A Quick but Comprehensive Protocol for Isolation and Short-Term Expansion of Tregs from Peripheral Blood Mononuclear Cells for Treg Therapy in Animal Models and Human In Vitro Studies

Abstract

Amir Hossein Mansourabadi39659, Farshid Noorbakhsh39660, Amir Ali Hamidieh39661 and Aliakbar Amirzargar39662*

Regulatory T cells (Tregs) are identified via a combination of different extracellular markers, mainly CD4⁺CD25⁺ CD127^dim/-, along with intracellular transcription factor FoxP3 and their main function is suppression followed by termination of pro-inflammatory immune responses. Tres represent approximately 1%-4% of PBMCs. Hence, therapeutic use of Tregs requires access to an appropriate number of these cells with stable inhibitory potential in the culture medium as well as controlled plasticity in inflammatory conditions. However, based on past studies and experiences, Treg expansion has always faced different challenges in cell biology studies. As the conventional protocols require a complicated and long period of in vitro expansion of Treg numbers. In this article, we described a feasible, quick, and simple protocol for isolation and expansion of Tregs from PBMCs for in vitro studies. We also evaluated their suppressive effects and their plasticity for proving their functions although, their conversion to Th17 cells under pro-inflammatory conditions remain a challenge.