Journal: The Journal of Immunology Author Choice

Article Title: Neoantigen Expression in Steady-State Langerhans Cells Induces CTL Tolerance

doi: 10.4049/jimmunol.1602098

Figure Lengend Snippet: Inducible expression and presentation of LC-restricted neoantigens. (A) huLang-CreERT2 transgenic mice were crossed with different ROSA26 reporter mouse lines harboring either the reversely orientated GFPOVA cds, or cds for YFP, GFP, or lacZ that are separated from the promoter by loxP-flanked STOP cassettes. Filled and open triangles indicate loxPwt and loxPmut L3 sequences, respectively. TAM treatment of bitransgenic mice results in Cre-mediated inversion of GFPOVA to sense orientation (LCre-GFPOVA mice) or excision of the STOP cassette in LCre-YPF, LCre-GFP, and LCre-lacZ mice and subsequent expression of the respective transgene in LCs. Time course of YFP expression in LCs, isolated from (B) skin or (C) SDLN of LCre-YFP mice, injected with 2 mg TAM i.p. or left untreated. Histograms and bar graphs show the percentage of YFP+ LCs at indicated time points. Total skin or LN cells were gated on CD45+ CD11c+ EpCam+ DCs or CD45+ CD11c+ CD207+ DCs, respectively. (D) Groups of LCre-GFPOVA mice were treated with 2 mg TAM i.p. or left untreated. TAM-treated Creneg littermates served as WT controls. On the same day, mice were injected with a mix of CFSE-stained OT-1 and WT cells (one million each) i.v. After 4 d, LN cells were isolated and gated on CD8+ CD45.1+ cells and analyzed for OT-1 cell activation relative to WT cells. Data represent mean ± SD of groups of three to five individually analyzed mice and are representative of two independent experiments.

Article Snippet: After 4 d, cells isolated from SDLN were stained with α-CD45.2–PerCP, α-CD45.1–APC-Cy7, α-CD8–APC and α-Vβ5.1, 5.2 (all from eBioscience).

Techniques: Expressing, Transgenic Assay, Isolation, Injection, Staining, Activation Assay

Journal: Cell stem cell

Article Title: Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow

doi: 10.1016/j.stem.2018.11.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: α-mouse CD45.1 PE-Cy7 , Tonbo Biosciences , A20; RRID:AB_2621850.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software

Journal: Molecular Therapy

Article Title: Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines

doi: 10.1038/mt.2012.202

Figure Lengend Snippet: DOTAP/DOPE complexed mRNA displays intrinsic adjuvant activity. (a) Expression pattern of maturation markers of bone marrow-derived DCs (BMDCs) transfected with DOTAP gag. BMDCs were transfected with 2.5 µg DOTAP gag and the expression of major histocompatibility complex (MHC)-II, CD80, CD86, and CD40 on Gag+ and Gag-CD11c+ cells was analyzed 24 hours later by flow cytometry. (b) Transcript levels of interleukin (IL)-6, IL-1β, IFN-α and interferon (IFN)-β mRNA were determined by RT-qPCR 24 hours (IL-6 and IL-1β) or 6 hours (IFN-α and IFN- β) after transfection of BMDCs with 2.5 µg DOTAP gag. Results represent mean n-fold induction levels compared to unstimulated control cells ± SD from triplicate PCR reactions. (c and d) Nuclear translocation of IFN regulatory factors (IRF)-3 and IRF-7 was visualized by confocal microscopy at different time points after stimulation of BMDCs with 2.5 µg DOTAP gag. (c) Confocal images taken 5 minutes after incubation with DOTAP or DOTAP gag and (d) ratios of protein present in the nucleus to total protein. Results represent mean ± SD of 100 cells. (e) Transcript levels of IFN-α and IFN-β mRNA were determined by RT-qPCR 6 hours after transfection of WT, MyD88−/− or TRIF−/− DCs with 2.5 µg DOTAP gag. Results represent mean n-fold induction levels compared to unstimulated control cells ± SD from triplicate PCR reactions. TRIF, TIR-domain-containing adapter-inducing interferon-β. (f) Mice were injected i.v. with 20 µg naked gag or DOTAP gag. Serum levels of IFN-α were determined 1, 3, 6, and 24 hours later by enzyme-linked immunosorbent assay. Mean ± SEM of 4 mice per group is shown. (g) Flowcytometric analysis of DC subsets and inflammatory monocytes present in the draining lymph nodes 1 and 3 days after footpad injection of 4µg of DOTAP gag. Conventional DCs were defined as CD11chi MHCIIint, Tissue-derived DCs were CD11cint MHCIIhi and inflammatory monocytes were MHC-II+ Ly6chi CD11bhi. Mean ± SEM 6 mice per group is shown. ***P < 0.001 as compared with the corresponding inflammatory monocyte numbers in DOTAP injected control mice and mice injected with naked gag.

