Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Immunology volume 16, pages 609–617 (2015)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Rothenberg, M.E. & Hogan, S.P. The eosinophil. Annu. Rev. Immunol. 24, 147–174 (2006).

    CAS  PubMed  Google Scholar 

  2. Rosenberg, H.F., Dyer, K.D. & Foster, P.S. Eosinophils: changing perspectives in health and disease. Nat. Rev. Immunol. 13, 9–22 (2013).

    CAS  PubMed  Google Scholar 

  3. Kita, H. Eosinophils: multifaceted biological properties and roles in health and disease. Immunol. Rev. 242, 161–177 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Melo, R.C., Liu, L., Xenakis, J.J. & Spencer, L.A. Eosinophil-derived cytokines in health and disease: unraveling novel mechanisms of selective secretion. Allergy 68, 274–284 (2013).

    CAS  PubMed  Google Scholar 

  5. Reinbach, G. Ueber das Verhalten der Leukocyten bei malignen Tumoren. Arch. Klin. Chir 46, 486–562 (1893).

    Google Scholar 

  6. Simson, L. et al. Regulation of carcinogenesis by IL-5 and CCL11: a potential role for eosinophils in tumor immune surveillance. J. Immunol. 178, 4222–4229 (2007).

    CAS  PubMed  Google Scholar 

  7. Cormier, S.A. et al. Pivotal advance: eosinophil infiltration of solid tumors is an early and persistent inflammatory host response. J. Leukoc. Biol. 79, 1131–1139 (2006).

    CAS  PubMed  Google Scholar 

  8. Lotfi, R. et al. Eosinophils oxidize damage-associated molecular pattern molecules derived from stressed cells. J. Immunol. 183, 5023–5031 (2009).

    CAS  PubMed  Google Scholar 

  9. Lotfi, R., Lee, J.J. & Lotze, M.T. Eosinophilic granulocytes and damage-associated molecular pattern molecules (DAMPs): role in the inflammatory response within tumors. J. Immunother. 30, 16–28 (2007).

    CAS  PubMed  Google Scholar 

  10. Davis, B.P. & Rothenberg, M.E. Eosinophils and cancer. Cancer Immunol. Res. 2, 1–8 (2014).

    CAS  PubMed  Google Scholar 

  11. Gatault, S., Legrand, F., Delbeke, M., Loiseau, S. & Capron, M. Involvement of eosinophils in the anti-tumor response. Cancer Immunol. Immunother. 61, 1527–1534 (2012).

    PubMed  Google Scholar 

  12. Tepper, R.I., Coffman, R.L. & Leder, P. An eosinophil-dependent mechanism for the antitumor effect of interleukin-4. Science 257, 548–551 (1992).

    CAS  PubMed  Google Scholar 

  13. Golumbek, P.T. et al. Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4. Science 254, 713–716 (1991).

    CAS  PubMed  Google Scholar 

  14. Noffz, G., Qin, Z., Kopf, M. & Blankenstein, T. Neutrophils but not eosinophils are involved in growth suppression of IL-4-secreting tumors. J. Immunol. 160, 345–350 (1998).

    CAS  PubMed  Google Scholar 

  15. Mattes, J. et al. Immunotherapy of cytotoxic T cell-resistant tumors by T helper 2 cells: an eotaxin and STAT6-dependent process. J. Exp. Med. 197, 387–393 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Samoszuk, M. et al. Increased blood clotting, microvascular density, and inflammation in eotaxin-secreting tumors implanted into mice. Am. J. Pathol. 165, 449–456 (2004).

    PubMed  PubMed Central  Google Scholar 

  17. Krüger-Krasagakes, S., Li, W., Richter, G., Diamantstein, T. & Blankenstein, T. Eosinophils infiltrating interleukin-5 gene-transfected tumors do not suppress tumor growth. Eur. J. Immunol. 23, 992–995 (1993).

    PubMed  Google Scholar 

  18. Huland, E. & Huland, H. Tumor-associated eosinophilia in interleukin-2-treated patients: evidence of toxic eosinophil degranulation on bladder cancer cells. J. Cancer Res. Clin. Oncol. 118, 463–467 (1992).

    CAS  PubMed  Google Scholar 

  19. Sosman, J.A. et al. Evidence for eosinophil activation in cancer patients receiving recombinant interleukin-4: effects of interleukin-4 alone and following interleukin-2 administration. Clin. Cancer Res. 1, 805–812 (1995).

    CAS  PubMed  Google Scholar 

  20. Galdiero, M.R. et al. Tumor associated macrophages and neutrophils in cancer. Immunobiology 218, 1402–1410 (2013).

    CAS  PubMed  Google Scholar 

  21. Li, X., Kostareli, E., Suffner, J., Garbi, N. & Hammerling, G.J. Efficient Treg depletion induces T-cell infiltration and rejection of large tumors. Eur. J. Immunol. 40, 3325–3335 (2010).

    CAS  PubMed  Google Scholar 

  22. Zimmermann, N. et al. Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils. Allergy 63, 1156–1163 (2008).

    CAS  PubMed  Google Scholar 

  23. Chu, V.T. et al. Eosinophils are required for the maintenance of plasma cells in the bone marrow. Nat. Immunol. 12, 151–159 (2011).

    CAS  PubMed  Google Scholar 

  24. Hamzah, J. et al. Vascular normalization in Rgs5-deficient tumours promotes immune destruction. Nature 453, 410–414 (2008).

