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Cited by 120Cancer Immunotherapy and BiomarkersHepatocellular Carcinoma Treatment and PrognosisT-cell and B-cell Immunology

The DOI matches its Crossref record; 41 of the 42 references it cites were found in a registry or an index; 17 of the 25 sources its claims cite were checked for support.

PRDM1/BLIMP1 induces cancer immune evasion by modulating the USP22-SPI1-PD-L1 axis in hepatocellular carcinoma cells

Open access
Li, Qing, Zhang, Liren, You, Wenhua, Xu, Jiali, Dai, Jingjing, Hua, Dongxu, Zhang, Ruizhi, Yao, Feifan, Zhou, Suiqing, Huang, Wei, Dai, Yongjiu, Zhang, Yu, Baheti, Tasiken, Qian, Xiaofeng, Pu, Liyong, Xu, Jing, Xia, Yongxiang, Zhang, Chuanyong, Tang, Jinhai, Wang, Xuehao·2022·Nature Communications
DOI 10.1038/s41467-022-35469-xPMC PMC9744896
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Abstract

Programmed death receptor-1 (PD-1) blockade have achieved some efficacy but only in a fraction of patients with hepatocellular carcinoma (HCC). Programmed cell death 1 ligand 1 (PD-L1) binds to its receptor PD1 on T cells to dampen antigen-tumor immune responses. However, the mechanisms underlying PD-L1 regulation are not fully elucidated. Herein, we identify that tumoral Prdm1 overexpression inhibits cell growth in immune-deficient mouse models. Further, tumoral Prdm1 overexpression upregulates PD-L1 levels, dampening anti-tumor immunity in vivo, and neutralizes the anti-tumor efficacy of Prdm1 overexpression in immune-competent mouse models. Mechanistically, PRDM1 enhances USP22 transcription, thus reducing SPI1 protein degradation through deubiquitination, which enhances PD-L1 transcription. Functionally, PD-1 mAb treatment reinforces the efficacy of Prdm1-overexpressing HCC immune-competent mouse models. Collectively, we demonstrate that the PRDM1-USP22-SPI1 axis regulates PD-L1 levels, resulting in infiltrated CD8+ T cell exhaustion. Furthermore, PRDM1 overexpression combined with PD-(L)1 mAb treatment provides a therapeutic strategy for HCC treatment.

Overview

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TL;DR

PRDM1/BLIMP1 promotes HCC immune evasion by transcriptionally inducing USP22, which deubiquitinates and stabilizes SPI1, increasing PD-L1 transcription and CD8+ T cell exhaustion. Combining Prdm1 overexpression with PD-1 mAb improves antitumor efficacy in mouse HCC models.

Key findings

  • PRDM1/BLIMP1 upregulates PD-L1 in HCC cells; in cohort 1 (40 patients with HCC), tumoral BLIMP1 was inversely correlated with CD8+ T cells (R = −0.4877, P = 0.0014) and GZMB+ CD8+ T cells (R = −0.4882, P = 0.0014), and positively correlated with PD1+ exhausted CD8+ TILs (R = 0.3722, P = 0.0181).
  • Mechanistically, PRDM1 enhances USP22 transcription; a DUB siRNA library among 98 known human DUBs found that USP22 and USP33 suppression decreased SPI1 protein by more than 50%. USP22 deubiquitinates SPI1, and SPI1 binds PD-L1 promoter sites SBS 3 and 4 to enhance PD-L1 transcription.
  • In immune-competent mice, Prdm1 overexpression alone did not reduce tumor size or overall survival but reduced infiltrated CD8+ T cells and their GZMB+ activity and increased PD-1+ exhaustion; in immunodeficient mice, Prdm1 overexpression slowed tumor growth and prolonged survival. PD-1 mAb plus Prdm1 overexpression further impaired tumor proliferation and prolonged survival versus either alone (n = 6 mice per group).
  • In cohort 2 (90 patients with HCC), BLIMP1, USP22, and SPI1 were positively associated with PD-L1 and negatively associated with CD8+ T cell infiltration; scRNA-seq of 8,277 cells from two patients showed that the PRDM1-high tumor had more CD8+LAG3+ exhausted T cells and CD4+FOXP3+ Tregs.

