Identification of a CD4⁺ T cell line with Treg-like activity

Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses, development of therapeutics that target Tregs is limited by their low abundance, heterogeneity, and lack of specific cell surface markers. We isolated human PBMC-derived CD4⁺ CD25^high Foxp3⁺ Tregs and demonstrate they suppress stimulated CD4⁺ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining, we identified a human T cell line, MoT, as a model of human Foxp3⁺ Tregs. Unlike Jurkat T cells, MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4, CD25, GITR, LAG-3, PD-L1, CCR4. PBMC-derived Tregs and MoT cells, but not Jurkat cells, inhibited proliferation of human CD4⁺ PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4⁺ PBMC proliferation by MoT cells and Tregs, suggesting cell–cell contact is required for suppressive activity. Blocking antibodies against PD-L1, LAG-3, GITR, CCR4, HLA-DR, or CTLA-4 did not reverse the suppressive activity. We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4⁺ PBMCs in a cell contact-dependent manner, suggesting that a Foxp3⁺ Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism.