Article | Published:

Translational Therapeutics

Increased expression of the immunosuppressive interleukin-35 in patients with non-small cell lung cancer

British Journal of Cancer (2019) | Download Citation



The immunosuppressive role of the cytokine IL-35 in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, we analysed the localisation and regulation of IL-35 in the lung of patients with non-small cell lung cancer (NSCLC) to further elucidate the immune-escape of cancer cells in perioperative course of disease.


Interleukin 35 (IL-35) was measured by ELISA in postoperative serum from 7 patients with NSCLC as well as 8 samples from healthy controls. Immunohistochemistry, FACS analysis, real-time PCR, as well as western blot from samples of the control (CTR), peri-tumoural (PT) and the tumoural (TU) region of the lung derived from patients with NSCLC and 10 controls were performed.


Here we found elevated levels of IL-35 in the TU region as well as postoperative serum from patients with lung adenocarcinoma. Consistently, we found an increased expression of IL-35+Foxp-3+ cells, which associated with ARG1 mRNA expression and decreased TNFA in the TU region of the lung of patients with NSCLC as compared to their CTR region. Furthermore, in the CTR region of the lung of patients with NSCLC, CD68+ macrophages were induced and correlated with IL-35+ cells. Finally, IL-35 positively correlated with TTF-1+PD-L1+ cells in the TU region of NSCLC patients.


Induced IL-35+Foxp3+ cell numbers in the TU region of the lung of patients with NSCLC associated with ARG1 mRNA expression and with TTF-1+PD-L1+ cells. In the tumour-free CTR area, IL-35 correlated with CD68+ macrophages. Thus inhibitors to IL-35 would probably succeed in combination with antibodies against immune checkpoints like PD-L1 and PD-1 currently used against NSCLC because they would inhibit immunosuppressive macrophages and T regulatory cells while promoting T cell-mediated anti-tumoural immune responses in the microenvironment as well as the TU region of NSCLC patients.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Kirk, R. A first for first-line therapy in NSCLC. Nat. Rev. Clin. Oncol. 8, 691 (2011).

  2. 2.

    Ni, X. Y., Sui, H. X., Liu, Y., Ke, S. Z., Wang, Y. N. & Gao, F. G. TGF-beta of lung cancer microenvironment upregulates B7H1 and GITRL expression in dendritic cells and is associated with regulatory T cell generation. Oncol. Rep. 28, 615–621 (2012).

  3. 3.

    Ilie, M., Benzaquen, J., Hofman, V., Lassalle, S., Yazbeck, N., Leroy, S. et al. Immunotherapy in non-small cell lung cancer: biological principles and future opportunities. Curr. Mol. Med. 17, 527–540 (2017).

  4. 4.

    Gadgeel, S., Shaw, A. T., Barlesi, F., Crino, L., Yang, J. C., Dingemans, A. C. et al. Cumulative incidence rates for CNS and non-CNS progression in two phase II studies of alectinib in ALK-positive NSCLC. Br. J. Cancer 118, 38–42 (2018).

  5. 5.

    Besse, B., Massard, C., Haddad, V., Andre, F., Dunant, A., Pirker, R. et al. ERCC1 influence on the incidence of brain metastases in patients with non-squamous NSCLC treated with adjuvant cisplatin-based chemotherapy. Ann. Oncol. 22, 575–581 (2011).

  6. 6.

    Faehling, M., Schwenk, B., Kramberg, S., Fallscheer, S., Leschke, M., Strater, J. et al. Second malignancy in non-small cell lung cancer (NSCLC): prevalence and overall survival (OS) in routine clinical practice. J. Cancer Res. Clin. Oncol. 144, 2059–2066 (2018).

  7. 7.

    Heim, L., Friedrich, J., Engelhardt, M., Trufa, D. I., Geppert, C. I., Rieker, R. J. et al. NFATc1 promotes anti-tumoral effector functions and memory CD8+ T cell differentiation during non-small cell lung cancer development. Cancer Res. 78, 3619–3633 (2018).

  8. 8.

    Collison, L. W., Chaturvedi, V., Henderson, A. L., Giacomin, P. R., Guy, C., Bankoti, J. et al. IL-35-mediated induction of a potent regulatory T cell population. Nat. Immunol. 11, 1093–1101 (2010).

  9. 9.

    Turnis, M. E., Sawant, D. V., Szymczak-Workman, A. L., Andrews, L. P., Delgoffe, G. M., Yano, H. et al. Interleukin-35 limits anti-tumor immunity. Immunity 44, 316–329 (2016).

  10. 10.

    Collison, L. W., Workman, C. J., Kuo, T. T., Boyd, K., Wang, Y., Vignali, K. M. et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 450, 566–569 (2007).

  11. 11.