    CAS  PubMed  Google Scholar 

  25. Munitz, A. & Levi-Schaffer, F. Eosinophils: 'new' roles for 'old' cells. Allergy 59, 268–275 (2004).

    CAS  PubMed  Google Scholar 

  26. Restifo, N.P., Dudley, M.E. & Rosenberg, S.A. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12, 269–281 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Jain, R.K. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 307, 58–62 (2005).

    CAS  PubMed  Google Scholar 

  28. Dalton, D.K. et al. Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science 259, 1739–1742 (1993).

    CAS  PubMed  Google Scholar 

  29. Stockmann, C. et al. Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis. Nature 456, 814–818 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Stockmann, C., Schadendorf, D., Klose, R. & Helfrich, I. The impact of the immune system on tumor: angiogenesis and vascular remodeling. Front. Oncol. 4, 69 (2014).

    PubMed  PubMed Central  Google Scholar 

  31. Huang, Y., Goel, S., Duda, D.G., Fukumura, D. & Jain, R.K. Vascular normalization as an emerging strategy to enhance cancer immunotherapy. Cancer Res. 73, 2943–2948 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Ganss, R., Ryschich, E., Klar, E., Arnold, B. & Hammerling, G.J. Combination of T-cell therapy and trigger of inflammation induces remodeling of the vasculature and tumor eradication. Cancer Res. 62, 1462–1470 (2002).

    CAS  PubMed  Google Scholar 

  33. Ryschich, E., Schmidt, J., Hammerling, G.J., Klar, E. & Ganss, R. Transformation of the microvascular system during multistage tumorigenesis. Int. J. Cancer 97, 719–725 (2002).

    CAS  PubMed  Google Scholar 

  34. Facciabene, A. et al. Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells. Nature 475, 226–230 (2011).

    CAS  PubMed  Google Scholar 

  35. Gabrilovich, D.I., Ostrand-Rosenberg, S. & Bronte, V. Coordinated regulation of myeloid cells by tumours. Nat. Rev. Immunol. 12, 253–268 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Klug, F. et al. Low-dose irradiation programs macrophage differentiation to an iNOS+/M1 phenotype that orchestrates effective T cell immunotherapy. Cancer Cell 24, 589–602 (2013).

    CAS  PubMed  Google Scholar 

  37. Garbi, N., Arnold, B., Gordon, S., Hammerling, G.J. & Ganss, R. CpG motifs as proinflammatory factors render autochthonous tumors permissive for infiltration and destruction. J. Immunol. 172, 5861–5869 (2004).

    CAS  PubMed  Google Scholar 

  38. Finlay, D.K. et al. PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells. J. Exp. Med. 209, 2441–2453 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Shi, S. et al. Combining antiangiogenic therapy with adoptive cell immunotherapy exerts better antitumor effects in non-small cell lung cancer models. PLoS ONE 8, e65757 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Huang, Y. et al. Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Proc. Natl. Acad. Sci. USA 109, 17561–17566 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Liu, L.Y. et al. Generation of Th1 and Th2 chemokines by human eosinophils: evidence for a critical role of TNF-α. J. Immunol. 179, 4840–4848 (2007).

    CAS  PubMed  Google Scholar 

  42. Wu, D. et al. Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science 332, 243–247 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Delyon, J. et al. Experience in daily practice with ipilimumab for the treatment of patients with metastatic melanoma: an early increase in lymphocyte and eosinophil counts is associated with improved survival. Ann. Oncol. 24, 1697–1703 (2013).

    CAS  PubMed  Google Scholar 

  44. Motz, G.T. & Coukos, G. Deciphering and reversing tumor immune suppression. Immunity 39, 61–73 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Fridman, W.H. et al. Prognostic and predictive impact of intra- and peritumoral immune infiltrates. Cancer Res. 71, 5601–5605 (2011).

    CAS  PubMed  Google Scholar 

  46. Suffner, J. et al. Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice. J. Immunol. 184, 1810–1820 (2010).

    CAS  PubMed  Google Scholar 

  47. Kohlmeyer, J. et al. Complete regression of advanced primary and metastatic mouse melanomas following combination chemoimmunotherapy. Cancer Res. 69, 6265–6274 (2009).

    CAS  PubMed  Google Scholar 

  48. Domschke, C. et al. Intratumoral cytokines and tumor cell biology determine spontaneous breast cancer-specific immune responses and their correlation to prognosis. Cancer Res. 69, 8420–8428 (2009).

    CAS  PubMed  Google Scholar 

Download references

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.).

Author information

Author notes

  1. Natalio Garbi

    Present address: Present address: Institutes of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany.,

Authors and Affiliations

  1. Division of Molecular Immunology, German Cancer Research Center, Heidelberg, Germany

    Rafael Carretero, Ibrahim M Sektioglu, Natalio Garbi, Oscar C Salgado & Günter J Hämmerling

  2. Division of Translational Immunology, German Cancer Research Center, Heidelberg, Germany

    Philipp Beckhove

  3. Regensburg Center for Interventional Immunology, University Clinic and University of Regensburg, Regensburg, Germany.,

    Philipp Beckhove

Authors
  1. Rafael Carretero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ibrahim M Sektioglu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Natalio Garbi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Oscar C Salgado
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Philipp Beckhove
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Günter J Hämmerling
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Ethics declarations

Competing interests

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1 (PDF 2247 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3159

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing