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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 immune responses against pancreatic cancer in vivo. DT6606 tumors were established in the flanks of immune-competent C57/Bl6 mice (n=3–4/group). Once palpable, mice were injected intratumorally (i.t) once with 1×10⁸ PFU VVΔTK, VVΔTKΔN1L or PBS. Splenocytes were collected 7 or 14 days after treatment and analyzed. (A) Splenocytes were analyzed ex vivo for response to growth-arrested DT6606 cells using coculture for 72 hours followed by interferon γ (IFNγ) ELISA. (B) Splenocytes were analyzed ex vivo for response to mesothelin peptide using IFNγ ELISA after a 72-hour stimulation. (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 immunohistochemistry (IHC) images are shown (original magnification x200) and cells/higher power field (HPF) shown graphically after 15 HPFs were counted. Of note, CD4+ staining cannot exclude the presence of TReg cells within the tumor. (E) and (F) Dendritic cells (DCs) and macrophages were matured from bone marrow precursors taken from C57/Bl6 mice using granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor, respectively. Enriched populations were validated using FACS and infected with the indicted viruses for 24 hours at an MOI of 1 PFU/cell (n=3/group). (E) Expression of CD80 and CD86 markers on enriched DCs was examined after in vitro infection using FACS and normalized to expression levels on mock (PBS)-infected cells. (F) Expression of CD80, CD86 and major histocompatibility complex II (MHCII) markers on enriched macrophages was examined after infection using FACS and normalized to expression levels on mock (PBS)-infected cells. In all cases, one-way analysis of variance (ANOVA) with post hoc Tukey tests was used to assess significance at each time point. (G) Cell depleting or IgG control antibodies were commenced intraperitoneally in mice bearing DT6606 flank tumors a day prior to the first i.t treatment of virus (n=7/group). Mice were treated as above and tumor growth monitored. A two-way ANOVA with post hoc Tukey tests was used to assess significance. In all cases, the mean±SEM is shown. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.