Fig 2: Regulation of IRF4 in CD8+ T cells by NAC1. CD8+ T cells were isolated from WT or NAC1−/− mice and analyzed for expression of IRF4. (A) Protein expression by Western blot. For the day 0 sample, T cells were not activated. For the day 3 sample, T cells were activated and cultured for 3 days. (B) mRNA expression by Q-PCR. CD8+ T cells had been cultured for 3 days, and then RNA was extracted, and qPCR was performed (ns, p > 0.05). (C) CHIP-seq analysis. WT CD8+ T cells were isolated from mice and CHIP was performed with anti-NAC1 and anti-IgG Abs (Input control). The sequenced data was visualized by IGV.

Fig 3: (a) Analysis of differences in tumor purity between three immune subtypes. Tumor purity was importantly more down in Immunity_H group and importantly more excellent in Immunity_L (p < 0.001, Kruskal-Wallis test). (b) Survival analysis of three immune subgroups. The survival curves of Immunity_L, Immunity_M, and Immunity_H subgroups were significantly different (p = 4.396E − 08). It also proved that immune grouping had a good predictive effect on the survival of DLBCL. Patients in Immunity_H had the best prognosis, patients in Immunity_L had got the poorest prognosis, and the Immunity_M was between them. (c–h) The expression of PD-1, PD-L1, CD3D, HIF1A, IRF4, and other genes was meaningfully correlated with the immune subgroup. The expressions of PD-1, PD-L1, CD3D, HIF1A, IRF4, and other genes were meaningfully dissimilar between Immunity_L and Immunity_H (ANOVA text, p < 0.001).

Fig 4: (a) IHC was used to detect the expression of IRF4 and PD-L1 in DLBCL. Two cases were stained with IRF4 and PD-L1 immunohistochemistry. Examine the section under a microscope. (b) The images described are representative of 30 cases of DLBCL. Correlation between IRF4 IHC score and IRF4 IHC score in 30 DLBCL patients, calculated by Spearman's rank correlation methods, in 30 DLBCL cases. (c) We transfected siControl and siIRF4 into DS cell line by transient transfection method. Proteins were collected and lysed, and the displayed proteins were analyzed by western blotting. (d) The expression of PD-L1 was detected by real-time fluorescence quantitative PCR. The error bar represents three separate experiments.

Fig 5: DNAs in T-MPs stimulate monocytes to upregulate IRF4 expression. a BALB/c mouse monocytes were co-incubated with PKH26-stained H22-MPs, cytochalasin D or PKH26-stained H22-MPs mixed with cytochalasin D. After 2, 4, 6 or 12 h, PKH26 fluorescence intensity of monocytes was measured via flow cytometry, and the IRF4 expression was analyzed at the levels of both gene and protein. b Monocytes pretreated with or without 2-DG for 30 min were co-incubated with H22-MPs for 24 h or 48 h. IRF4 expression was measured at the levels of both gene and protein. c Monocytes were co-cultured with PKH26 or PKH67-labeled H22-MPs for 14 h. Then the monocytes were analyzed with ER, Golgi, mitochondria, endosome or lysosome fluorescent trackers using a two-photon confocal microscope. d Monocytes were treated with DNAs or RNAs extracted from H22-MPs. After 24 or 48 h, the IRF4 expression was analyzed at the levels of both gene and protein. e Monocytes were co-cultured with H22-MP-derived DNAs or H22-MP-derived DNAs pretreated with DNase. 24 or 48 h later, IRF4 expression was analyzed at the levels of both gene and protein. f Monocytes transfected with TLR9 siRNA were co-cultured with H22-MPs. After 24 or 48 h, IRF4 expression was analyzed at the levels of both gene and protein. Mean ± s.e.m. is represented in the data and two-tailed unpaired Student's t test was used to statistically analyze the P values. **P < 0.01; ***P < 0.001; NS, not significant