supported an alternative hypothesis, that CD27lo cells are a transient pre-plasmablast population, expressing genes associated with Bc receptor editing. Undivided cells had an active transcriptional program of non-ASC B cell functions, including cytokine secretion and costimulation, suggesting a link between innate and adaptive Bc responses. Transcriptome analysis suggested a gene regulatory network for CD27lo and CD27hi Bc differentiation. 18 B cell samples from 6 subjects. From each subject, 3 in vitro B cell populations were isolated: undivided cells, proliferating cells with low CD27 (marker of antibody secretion), proliferating cells with high CD27
Project description:During the human B cell (Bc) recall response, rapid cell division results in multiple Bc subpopulations. The TLR-9 agonist CpG oligodeoxynucleotide, combined with cytokines, causes Bc activation and division in vitro and increased CD27 surface expression in a sub-population of Bc. We hypothesized that the proliferating CD27lo subpopulation, which has a lower frequency of antibody-secreting cells (ASC) than CD27hi plasmablasts, provides alternative functions such as cytokine secretion, costimulation, or antigen presentation. We performed genome-wide transcriptional analysis of CpG activated Bc sorted into undivided, proliferating CD27lo and proliferating CD27hi subpopulations. Our data supported an alternative hypothesis, that CD27lo cells are a transient pre-plasmablast population, expressing genes associated with Bc receptor editing. Undivided cells had an active transcriptional program of non-ASC B cell functions, including cytokine secretion and costimulation, suggesting a link between innate and adaptive Bc responses. Transcriptome analysis suggested a gene regulatory network for CD27lo and CD27hi Bc differentiation.
Project description:The adaptor protein ASC is known to facilitate caspase-1 activation essential for innate host immunity via the formation of the inflammasome complex - a multi-protein structure responsible for processing IL-1beta and IL-18 to their active moieties. Here we demonstrate that ASC-deficient CD8+ T cells fail to induce graft-versus-host disease (GVHD) and have impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects are the result of an inability to differentiate into fully cytolytic, granzyme B-expressing effector cells, with a developmental bias instead towards CD127+KLRG1- memory CD8+ T cells. These alterations in differentiation are inflammasome-independent, since GVHD lethality and CTL differentiation are not altered in recipients of caspase-1-deficient T cells. We demonstrate that ASC binds to T-bet in CD8+ T and in the absence of ASC, the binding of T-bet to the granzyme B promoter is impaired. Thus, the inhibition of ASC represents an attractive therapeutic target to manipulate transplant outcomes. Single colour, Illumina MouseRef-8 v2.0 Beadarrays.
Project description:Background: Hypoxia is a potent molecular signal for cellular metabolism, mitochondrial function, and migration. Conditions of low oxygen tension trigger regulatory cascades mediated via the highly conserved HIF-1 α post-translational modification system. In the adaptive immune response, B cells (Bc) are activated and differentiate under hypoxic conditions within lymph node germinal centers, and subsequently migrate to other compartments. During migration, they traverse through changing oxygen levels, ranging from 1-5% in the lymph node to 5-13% in the peripheral blood. Interestingly, the calcineurin inhibitor cyclosporine A is known to stimulate prolyl hydroxylase activity, resulting in HIF-1 α destabilization and may alter Bc responses directly. Over 60% of patients taking calcineurin immunosuppressant medications have hypo-gammaglobulinemia and poor vaccine responses, putting them at high risk of infection with significantly increased morbidity and mortality. Results: We demonstrate that oxygen tension is a previously unrecognized Bc regulatory switch, altering CXCR4 and CXCR5 chemokine receptor signaling in activated Bc through HIF-1 α expression, and controlling critical aspects of Bc migration. Our data demonstrate that calcineurin inhibition hinders this oxygen regulatory switch in primary human Bc. Conclusion: This previously unrecognized effect of calcineurin inhibition directly on human Bc has significant and direct clinical implications.