Article Snippet: Antibodies used are α-CD3 Pacific Blue or PE, α-CD4 FITC, α-CD8 PerCP, α-CD69 PE-Cy7, α-CD45 Horizon-V450, α-CD19 APC-Cy7, α-GL-7 FITC, α-CD95 PE-Cy7, α-CD11c APC, α-CD40 biotin/streptavidin-PerCP, α-CD80 FITC, α-CD86 PE-Cy7, α-MHC-II PE-Cy7, α-CD11b APC-Cy7, α-Ly-6C Horizon-V450 (all BD Biosciences), and α-MHC-II eFluor450 (eBioscience, Vienna, Austria).

Techniques: Activity Assay, Expressing, Derivative Assay, Transfection, Flow Cytometry, Quantitative RT-PCR, Translocation Assay, Confocal Microscopy, Incubation, Injection, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines

doi: 10.1038/mt.2012.202

Figure Lengend Snippet: Type I IFN inhibitexpression of antigen-encoding mRNA and subsequent induction of immune responses. (a) Wild-type (WT) and interferon (IFN)αR−/− mice were immunized with 20µg DOTAP gag as previously described. Gag-specific IFN-γ and interleukin-2 secreting T cells were determined by enzyme-linked immunosorbent spot on isolated spleens. Mean of 5 mice per group is shown. ***P < 0.001. (b and c) WT, MyD88−/−, TRIF−/−, and IFNαR−/− bone marrow-derived DCs were transfected with 2.5 µg of DOTAP/DOPE-complexed Gag-encoding mRNA. Mean percentage ± SD of Gag+ CD11c+ cells (b) and expression pattern of the maturation markers major histocompatibility complex (MHC)-II, CD80, CD86, and CD40 on Gag+ and Gag- CD11c+ cells (c) was determined 24 hours later by flowcytometry. TRIF, TIR-domain-containing adapter-inducing interferon-β. (d) Flowcytometric analysis of DC subsets and inflammatory monocytes present in the draining lymph nodes of WT and IFNαR−/− mice 1 day post footpad injection of 4µg DOTAP gag. Conventional DCs were defined as CD11chi MHCIIint, tissue-derived DCs were CD11cint MHCIIhi and inflammatory monocytes were MHC-II+ Ly6chi CD11bhi. Mean ± SEM of 6 mice per group is shown.

Article Snippet: Antibodies used are α-CD3 Pacific Blue or PE, α-CD4 FITC, α-CD8 PerCP, α-CD69 PE-Cy7, α-CD45 Horizon-V450, α-CD19 APC-Cy7, α-GL-7 FITC, α-CD95 PE-Cy7, α-CD11c APC, α-CD40 biotin/streptavidin-PerCP, α-CD80 FITC, α-CD86 PE-Cy7, α-MHC-II PE-Cy7, α-CD11b APC-Cy7, α-Ly-6C Horizon-V450 (all BD Biosciences), and α-MHC-II eFluor450 (eBioscience, Vienna, Austria).

Techniques: ELISpot Assay, Isolation, Derivative Assay, Transfection, Expressing, Injection