CD25 genetic ablation on lymphoid cell lines to obtain models of stimulation through the Interleukin-2 beta/gamma receptor.

Interleukin-2 (IL-2) has pleiotropic roles within the immune system. Its functional versatility depends upon the interactions with diverse arrays of receptor subunits differentially expressed on immune cell subsets. The balance between stimulation of regulatory T cells (Tregs) and effector lymphocytes determines the immunological outcome. While Tregs display constitutively a trimeric IL-2 receptor composed by alpha (also known as CD25), beta and gamma subunits, resting and memory CD8 + T cells express a dimeric beta/gamma receptor. The effects of IL-2 signaling through the trimeric receptor are routinely studied in vitro using either mouse CTLL-2 or human Kit225 lymphoid cell lines. We obtained equivalent IL-2-responsive cellular models displaying dimeric beta/gamma receptors. The purpose was achieved through genetic ablation of CD25 expression on CTLL-2 and Kit225 cells using CRISPR/Cas9 edition. The usefulness of the stable CD25-KO cell lines thus generated was illustrated by setting up proliferation assays, and characterizing a panel of IL-2-derived recombinant muteins, including agonists, super-agonists and antagonists. Problems and pitfalls of such assays, as well as the ways to mitigate them, were depicted. Stat5 phosphorylation assays based on KO cell lines were also performed. These cellular models, and the experience accumulated during their use, could contribute to the evaluation of the output of IL-2 engineering strategies developed across different laboratories.