The Effect of Nintedanib on T-Cell Activation, Subsets and Functions

Abstract

Background

T cells are important regulators of inflammation and, via release of mediators, can contribute to pulmonary fibrosis. Nintedanib is approved for the treatment of idiopathic pulmonary fibrosis, systemic sclerosis-associated interstitial lung disease (ILD) and chronic fibrosing ILDs with a progressive phenotype. However, how nintedanib targets T cells has not been elucidated.

Materials and Methods

We investigated the immunomodulatory effects of nintedanib on T cells and peripheral blood mononuclear cells isolated from healthy donors. Cells were pre-incubated with different concentrations of nintedanib and then stimulated for 24 hours with anti-CD3 with or without anti-CD28 and with or without different cytokines. Levels of interferon gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12p70 and IL-13 were quantitated. Western blotting with primary antibodies against phospho-Lck-Y394, phospho-Lck-Y505, Lck-total and Cofilin examined the phosphorylation level of the Lck protein. In vitro T-cell proliferation, T-cell clustering and different T-cell populations were also assessed.

Results

Nintedanib blocked T-cell activation through inhibiting Lck-Y394 phosphorylation. Pretreatment of T cells with nintedanib reduced cluster formation as a marker of activation and inhibited the release of IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12p70 and IL-13 at clinically relevant concentrations ranging from 5–77 nmol/L. Nintedanib did not alter T-cell proliferation or numbers of CD4+ and CD8+ T cells, but did increase stimulated Th17-like cells without increasing IL-17A levels.

Conclusion

These immunomodulatory effects may further explain how nintedanib slows the progression of pulmonary fibrosis in various ILDs.

Keywords:

• cytokines
• fibrosis
• inflammation
• nintedanib
• T cells
• tyrosine kinase

Acknowledgments

The technical assistance of Angela Ostermann and Verena Brauchle in conducting the study is gratefully acknowledged. Under the authors’ conceptual direction and feedback, editorial support was provided by Henry Chung (PhD) of MediTech Media, and funded by Boehringer Ingelheim International GmbH, in accordance with Good Publication Practice (GPP3) guidelines. This paper was presented as a poster at the American Thoracic Society 2019 International Conference. The poster’s abstract was published in the American Journal of Respiratory and Critical Care Medicine 2019;199 Abstract Issue: Abstract A4600.

Abbreviations

BrdU, bromodeoxyuridine; cHP, chronic hypersensitivity pneumonitis; ECM, extracellular matrix; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; IC₅₀, half maximal inhibitory concentration; IFN-γ, interferon gamma; IL, interleukin; ILD, interstitial lung disease; IPF, idiopathic pulmonary fibrosis; PAH, pulmonary arterial hypertension; PBMC, peripheral blood mononuclear cell; RA, rheumatoid arthritis; SSc, systemic sclerosis; TCR, T-cell antigen receptor; Th, T helper cells; Treg, regulatory T cells.

Ethics Approval and Informed Consent

Blood was donated by internal donors at the center for occupational health at Boehringer Ingelheim in Biberach; the donors provided signed informed consent that allows use for scientific purposes. All research was performed in accordance with the principles stated in the Declaration of Helsinki. Ethical approval was provided by the Federal State Medical Association of Baden-Württemberg, reference no. F-2016-121.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

Matthew James Thomas and Lutz Wollin are employees of Boehringer Ingelheim, the manufacturer of nintedanib. Kenia Ubieta was an employee of Boehringer Ingelheim at the time the study was conducted. His current affiliation is at Siemens Healthineers, Siemens Healthcare GmbH, Henkestr. 127, 91052 Erlangen, Germany. The authors report no other conflicts of interest in this work.

Additional information

Funding

This study was conducted at Boehringer Ingelheim, Biberach, Germany.