TLR-activated plasmacytoid dendritic cells inhibit breast cancer cell growth in vitro and in vivo

Abstract

Plasmacytoid dendritic cells (pDCs) are a unique subset of naturally occurring dendritic cells, which triggers the production of large amounts of type I interferons (IFNs) after viral infections through Toll-like receptor (TLR) 7 and TLR9. Recent studies have demonstrated that the activation of pDCs kills melanoma cells. However, the role of activated pDCs in breast cancer remains to be determined. In the present study, we generated mouse models of breast cancer and demonstrated that activated pDCs can directly kill breast tumor cells through TRAIL and Granzyme B. Furthermore, we established that pDCs initiate the sequential activation of NK cells and CD8+ T cells, and ultimately inhibit breast tumor growth. Understanding the role of activated pDCs in breast cancer may help to develop new strategies for manipulating the function of pDCs and induce anti-tumor immunity in breast cancer.

Keywords: CpG; Imiquimod; Toll-like receptor; breast cancer; plasmacytoid dendritic cells.

Figure 1. Morphologic, phenotypic and functional changes of pDCs after activation with IMQ and CpG

pDCs were harvested after activation with IMQ and CpG for 48 hours, and assessed for morphologic changes by Giemsa staining A. and for phenotypic changes by flow cytometry B. C. Supernatant from pDCs culture medium were collected after activation with IMQ and CpG for 36 hours to detect the release of IFN-α by ELISA and IL-12p70, TNF-α, and IL-6 by CBA. Data shown are expressed as mean ± SEM, and represent three independent experiments with similar results. Paired t-test was used for statistical comparison, ^*P<0.01, ^***P<0.001.

Figure 2. IMQ or CpG-activated pDCs kill tumor cells in vitro

A. Sorted pDCs were stimulated by IMQ or CpG for 36 hours, and were co-cultured with TUBO cells for another 5 hours. Unstimulated pDCs were used as control. The killing activity was determined by cytotoxicity assay. B. Supernatant from activated pDCs were harvested after 36 hours, and added to TUBO cells for 5 hours. Killing activity was determined by cytotoxicity assay. Specific lysis means of 3 independent experiments performed in triplicates are represented ± SEM. Paired t-test was used for statistical comparison, *P<0.05, ^*P<0.01.

Figure 3. IMQ or CpG-activated pDCs kill TUBO cells in a TRAIL and Granzyme B-dependent fashion

A. pDCs were stimulated by IMQ or CpG for 36 hours, and the expression of cytotoxic molecules TRAIL and Granzyme B on pDCs was assessed by flow cytometry and B. release of Granzyme B from pDCs by ELISA. Data shown are expressed as mean ± SEM. C. pDCs were stimulated by IMQ or CpG for 36 hours, and pre-incubated with neutralizing anti-TRAIL, anti-Granzyme B (10 ug/ml each) alone or combined, and isotype-matched control Abs for 30 minutes prior to the addition of TUBO cells. Cytotoxic activity was determined 5 hours later. Specific lysis means of 3 independent experiments performed in triplicates are represented ± SEM. Paired t-test was used for statistical comparison, *P<0.05, ^*P<0.01.

Figure 4. Administration of activated pDCs induces systemic anti-tumor activity

TUBO or 4T1 bearing mice were treated by i.t. injection of IMQ or CpG activated pDCs, resting pDCs, or saline on day 0. A, D. Tumor growth and B, E. mice survival monitored over time is shown. C, F. Mice bearing subcutaneous TUBO or 4T1 tumors after injection of TUBO cells at day -7 into the right flank and at day -2 into the left flank were treated by i.t. injection of pDCs into the tumor on the right flank on day 0 and day 2. The graph depicts the growth of tumors on both flanks over time following treatment. Data show a representative of three independent experiments with similar results.

Figure 5. NK cells mediate the anti-tumor activity of pDCs

A. Tumor-bearing mice were injected i.t. with IMQ or CpG-activated pDCs, resting pDCs, or with saline. Infiltration of NK cells at the tumor microenvironment was determined by flow cytometry on day 2. B. The expression of NK cell receptors NKG2D, NKG2A and cytotoxic molecule TRAIL were detected by flow cytometry on day 2. C. Chemotaxis of NK cells to tumor sites was induced by pDCs activated with IMQ or CpG for 36 hours. The levels of cytokine CCL3 and CCL5 in supernatant from pDCs were determined using CBA. D. Tumor-bearing mice were injected i.t. with IMQ or CpG-activated pDCs, resting pDCs, or saline. The level of cytokine CCL3 and CCL5 in serum of mice were determined using CBA. Data shown are expressed as mean ± SEM, and represent three independent experiments with similar results. Paired t-test was used for statistical comparison, *P<0.05, ^*P<0.01.

Figure 6. The role of CD8⁺ T cells in the anti-tumor activity of pDCs

A. Tumor-bearing mice were injected with IMQ or CpG-activated pDCs, resting pDCs, or saline. The infiltration of CD8⁺ T cells at the tumor microenvironment were determined by flow cytometry on day 5. B. The expression of cytotoxic molecule CD107a on CD8⁺T cells was determined by flow cytometry on day 5. Data were shown as mean ± SEM. One of these three experiments is presented.