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. 2009 Jan 13;6(1):e1000010. doi: 10.1371/journal.pmed.1000010

Figure 1. Flt3L and TK Recruits DC into Brain Tumors and Mediates T Cell–Dependent Brain Tumor Regression.

Figure 1

(A) GL26 cells were implanted in the striatum of C57BL/6 mice. Tumor-bearing mice were treated 17 d later with Ad-Flt3L and Ad-TK (black solid line, n = 6), Ad-TK (blue solid line, n = 6), Ad-Flt3L (purple solid line, n = 5), Ad0 (orange solid line, n = 4), or saline (black broken line, n = 6). Treatment with Ad-Flt3L and Ad-TK significantly improved survival; *, p < 0.05 versus saline (Mantel log-rank test).

(B) GL26 cells were implanted in the striatum of WT (black, n = 6), or Rag1 −/− (Rag10, blue , n = 5), CD4 −/− (CD40, purple, n = 4), or CD8 −/− (CD80, orange, n = 5) knockout mice. 17 d later, mice were treated with Ad-Flt3L and Ad-TK (solid lines) or saline (broken lines). Treatment with Ad-Flt3L and Ad-TK significantly improved survival of WT animals when compared to all other strains; (*, p < 0.05; Mantel log-rank test).

(C) Long-term survivors (>150 d post-tumor cell implantation) were rechallenged with GL26 cells in the contralateral hemisphere (solid line, n = 4). Naïve, age matched C57BL/6 mice were also implanted with GL26 cells as a control (broken line, n = 5). Only long-term survivors showed increased survival following tumor rechallenge (*, p < 0.05 versus saline; Mantel log-rank test).

(D) Tumor antigen specific, IFNγ-producing T lymphocytes were quantified using an IFNγ ELISPOT. GL26 cells were implanted in the brains of C57BL/6 mice and treated 17 d later with Ad-Flt3L and Ad-TK (TF) or Saline (S). T cells were isolated and incubated with DC loaded with either GL26 tumor antigen (GL26) or LLc1 tumor antigen (LLc1) before quantification of IFNγ production. Inset: Concanavalin A (ConA)-stimulated splenocytes (+) are shown as a positive control. *, p < 0.05 versus saline (Mann-Whitney U-test).

(E) A T cell proliferation assay was used to determine the percentage of T cells that recognize the tumor antigen Trp2180–188. GL26 cells were implanted in the brains of mice, and treated after 17 d with Ad-Flt3L and Ad-TK or with saline. Splenocytes were isolated 7 d later and stained with CFSE, then incubated with (+) or without (−) Trp2180–188. CD8+ T cell proliferation was calculated using flow cytometry by quantifying the percentage of cells that had divided (sequential dilution of CFSE fluorescence). *, p < 0.05 versus saline (Mann-Whitney U-test). Inset: Reverse transcriptase PCR confirms the expression of Trp2 mRNA by GL26 cells. GAPDH primers were used as a control.

(F) GL26 cells were implanted in the brain of C57BL/6 mice and treated 17 d later with Ad-Flt3L and Ad-TK, Ad0, or saline. The total number of tumor-infiltrating DCs (CD11c+ CD45High MHC II+) was quantified by flow cytometry. *, p < 0.05 versus saline (Kruskal-Wallis test followed by Dunn's test). Representative dot plots display tumor-infiltrating CD45+ immune cells stained with CD11c (x-axis) and MHC II (I-Ab; y-axis). DCs were identified as (CD11c+ CD45High MHC II+). The total number of tumor-infiltrating mDC is indicated for each plot.