Fig 2: SLC7A5 knockdown in target cells rescues functions of B7H3-DAP12-CAR-T at low Met concentration.A Normalized cell index of B7H3-DAP12-CAR-T co-cultured with normal or SLC7A5 downregulated target cells (E:T = 0:1 and 2:1) in 25 or 100 μM Met within 30 h. B IL-2 and IFN-γ secreted by B7H3-DAP12-CAR-T co-cultured with normal or SLC7A5 downregulated target cells (2 × 105) at E:T = 2:1 for 48 h in 25 or 100 μM Met measured by ELISA. C Tcm subsets of B7H3-DAP12-CAR-T co-cultured with normal or SLC7A5 downregulated target cells at E:T = 2:1 for 48 h at 25 or 100 μM shown by flow cytometry. D Exhaustion markers (PD1 and LAG3) on B7H3-DAP12-CAR-T co-cultured with normal or SLC7A5 downregulated target cells at 25 or 100 μM for 48 h measured by flow cytometry. E Cytotoxicity of normal or SLC7A5 upregulated B7H3-DAP12-CAR-T co-cultured with 1 × 104 LUSC cells (E:T = 2:1) for 24 h at 25 or 100 µM Met shown by LDH cytotoxicity assay. F In a Met-sufficient environment, CAR-T cells equipped with B7H3 scFv can recognize B7H3-positive LUSC, activate the DAP12 stimulating domain, and exert killing function. In a Met-deficient environment, LUSC cells with high expression of SLC7A5 would compete for Met uptake, resulting in reduced Met absorption by CAR-T cells and a subsequent decline in the m5C modification within CAR-T cells. The reduction in m5C would compromise the stability of NKG7 mRNA, leading to the downregulation of NKG7 expression and subsequently inhibiting the cytotoxicity of CAR-T cells. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Variables are presented as mean ± SD.

Fig 3: Up-regulated infiltration of CD4+T cells in livers from patients with ALD. (A) Heatmap showing the altered cell abundance in ALD samples compared with healthy and MASLD samples. (B) Box plots illustrating the proportions of NK & T-cell subsets in the livers of 3 different donors. The Wilcoxon test was used to analyze differences between groups. (C) Bar plots indicating the proportions of 5 NK & T-cell subclusters in the livers of different donors. (D) IHC staining and quantification of NK, CD4, and CD8 in the 3 types of liver samples. (E) Representative photomicrographs of the human liver tissue sections from healthy donors (Ctrl) and patients with MASLD and ALD after IF staining with antibodies against CD8 (green) and NKG7 (red), CD4 (green) and ANXA1 (red), CD8 (green) and ANXA1 (red). For B and D–E, data are shown in mean ± SEM; P-value was obtained by Student’s t-test.

Fig 4: NSUN4 was screened as a key m5C regulator in SLE through conjoint analysis of scRNA‐seq and m5C‐seq. (A) Average expression of m5C regulators in the single cells based on different clinical variables containing cell types by using z‐score. (B) NMF clustering for the differentially expressed m5C regulators in different cell types. (C) Heatmap distribution of differentially expressed m5C regulators in different cell types. (D) Immunofluorescence staining was used to evaluate the NSUN4 levels in CD8+NKG7+ T cells from Controls and SLEs. ×400.

Fig 5: Knockdown of NSUN4 inactivates the CD44/mTOR/S6K pathway and improves mitophagy in CD8+ T cells in vitro. (A) The screening process of the interacting proteins of CD74. (B) Coimmunoprecipitation (co‐IP) was used to verify the binding capacity of CD74 and CD44. 50 nM NSUN4 siRNAs or scrambled siRNA was transfected into cultured CD8+NKG7+ T cells by using the PEI–PBA system. After 72 h, cells were harvested for functional evaluation. (C) Western blotting was used to detect the expression of NSUN4, CD74, and CD44. (D) Autophagic flux assay was used to evaluate the level of gross autophagy. DAPI was used to mark the nucleus (blue). ×1000. (E) Transmission electron microscopy was used to observe damaged mitochondria and mitochondrial autophagosomes in the cells. ×8000. The arrows indicate the mitochondrial autophagosomes. (F) MitoSOXTM Red was used to evaluate the level of mitochondrial oxidative stress. (G) JC‐1 fluorescence probe was used to evaluate the mitochondrial membrane potential. N = 3, *p < 0.05, **p < 0.01 compared with control and scramble.