Fig 2: Lycorine treatment inhibits human T‐cells reactivity in response to stimulus. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by density gradient centrifugation. A,B) A mixed lymphocyte reaction was performed at various mixing ratios, and cell proliferation and cytokine levels were measured on day 6 and day 3, respectively. Lycorine (200 nm) or FK506 (200 nm) was added simultaneously with allogenic PBMCs. A) Percentages of proliferated PBMCs and B) levels of cytokines in the culture supernatant from the various groups are shown (n = 9). C,D) Dynabeads Human T‐Activator CD3/anti‐CD28 were used for stimulating PBMCs. After 72 h, C) PBMC proliferation and D) cytokine production were analyzed by flow cytometry (n = 4). E) Representative images of 5‐ethynyl‐2′‐deoxyuridine (EdU) staining and F) numbers of cells positively stained for EdU (per 1000 cell) in PBMCs without stimulation (Control), with anti‐CD3/anti‐CD28 stimulation (αCD3/αCD28), or with stimulation in the presence of lycorine (200 nm) (αCD3/αCD28+Lycorine). Scale bars, 50 µm. Data are shown as means ± SEM and pooled from at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, one‐way ANOVA.

Fig 3: Lycorine‐mediated inhibition of ERK in human T cells downregulates the immune response and changes metabolic processes occurring in response to stimulation. CD4+ and CD8+ T cells were purified from four healthy individuals and stimulated with anti‐CD3/anti‐CD28 beads (αCD3/αCD28) or anti‐CD3/anti‐CD28 beads in the presence of lycorine (200 nm) (αCD3/αCD28+Lycorine). A) Clustered heatmap of significantly differentially expressed mRNAs in CD4+/CD8+ T cells receiving various treatments. B) Volcano plots indicate the significance of upregulated genes (red) and downregulated genes (blue) in αCD3/αCD28 and αCD3/αCD28+Lycorine‐treated CD4+ (upper)/CD8+ (bottom) T cells, with the number of differentially expressed genes. C) Venn diagram showing the overlapping of the downregulated mRNAs in CD4+ and CD8+ T cells after lycorine treatment. D) Scatter plot showing the GO enrichment results in the downregulated genes. The dot size indicates the relative number of differentially expressed genes contained in the GO terms, and the shade of the dots indicates the adjusted P‐value (padj) of the enrichment. E) Heat maps show standardized log expression (gene‐wise Z‐score) of selected DEGs between CD4+ and CD8+ T cells stimulated with anti‐CD3/anti‐CD28 + Lycorine versus CD4+ and CD8+ T cells stimulated with anti‐CD3/anti‐CD28. F,G) Human PBMCs were stimulated with anti‐CD3/anti‐CD28 beads in the presence/absence of lycorine (200 nm). After 24 h, cells were collected and stained with MG, MDR, and MitoSOX. F) Bar graph for the percentage of MGhighMDRlow subsets (n = 4). G) Summary bar graphs depicting the MFI of MitoSOX in MGhighMDRhigh subsets in CD3+ T cells receiving the various treatments (n = 4). Data are shown as means ± SEM and from one of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, Student's t‐test.

Fig 4: Identification lycorine as an ERK‐specific inhibitor. A) In SPR assays, recombinant human ERK1 protein was immobilized on a BIAcore CM5 sensor chip, followed by analysis its interaction with lycorine at the indicated concentrations. B) Structure of biotinylated lycorine. C) The schematic depicts the procedure for the affinity‐based approach to identify target(s) of lycorine from protein extracts derived from murine splenocytes. MS, mass spectrometry. D) The Venn diagram shows the percentages of overlapping and unique proteins pulled‐down by biotin and biotinylated lycorine. E,F) Flow cytometry staining for phosphorylated‐ERK1/2 (p‐ERK1/2) in splenocytes without stimulation, with anti‐CD3/anti‐CD28 bead stimulation, or with anti‐CD3/anti‐CD28 beads stimulation in the presence of lycorine (200 nm) for 15 min (n = 3 for each group). E) Representative histogram with mean fluorescence intensity and F) bar graph with mean ± SEM. G–H) Flow cytometry staining for phosphorylated‐c‐Fos (p‐c‐Fos) in CD4+ cells receiving the various treatments (n = 3 for each group), after 48 or 72 h later. G) A representative histogram with mean fluorescence intensity and H) bar graph are shown. Data are shown as means ± SEM and from one of three independent experiments. *P < 0.05, and ***P < 0.001, Student's t‐test.

Fig 5: Lycorine treated T cells exhibit mitochondrial dysfunction. A–D) Purified CD4+ T cells were stimulated with anti‐CD3/anti‐CD28 beads in the presence/absence of lycorine (200 or 400 nm). After 24 h, cells were collected and stained with MitoTracker Green (MG), MitoTracker Deep Red (MDR), and MitoSOX. A) Representative zebra plot and B) bar graph for the percentage of MGhighMDRlow subsets (n = 6). C,D) Summary bar graphs depicting the MFI of MitoSOX in MGhighMDRhigh (left panel) and MGhighMDRlow (right panel) subsets in CD4+ T cells receiving the various treatments (n = 6). E–H) OCR of purified CD4+ cells with different treatment were measured under basal conditions and in response to oligomycin (oligo), FCCP, and rotenone plus antimycin A (Rot/AM). E) Real‐time OCR levels, F) baseline OCR levels, G) maximal OCR levels, and H) spare respiratory capacity (percent maximum OCR after FCCP injection of baseline OCR) in the indicated groups (n = 3–4 per group). I) Transmission electron microscope sections of mitochondria from CD4+ T cells in an in vitro experiment or J) graft‐infiltrating lymphocytes. K) Quantitative plots for crista number per mitochondrion and L) crista length per mitochondrion area in each group (n = 6). Scale bars, 500 nm. Bar graphs show the means ± SEM from one of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, B,F,G,H) one‐way ANOVA, C,D) two‐way ANOVA, and K,L) Student's t‐test.