## 4 Discussion  
In this study, we performed an ELISA-binding assay of CME and CMW on the blocking of the CTLA-4/CD80 interaction in a concentration-dependent manner. Additionally, to evaluate whether the CME and CMW can promote T cell functional activity using the CTLA-4/CD80 blockade bioassay, both extracts were used to enhance T cell activation and increase the expression of luciferase. Moreover, we confirmed the constituents of the Chaga mushroom/70% ethanol extract via HPLC-CAD analysis.  
Chaga mushroom (_I. obliquus_) has been used as a traditional therapy in Asia for a long time [1]. The anticancer effects of _I. obliquus_ extract on various types of cells, including melanoma cells, human lung carcinomas, and human liver cancer cells, have been researched and reported [15; 16; 17]. However, the blocking effect of the immune checkpoint against Chaga mushroom extract is not understood.  
The CTLA-4/CD80 inhibitory effect and T cell activation effects of CME and CMW extracts were confirmed using several cell-based assays. Competitive ELISA results showed that CME and CMW blocked CTLA-4/CD80 interaction in a concentration-dependent manner. It was also confirmed through CTLA-4 blocking assays that CMW and CME interfere with CTLA-4/CD80 interaction and activate T cells. CMW and CME were concentration-dependent, and did not show cell cytotoxicity in CTLA-4 Jurkat T and CD80/aAPC CHO-K1 cells. We conducted HPLC-CAD analysis to confirm the content of lanosterol. According to the standard curve solution, the amount of lanosterol (1) was diluted with methanol at each concentration. The established HPLC-CAD method was suitable for analysis of lanosterol (Figure 4). Lanosterol is present in the Chaga mushroom/ethanol extract at 0.41%. Lanosterol is one of the tetracyclic triterpenoids, and is the precursor to all animal and fungal steroids. Steroids  
Figure 4: Structure of lanosterol (**1**) (**A**); UV spectra of the standard compound of lanosterol (**1**) and 70% ethanol extract (**B**); regression equations, LODs, and LOQs of lanosterol (**C**); and HPLC–CAD chromatograms (**D**).  
are hydrophobic compounds that have very low solubility in water, and we showed that lanosterol was detected only in the ethanol extract but not the water extract. As shown in Supplementary Material Figure S1, lanosterol tested competitive ELISA and it also blocked the interaction of CTLA-4/CD80. Although lanosterol has been suggested as one of active compound for Chaga mushroom ethanol extract, however, we will investigate other immune checkpoint inhibitors in the CMW extract in a future study.  
Chaga mushrooms stimulate the immune system and show antitumor activity against diverse types of cancer [15; 16; 17; 18; 19; 20]. In addition, the Chaga mushroom blocks CTLA-4/CD80 interaction and stimulates T cell activity. Terpenoids isolated from the herb medicinal plant showed immunological activity. [21] Triterpenoids isolated from the Chaga mushroom have been reported to show a variety of bioactivities, including those mentioned in ref. [22; 23]. Thus, we suggest that the blocking effect of CME on the CTLA-4/CD80 interaction can be attributed to the abundant triterpenoid constituents in the CME. According to a previous study [24], adverse effects, such as oxalate nephropathy, can occur after the long-term usage of Chaga mushrooms. Administration of Chaga mushroom should be carefully monitored in patients with kidney function disorders, due to contraindications and side effects. In addition, Chaga mushrooms may interact with drugs such as aspirin and clopidogrel, given that the Chaga mushroom inhibits platelet aggregation [25].