Methods

  • Human Hep3B/Huh7 and murine Hepa1-6/H22 HCC cells were used with PRDM1/Prdm1 overexpression or CRISPR knockout; assays included qRT-PCR, western blotting, flow cytometry, 3D co-culture, T cell-mediated killing, PD1 binding, luciferase reporter, ChIP, co-IP/mass spectrometry, TMT proteomics, and a DUB siRNA library.
  • Mouse models used immune-competent C57BL/6 and immunodeficient BALB/c nude mice with subcutaneous or orthotopic tumors, plus a DEN/CCl4-induced HCC model; treatments included PD-1 mAb, IgG control, CD8α mAb, and AAV8-Prdm1, with n = 6 mice per group in tumor/survival experiments.
  • Clinical samples included cohort 1 (40 fresh HCC tissues), cohort 2 (90 paired HCC and adjacent normal tissues), and cohort 3 (22 HCC biopsies before PD-1 mAb-based therapy); scRNA-seq analyzed 8,277 cells from two patients. Statistical analyses used Student’s t test, Pearson correlation, and Kaplan–Meier/log-rank methods.

Limitations

  • Stated: the authors could not fully exclude that PRDM1 may also influence SPI1 protein stability through a specific E3 ligase; the mechanism separating PRDM1/BLIMP1’s growth-inhibitory and immune-evasion effects needs further investigation; and ideal therapeutic vectors such as AAV carrying PRDM1 still need safety and efficacy evaluation.
  • Evident: the scRNA-seq analysis included only two patients; the clinical cohorts came from a single center; and the proposed PRDM1 overexpression plus PD-(L)1 combination was tested in preclinical models, not in a human trial.

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Crossref · 10.1038/s41467-022-35469-x

