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Eosinophils orchestrate cancer rejection by normalizing tumor vessels and enhancing infiltration of CD8+ T cells

A Corrigendum to this article was published on 19 January 2016

This article has been updated

Abstract

Tumor-associated eosinophilia is frequently observed in cancer. However, despite numerous studies of patients with cancer and mouse models of cancer, it has remained uncertain if eosinophils contribute to tumor immunity or are mere bystander cells. Here we report that activated eosinophils were essential for tumor rejection in the presence of tumor-specific CD8+ T cells. Tumor-homing eosinophils secreted chemoattractants that guided T cells into the tumor, which resulted in tumor eradication and survival. Activated eosinophils initiated substantial changes in the tumor microenvironment, including macrophage polarization and normalization of the tumor vasculature, which are known to promote tumor rejection. Thus, our study presents a new concept for eosinophils in cancer that may lead to novel therapeutic strategies.

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Figure 1: Treg cell depletion results in eosinophil infiltration and tumor rejection.
Figure 2: Tumor rejection after Treg cell depletion is dependent on infiltrating eosinophils.
Figure 3: Changes in the tumor microenvironment after depletion of Treg cells and eosinophils.
Figure 4: Eosinophil-derived chemokines induce T cell migration and vascular normalization.
Figure 5: Adoptive transfer of tumor-specific CD8+ T cells alone fails to reject tumors, whereas transfer of those cells together with activated eosinophils leads to substantial T cell infiltration and tumor rejection.
Figure 6: Cotransfer of cells promotes a reduction in vessel size and increases VCAM-1 expression.
Figure 7: Normalization of tumor vasculature.
Figure 8: Cotransfer of cells results in the M1-like polarization of tumor-associated macrophages.

Change history

  • 21 May 2015

    In the version of this article initially published, the description of the data presented in Figure 4f–i was incorrect. That section of Results should read as follows: "The additional depletion of eosinophils...resulted in increased tumor hypoxia....(Fig. 4f and Supplementary Fig. 6). Depletion of eosinophils also increased vascular leakiness and diminished vascular perfusion...(Fig. 4g–i and Supplementary Fig. 6)." The errors have been corrected in the HTML and PDF versions of the article.

  • 13 November 2015

    In the version of this article initially published, the graph in Figure 2e was incorrect. This has been replaced with the correct graph. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank S. Schmitt for technical assistance; L. Umansky for multiplex analysis of cytokines; and T. Tüting (University of Bonn) for HCmel melanoma cells. Supported by Wilhelm Sander Stiftung (2009.022.2 to G.J.H.), the Cooperation Program in Cancer Research by the German Cancer Research Center, the Israel Ministry of Science, Technology and Space (G.J.H.) and the Bonn Cluster of Excellence to (N.G.).

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R.C., I.M.S., N.G., P.B. and G.J.H. designed the experimental plan; R.C., I.M.S. and O.C.S. performed the experiments; and R.C., I.M.S. and G.J.H. wrote the manuscript.

Corresponding author

Correspondence to Günter J Hämmerling.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Flow cytometry gating strategy for determination of tumor-infiltrating leukocyte subpopulations.

Abbreviations used: Singlets- single events. Leu- leukocytes. B- B cells. DCs- dendritic cells. Mye- Myeloid cells. NKT- NKT cells, NK- natural killer cells Neu- neutrophils. Mon- monocytes. Mac- macrophages. Eos- eosinophils. T- T cells. CD4- T helper cells. CD8- T cytotoxic cells.

Supplementary Figure 2 Characterization of tumor-associated eosinophils.

a) Q-PCR analysis of sorted tumor CD11b+, Gr-1low, F4/80+ cells for Mbp and Epo eosinophil markers. b) Sorted tumor infiltrating eosinophils stained with hematoxilin-eosin after Treg depletion. Results are shown as mean ±SEM. n=6 mice per group. 1 of 3 independent experiments is shown. *=p<0.05; **=p<0.01; ***=p<0.001; NS= not significant (unpaired T-test).

Supplementary Figure 3 Efficiency of eosinophil depletion with mAb to Siglec-F.

