Intratumoral delivery of mTORC2-deficient dendritic cells inhibits B16 melanoma growth by promoting CD8⁺ effector T cell responses

Figures & data

Figure 1. Rictor^−/− BMDC display pro-inflammatory properties. (A) CD11c⁺-gated BMDC were analyzed for cell surface MHC class-II (I-A^b), CD40, CD86, B7-H1 (PD-L1) and CCR7 expression by flow cytometry following 6 d culture in the absence of stimulation (−) or after LPS stimulation (LPS) for the last 18 h of culture. Plots show the means and individual values of n = 3–6 mice. (B) Cytokine levels in supernatants were assessed by cytokine bead array (IL-6, TNFα, MCP-1) or ELISA (IL-12p70 and IFNγ). Data are from n = 4–6 mice. #p < 0.05 compared to corresponding non-stimulated cells; *p < 0.05 and **p < 0.01 between control and Rictor^−/− DC.

Figure 2. I.t. injection of LPS-activated Rictor^−/− DC markedly reduces B16 melanoma growth. C57BL/6 mice bearing day 7 s.c. B16 melanomas were given an i.t. injection of 10⁶ control DC or Rictor^−/− DC, that was repeated at day 14 post-tumor inoculation. Tumor growth was monitored every 3–4 d and is shown as mean + SD for five animals per group. p < 0.05 when comparing Rictor^−/− DC-injected mice with untreated or control DC-injected mice.

Figure 3. I.t.-delivered rictor^−/− DC show similar migration to draining lymphoid tissue, but reduce the frequency of MDSC within the tumor. 5 × 10⁶ CFSE-labeled control DC or Rictor^−/− DC were injected i.t. on day 7 post-tumor inoculation in B16-melanoma-bearing B6 mice. After 3 d, tumors, spleens and tumor-draining inguinal lymph nodes were harvested and cells isolated. (A) Plots show the percentages of CFSE⁺ DC recovered from the tumor. (B, C) Absolute numbers of CFSE⁺ DC recovered from the tumor (B) and inguinal lymph nodes (C). Box plots show median, 25%- and 75%-quartiles, and both extreme values. (D) Percent CD11c⁺ and CD11b⁺Gr1⁺ cells in the tumor shown as means + SD for three animals per group. *p < 0.05.

Figure 4. I.t.-delivery of Rictor^−/− DC results in a therapeutic antitumor response in a contralateral tumor site. Mice bearing 7-d B16 tumors in each flank, with similar mean total tumor sizes, were injected with 50 µL of PBS or 50 µL of PBS containing 10⁶ control DC or 10⁶ Rictor^−/− DC on the right flanks. Tumors on the left flank of each animal were left untreated. Animals received an identical treatment on day 14 post-tumor implantation. Treated and untreated tumor sizes (in mm²) for each animal were then monitored longitudinally every 3–4 d, and are reported as means + SD for five animals per group. *p < 0.05.

Figure 5. Reduction of B16 melanoma growth is dependent mainly on CD8⁺ T cells. (A) Rag1^−/− mice bearing day 7 s.c. B16 melanomas were given i.t. injections of 10⁶ control DC or Rictor^−/− DC. DC injection was repeated at day 14 post-tumor inoculation. Tumor growth was monitored every 3–4 d and is shown as means + SD for five animals per group. (B) C57BL/6 mice bearing s.c. B16 melanomas were treated on days 7 and 14 post-tumor inoculation by i.t. injection of 10⁶ Rictor^−/− DC. On days 6, 13, and 20 after tumor inoculation, different groups of mice were injected i.p. with control IgG, anti-CD4, anti-CD8⁺, or anti-NK Ab to specifically deplete these cell populations in vivo. Tumor growth was monitored every 3–4 d and is reported as mean + SD for five animals per group.

Figure 6. Rictor^−/− DC administration promotes increased frequencies of tumor-infiltrating cytotoxic T cells. C57BL/6 mice bearing s.c. B16 melanomas were injected i.t. at days 7 and 14 post-tumor inoculation with 10⁶ control DC or Rictor^−/− DC. At day 21 post-tumor inoculation, tumors were harvested and tumor-infiltrating lymphocytes obtained and stained for the indicated surface markers and intracellular cytokines. (A) Plots show absolute numbers of CD4⁺ and CD8⁺ tumor-infiltrating T cells divided by the respective tumor weight. (B) Frequencies of IFNγ⁺, IL-17⁺ and CD25⁺Foxp3⁺ cells in the total CD4⁺ T cell population. (C) Frequencies of IFNγ⁺, granzyme-B⁺ and PD-1⁺ cells in the total CD8⁺ T cell population. Data are shown as means + SD for three to four animals per group, and two independent experiments. *p < 0.05 and **p < 0.01.

Figure 7. Rictor^−/− DC administration enhances activation of antitumor CD8⁺ T cells in the periphery. C57BL/6 mice bearing s.c. B16 melanomas were treated at days 7 and 14 post-tumor inoculation with i.t. injection of 10⁶ control DC or Rictor^−/− DC. At day 21 post-tumor inoculation, spleens were harvested and (A, B, C) splenocytes stained with CFSE and then stimulated with irradiated (100 Gy) B16 cells (ratio 10:1 respectively) in the presence of 30 IU/mL recombinant human IL-2 for 5 d in 24-well culture plates. (A, B) Responder T cells were analyzed for CD8⁺ and intracellular IFNγ and granzyme-B, showing (A) representative plots and (B) means + SD for six animals per group reported from three independent experiments performed. (C) Cell-free supernatants of these cultures were analyzed for IFNγ, IL-6 and TNF-α. Box plots show median, 25%- and 75%-quartiles, and both extreme values. (D, E) Splenic CD8⁺ T cells isolated from untreated (PBS), control-DC- or Rictor^−/− DC-treated mice were cultured with CFSE-labeled B16 melanoma cells and violet-labeled irrelevant control EL4 thymoma cells at ratios of 5:1:1, 2:1:1 or 1:1:1 (where a unit of 1 = 5 × 10⁴ cells) for 18 h. Cells were analyzed by flow cytometry to determine the percentage of viable B16 (green) or EL4 (violet) cells, as shown in (D), versus a control consisting of a 1:1 mixture of each of the labeled tumor cell lines in the absence of T cells. (E) Results are reported as means +/− SD of data obtained from five mice/cohort. Percent killing was determined based on the formula: 100% × [1 – (percentage of viable tumor cells in the presence of T cells/percentage of viable tumor cells in the absence of T cells)]. *p < 0.05, **p < 0.01, ***p < 0.001.