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 6

Tregs are selectively killed by CTX and prevent TRAIL-dependent cytotoxicity of TIDCs.

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Tregs are selectively killed by CTX and prevent TRAIL-dependent cytotoxi...
(A) FACS analysis of CD4+CD25+ (Treg) cells in the CD3+CD4+CD127– tumor-infiltrating T cell subsets. The percentage of Tregs and dead cells in tumors were determined by DAPI labeling 2 days after CTX injection. Numbers indicate the percentage of cells ± SD. (B) The day of the first BCG injection, 2 groups of mice were adoptively transferred with 1 × 106 Tregs or 1 × 106 CD4+CD25– (Tconv) cells (n = 5 in each group). (C) CD11b+ TIDCs from untreated CT26 tumor-bearing mice were incubated overnight with BCG and with Tregs or Tconvs at a ratio 1:1. TRAIL expression was determined by FACS analysis. (D) Same experiment as in C but using mouse BM-DCs instead of CD11b+ TIDCs. (E) Same experiment as in D but in some wells DCs were separated from T cells by a 0.4-μm Transwell insert. TRAIL and GAPDH mRNA levels were determined by quantitative RT-PCR on CD11c cells sorted by magnetic isolation. BM-DCs incubated with IFN-γ represent a positive control of TRAIL mRNA expression. Each value is expressed as fold increase from medium control after normalization with GAPDH. (F) CD11b+ TIDCs from untreated CT26 tumor-bearing animals were incubated overnight with BCG and with Tregs or Tconvs at a ratio 1:1. Then these cells (5 × 104 cells) were cultured with 1 × 104 CT26 cells for 48 hours. TIDC cytotoxicity on CT26 cells was determined using crystal violet staining. (G) Same experiment as in F but using mouse BM-DCs instead of CD11b+ TIDCs. (H) Same experiment as in F but using mouse Mo-DCs instead of CD11b+ TIDCs and M45 melanoma cells instead of CT26 cells. Values from 1 experiment out of 2 represent mean ± SEM. *P < 0.05.