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. 2020 Mar 26;8(1):e000415. doi: 10.1136/jitc-2019-000415

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© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See https://creativecommons.org/licenses/by/4.0/.

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VVΔTKΔN1L induces adaptive and innate immunity against lung cancer in vivo. Lewis lung carcinoma (LLC) tumors were established in the flanks of immune-competent C57/Bl6 mice. (A) Once palpable, mice were injected intratumorally (i.t) daily for 5 days with 1×10⁸ PFU VVΔTK, VVΔTKΔN1L or PBS (n=14/group). Mice were sacrificed at day 15, lungs H&E stained and analyzed for the presence of metastasis by a pathologist blinded to treatment groups. Lungs were scored as negative or positive and a Fishers exact test used to determine significance. (B)–(H) Once palpable, mice were injected i.t once with 1×10⁸ PFU VVΔTK, VVΔTKΔN1L or PBS (n=3–4/group). (B) Splenocytes were analyzed ex vivo for response to growth-arrested LLC cells using coculture for 72 hours followed by interferon γ (IFNγ) ELISA. (C) Splenocytes were collected and analyzed using FACS for expression of CD45, CD3, CD8, CD44 and CD62L. Percentage CD44^hiCD62L^lo cells (of live, CD45+/CD3+/CD8+ cells) are shown. (D) Tumors were collected 14 days post treatment and stained for CD4+ or CD8+ cells. Representative IHC images are shown (magnification x200) and cells/HPF shown graphically after 15 HPFs were counted. Of note, CD4+ staining cannot exclude the presence of TReg cells within the tumor. (E) FACS analysis was used to assess CD11b+Gr1+ neutrophils in tumors at the indicated time points. (F) FACS analysis was used to assess CD11b+F4/80+ macrophages in the tumors at the indicated time points. (G) Fourteen days post infection tumor sections were stained with F4/80 to assess macrophage infiltration. Representative images are shown. Magnification x200. (H) FACS analysis was used to assess CD3−CD49b+ natural killer cells in blood at the indicated time points. In all cases, one-way analysis of variance with post hoc Tukey tests was used to assess significance at each time point. In all cases, the mean±SEM is shown. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.