Journal: EMBO Molecular Medicine

Article Title: Tumor endothelial cell autophagy is a key vascular‐immune checkpoint in melanoma

doi: 10.15252/emmm.202318028

Figure Lengend Snippet: A Gating strategy for analyzing immune cell subsets in subcutaneous B16‐F10 melanoma from WT and Atg5 BECKO mice. B Gene expression of CD8 , CD3g , CD45 , and Nkp46 in blood collected from WT and Atg5 BECKO mice with subcutaneous B16‐F10 tumors and injected with αCD8 antibody. Each point represents 1 mouse; with at least n = 4 mice per group. Data Information: All data represent mean ± s.e.m. Statistical differences were determined using one‐way Anova with Tukey corrections for multiple comparisons (B).

Article Snippet: Cell surface immunostaining used the following antibodies: CD31 (1:400, BV421; BD#562939), CD45 (1:200, APC‐Cy7; BD#561037), VCAM1 (1:200, FITC; Biolegend#105705), ICAM1 (1:200, PE; BD #568539), MHCI (1:100 dilution, APC; Biolegend #107613), and MHCII (1:100 dilution, FITC; Biolegend#107605). eBioscience™ Fixable Viability Dye eFluor™ 780 (1:1,000,Thermo#65‐0865‐14) was used to discriminate dead cells.

Techniques: Expressing, Injection

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