Monocyte-Derived Dendritic Cells Are Essential for CD8(+) T Cell Activation and Antitumor Responses After Local Immunotherapy

Abstract

Tumors harbor several populations of dendritic cells (DCs) with the ability to prime tumor-specific T cells. However, these T cells mostly fail to differentiate into armed effectors and are unable to control tumor growth. We have previously shown that treatment with immunostimulatory agents at the tumor site can activate antitumor immune responses and is associated with the appearance of a population of monocyte-derived DCs (moDCs) in the tumor and tumor-draining lymph node (dLN). Here, we use depletion of DCs or monocytes and monocyte transfer to show that these moDCs are critical to the activation of antitumor immune responses. Treatment with the immunostimulatory agents monosodium urate crystals and Mycobacterium smegmatis induced the accumulation of monocytes in the dLN, their upregulation of CD11c and MHCII, and expression of iNOS, TNFα, and IL12p40. Blocking monocyte entry into the lymph node and tumor through neutralization of the chemokine CCL2 or inhibition of colony-stimulating factor-1 receptor signaling prevented the generation of moDCs, the infiltration of tumor-specific T cells into the tumor, and antitumor responses. In a reciprocal fashion, monocytes transferred into mice depleted of CD11c(+) cells were sufficient to rescue CD8(+) T cell priming in lymph node and delay tumor growth. Thus, monocytes exposed to the appropriate conditions become powerful activators of tumor-specific CD8(+) T cells and antitumor immunity.

Keywords: CSF1R; T cells; dendritic cells; monocyte-derived dendritic cells; mouse models; tumor immunotherapy.

Figure 1

Dendritic cells are required for the activation of antitumor immunity by MSU + Msmeg. C57BL/6 WT (C57WT) or CD11c-DTR BM chimeras were injected with B16 melanoma tumors, treated every second day for four times with MSU + Msmeg or PBS, and at the same time depleted of CD11c⁺ cells by i.p. DT treatment as indicated. (A) Individual tumor sizes in each group, as measured on the day when average tumor size in the C57 WT control group approached 150 mm² (days 17–23 after tumor challenge). Horizontal lines show mean ± SEM. (B) Survival of tumor challenged mice. Data in (A,B) are pooled from two independent experiments each with 3–5 mice/group. (C) Numbers of total DCs (CD11c⁺MHCII⁺) and (D) numbers of moDCs (CD11c⁺MHCII⁺CD11b⁺CD64⁺Ly6C⁺) in draining and contralateral LNs 2 days after the completion of MSU + Msmeg treatment. Data refer to one of two independent experiments that gave similar results. Graphs show mean + SEM for 5 mice/group. Statistical analysis in (A,C,D) was by one-way ANOVA with Tukey’s post-test. (B) Log-rank test with Bonferroni’s correction for multiple testing.

Figure 2

Monocyte accumulation and upregulation of DCs markers in the dLN of MSU + Msmeg-treated mice. (A,B) Mice were injected s.c. with MSU + Msmeg or PBS on days 0 and 2. About 17 h later dLNs were harvested and analyzed by flow cytometry. (A) Expression of DC and monocyte lineage markers on total DCs (MHCII⁺CD11c⁺), CD11b⁺ DC (CD11b⁺CD8⁻Ly6B⁻Ly6C⁻ DCs), CD8a⁺ DC (CD8a⁺CD11b⁻ DCs), and moDCs (CD11b⁺Ly6B⁺Ly6C⁺ DCs). (B) Frequency and number of moDCs (CD11b⁺Ly6B⁺Ly6C⁺CD11c⁺MHCII⁺) and monocytes (CD11b⁺Ly6B⁺Ly6C⁺CD11c⁻) in dLN. Data are pooled from two independent experiments each with 3–5 mice/group. (C,D,E) As in (A) but mice received two million CD45.1⁺ BM monocytes i.v. 17 h before analysis. (C) Identification of transferred monocytes in dLN. (D) Number of transferred monocytes and (E) expression of CD11c and MHCII on transferred monocytes, expressed as median fluorescence intensity (MFI). Data in (C–E) are from one of two independent experiments, each with 3–5 mice/group, that gave similar results. All graphs show mean + SEM. Statistical analyses used a Student’s t-test.

Figure 3

CCL2 is necessary for the recruitment of monocytes to MSU + Msmeg-treated dLNs and tumors. (A) Expression of CCR2 on monocytes (CD11b⁺Ly6B⁺Ly6C⁺CD11c⁻), moDCs (CD11c⁺MHCII⁺Ly6B⁺Ly6C⁺), and other DCs (CD11c⁺MHCII⁺Ly6B⁻Ly6C⁻) in dLNs of mice treated with MSU + Msmeg once 45 h earlier. (B) Frequency of CCR2⁺ monocytes in blood 15 h after MSU + Msmeg treatment. (C,D) Mice bearing palpable B16.OVA tumors were treated with anti-CCL2 or control antibody. About 2 h later, mice received 2 million purified CD45.1⁺ naive monocytes and were treated with MSU + Msmeg. The recruitment of transferred (CD45.1⁺) or host (CD45.2⁺) monocytes (Ly6B⁺Ly6C⁺CD11c⁻) to LN (C) and tumors (D) was examined 18 h later. Data are pooled from two independent experiments each with 3–5 mice/group. All graphs show mean + SEM. Statistical analyses used a Student’s t-test (B) or a Kruskal–Wallis with Dunn’s post-test (C,D).