Cites

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Figures

Fig. 1 PRDM1 upregulates PD-L1 expression in HCC. a qRT-PCR of PD-L1 expression in Hep3B cells or Huh7 cells with or without IFN-γ treatment. Data presented as mean ± SEM (n = 3 independent biological replicates). b, c Western blotting of PD-L1 expression in Hep3B cells or Huh7 cells with or without IFN-γ treatment. Representative of n = 3 independent biological replicates. d, e Flow cytometry analysis of PD-L1 expression in Hep3B cells (d) or Huh7 cells (e) with or without IFN-γ treatment. n = 3 independent biological replicates. f 3D-co-culture model using pre-activated T cells and Hep3B or Huh7 cells. g Flow cytometry analysis of CD8+GZMB+ cell content in pre-activated T cells following 72 h of co-culture with Hep3B or Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). h Flow cytometry analysis of CD8+TNFα+ cell content in pre-activated T cells following 72 hours of co-culture with Hep3B or Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). i Time-lapse microscopic images revealing the binding of Hep3B or Huh7 cells with PD1 at 12 h. Scale bars, 100 µm. j Analysis of PD1/Fc protein binding on Hep3B and Huh7 cells at 2 h. Data presented as mean ± SEM (n = 3 independent biological replicates). k Immunohistochemical staining of BLIMP1 protein in HCC samples (Cohort 1). Scale bars, 100 µm. l Correlation analysis of tumoral BLIMP1 protein expression and tumor-infiltrating T cell content. Pearson analysis revealed a negative correlation between tumoral BLIMP1 protein expression and CD8+ T cells and the activity (GZMB+) of infiltrated CD8+ T cells in infiltrating CD45+ cells and a positive correlation between tumor BLIMP1 protein expression and the percentages of PD1+ cells in CD8+ TILs. n = 40 patients. P value was determined by unpaired two-sided Student’s t test (a, c, g, h, j) and Pearson correlation analysis (l). Source data are provided as a Source data file.
Fig. 1 PRDM1 upregulates PD-L1 expression in HCC. a qRT-PCR of PD-L1 expression in Hep3B cells or Huh7 cells with or without IFN-γ treatment. Data presented as mean ± SEM (n = 3 independent biological replicates). b, c Western blotting of PD-L1 expression in Hep3B cells or Huh7 cells with or without IFN-γ treatment. Representative of n = 3 independent biological replicates. d, e Flow cytometry analysis of PD-L1 expression in Hep3B cells (d) or Huh7 cells (e) with or without IFN-γ treatment. n = 3 independent biological replicates. f 3D-co-culture model using pre-activated T cells and Hep3B or Huh7 cells. g Flow cytometry analysis of CD8+GZMB+ cell content in pre-activated T cells following 72 h of co-culture with Hep3B or Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). h Flow cytometry analysis of CD8+TNFα+ cell content in pre-activated T cells following 72 hours of co-culture with Hep3B or Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). i Time-lapse microscopic images revealing the binding of Hep3B or Huh7 cells with PD1 at 12 h. Scale bars, 100 µm. j Analysis of PD1/Fc protein binding on Hep3B and Huh7 cells at 2 h. Data presented as mean ± SEM (n = 3 independent biological replicates). k Immunohistochemical staining of BLIMP1 protein in HCC samples (Cohort 1). Scale bars, 100 µm. l Correlation analysis of tumoral BLIMP1 protein expression and tumor-infiltrating T cell content. Pearson analysis revealed a negative correlation between tumoral BLIMP1 protein expression and CD8+ T cells and the activity (GZMB+) of infiltrated CD8+ T cells in infiltrating CD45+ cells and a positive correlation between tumor BLIMP1 protein expression and the percentages of PD1+ cells in CD8+ TILs. n = 40 patients. P value was determined by unpaired two-sided Student’s t test (a, c, g, h, j) and Pearson correlation analysis (l). Source data are provided as a Source data file.CC BY · PubMed Central
Fig. 2 Prdm1 attenuates its effects on tumor growth inhibition through PD-L1-induced tumor immune evasion in immune-competent mice. a Representative subcutaneous tumors (left)/orthotopic transplantation tumors (right) collected from Hepa1-6-bearing C57BL/6 mice. b Tumor proliferation curves of subcutaneous xenografts in Hepa1-6-bearing C57BL/6 mice. Data presented as mean ± SEM. n = 6 mice per group. c Kaplan–Meier survival curves of Hepa1-6-bearing C57BL/6 mice. n = 6 mice per group. d Representative subcutaneous tumors (left)/orthotopic transplantation tumors (right) collected from H22-bearing C57BL/6 mice. e Tumor proliferation curves of subcutaneous xenografts in H22-bearing C57BL/6 mice. Data presented as mean ± SEM. n = 6 mice per group. f Kaplan-Meier survival curves of H22-bearing C57BL/6 mice. n = 6 mice per group. g–i