Project description:Engagement of CD27 during naïve CD8+ T (TN) cell activation is critical for T cell memory generation through poorly understood mechanisms. To examine the effects of CD27 signaling during TN cell activation we designed a synthetic trimeric CD70 ligand. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 resulted in immediate receptor internalization, recruitment of TRAF2/SHP-1, and modulation of Lck and ERK/AKT signaling in cells receiving concurrent CD28 costimulation. Independent of this modulatory effect on CD28 costimulated T cells, CD27 signaling enhanced ATF2, FOXO1, and FOXP1 transcription factor circuits, which induced T cell memory rather than the effector associated gene programs observed with CD28 costimulation alone. CD27-costimulated T cells engineered with a chimeric antigen receptor (CAR) exhibited improved tumor control. CD27 signaling during TN cell activation modulates activation strength and directs memory T cell properties that may benefit therapeutic T cells engineered with CARs or T cell receptors.
Project description:MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been previously reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome. The pre-treatment sera of 42 stage II–III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs.
Project description:Rationale: Genome-wide association studies (GWAS) and candidate gene studies have identified a number of loci linked to susceptibility of chronic obstructive pulmonary disease (COPD), a smoking-related disorder that originates in the airway epithelium. Objectives: Since airway basal cell (BC) stem/progenitor cells exhibit the earliest abnormalities associated with smoking (hyperplasia, squamous metaplasia), we hypothesized that smoker BC have a dysregulated transcriptome linked, in part, to known GWAS/candidate gene loci. Methods: Massive parallel RNA sequencing was used to compare the transcriptome of BC purified from the airway epithelium of healthy nonsmokers (n=10) and smokers (n=7). The chromosomal location of the differentially expressed genes was compared to loci identified by GWAS and candidate gene studies to confer risk for COPD. Measurements and Main Results: Smoker BC have 676 known genes differentially expressed compared to nonsmoker BC, dominated by smoking up-regulation. Strikingly, 166 (25%) of these genes are located on chromosome 19, with 13 localized to 19q13.2 (p<10-4 compared to chance), including TGFB1, LTBP4, EGLN2 and NFKBIB, genes associated with risk for COPD. Conclusions: These observations provide the first direct link of known genetic risks for smoking-related lung disease with the specific population of lung cells that undergoes the earliest changes associated with smoking. The human airway basal cell transcriptome of 7 smokers versus 10 nonsmokers was compared using massive parallel RNA sequencing (Illumina HiSeq 2000).
Project description:Exploring epigenetic gene essentiality identifying key regulators of cell survival represents a promising way to exploit new targets to overcome resistance to current pharmacological regimens. In breast cancer (BC), endocrine therapy (ET) resistance arises from aberrant Estrogen Receptor alpha (ERα) signaling and targeting cancer cell vulnerability within the ERα pathway represents a rationale for development of effective new drugs against this disease. By combining bioinformatics analysis of genome-wide ‘drop-out’ screenings and siRNA-mediated gene knock-down (kd) we identified a set of essential genes that includes, as the most effective, the bromodomain containing protein BRPF1. BRPF1 belongs to a family of epigenetic readers acting as chromatin remodelers to control gene transcription. To gather mechanistic insight into the role of this epienzyme in BC, we applied chromatin and transcriptome profiling, gene ablation and specific pharmacological inhibition followed by cellular and functional assays. Experimental evidences indicate that BRPF1 associates with ERa in MCF-7 cells chromatin and its blockade inhibits cell cycle progression, reduces cell proliferation and mediates transcriptome changes through modulation of chromatin accessibility. This results in a widespread inhibition of ER-dependent hormonal signaling obtained by ERa gene silencing in AE-sensitive and -resistant BC cells and pre-clinical patient-derived models (PDO). The characterization of functional interplays between ERα and BRPF1 revealed a new master regulator of BC survival, providing a rationale for a BRPF1 targeted cancer diagnosis and highlighting a new therapeutic vulnerability for the treatment of these aggressive tumors.