    Song, M. & Ma, X. The immunobiology of interleukin-35 and its regulation and gene expression. Adv. Exp. Med. Biol. 941, 213–225 (2016).

  12. 12.

    Trinchieri, G., Pflanz, S. & Kastelein, R. A. The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 19, 641–644 (2003).

  13. 13.

    Pflanz, S., Timans, J. C., Cheung, J., Rosales, R., Kanzler, H., Gilbert, J. et al. IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4+T cells. Immunity 16, 779–790 (2002).

  14. 14.

    Dixon, K. O., van der Kooij, S. W., Vignali, D. A. & van Kooten, C. Human tolerogenic dendritic cells produce IL-35 in the absence of other IL-12 family members. Eur. J. Immunol. 45, 1736–1747 (2015).

  15. 15.

    Xiang, X. G. & Xie, Q. IL-35: a potential therapeutic target for controlling hepatitis B virus infection. J. Dig. Dis. 16, 1–6 (2015).

  16. 16.

    Dambuza, I. M., He, C., Choi, J. K., Yu, C. R., Wang, R., Mattapallil, M. J. et al. IL-12p35 induces expansion of IL-10 and IL-35-expressing regulatory B cells and ameliorates autoimmune disease. Nat. Commun. 8, 719 (2017).

  17. 17.

    Vahl, J. M., Friedrich, J., Mittler, S., Trump, S., Heim, L., Kachler, K. et al. Interleukin-10-regulated tumour tolerance in non-small cell lung cancer. Br. J. Cancer 117, 1644–1655 (2017).

  18. 18.

    Kachler, K., Bailer, M., Heim, L., Schumacher, F., Reichel, M., Holzinger, C. D. et al. Enhanced acid sphingomyelinase activity drives immune evasion and tumor growth in non-small cell lung carcinoma. Cancer Res. 77, 5963–5976 (2017).

  19. 19.

    Eisenhut, F., Heim, L., Trump, S., Mittler, S., Sopel, N., Andreev, K. et al. FAM13A is associated with non-small cell lung cancer (NSCLC) progression and controls tumor cell proliferation and survival. Oncoimmunology 6, e1256526 (2017).

  20. 20.

    Andreev, K., Trufa, D. I., Siegemund, R., Rieker, R., Hartmann, A., Schmidt, J. et al. Impaired T-bet-pSTAT1alpha and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma. Br. J. Cancer 115, e11 (2016).

  21. 21.

    Vignali, D. A. & Kuchroo, V. K. IL-12 family cytokines: immunological playmakers. Nat. Immunol. 13, 722–728 (2012).

  22. 22.

    Kachroo, P., Lee, M. H., Zhang, L., Baratelli, F., Lee, G., Srivastava, M. K. et al. IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer. J. Exp. Clin. Cancer Res. 32, 97 (2013).

  23. 23.

    Chaturvedi, V., Collison, L. W., Guy, C. S., Workman, C. J. & Vignali, D. A. Cutting edge: Human regulatory T cells require IL-35 to mediate suppression and infectious tolerance. J. Immunol. 186, 6661–6666 (2011).

  24. 24.

    Solis, L. M., Behrens, C., Raso, M. G., Lin, H. Y., Kadara, H., Yuan, P. et al. Histologic patterns and molecular characteristics of lung adenocarcinoma associated with clinical outcome. Cancer 118, 2889–2899 (2012).

  25. 25.

    Collison, L. W. & Vignali, D. A. Interleukin-35: odd one out or part of the family? Immunol. Rev. 226, 248–262 (2008).

  26. 26.

    Sui, H., Ma, N., Wang, Y., Li, H., Liu, X., Su, Y. et al. Anti-PD-1/PD-L1 therapy for non-small-cell lung cancer: toward personalized medicine and combination strategies. J. Immunol. Res. 2018, 6984948 (2018).

  27. 27.

    Noy, R. & Pollard, J. W. Tumor-associated macrophages: from mechanisms to therapy. Immunity 41, 49–61 (2014).

  28. 28.

    Aguiar, P. N. Jr., Perry, L. A., Penny-Dimri, J., Babiker, H., Tadokoro, H., de Mello, R. A. et al. The effect of PD-L1 testing on the cost-effectiveness and economic impact of immune checkpoint inhibitors for the second-line treatment of NSCLC. Ann. Oncol. 29, 1078 (2018).

  29. 29.

    Mencoboni, M., Filiberti, R. A., Taveggia, P., Bruzzone, A., Garuti, A., Del Corso, L. et al. Outcome of EGFR inhibitors treatment in advanced NSCLC patients, not enrolled in clinical trials. Neoplasma 64, 253–261 (2017).

  30. 30.

