CD4+CD25+ Tregs control the TRAIL-dependent cytotoxicity of tumor-infiltrating DCs in rodent models of colon cancer
Stephan Roux, Lionel Apetoh, Fanny Chalmin, Sylvain Ladoire, Grégoire Mignot, Pierre-Emmanuel Puig, Gregoire Lauvau, Laurence Zitvogel, François Martin, Bruno Chauffert, Hideo Yagita, Eric Solary, François Ghiringhelli
Stephan Roux, Lionel Apetoh, Fanny Chalmin, Sylvain Ladoire, Grégoire Mignot, Pierre-Emmanuel Puig, Gregoire Lauvau, Laurence Zitvogel, François Martin, Bruno Chauffert, Hideo Yagita, Eric Solary, François Ghiringhelli
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Research Article

CD4+CD25+ Tregs control the TRAIL-dependent cytotoxicity of tumor-infiltrating DCs in rodent models of colon cancer

Abstract

Tumors that progress do so via their ability to escape the antitumor immune response through several mechanisms, including developing ways to induce the differentiation and/or recruitment of CD4+CD25+ Tregs. The Tregs, in turn, inhibit the cytotoxic function of T cells and NK cells, but whether they have an effect on the cytotoxic function of tumor-infiltrating DCs (TIDCs) has not been determined. Here we have shown, in 2 rodent models of colon cancer, that CD4+CD25+ Tregs inhibit the ability of CD11b+ TIDCs to mediate TNF-related apoptosis-inducing ligand–induced (TRAIL-induced) tumor cell death. In both models of cancer, combination treatment with Mycobacterium bovis Bacillus Calmette-Guérin (BCG), which activates the innate immune system via TLR2, TLR4, and TLR9, and cyclophosphamide (CTX), which depletes Tregs, eradicated the tumors. Further analysis revealed that the treatment led to a marked increase in the number of CD11b+ TIDCs that killed the tumor cells via a TRAIL-dependent mechanism. Furthermore, acquisition of TRAIL expression by the CD11b+ TIDCs was induced by BCG and dependent on signaling through TLR2, TLR4, and TLR9. In vivo transfer of Tregs abrogated the ability of BCG to induce CD11b+ TIDCs to express TRAIL and thereby nullified the efficacy of the CTX-BCG treatment. Our data have therefore delineated what we believe to be a novel mechanism by which Tregs inhibit the antitumor immune response.

Authors

Stephan Roux, Lionel Apetoh, Fanny Chalmin, Sylvain Ladoire, Grégoire Mignot, Pierre-Emmanuel Puig, Gregoire Lauvau, Laurence Zitvogel, François Martin, Bruno Chauffert, Hideo Yagita, Eric Solary, François Ghiringhelli

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Figure 4

BCG endows DCs with killing capacities via TRAIL upregulation.

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BCG activated human Mo-DCs are cytotoxic against human cell lines in a T...
(A) For 48 hours, 5 × 104 CD11b+ TIDCs isolated from CTX-BCG–treated mice were incubated with 1 × 104 CT26 cells. In some wells, Z-VAD-fmk (Z-VAD), blocking anti–Fas ligand (FasL), anti-TRAIL mAbs, l-NMMA, or concanamycin A (CcmA) were added. CD11b+ TIDC cytotoxicity against CT26 cells was determined using a crystal violet assay. (B) FACS analysis of TRAIL expression on CD11b+ TIDCs (CD45+) that infiltrated untreated CT26 tumor and tumor treated with CTX-BCG. (C) CD11b+ TIDCs isolated from untreated CT26 tumors or BM-DCs were either untreated or cultured overnight with BCG (8 × 105 CFU/ml). TRAIL expression was determined by FACS analysis on CD11c+ I-A/I-E+ gated cells and (D) 5 × 104 CD11b+ TIDCs or BM-DCs were incubated with 1 × 104 CT26 cells for 48 hours. A blocking anti-TRAIL mAb was also added to some of the wells containing BM-DCs activated by BCG. Cytotoxic effect on the CT26 cells was determined using a crystal violet assay. (E) BM-DCs were incubated overnight in the absence or presence of ligands for TLR2 (Pamv3CSK4 [Pam3]), TLR3 [Poly(I:C)], TLR4 (LPS), TLR9 (CpG ODN 1826 [CpG 1826]), or with BCG (8 × 105 CFU/ml). TRAIL expression was determined by FACS analysis on CD11c+ I-A/I-E+ gated cells. (F) Wild-type, Myd88–/–, Tlr2–/–, Tlr4–/–, or wild-type mice treated with i.p. injections of TLR9 inhibitor ODN (IRS 954) every 3 days received a subcutaneous injection of 5 × 105 CT26 tumor cells. In the same experiment, 2 groups of mice received either saline or the combined treatment. Data represent mean ± SEM. *P < 0.05.