Figure 4

Treatment with the CSF1-signaling inhibitor GW2580 impairs monocyte and moDCs recruitment and abrogates the antitumor activity of MSU + Msmeg. Mice bearing B16.OVA tumors were treated with MSU + Msmeg, and at the same time they received daily oral doses of GW2580 or vehicle control. LN and tumors were analyzed 2 days after the last MSU + Msmeg treatment. (A) Numbers of monocytes (CD11b⁺Ly6B⁺Ly6C⁺CD64⁺CD11c⁻) and moDCs (CD11b⁺Ly6B⁺Ly6C⁺CD11c⁺MHCII⁺) in dLN. (B) Frequencies of monocytes (CD45⁺CD11b⁺Ly6C^highLy6G⁻CD11c⁻), total DCs (CD45⁺CD11c⁺MHCII⁺), and moDCs (CD45⁺CD11c⁺MHCII⁺CD11b⁺Ly6C⁺Ly6B⁺) in tumors. (C) Tumor sizes on day 13. All data are pooled from two independent experiments each with 5 mice/group. Graphs show mean + SEM. Statistical analyses used ANOVA with Tukey’s post-test (A) or a Kruskal–Wallis with Dunn’s post-test (B,C).

Figure 5

Monocytes and moDCs in dLN express proinflammatory mediators after MSU + Msmeg treatment. Mice were treated with a single dose of MSU + Msmeg s.c., and 19 h later dLNs were collected and analyzed by intracellular staining for the indicated markers. Staining controls included an isotype control (isotype) and stained samples that were not treated with GolgiStop/Brefeldin A (no block). (A) Representative flow plots and (B) frequencies of iNOS-, IL-12-, and TNFα-expressing moDCs (CD11c⁺MHCII⁺CD11b⁺Ly6B⁺Ly6C⁺). (C) Frequencies of monocytes (CD11b⁺Ly6B⁺Ly6C⁺CD11c⁻) expressing proinflammatory molecules. Data are from one of two independent experiments, each with 3–5 mice/group that gave similar results. Graphs show means + SEM. Statistical analyses used a Student’s t-test.

Figure 6

Monocyte-derived DCs are necessary for the proliferation of tumor-specific CD8⁺ T cells in dLN and their infiltration into tumors. Mice bearing B16.OVA tumors were treated with MSU + Msmeg, and at the same time they were given daily oral doses of GW2580 or vehicle. LN and tumors were analyzed 2 days after the last MSU + Msmeg treatment. (A) Numbers of total and divided OTI T cells (CD45.1⁺CD8α⁺ lymphocytes) in tumor dLN, as assessed by CFSE dilution. (B) Total CD8⁺ and OTI T cells (CD45⁺CD8α⁺ and CD45⁺CD8α⁺CD45.1⁺, respectively) expressed as frequencies of total live cells in tumors. Data are pooled from two independent experiments, each with 5 mice/group. All graphs show mean + SEM. Statistical analyses were by one-way ANOVA with Tukey’s post-test (A) and Kruskal–Wallis test with Dunn’s post-test (B).

Figure 7

Monocyte-derived DCs are sufficient for the recruitment of CD8⁺ T cells to tumors and the activation of antitumor immunity. (A) Schematic representation of experimental design. C57BL/6 WT (C57WT) or CD11c-DTR BM chimeras were injected with B16 melanoma tumors, treated every second day for four times with MSU + Msmeg or PBS, and at the same time depleted of CD11c⁺ cells by DT treatment as indicated. Some of these mice were also injected with WT BM monocytes to obtain mice where moDC populations could only originate from the injected monocytes. (B) Mice were treated as in (A), except that tumors were B16.OVA, and all mice also received naive OTI cells given at the time of the second dose of immunotherapy. Graphs show the percentage of CD8⁺ T cells (CD45⁺CD4⁻CD8⁺) and OTI cells (CD45⁺CD4⁻CD8⁺CD45.1⁺) among total live cells in tumors 2 days after the fourth MSU + Msmeg treatment. Data are pooled from two independent experiments each with 5 mice/group. Graphs show mean + SEM. Statistical analysis used a Kruskal–Wallis test with Dunn’s post-test. (C) Mean tumor sizes + SEM on day 21 after tumor challenge. Data are pooled from three independent experiments each with 5 mice/group. Statistical analysis was by ANOVA with Tukey’s post-test. (D) Survival data, pooled from two independent experiments each with 5 mice/group. Statistical significance was evaluated using a log-rank test and Bonferroni’s correction.