Background: There is an ongoing need for development of novel immune suppressants, particularly those that enhance regulatory T cells (Treg). QMAD is a soluble fraction of the Chinese herb Qu Mai, and HPLC analysis showed QMAD contains 3 distinct ∼600 dalton non-polar components (characterization ongoing). We previously showed that QMAD inhibits human effector T cell proliferation and IFNΓ secretion. Effects on human Treg induction and function are unknown.

Methods: Human PBMC were separated by Ficoll, and CD3+ and CD4CD45RA+ cells were isolated with magnetic beads (negative selection). Treg inductions were performed with ΑCD3/CD28, IL-2 ± TGFΒ ± QMAD. Suppressive capacity was tested using in vitro suppression assays (CFSE dilution). Intracellular Foxp3 and phospho-AKT (pAKT) were quantified by flow cytometry and IL-10 and TGFΒ were measured by ELISA.

Results: When we added QMAD to ΑCD3/CD28 stimulated PBMC we initially observed increased Foxp3+CD4+ cells (3.5-fold increase vs. control, n=4), raising the hypothesis that QMAD induces Treg. To test this we stimulated naÏve CD4+CD45RA+ T cells with ΑCD3/CD28, IL-2 and TGFΒ and observed that QMAD upregulated the percentage of Foxp3+ cells (50% Foxp3+ with QMAD vs 25% in vehicle controls, n=6, p<0.05) as well as the absolute number of Foxp3+ CD4 cells (9.63 vs. 4.98 E5 cells, n=4, p<0.05). Remarkably, QMAD also induced Foxp3 expression in the absence of TGFΒ (18.7% with QMAD vs 8.8% in vehicle controls, n=6, p<0.05). The QMAD-induced (without TGFΒ) Foxp3+ CD4 cells potently suppressed effector T cell proliferation, while CD4+ control cells did not (n=3, p<0.05), confirming that QMAD generates bona fide Treg. QMAD did not upregulate IL-10 or TGFΒ production by CD4 T cells. Because pAKT has been shown to prevent T cell Foxp3 expression and absent pAKT is required for Treg function, we tested whether QMAD regulates pAKT. QMAD inhibited ΑCD3/CD28 induced pAKT in CD3 T cells ∼6 fold vs. controls (n=3, p<0.05).

Conclusion: Our data provide an explanation to account for the observed immune suppressive effects of the Chinese herb Qu Mai, and suggest that small molecular component(s) of QMAD directly induce Treg, in part by inhibiting pAKT during T cell activation. The novel findings show human Treg can be induced without TGFΒ and support further testing of QMAD and its molecular constituents as Treg-sparing immunosuppressants for use in humans.

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