3.3. Effects of MQL on CD4+ T cell proliferation and HO-1 expression
IL-2 is an essential factor for CD4+ T cell proliferation and is produced following antigen recognition by TCR with co-stimulation by CD28. IL-2 then binds its receptor via paracrine and autocrine mechanisms and causes clonal expansion of CD4+ T cells3. In this experiment, naïve CD4+ T cells were isolated and stimulated with anti-CD3 (TCR stimulation) and anti-CD28 antibodies (co-stimulation). We then investigated the effects of MQL on CD4+ T cell proliferation and IL-2 production, and found that MQL treatment suppressed both proliferation and IL-2 production in a dose-dependent manner (Fig. 3A and B) .
p21 (known as p21WAF1/Cip1) is a cyclin-dependent kinase (CDK) inhibitor and arrests cell cycle progression in the G1 phase27. Furthermore, it was reported that up-regulation of HO-1 is able to increase = p21 levels, resulting in reduced cell proliferation12. Therefore, we investigated whether MQL affects the cell cycle in CD4+ T cells, and observed that MQL treatment increased p21 levels and arrested the cell cycle at the G1 stage compared with untreated CD4+ T cells (Fig. 3C and D) .
Nrf2 is a transcription factor of HO-1 that is sequestered in the cytoplasm by binding with Kelch-like ECH-associated protein 1 (Keap1). When Nrf2 is released from Keap1, it translocates into the nucleus then increases transcription of the HO-1 gene by binding to its antioxidant response element (ARE)28,29. We thus investigated Nrf2 translocalization following MQL and found that NRF2 nuclear translocation and consequent HO-1 were increased by MQL in CD4+ T cells (Fig. 3E and F) . Collectively, these results demonstrate that MQL upregulates p21, arrests the cell cycle, and activates the NRF2-HO-1 pathway, resulting in reduced CD4+ T cell proliferation.