Cannabidiol (CBD) induces functional CD4+ CD25+ FOXP3+ Tregs

Abstract

Abstract We previously reported that cannabidiol (CBD), one of the Cannabis sativa-derived cannabinoid compounds and a potent immunosuppressive agent, increased CD25 and FOXP3 expression on CD4+ cells under “ultrasuboptimal” T cell stimulation (1 nM PMA/0.0125 μM Ionomycin, designated “Us/o”). We also observed that CBD increased IL-10 and il10 mRNA expression under Us/o stimulation. Thus, we tested the hypothesis that CBD-induced CD4+ CD25+ FOXP3+ cells are functional Tregs. First, we determined that the expression of CD25 and FOXP3 was induced on CD4-enriched cells in response to CBD (10 μM) plus Us/o activation as compared to CBD plus low level anti-CD3/28 stimulation (soluble anti-CD3 and anti-CD28 antibodies at 400 or 800 ng each) for 5 days. Second, we determined the functionality of the CBD-induced Tregs using a mixed lymphocyte response assay. For this study, splenocytes were treated with CBD (10 μM) plus Us/o activation for 5 days. CBD-induced CD4+ CD25+ and CD4+ CD25− cells were then purified and treated with mitomycin C (MMC). Fresh, naïve responder splenocytes were activated with anti-CD3/28-coated beads to induce proliferation of the responder cells, then mixed with the MMC-treated CD4+ CD25+ or CD4+ CD25− cells. CBD-induced CD4+ CD25+ cells, but not CBD-induced CD4+ CD25− cells, suppressed responder cell proliferation. Thus, CBD induced functional Tregs under conditions of low level activation, which is consistent with other studies demonstrating that CBD can induce Tregs under optimal stimulation conditions.