biotinylated anti- mouse il- 15  (PeproTech)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1073/pnas.2306782120

Figure Lengend Snippet: Endogenous IL-15 participates in the therapeutic activity of CD40 agonism. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody and/or anti-IL-15 blocking antibody or isotype-matched control antibody. ( B ) Representative intravital luciferase imaging ( Left ) and quantification of luminescence ( Center ) and bladder weights ( Right ) across mice at day 12 post-tumor implantation (n = 5 mice per group; bars represent SD). ( C ) Representative flow cytometry plots and quantification across mice (n = 5 mice per group) of CD44 hi CD122 + CD8 T cells ( Top ) and CXCR6 + CD44 hi CD122 + CD8 T cells ( Bottom ) in the bladders of mice treated as outlined in A . * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Surface IL-15 was stained with biotinylated anti-mouse IL-15 (polyclonal rabbit; PeproTech) followed by streptavidin-APC (R&D).

Techniques: Activity Assay, Blocking Assay, Luciferase, Imaging, Tumor Implantation, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1073/pnas.2306782120

Figure Lengend Snippet: CD40 agonism induces IL-15/IL-15Rα upregulation on DCs in the bladder tumor microenvironment. Mice bearing orthotopic MB49 bladder tumors were treated intravesically with anti-CD40 antibody, BCG, or isotype-matched control antibody on days 6 and 9 post-tumor implantation. ( A ) Representative histogram and ( B ) quantification across mice of IL-15Rα mean fluorescence intensity on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). ( C ) Representative flow cytometry plots and ( D ) quantification across mice of IL-15 and IL-15Rα surface expression on cDC1s in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). The left graph is gated on cDC1s and depicts proportions of cDC1s that are double-positive for surface IL-15 and IL-15Rα. The right graph is gated on IL-15Rα-expressing cDC1s and depicts proportions of cDC1s expressing IL-15Rα that is occupied by IL-15. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Surface IL-15 was stained with biotinylated anti-mouse IL-15 (polyclonal rabbit; PeproTech) followed by streptavidin-APC (R&D).

Techniques: Tumor Implantation, Fluorescence, Flow Cytometry, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic Combination of Oncolytic Virotherapy and Immunotherapy for Glioma

doi: 10.1158/1078-0432.CCR-18-3626

Figure Lengend Snippet: In vitro characterization of vMyx-tdTr, vMyx-IL15Rα-tdTr, vvDD-RFP, and vvDD-IL15Rα-YFP. A total of 2 × 105 GL261WT cells were cultured per well of a 24-well plate, then infected with PBS, vMyx-tdTr, vMyx-IL15Rα-tdTr, vvDD-RFP, or vvDD-IL15Rα-YFP at MOI 5 for 24, 48, or 72 hours. A, Viable cell counts. B, Flow cytometry analysis of tdTomato Red (vMyx-tdTr and vMyx-IL15Rα-tdTr), RFP (vvDD-RFP), and YFP (vvDD-IL15Rα-YFP). Mean values and SEM are shown. C, Brightfield and fluorescence images of GL261 WT cells were taken 48 hours after virus infection. Red channel: tdTomato Red (vMyx-tdTr and vMyx-IL15Rα-tdTr), RFP (vvDD-RFP); green channel: YFP (vvDD-IL15Rα-YFP). Scale bar, 100 μm. D, Supernatants of cells from each treatment were collected 48 hours after virus infection. The production of IL15Rα-IL15 was measured by ELISA. Mean ELISA values and SEM are shown. There was a significant increase in IL15Rα-IL15 expression in the supernatants of vMyx-IL15Rα-tdTr or vvDD-IL15Rα-YFP–treated cells compared with vMyx-tdTr or vvDD-RFP–treated cells. P values: **, < 0.01; ****, < 0.0001. E, GL261 WT cells were treated with or without 100 nmol/L rapamycin 2 hours prior to the 1-hour inoculation of vMyx-IL15Rα-tdTr or vvDD-IL15Rα-YFP at MOI 5. Cells were fixed and analyzed for tdTomato red (vMyx-IL15Rα-tdTr) or YFP (vvDD-IL15Rα-YFP) expression by flow cytometry at 48 hours p.i. Mean ± SEM, n = 3, P value: ***, < 0.001.

Article Snippet: Primary antibodies: rabbit anti-M-T7 (Myxoma; generated in the lab of Dr. Grant McFadden), rabbit anti-vaccinia (Vaccinia; YVS8101, Accurate Chemical & Scientific Corporation), rat anti-mouse Ly-49G2 (NK cell; 555314, BD Pharmingen), rat anti-mouse CD8a (CD8 + T cells; 14080882, Invitrogen), rat anti-mouse FOXP3 (Treg; 14477180, Invitrogen), biotinylated anti-mouse IL15 (IL15Rα-IL15; BAF447, R&D Systems).

Techniques: In Vitro, Cell Culture, Infection, Flow Cytometry, Fluorescence, Virus, Enzyme-linked Immunosorbent Assay, Expressing