Project description:The Piedmont study is a prospectively designed retrospective evaluation of a new 48-gene antifolate response signature (AF-PRS) in patients with locally advanced/metastatic NS-NSCLC treated with pemetrexed-containing platinum doublet chemotherapy (PMX-PDC). The study tested the hypothesis that the AF-PRS selects for patients with NS-NSCLC that preferentially respond to PMX-PDC, with a goal of providing clinical support for AF-PRS as potential diagnostic test. Overall, 53% of patients were AF-PRS(+), which was associated with extended PFS, but not OS, vs. AF-PRS(-) (16.6 vs. 6.6 mo; p = 0.025). In patients who were Stage I-III patients at time of treatment, PFS, but not OS, was further extended in AF-PRS(+) vs. AF-PRS(-) (36.2 vs. 9.3 mo; p = 0.03). A complete response (CR) to therapy was noted in 14 of 95 patients. AF-PRS(+) preferentially selected a majority (79%) of CRs, which were evenly split between patients Stage I-III (6 of 7) and Stage IV (5 of 7) at time of treatment. AF-PRS identified a significant population of patients with extended survival and/or clinical response following PMX-PDC treatment. AF-PRS may be a useful diagnostic test for patients indicated for systemic chemotherapy, especially when determining the optimal PDC regimen for locally advanced disease.
Project description:Plasmacytoid dendritic cells (pDC) are the main source of type I interferon (IFN) during viral infections. Their other functions are debated, due to a lack of tools to identify and target them in vivo without affecting pDC-like cells and transitional DC (tDC), which harbor overlapping phenotypes and transcriptomes but a higher efficacy for T cell activation. To overcome this bottleneck, we designed, generated and validated a pDC-Tomato reporter mouse. We bred pDC-Tomato with Zbtb46 GFP mice to yield the he ZeST mouse strain that enabled transcriptomic profiling of all splenic DC types, by single cell RNA sequencing, both at steady state and during the course of the infection with mouse cytomegalovirus (MCMV). Analyses of the transcriptomic dataset unraveled diverging activation of pDC-like cells vs tDC during the infection. This dataset and the associated specific gene modules will be useful to delineate the physiological functions of pDC versus other DC types.
Project description:Here we confirm an essential requirement for the BCL11A transcription factor in fetal pDC development, and for the first time demonstrate this lineage specific requirement in the adult organism. Genome-wide analyses of BCL11A DNA binding and expression revealed that BCL11A regulates transcription of E2-2 and other pDC differentiation modulators including ID2 and MTG16. Our results identify BCL11A as an essential, lineage-specific factor that regulates pDC development. ChIP sequenicng was performed for a transcription factor of BCL11A in Cal1 cell line. Input was sequenced and used as a control.
Project description:Plasmacytoid dendritic cells (pDC) are the major source of type I and type III interferons (IFN-I/III) during viral infections, in response to triggering of endosomal Toll Like Receptors (TLRs) 7 or 9 by viral single-stranded RNA or unmethylated CpG DNA, respectively. Interestingly, this function is restricted to a minor fraction of pDC (Zucchini et al. Int. Immunol. 2008). In this project, we aimed at identifying the molecular pathways involved in inducing IFN-I/III production in this minor faction of pDC during in vivo infection by the mouse cytomegalovirus (MCMV). To achive this goal, we infected with MCMV Ifnb1Eyfp mice, in which IFN-producing pDC can be detected by YFP expression (Scheu et al. PNAS 2008). Thanks to this model, we were able to sort three distinct subsets of pDC: CD86-YFP- (not activated, non IFN-producing), CD86+YFP- (activated, non IFN-producing) and CD86+YFP+ (activated, IFN-producing) and to perform microarray analysis. This allowed us to select genes differentially expressed among these three subsets and to mine these data in order to identify the related signaling pathways.
Project description:Dendritic cells (DC) are professional antigen presenting cells that develop from hematopoietic stem cells in bone marrow by successive steps of lineage commitment and differentiation. Different DC subsets were identified based on phenotype, localisation and function: (i) classical DC (cDC) and plasmacytoid DC (pDC) are found in lymphoid organs and (ii) migratory tissue DC are spread throughout peripheral organs, including Langerhans cells, the cutaneous contingent of DC. We have developed a two-step culture system that recapitulates DC development in vitro (Felker et al., J. Immunol. 185, 5326-5335, 2010). In this system multipotent hematopoietic progenitors (MPP) progress into DC-restricted common DC progenitors (CDP) and further into the two major DC subsets cDC and pDC. We employed chromatin immunoprecipitation (ChIP) with deep sequencing (ChIP-seq) to determine the dynamics of H3K27ac occupancy in MPP, CMP, cDC and pDC. Histone modification H3K27ac and RNA-Seq in MPP, CDP, cDC and pDC
Project description:Type I interferons (IFN) are crucial mediators of human innate and adaptive immunity and are massively produced from plasmacytoid dendritic cells (pDC). IRF7 is a critical regulator of type I IFN production when pathogens are detected by TLR7/9 in pDC. However, hyperactivation of pDC can cause life-threatening autoimmune diseases. To avoid the deleterious effects of aberrant pDC activation, tight regulation of IRF7 is required. Nonetheless, the detailed mechanisms of how IRF7 transcription is regulated in pDC are still elusive. To this end, we identified the global gene expression changes after stimulation of human primary pDC with the TLR9 agonist CpGB. We identified that the transcription factor MYC is prominently upregulated upon CpGB engagement in pDC. Moreover, when we knocked down MYC in the pDC-like cell line GEN2.2, production of interferon-stimulated genes (ISGs) was dramatically increased and was further enhanced by CpGB. Interestingly, MYC is shown to be recruited to the IRF7 promoter region through interaction with NCOR2/HDAC3 for its repression, and HDAC3 inhibition enhanced IRF7 expression and IFNβ production. Interestingly, activation of TLR9-mediated NF-kB and MAPK and nuclear translocation of IRF7 were greatly enhanced by MYC depletion. Pharmaceutical inhibition of MYC recovered IRF7 expression, further confirming the negative role of MYC in the antiviral response by pDC. Furthermore, the inverse correlation of MYC and IRF7 was validated in psoriasis skin sample datasets. Therefore, our results identify the novel immunomodulatory role of MYC in human pDC and may add to our understanding of aberrant pDC function in autoimmune diseases.