    Barnet, M. B., O’Toole, S., Horvath, L. G., Selinger, C., Yu, B., Ng, C. C. et al. EGFR-co-mutated advanced NSCLC and response to EGFR tyrosine kinase inhibitors. J. Thorac. Oncol. 12, 585–590 (2017).

  31. 31.

    Maemondo, M. Tyrosine kinase inhibitors as first-line treatment in NSCLC. Lancet Oncol. 17, 541–543 (2016).

  32. 32.

    Remon, J., Vilarino, N. & Reguart, N. Immune checkpoint inhibitors in non-small cell lung cancer (NSCLC): Approaches on special subgroups and unresolved burning questions. Cancer Treat. Rev. 64, 21–29 (2018).

  33. 33.

    Kachler, K., Holzinger, C., Trufa, D. I., Sirbu, H. & Finotto, S. The role of Foxp3 and Tbet co-expressing Treg cells in lung carcinoma. Oncoimmunology 7, e1456612 (2018).

  34. 34.

    Cai, Z., Wong, C. K., Kam, N. W., Dong, J., Jiao, D., Chu, M. et al. Aberrant expression of regulatory cytokine IL-35 in patients with systemic lupus erythematosus. Lupus 24, 1257–1266 (2015).

  35. 35.

    Cai, Z., Wong, C. K., Dong, J., Chu, M., Jiao, D., Kam, N. W. et al. Remission of systemic lupus erythematosus disease activity with regulatory cytokine interleukin (IL)-35 in Murphy Roths Large (MRL)/lpr mice. Clin. Exp. Immunol. 181, 253–266 (2015).

  36. 36.

    Steward-Tharp, S. M., Song, Y. J., Siegel, R. M. & O’Shea, J. J. New insights into T cell biology and T cell-directed therapy for autoimmunity, inflammation, and immunosuppression. Ann. NY Acad. Sci. 1183, 123–148 (2010).

Download references


The authors thank Rebekka Springel and Sonja Trump for the qPCR analysis and their excellent experimental help, as well as all the supporting team in the Molecular Pneumology Department, the Thoracic Surgery and the Institute of Pathology in Erlangen. This work was financially supported by an IZKF grant number A59 awarded to S.F. D.I.T. was supported by a 2-year 50% time rotation fellowship at MP, financed by the IZKF in Erlangen.

Author contributions

D.I.T. and H.S. were involved in the recruitment of the patients of the cohort and performed surgery. Further, D.I.T. made a 2-year period rotation at the MP and contributed to cell isolation, tissue storage, and FACS analysis of the patients as reported in Fig. 2. D.I.T. did also the IL-35 serum analysis and contributed to the patient clinical analysis. R.S. did the qPCR analysis of the patients as reported in Fig. S2, contributed to cell culture, and analysed the IL-35 immunohistochemistry. L.H. contributed to all figures, supplementary figures and tables and to the recruitment of all patients. S.M. contributed to patients’ sample recruitment and did the immunohistochemistry. K.K. contributed to most of the patient tissue sample recruitment and performed the FACS analysis reported in Fig. 2. J.F. was involved in patient sample recruitment and storage and performed the analysis presented in Figs. S2 and S3. S.F. analysed the data reported in Fig. S4. S.F. and L.H. wrote the manuscript. The pathologists C.-I.G. made the diagnosis and did the scanning of the slides and R.J.R. did the TMA, additionally. All authors have read and approved the final manuscript.

Author information


  1. Department of Molecular Pneumology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany

    • Lisanne Heim
    • , Katerina Kachler
    • , Raphaela Siegmund
    • , Denis I. Trufa
    • , Susanne Mittler
    • , Juliane Friedrich
    •  & Susetta Finotto
  2. Department of Thoracic Surgery, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany

    • Denis I. Trufa
    •  & Horia Sirbu
  3. Institute of Pathology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany

    • Carol-Immanuel Geppert
    •  & Ralf J. Rieker


  1. Search for Lisanne Heim in:

  2. Search for Katerina Kachler in:

  3. Search for Raphaela Siegmund in:

  4. Search for Denis I. Trufa in:

  5. Search for Susanne Mittler in:

  6. Search for Carol-Immanuel Geppert in:

  7. Search for Juliane Friedrich in:

  8. Search for Ralf J. Rieker in:

  9. Search for Horia Sirbu in:

  10. Search for Susetta Finotto in:

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was performed at the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) and was approved by the ethics review board of the University of Erlangen (Re-No: 56_12B; DRKS-ID: DRKS00005376). Patients who suffered from NSCLC underwent surgery and gave their approval to be enrolled in this study in an informed written consent. The patient studies were conducted in accordance with the ethical guidelines of the Declaration of Helsinki.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.


This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).

Corresponding author

Correspondence to Susetta Finotto.

Supplementary information

About this article

Publication history