Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds

Abstract

CD4 + CD25+ regulatory T cells (Tregs) are believed to be pivotal in controlling chronic inflammation as well as in opposing the effect of cancer immunotherapy. Therefore, identification of novel drug compounds that interfere with Treg function is of high priority together with research that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell sorting (FACS) sorted CD4 + CD25^highCD127^dim/−CD45RA+ naïve Treg cells followed by in vitro expansion. We report on the use of these cells in a short-term assay based on Treg mediated inhibition of the early effector T cell activation markers CD69 and CD154. Additionally, we investigate the use of highly pure Tregs in a functional assay based on Treg mediated inhibition of effector T cell proliferation. We report highly reproducible Treg function in assays that test the effect of well-known model compounds such as CpG-A, anti-IL-6R (tocilizumab), anti-TNF-α (adalimumab) or a combination of IL-6 and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology.

• Drug screening
• in vitro assay
• pharmacology
• regulatory T cells

Acknowledgements

The authors wish to thank Dorte Lauritsen, Bente Smith Thorup and Trine Møller for excellent technical assistance. We are grateful to Vivianne Malmström, The Karolinska Institute, Rheumatology unit, who provided the drugs Adalimumab and Tocilizumab.

Declaration of interest

The authors declare no personal financial or commercial conflict of interest. Bioneer A/S develops in vitro assays for commercial use.

The research was funded by the Danish Ministry of Higher Education and Science.