Flow cytometry analysis of CD8+, GZMB+CD8+, and PD1+CD8+ in CD3+ TILs from Hepa1-6 (g) or H22 xenografts (h) in C57BL/6 mice and their quantification (i). Data presented as mean ± SEM. n = 3 mice per group. j Representative subcutaneous tumors (left)/orthotopic transplantation tumors (right) collected from Hepa1-6-bearing BALB/c nude mice. k Tumor proliferation curves of subcutaneous xenografts in Hepa1-6-bearing BALB/c nude mice. Data presented as mean ± SEM. n = 6 mice per group. l Kaplan–Meier survival curves of Hepa1-6-bearing BALB/c nude mice. n = 6 mice per group. m Representative subcutaneous tumors (left)/orthotopic transplantation tumors (right) collected from H22-bearing BALB/c nude mice. n Tumor proliferation curves of subcutaneous xenografts in H22-bearing BALB/c nude mice. Data presented as mean ± SEM. n = 6 mice per group. o Kaplan–Meier survival curves of H22-bearing BALB/c nude mice. n = 6 mice per group. p, q T cell-mediated cancer cell-killing assay results. Data presented as mean ± SEM (n = 3 independent biological replicates). P value was determined by unpaired two-sided Student’s t test with no correction for multiple comparisons (b, e, i, k, n, p, q) and Kaplan-Meier method (c, f, l, o). Schematic diagrams (a, d, j, m) were created with BioRender.com. Source data are provided as a Source data file.
Fig. 2 Prdm1 attenuates its effects on tumor growth inhibition through PD-L1-induced tumor immune evasion in immune-competent mice. a Representative subcutaneous tumors (left)/orthotopic transplantation tumors (right) collected from Hepa1-6-bearing C57BL/6 mice. b Tumor proliferation curves of subcutaneous xenografts in Hepa1-6-bearing C57BL/6 mice. Data presented as mean ± SEM. n = 6 mice per group. c Kaplan–Meier survival curves of Hepa1-6-bearing C57BL/6 mice. n = 6 mice per group. d Representative subcutaneous tumors (left)/orthotopic transplantation tumors (right) collected from H22-bearing C57BL/6 mice. e Tumor proliferation curves of subcutaneous xenografts in H22-bearing C57BL/6 mice. Data presented as mean ± SEM. n = 6 mice per group. f Kaplan-Meier survival curves of H22-bearing C57BL/6 mice. n = 6 mice per group. g–i Flow cytometry analysis of CD8+, GZMB+CD8+, and PD1+CD8+ in CD3+ TILs from Hepa1-6 (g) or H22 xenografts (h) in C57BL/6 mice and their quantification (i). Data presented as mean ± SEM. n = 3 mice per group. j Representative subcutaneous tumors (left)/orthotopic transplantation tumors (right) collected from Hepa1-6-bearing BALB/c nude mice. k Tumor proliferation curves of subcutaneous xenografts in Hepa1-6-bearing BALB/c nude mice. Data presented as mean ± SEM. n = 6 mice per group. l Kaplan–Meier survival curves of Hepa1-6-bearing BALB/c nude mice. n = 6 mice per group. m Representative subcutaneous tumors (left)/orthotopic transplantation tumors (right) collected from H22-bearing BALB/c nude mice. n Tumor proliferation curves of subcutaneous xenografts in H22-bearing BALB/c nude mice. Data presented as mean ± SEM. n = 6 mice per group. o Kaplan–Meier survival curves of H22-bearing BALB/c nude mice. n = 6 mice per group. p, q T cell-mediated cancer cell-killing assay results. Data presented as mean ± SEM (n = 3 independent biological replicates). P value was determined by unpaired two-sided Student’s t test with no correction for multiple comparisons (b, e, i, k, n, p, q) and Kaplan-Meier method (c, f, l, o). Schematic diagrams (a, d, j, m) were created with BioRender.com. Source data are provided as a Source data file.CC BY · PubMed Central
Fig. 3 SPI1 serves as a downstream effector of PRDM1 to enhance PD-L1 transcription. a Western blotting of PD-L1/Flag levels in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. b TMT-based quantitative proteomic analysis of Hep3B cells stably overexpressing PRDM1 and the corresponding control cells, (PRDM1 and vector cells, respectively), with 3 replicates per group. Heat map showing the top 20 upregulated proteins and the top 20 downregulated proteins between PRDM1-overexpressing Hep3B cells and control cells in the proteomics analysis. c qRT-PCR of SPI1 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). d Western blotting of SPI1 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. e Western blotting of PD-L1 expression in SPI1-overexpressing and control Hep3B cells or SPI1-knockdown and shNC Huh7 cells with or without IFN-γ treatment. n = 3 independent biological replicates. f Putative SPI1-binding sites (SBS) within the genomic sequence adjacent to the transcription start site (TSS) of the PD-L1 gene. g, h Luciferase activities of PD-L1 promoter reporter vectors in Hep3B (g) and Huh7 (h) cells. Red characters in the binding regions suggest the putative or mutated SPI1-binding sequences. Data presented as mean ± SEM (n = 3 independent biological replicates). i, j ChIP analysis of SPI1 binding to the PD-L1 promoter in Hep3B (i) and Huh7 (j) cells. Two promoter regions of PD-L1 not expected to be bound by SPI1 were employed as negative controls (negative control#1 and #2). Data presented as mean ± SEM (n = 3 independent biological replicates). k Flow cytometry analysis of PD-L1 expression in Hep3B cells with or without IFN-γ treatment. n = 3 independent biological replicates. P value was determined by unpaired two-sided Student’s t test (c, g, h, i, j). Source data are provided as a Source data file.
