Melanoma is the most aggressive skin cancer and tends to metastasize to other tissues; therefore appropriate treatment in the early stage is very important due to its low therapeutic efficiency after metastasis (Nicholas and Lesinski, 2011). A molecular understanding of cancer biology has revealed that melanoma formation and progression are closely related to altered oncogenic signaling pathways and immune cells (Davis et al., 2019). Recent clinical trials for cancer therapy have demonstrated that immune-based therapies are the most promising option, and combination therapy is considered a promising approach for cancer patients because it elicits synergistic or additive efficacy (Ayoub, 2021; Murciano-Goroff et al., 2020; Narayan et al., 2020).

Interleukin 2 (IL-2) acts as a key regulator of immune cells by binding to the IL-2 receptor (IL-2R) (Jiang et al., 2016; Mitra and Leonard, 2018; Skrombolas and Frelinger, 2014). Although IL-2 therapy elicits excellent anticancer efficacy, its pleiotropic properties expand regulatory T cells (Tregs) with high CD25 and Foxp3 expression, hampering the effective antitumor action induced by CD8+ T cell responses through immunosuppressive activity. Therefore, IL-2 and CD25 have attracted attention as therapeutic targets for cancer immunotherapy, and several agents that modify IL-2/IL-2 receptor binding have been developed (Shanafelt et al., 2000). IL-2 antibody and IL-2 muteins, that can block CD25 binding, increase the ratio of CD8+ T cells/Tregs in the tumor microenvironment (TME) and suppress tumor growth in various cancers (Carmenate et al., 2013; Spangler et al., 2015).

Tumor necrosis factor receptor 2 (TNFR2) is characterized by high expression on Tregs in the TME; TNFR2+ Tregs at tumor sites exhibit strong suppressive activity against cytotoxic CD8+ T cells and lead to negative progression in cancer patients (Okuzono et al., 2022). TNFR2-blocking antibodies showed tumor suppression by inhibiting survival and infiltration of Tregs in the TME, and are recognized as potential immune checkpoints in cancers (Chen et al., 2022; Torrey et al., 2017).

Celastrol (CEL) is a natural pentacyclic triterpenoid present in the roots of Tripterygium wilfordii Hook F. that possesses various beneficial bioactivities against inflammation, autoimmune diseases, and cancer (Kannaiyan et al., 2011; Shrivastava et al., 2015; Venkatesha et al., 2016). Despite the potential pharmacological activity of CEL, its therapeutic application is strictly limited owing to its poor water solubility and high toxicity, and research on the immune-based mechanisms of its anticancer efficacy is still insufficient.

Here, we identified CEL, a natural bioactive small molecule, as an inhibitor of the IL-2/CD25 interaction that targets IL-2 and investigated the effects of immune cells on its anticancer efficacy in a mouse melanoma model. In addition, the therapeutic effects of combination therapy with low-dose CEL and anti-TNFR2 antibody were investigated in vivo.