Data_Sheet_1_BCAP Regulates Dendritic Cell Maturation Through the Dual-Regulation of NF-κB and PI3K/AKT Signaling During Infection.pdf

The maturation of dendritic cells (DCs) is essential in adaptive immunity. B cell adapter for phosphoinositide 3-kinase (BCAP) has been shown a divergent activities in cell type dependent manner including B cells, NK cells, macrophages, and plasmacytoid DCs (pDCs), however, its role in conventional DCs (cDCs) remains unknown. Here, we report that BCAP negatively regulates Toll-like receptor-induced cDC maturation and inhibits cDCs from inducing antigen-specific T cell responses, thereby weakening the antibacterial adaptive immune responses of mice in a Listeria monocytogenes-infection model. Furthermore, we demonstrate that BCAP simultaneously modulates the activation of the NF-κB and PI3K/AKT signaling by dynamically interacting with, respectively, MyD88 and the p85α subunit of PI3K. Our study thus reveals non-redundant roles for BCAP in regulating cDC maturation and reveals a bilateral signal transduction mechanism.</p

Data_Sheet_1_HMGN1 and R848 Synergistically Activate Dendritic Cells Using Multiple Signaling Pathways.pdf

High mobility group nucleosome-binding protein 1 (HMGN1 or N1) is a Th1-polarizing alarmin, but alone is insufficient to induce antitumor immunity. We previously showed that combination of N1 and R848, a synthetic TLR7/8 agonist, synergistically activates dendritic cells (DCs) and induces therapeutic antitumor immunity, however, it remained unclear how N1 and R848 synergistically activate DCs. Here, we show that co-stimulation with N1 and R848 of human monocyte-derived DCs (MoDCs) markedly upregulated DC's surface expression of CD80, CD83, CD86, and HLA-DR, as well as synergistic production of pro-inflammatory cytokines including IL-12p70, IL-1β, and TNF-α. This combination also synergistically activated NF-κB and multiple MAPKs that are involved in DC maturation. Moreover, N1 and R848 synergistically increased nuclear translocation of interferon (IFN) regulatory transcription factors (e.g., IRF3 and IRF7) and promoted the expression of type 1 IFNs such as IFN-α2, IFN-α4, and IFN-β1. Similar signaling pathways were also induced in mouse bone marrow-derived DCs (BMDCs). RNA-seq analysis in human MoDCs revealed that N1 plus R848 synergistically upregulated the expression of genes predominantly involved in DC maturation pathway, particularly genes critical for the polarization of Th1 immune responses (e.g., IL12A, IL12B, and IFNB1, etc.). Overall, our findings show that (1) N1 synergizes with R848 in activating human and mouse DCs and (2) the synergistic effect based on various intracellular signaling events culminated in the activation of multiple transcriptional factors. These findings have important implications for future clinical trials since N1 and R848 synergistically promoted optimal Th1 lineage immune responses resulting in tumor rejection in mice.</p

Reactive Oxygen Species-Sensitive Biodegradable Mesoporous Silica Nanoparticles Harboring TheraVac Elicit Tumor-Specific Immunity for Colon Tumor Treatment

Immunotherapy has revolutionized the field of cancer treatment through invigorating robust antitumor immune response. Here, we report the development of a therapeutic vaccine [consisting of high mobility group nucleosome-binding protein 1 (HMGN1), resiquimod/R848, and anti-PD-L1 (αPD-L1)]-loaded reactive oxygen species (ROS)-responsive mesoporous silica nanoparticle (MSN@TheraVac) for curative therapy of colon cancer. In MSN@TheraVac, αPD-L1 conjugated onto the surface of MSNs via a diselenide bond, which can be rapidly released under the oxidative condition of the tumor microenvironment to avert immunosuppression and effector T cell exhaustion while coloaded HMGN1 and R848 would cooperatively trigger robust tumor-infiltrating dendritic cell (TiDC) maturation and elicitation of antitumor immune responses. Indeed, MSN@TheraVac induced the maturation and activation of dendritic cells (DCs) by promoting the surface expression of CD80, CD86, and CD103 as well as the production of pro-inflammatory cytokines, including TNFα, IL-12, and IL-1β. Importantly, treatment with intravenous MSN@TheraVac led to a complete cure of 100% of BALB/c mice bearing large colon tumors and induced the generation of tumor-specific protective memory without apparent toxicity. Thus, MSN@TheraVac provides a timely release of TheraVac for the curative treatment of colon tumors and holds potential for translation into a clinical therapy for patients with immunologically “cold” colorectal cancers. This ROS-responsive MSN platform may also be tailored for the selective delivery of other cancer vaccines for effective immunotherapy

Iron-Catalyzed Synthesis of Peroxylpyrrolo[2,1‑<i>a</i>]isoquinolines through Oxidative Dearomatization

A mild late-stage modification of pyrrolo[2,1-a]isoquinolines was established through iron-catalyzed oxidative dearomatization and peroxidation. Peroxylated pyrroloisoquinolines have been prepared readily with hydroperoxide in low to good yields (up to 72%) at room temperature. Interestingly, the treatment of fully aromatized pyrrolo[1,2-a]quinolines under the current reaction system resulted in the formation of ring-opening products

