Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells
Fanny Chalmin, Sylvain Ladoire, Grégoire Mignot, Julie Vincent, Mélanie Bruchard, Jean-Paul Remy-Martin, Wilfrid Boireau, Alain Rouleau, Benoit Simon, David Lanneau, Aurélie De Thonel, Gabriele Multhoff, Arlette Hamman, François Martin, Bruno Chauffert, Eric Solary, Laurence Zitvogel, Carmen Garrido, Bernhard Ryffel, Christophe Borg, Lionel Apetoh, Cédric Rébé, François Ghiringhelli
Fanny Chalmin, Sylvain Ladoire, Grégoire Mignot, Julie Vincent, Mélanie Bruchard, Jean-Paul Remy-Martin, Wilfrid Boireau, Alain Rouleau, Benoit Simon, David Lanneau, Aurélie De Thonel, Gabriele Multhoff, Arlette Hamman, François Martin, Bruno Chauffert, Eric Solary, Laurence Zitvogel, Carmen Garrido, Bernhard Ryffel, Christophe Borg, Lionel Apetoh, Cédric Rébé, François Ghiringhelli
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Research Article Immunology

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells

Abstract

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.

Authors

Fanny Chalmin, Sylvain Ladoire, Grégoire Mignot, Julie Vincent, Mélanie Bruchard, Jean-Paul Remy-Martin, Wilfrid Boireau, Alain Rouleau, Benoit Simon, David Lanneau, Aurélie De Thonel, Gabriele Multhoff, Arlette Hamman, François Martin, Bruno Chauffert, Eric Solary, Laurence Zitvogel, Carmen Garrido, Bernhard Ryffel, Christophe Borg, Lionel Apetoh, Cédric Rébé, François Ghiringhelli

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Figure 3

TDEs trigger pStat3 expression in MDSCs through autocrine production of IL-6.

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TDEs trigger pStat3 expression in MDSCs through autocrine production of ...
MDSCs from naive mice were treated with PBS or with TDSFs or TDEs. (A) IL-6 concentration in the supernatant was determined by ELISA. (B) In some wells, rIL-6 or blocking anti–IL-6 mAb (aIL-6) were added. Data represent pStat3 MFI ± SD. (C) Naive mice were i.v. injected with PBS, EL4 TDEs, or rIL-6. EL4 TB mice were i.v. injected with PBS or IL-6 siRNA. 24 hours later, spleens were harvested. MDSC percentage (denoted) and pStat3 expression were determined by FACS on gated MDSCs. (D) EL4 cells were cultured in vitro for 24 hours and treated with PBS, DMA, or omeprazole. In vivo EL4 TB mice were injected daily with DMA for 1 week. Acetylcholine esterase activity was assayed in supernatant or sera. (E) IL-6 concentration in sera was determined by ELISA in naive mice, which received a single injection of PBS or EL4 TDEs (left), or in EL4 TB mice, which received 1 daily injection of PBS, DMA, or omeprazole for 1 week (right). (F) EL4 TB mice were injected with PBS alone or DMA, omeprazole, or IL-6 siRNA (si). Spleens were harvested 24 hours later, and pStat3 expression was determined by FACS. Data are shown as MFI ± SD. (G) Mice (n = 5) were vaccinated with frozen/thawed CT26 cells 1 week before i.v. injection of live CT26 admixed or not with MDSCs isolated from PBS- or DMA-treated CT26 TB mice. Twelve days later, lung metastasis number was evaluated. Each experiment was done in triplicate. For box and whisker plots, bottoms and tops of boxes show the 25th and 75th percentiles, respectively, and middle bands show the median; whiskers show extrema. *P < 0.05.