Project description:Exploring epigenetic gene essentiality identifying key regulators of cell survival represents a promising way to exploit new targets to overcome resistance to current pharmacological regimens. In breast cancer (BC), endocrine therapy (ET) resistance arises from aberrant Estrogen Receptor alpha (ERα) signaling and targeting cancer cell vulnerability within the ERα pathway represents a rationale for development of effective new drugs against this disease. By combining bioinformatics analysis of genome-wide ‘drop-out’ screenings and siRNA-mediated gene knock-down (kd) we identified a set of essential genes that includes, as the most effective, the bromodomain containing protein BRPF1. BRPF1 belongs to a family of epigenetic readers acting as chromatin remodelers to control gene transcription. To gather mechanistic insight into the role of this epienzyme in BC, we applied chromatin and transcriptome profiling, gene ablation and specific pharmacological inhibition followed by cellular and functional assays. Experimental evidences indicate that BRPF1 associates with ERa in MCF-7 cells chromatin and its blockade inhibits cell cycle progression, reduces cell proliferation and mediates transcriptome changes through modulation of chromatin accessibility. This results in a widespread inhibition of ER-dependent hormonal signaling obtained by ERa gene silencing in AE-sensitive and -resistant BC cells and pre-clinical patient-derived models (PDO). The characterization of functional interplays between ERα and BRPF1 revealed a new master regulator of BC survival, providing a rationale for a BRPF1 targeted cancer diagnosis and highlighting a new therapeutic vulnerability for the treatment of these aggressive tumors.
Project description:Breast Cancer (BC) is one of the main causes of cancer-related death in women. The most widespread BC subtype (75%) is the hormone-dependent, characterized by high expression of Estrogen Receptor Alpha (ERa). ERa exerts its oncogenic activity through the interaction with different co-regulators, carrying out a direct control on gene transcription and thus being the target of specific anticancer therapies. Despite the efficiency of these drugs, 30% of patients develop de novo or acquired resistance mechanisms, caused by alternative mechanisms that bypass antiestrogenic effects determining the onset of relapse. In order to identify new and potentially drugable Era molecular partners, essential genes for BC progression in luminal cell lines assessed by CRISPR-cas assay were compared to ERa interactome, dropping our focus on the tyrosine-protein kinase BAZ1B. BAZ1B is an essential component of the WICH complex and plays a crucial role in chromatin remodelling acting also as transcription regulator. In order to characterize the functional role of BAZ1B in luminal BC, we first employed a TCGA analysis that showed higher expression of BAZ1B in luminal BC associated with a worst overall survival and progression free survival of patient affected by this disease. A transcriptome profiling after BAZ1B silencing was performed in MCF-7 cells, highlighting an impact of modulated genes in key BC pathways including the estrogen signalling. BAZ1B silencing showed also an effect on cell growth, inhibiting cell proliferation and increasing apoptosis activation mechanisms promoted by the loss of ERa protein expression coupled to a reduction of the receptor transactivation activity. Interesting, these effects were confirmed also in antiestrogen (Tamoxifen and Fulvestrant) resistant BC cell models that retain ERa expression, suggesting BAZ1B as a novel and potentially exploitable therapeutic target in the treatment of both hormone dependent and resistant mammary tumours.
Project description:Cancer cells adapt to harsh environmental conditions by inducing the Unfolded Protein Response (UPR), of which ERO1A is one of the mediators. ERO1A aids protein folding by acting as a protein disulfide oxidase, and under cancer-related hypoxia conditions, it favors the folding of angiogenic VEGFA, leading tumor cells to thrive and spread. The upregulation of ERO1A in cancer and the dispensability of ERO1A activity in healthy cells render ERO1 a perfect target for cancer therapy. Here, we report the upregulation of ERO1 in aggressive triple-negative breast cancer (TNBC) patients. Thus, we designed new ERO1A inhibitors in a campaign of lead compound optimization on the prototype EN460 to be tested in TNBC treatment. Cell-based screenings showed that I2 and I3, two novel EN460 analogs, inhibited ERO1A efficiently, thus blunting VEGFA secretion. Accordingly, in vitro assays to measure ERO1A engagement confirmed that I2 and I3 bind ERO1A. EN460, I2 and I3 triggered breast cancer cytotoxicity while specifically inhibiting ERO1A in a dose-dependent manner. I2 more efficiently impaired cancer-relevant features such as VEGFA secretion and the related cell migration. Furthermore, I2 impinged on the tumor microenvironment and viability of xenografts and syngeneic TNBC. Thus, ERO1A pharmacological inhibition promises to lead to an effective anti-cancer therapy for the yet incurable TNBC.