Supplementary Data from Down-Regulation of 3-Phosphoinositide–Dependent Protein Kinase-1 Levels Inhibits Migration and Experimental Metastasis of Human Breast Cancer Cells

Supplementary Data from Down-Regulation of 3-Phosphoinositide–Dependent Protein Kinase-1 Levels Inhibits Migration and Experimental Metastasis of Human Breast Cancer Cell

Quantitative Study of the Interactome of PKCζ Involved in the EGF-induced Tumor Cell Chemotaxis

Chemotaxis plays an important role in metastasis. In our previous studies, we reported that protein kinase C ζ (PKCζ) mediated cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. To further study the molecular mechanism of chemotaxis, mass spectrometry-based approaches were employed to investigate the interactome of PKCζ and its changes upon stimulation by epidermal growth factor (EGF). As a result, 233 proteins were identified as potential PKCζ binding partners. Label free quantification was applied to examine the quantitative changes of these interactions involved in the EGF induced chemotaxis. Fifteen identified proteins were enriched and 9 proteins were reduced in the presence of EGF (≥1.5 folds, <i>p</i> ≤ 0.05). The interaction between cofilin-1 (CFL1) and PKCζ was evidenced and this interaction was enhanced in the EGF induced chemotaxis signaling transduction. In addition, novel PKCζ interacting proteins potentially related with chemotaxis were characterized, such as isoform 1 of nucleophosmin (NPM1). Furthermore, Western blotting and chemotaxis assays were also applied to validate the proteomics result and explore its biological implications. Collectively, the combination of quantitative proteomics and biological assays provides a powerful strategy for elucidating the signaling pathway of tumor cell chemotaxis

Two new iridoid glycosides from <i>Odontites vulgaris</i>

Two new iridoid glycosides, named 3′-O-benzoyl-dolichocymboside D (1) and dolichocymboside E (2), along with ten known glycosides (3–12), were isolated from the ethanol extract of the whole plants of Odontites vulgaris Moench. The structures of the isolated compounds were elucidated by 1D and 2D NMR and HR-ESI-MS spectra and by comparison with those reported in the literature. This is the first report on compounds 11 and 12 isolated from the family Scrophulariaceae, and compounds 8–10 were isolated from the genus Odontites.</p

HIV-1 co-receptor expression in DCs and HIV-1 infection rates in DC subsets.

<p>A: The expression level of CD4, CXCR4 and CCR5 was measured using flow cytometry in iDCs, mDCs, LLC and IL-27-treated iDCs. DC-SIGN was also measured but only in iDCs, mDCs and IL-27 treated iDCs. The red color in the plots indicates specific antibody staining whilst green represents the isotype control. B: iDCs, mDCs and LLCs were infected with either HIV-1_Ba-L or HIV-1_NL4.3 as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059194#s2" target="_blank">Materials and Methods</a>. HIV-1 replication was measured using HIV-1 p24 antigen capture kit as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059194#s2" target="_blank">Materials and Methods</a>. These graphs are representative data from a single donor from three independent experiments.</p

Inhibition of HIV-1 replication in DCs occurs after viral entry but before completion of reverse transcription.

<p>A: iDCs were pre-treated with IL-27 or mock treated for 48 hours. The treated cells were then infected with a HIV _{NL4.3-Luc-VSVG} virus followed by 4 days culture in the absence of IL-27 (Pre-IL-27 iDC). To compare the IL-27 effect, the mock-treated iDCs was also cultured in the presence of IL-27 (iDC+IL-27). Virus reporter activity was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059194#s2" target="_blank">Materials and Methods</a> B: The Late RT cDNA products were also semi quantitated using qPCR using the pseudotyped HIV infected cells. These experiments show the combined results of 4 separate donors. Error bars represent +/− SEM.</p

Additional file 1: of Combined treatment with HMGN1 and anti-CD4 depleting antibody reverses T cell exhaustion and exerts robust anti-tumor effects in mice

Figure S1. The purification of recombinant HMGN1 proteins. Figure S2. Administration of human HMGN1 (hH) with anti-CD4 depleting antibody (αCD4) exerted anti-tumor effects in mice. Figure S3. Administration of murine HMGN1 with anti-CD4 antibody exerted anti-tumor effects in melanoma model. (A). Figure S4. Flow cytometry analysis of T cell populations in the draining lymph node (dLN) from Colon26 tumor-bearing mice on day 13 after tumor inoculation. Figure S5. Gating scheme of CCR7+ CD11b+ migratory dendritic cells (DCs). Figure S6. Flow cytometry analysis of exhausted CD8+ T cell populations in the tumor from tumor-bearing mice on day 17 after treatment. Figure S7. CD8 T cell transcriptome analysis revealed the promotion of activation after HMGN1/αCD4 treatment. Method S1. Production and purification of HMGN1 in E. coli. Method S2. 3’end Serial Analysis of Gene Expression (SAGE) sequencing library preparation. Table S1. The list of recombinant proteins and peptides. Table S2. Antibodies for flow cytometry. Table S3. Primers for quantitative real-time PCR. Table S4. Gene ontology (GO) analysis of GO: biological processes and KEGG pathway. (PDF 2580 kb