Fig. 3 SPI1 serves as a downstream effector of PRDM1 to enhance PD-L1 transcription. a Western blotting of PD-L1/Flag levels in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. b TMT-based quantitative proteomic analysis of Hep3B cells stably overexpressing PRDM1 and the corresponding control cells, (PRDM1 and vector cells, respectively), with 3 replicates per group. Heat map showing the top 20 upregulated proteins and the top 20 downregulated proteins between PRDM1-overexpressing Hep3B cells and control cells in the proteomics analysis. c qRT-PCR of SPI1 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). d Western blotting of SPI1 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. e Western blotting of PD-L1 expression in SPI1-overexpressing and control Hep3B cells or SPI1-knockdown and shNC Huh7 cells with or without IFN-γ treatment. n = 3 independent biological replicates. f Putative SPI1-binding sites (SBS) within the genomic sequence adjacent to the transcription start site (TSS) of the PD-L1 gene. g, h Luciferase activities of PD-L1 promoter reporter vectors in Hep3B (g) and Huh7 (h) cells. Red characters in the binding regions suggest the putative or mutated SPI1-binding sequences. Data presented as mean ± SEM (n = 3 independent biological replicates). i, j ChIP analysis of SPI1 binding to the PD-L1 promoter in Hep3B (i) and Huh7 (j) cells. Two promoter regions of PD-L1 not expected to be bound by SPI1 were employed as negative controls (negative control#1 and #2). Data presented as mean ± SEM (n = 3 independent biological replicates). k Flow cytometry analysis of PD-L1 expression in Hep3B cells with or without IFN-γ treatment. n = 3 independent biological replicates. P value was determined by unpaired two-sided Student’s t test (c, g, h, i, j). Source data are provided as a Source data file.CC BY · PubMed Central
Fig. 4 USP22 inhibits ubiquitin-proteasomal degradation of SPI1. a, b SPI1 protein expression in PRDM1-overexpressing and vector Hep3B cells (a) or in PRDM1-knockout and sgCtrl Huh7 cells (b). n = 3 independent biological replicates. c SPI1 protein expression was determined in PRDM1 knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. d SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. e SPI1 was pulled down and ubiquitin-conjugated SPI1 was determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. f DUB siRNA library screening indicated that USP22 and USP33 suppression decreased SPI1 protein levels. g qRT-PCR of USP22 or USP33 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). h Western blotting of USP22 or USP33 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. i, j SPI1 protein expression in USP22-overexpressing and vector Hep3B cells (i) or in USP22-knockdown and control Huh7 cells (j). n = 3 independent biological replicates. k SPI1 protein expression was determined in USP22-knockdown or control Huh7 cells. n = 3 independent biological replicates. l SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. m SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. P value was determined by unpaired two-sided Student’s t test (g). Source data are provided as a Source data file.
Fig. 4 USP22 inhibits ubiquitin-proteasomal degradation of SPI1. a, b SPI1 protein expression in PRDM1-overexpressing and vector Hep3B cells (a) or in PRDM1-knockout and sgCtrl Huh7 cells (b). n = 3 independent biological replicates. c SPI1 protein expression was determined in PRDM1 knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. d SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. e SPI1 was pulled down and ubiquitin-conjugated SPI1 was determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. f DUB siRNA library screening indicated that USP22 and USP33 suppression decreased SPI1 protein levels. g qRT-PCR of USP22 or USP33 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). h Western blotting of USP22 or USP33 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. i, j SPI1 protein expression in USP22-overexpressing and vector Hep3B cells (i) or in USP22-knockdown and control Huh7 cells (j). n = 3 independent biological replicates. k SPI1 protein expression was determined in USP22-knockdown or control Huh7 cells. n = 3 independent biological replicates. l SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. m SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. P value was determined by unpaired two-sided Student’s t test (g). Source data are provided as a Source data file.CC BY · PubMed Central