1 day after intraperitoneal injection of Siglec-F mAb (15 ng/mouse) efficient depletion of eosinophils was observed in draining lymph nodes, (dLNs) and tumors. n=6 mice. 1 of 3 independent experiments is shown.

Supplementary Figure 4 Purification and survival of eosinophils.

a) Mice were injected i.p. with thioglycollate and peritoneal cells where harvested 3 days later. b) Eosinophils were purified from these peritoneal cells by MACS using beads coated with anti-PE antibodies and Siglec-F PE labeled antibodies. The purity obtained was above 80%. c) Enriched eosinophils were activated in vitro with complete RPMI medium containing IFNγ (15 ng/ml) and TNF (20 ng/ml) up to 16h. After activation the purity was 90%. d) Viability of eosinophil after in vitro culture. n=6. 1 of 3 independent experiments is shown.

Supplementary Figure 5 Titration of eosinophil-induced in vitro migration.

a) Multiplex protein analysis of lysates from activated or non-activated eosinophils. n=5. S Shown is 1 out of 2 independent experiments. b) Titration of eosinophil effect in CD8+ T cell in vitro migration. A range from 2,5x104 to 8x105 eosinophils in the lower chamber was used. Concentration of CD8+ T cells in the upper chamber was 1x105. c) Expression of angiogenesis factors by in vitro activated eosinophils. n=5 mice Results are shown as mean ±SEM. 1 of 2 independent experiments is shown. *=p<0.05; **=p<0.01; ***=p<0.001; NS= not significant (unpaired T-test).

Supplementary Figure 6 Examples of immunohistochemistry of tumors 6 d after depletion of Treg cells.

a) Hypoxia analysis. Hypoxiprobe (turquoise) and CD31 (magenta) staining. Bar =1mm b) Perfusion analysis. Tomatolectin (turquoise) and CD31 (magenta). Bar =100µm c) leakiness analysis. Dextran (turquoise) and CD31 (magenta). Bar =100µm d) Pericyte coverage. αSMA (turquoise) and CD31 (magenta). Bar =100µm. e-h) Quantification of IHC as shown in a-d. Each dot represents average of 10 fields evaluated per tumor. n=6 mice per group. Results are shown as mean ±SEM. *=p<0.05; **=p<0.01; ***=p<0.001; NS= not significant (unpaired T-test).

Supplementary Figure 7 Changes in the tumor microenvironment after transfer of eosinophils and OT-I cells.

a) Flow cytometric quantification of tumor infiltrating leukocyte subpopulations after transfer of activated eosinophils and OTI cells. The data shows significantly more T-cells infiltrating the tumor after cotransfer of activated eosinophils and OT-I as compamagenta to OTI cells alone. b) Representative confocal images of MO4 tumors 2 days after transfer of activated tumor-specific CD8+ T cells and activated eosinophils. Bar, 50µm. n=7 mice per group. c) Q-PCR analysis of cytokine and chemokine levels in the tumor microenvironment after transfer of activated eosinophils and OTI cells. d) Q-PCR analysis of angiogenesis factors in the tumor microenvironment after transfer of activated eosinophils and OTI cells. n=6 mice. Results are shown as mean ±SEM. 1 of 2 independent experiments is shown *=p<0.05; **=p<0.01; ***=p<0.001; NS=not significant (unpaired T-test).

Supplementary Figure 8 Effect of eosinophils in the Hcmel melanoma model.

a) Tumor growth after adoptive therapy of 5x106 eosinophils and 5x106 pmel CD8+ T cells. b) Survival of mice shown in a. n=6 mice. Results are shown as mean ±SEM. 1 of 2 independent experiments is shown. *=p<0.05; **=p<0.01; ***=p<0.001; ns= not significant. log Rank (Mantel-Cox) test.

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Carretero, R., Sektioglu, I., Garbi, N. et al. Eosinophils orchestrate cancer rejection by normalizing tumor vessels and enhancing infiltration of CD8+ T cells. Nat Immunol 16, 609–617 (2015). https://doi.org/10.1038/ni.3159

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