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Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33-dependent eosinophil-mediated control of tumor growth

Abstract

Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited.

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Fig. 1: T cells are not required for the DPP4i-mediated anti-tumor effect in HCC and breast cancer syngeneic models.
Fig. 2: DPP4i enhances eosinophilia in solid tumors.
Fig. 3: Eosinophils are required for DPP4i-mediated anti-tumor response.
Fig. 4: DPP4i enhances anti-tumor responses via a CCL11-mediated mechanism.
Fig. 5: IL-33 expression promotes eosinophil-mediated anti-tumor responses in the presence of DPP4i.
Fig. 6: Type 1 immunity and type 2 immunity collaborate to achieve tumor immunotherapy.

Data availability

The raw data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

Funding for the work was provided by Fondation ARC pour la recherche sur le cancer, Institut national de la santé et de la recherche médicale (Inserm), Fondation pour la recherche médicale (FRM, FDM 40432) and LabEx Immuno-Onco (ANR). Thanks to H. Saklani, M. A. Ingersoll and T. Canton for their help with mouse experimental work and ethical statement.

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C.H., S.P., R.B.d.S. and M.L.A. designed the study. C.H. and R.B.d.S. designed, carried out and analyzed experiments. J.B., T.N., D.D., V.M., J.M.S. and G.E. provided technical and intellectual assistance. V.B., W.P. and W.S. conducted mass spectrometry experiments. B.L. provided tumor cell lines. V.P. and J.Z. conducted histological experiments. C.H., R.B.d.S. and M.L.A. wrote the manuscript. M.L.A., R.B.d.S. and S.P. supervised the study.

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Correspondence to Matthew L. Albert.

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R.B.d.S., M.L.A., J.Z., T.N., W.P., W.S. and J.M.S. are current employees of Genentech, a member of the Roche group.

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Integrated supplementary information

Supplementary Figure 1 DPP4i diminishes tumor volume and DPP enzymatic activity in Hepa1-6 and EMT6 subcutaneous tumors.

(a, b) WT mice were fed with DPP4i or with Ctrl chow and subcutaneously injected with Hepa 1-6 (a) or EMT6 (b) cells. Tumors were collected at day 10 post-inoculation and tumor mass was determined. Bars represent median values (n = 17 (Ctrl, panel a), 18 (DPP4i, panel a) or 6 per group (b)). (c, d) Eight days after Hepa 1-6 (c) or EMT6 (d) tumor cell inoculation, tumors were collected and homogenates were prepared. DPP4 activity per tumor mass was determined and normalized to the amount of DPP4 protein (RLU, relative luminescence units). Bars represent median values (n = 4 per group (c), 6 (Ctrl, d) or 7 (DPP4i, d) mice). (e) Hepa 1-6 cells were cultured in the presence of 5 (C1), 0.05 (C2) or 0.005 (C3) μg/ml of DPP4i, or left untreated (). Cell confluence was measured over time using intra-incubator microscopy (mean ± SEM, n = 3 technical replicates). Each dot corresponds to one mouse. Data are representative of two independent experiments (be) or pooled from two (a) independent experiments. NS, not significant; *P < 0.05, ****P < 0.0001. Significance was determined using two-sided Mann–Whitney test (ad). (Allergy 63, 1156–1163, 2008)

Supplementary Figure 2 DPP4i enhances infiltration of eosinophils in the EMT6 breast model and enrichment for T cells in the parenchyma of Hepa1-6 subcutaneous tumors.

(a, b) Gating strategy for identification of tumor infiltrating leukocytes. (c, d) WT mice were fed with DPP4i or with Ctrl chow and subcutaneously injected with EMT6 cells. Tumors were collected at day 10 post-inoculation and tumor-associated leukocytes were analyzed by flow cytometry. Number of tumor infiltrating T cells and eosinophils is shown. Each dot corresponds to one mouse (n = 11 (Ctrl, c) or 12 (DPP4i, c) or 12 per group (d)). Bars represent median values. (e, f) WT mice were treated as in c and inoculated with Hepa1-6 cells. Representative images (scale, 40 μm) and quantification of CD3 expressing cells in Hepa 1-6 tumors collected 10 d after inoculation. Each dot corresponds to one mouse (n = 8 (Ctrl) or 7 (DPP4i) mice). Bars represent median values. (g) Rag2–/–γc–/– mice fed with Ctrl or DPP4i were inoculated with Hepa 1-6 cells. Tumor volumes were measured over time (mean ± SEM, n = 4 mice (Ctrl) or 5 mice (DPP4i). Data are pooled from two (c, d) independent experiments. Histological analysis on T cell distribution and tumor growth on Rag2–/–γc–/– was done once. NS, not significant; *P < 0.05, **P < 0.01. Significance was determined using or two-sided Mann–Whitney test (c, d, f) or two-way analysis of variance (g).

Supplementary Figure 3 DPP4 does not truncate mouse CCL24 but reduces CCL11-mediated internalization of CCR3 in eosinophils.

(a) Recombinant mCCL24 was incubated in the presence or absence of mDPP4 and analyzed by mass spectrometry. Numbers indicate the molecular weight (in Daltons). (b) WT mice fed with Ctrl or with DPP4i were injected intravenously with 1 μg of CCL11 and blood was collected 1 h after injection. Representative histogram of CCR3 expression on eosinophils is shown. Quantification was determined by flow cytometry. Each dot represents one mouse (n = 7 (Ctrl) or 8 (DPP4i) mice). Data are representative of two independent experiments yielding similar results. Bars represent median values. NS, not significant; **P < 0.01. Significance was determined using or two-sided Mann–Whitney test.

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Hollande, C., Boussier, J., Ziai, J. et al. Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33-dependent eosinophil-mediated control of tumor growth. Nat Immunol 20, 257–264 (2019). https://doi.org/10.1038/s41590-019-0321-5

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