Fig. 5 USP22 was identified as a SPI1-interacting protein. a Co-IP experiments indicated the interaction of endogenous USP22 and SPI1. n = 3 independent biological replicates. b Confocal microscopy showing colocalization of USP22 (red) with SPI1 (green). n = 3 independent biological replicates. Scale bars, 20 µm. c Schematic illustration of USP22 and its mutants. d USP22 and its mutants were immunoprecipitated and the bound SPI1 was determined. n = 3 independent biological replicates. e SPI1 ubiquitination was determined by SPI1 immunoprecipitation and western blotting using an anti-HA antibody. n = 3 independent biological replicates. f Schematic illustration of USP22 and its point mutants. g USP22 and its point mutants were immunoprecipitated and the bound SPI1 was determined. n = 3 independent biological replicates. h The effects of USP22 and its point mutants on SPI1 ubiquitination were confirmed. n = 3 independent biological replicates. i The protein levels of USP22 (Myc) and SPI1 were detected. Data presented as mean ± SEM (n = 3 independent biological replicates). j Schematic representation of full-length SPI1 and truncated SPI1. k Interactions between USP22 and full-length or truncated SPI1 were analyzed using co-IP in HEK-293T cells. n = 3 independent biological replicates. l Co-IP experiments indicated the interaction of Myc-USP22 (161-525) and His-SPI1 (D3) in HEK-293T cells. n = 3 independent biological replicates. m Putative BLIMP1-binding site (BBS) within the genomic sequence adjacent to TSS of USP22 gene. n, o Luciferase activities of USP22 promoter reporter vectors in Hep3B (n) and Huh7 (o) cells. Red characters in the binding regions suggest putative or mutated BLIMP1 binding sequences. Data presented as mean ± SEM (n = 3 independent biological replicates). p, q ChIP analysis of BLIMP1 binding to the USP22 promoter in Hep3B (p) and Huh7 (q) cells. Two promoter regions of USP22 not expected to be bound by BLIMP1 were employed as negative controls. Data presented as mean ± SEM (n = 3 independent biological replicates). P value was determined by unpaired two-sided Student’s t test (n, o, p, q). Source data are provided as a Source data file.
Fig. 5 USP22 was identified as a SPI1-interacting protein. a Co-IP experiments indicated the interaction of endogenous USP22 and SPI1. n = 3 independent biological replicates. b Confocal microscopy showing colocalization of USP22 (red) with SPI1 (green). n = 3 independent biological replicates. Scale bars, 20 µm. c Schematic illustration of USP22 and its mutants. d USP22 and its mutants were immunoprecipitated and the bound SPI1 was determined. n = 3 independent biological replicates. e SPI1 ubiquitination was determined by SPI1 immunoprecipitation and western blotting using an anti-HA antibody. n = 3 independent biological replicates. f Schematic illustration of USP22 and its point mutants. g USP22 and its point mutants were immunoprecipitated and the bound SPI1 was determined. n = 3 independent biological replicates. h The effects of USP22 and its point mutants on SPI1 ubiquitination were confirmed. n = 3 independent biological replicates. i The protein levels of USP22 (Myc) and SPI1 were detected. Data presented as mean ± SEM (n = 3 independent biological replicates). j Schematic representation of full-length SPI1 and truncated SPI1. k Interactions between USP22 and full-length or truncated SPI1 were analyzed using co-IP in HEK-293T cells. n = 3 independent biological replicates. l Co-IP experiments indicated the interaction of Myc-USP22 (161-525) and His-SPI1 (D3) in HEK-293T cells. n = 3 independent biological replicates. m Putative BLIMP1-binding site (BBS) within the genomic sequence adjacent to TSS of USP22 gene. n, o Luciferase activities of USP22 promoter reporter vectors in Hep3B (n) and Huh7 (o) cells. Red characters in the binding regions suggest putative or mutated BLIMP1 binding sequences. Data presented as mean ± SEM (n = 3 independent biological replicates). p, q ChIP analysis of BLIMP1 binding to the USP22 promoter in Hep3B (p) and Huh7 (q) cells. Two promoter regions of USP22 not expected to be bound by BLIMP1 were employed as negative controls. Data presented as mean ± SEM (n = 3 independent biological replicates). P value was determined by unpaired two-sided Student’s t test (n, o, p, q). Source data are provided as a Source data file.CC BY · PubMed Central
Fig. 6 Synergistic effect of Prdm1 overexpression and PD-1 mAb therapy in mice. a, b Schematic view of the treatment plan in subcutaneous tumors and orthotopic tumors. C57BL/6 mice were implanted with Hepa1-6/H22 cells subcutaneously or as orthotopic tumors and were treated with PD-1 mAb or IgG isotype control. c, d Representative xenograft tumors (left) and orthotopic tumors (right) obtained after euthanizing the mice. e, f Tumor proliferation curves (e) and Kaplan–Meier survival curves (f) of Hepa1-6-bearing C57BL/6 mice. Data presented as mean ± SEM (e). n = 6 mice per group. g, h Tumor proliferation curves (g) and Kaplan-Meier survival curves (h) of H22-bearing C57BL/6 mice. Data presented as mean ± SEM (g). n = 6 mice per group. i, j Flow cytometry analysis of CD8+, GZMB+CD8+, and PD1+CD8+ in CD3+ TILs from Hepa1-6 (i) or H22 (j) subcutaneous tumors in C57BL/6 mice. Data presented as mean ± SEM. n = 6 mice per group. k A schematic view of the treatment plan in the DEN/CCL4-induced HCC model. l Representative images of liver tumors (annotated by white arrows). m H&E staining of liver sections of sacrificed mice. Scale bars, 400 µm. n, o Quantification of tumor numbers (n) and maximal tumor sizes (o). Data presented as mean ± SEM. n = 6 mice per group. p Kaplan–Meier survival curves in the DEN-induced HCC model. n = 6 mice per group. P value was determined by unpaired two-sided Student’s t test (e, g, i, j, n, o) and Kaplan–Meier method (f, h, p) with no correction for multiple comparisons. Schematic diagrams (a, b, k) were created with BioRender.com. Source data are provided as a Source data file.
Fig. 6 Synergistic effect of Prdm1 overexpression and PD-1 mAb therapy in mice. a, b Schematic view of the treatment plan in subcutaneous tumors and orthotopic tumors. C57BL/6 mice were implanted with Hepa1-6/H22 cells subcutaneously or as orthotopic tumors and were treated with PD-1 mAb or IgG isotype control. c, d Representative xenograft tumors (left) and orthotopic tumors (right) obtained after euthanizing the mice. e, f Tumor proliferation curves (e) and Kaplan–Meier survival curves (f) of Hepa1-6-bearing C57BL/6 mice. Data presented as mean ± SEM (e). n = 6 mice per group. g, h Tumor proliferation curves (g) and Kaplan-Meier survival curves (h) of H22-bearing C57BL/6 mice. Data presented as mean ± SEM (g). n = 6 mice per group. i, j Flow cytometry analysis of CD8+, GZMB+CD8+, and PD1+CD8+ in CD3+ TILs from Hepa1-6 (i) or H22 (j) subcutaneous tumors in C57BL/6 mice. Data presented as mean ± SEM. n = 6 mice per group. k A schematic view of the treatment plan in the DEN/CCL4-induced HCC model. l Representative images of liver tumors (annotated by white arrows). m H&E staining of liver sections of sacrificed mice. Scale bars, 400 µm. n, o Quantification of tumor numbers (n) and maximal tumor sizes (o). Data presented as mean ± SEM. n = 6 mice per group. p Kaplan–Meier survival curves in the DEN-induced HCC model. n = 6 mice per group. P value was determined by unpaired two-sided Student’s t test (e, g, i, j, n, o) and Kaplan–Meier method (f, h, p) with no correction for multiple comparisons. Schematic diagrams (a, b, k) were created with BioRender.com. Source data are provided as a Source data file.CC BY · PubMed Central
Fig. 7 The PRDM1-USP22-SPI1 axis regulates PD-L1 levels in patients with HCC. a, b Immunohistochemical staining of BLIMP1, USP22, SPI1, PD-L1, and CD8α expression in patients with HCC. n = 90 patients. Scale bars, 100 mm. c, d Multi-color immunohistochemistry using BLIMP1, USP22, SPI1, and PD-L1 (c) or BLIMP1, PD-L1, CD8α, and GZMB (d) antibodies in patients with HCC. n = 90 patients. Scale bars, 100 µm. e Proposed model underlying the roles of PRDM1/BLIMP1 in promoting tumor immune evasion in HCC. A schematic diagram was designed using BioRender.
Fig. 7 The PRDM1-USP22-SPI1 axis regulates PD-L1 levels in patients with HCC. a, b Immunohistochemical staining of BLIMP1, USP22, SPI1, PD-L1, and CD8α expression in patients with HCC. n = 90 patients. Scale bars, 100 mm. c, d Multi-color immunohistochemistry using BLIMP1, USP22, SPI1, and PD-L1 (c) or BLIMP1, PD-L1, CD8α, and GZMB (d) antibodies in patients with HCC. n = 90 patients. Scale bars, 100 µm. e Proposed model underlying the roles of PRDM1/BLIMP1 in promoting tumor immune evasion in HCC. A schematic diagram was designed using BioRender.CC BY · PubMed Central
Fig. 8 Single-cell analysis of intra-tumoral cell populations. a In total, 8 cell types were identified and shown using UMAP. The colors were coded by cell types (top) and patients (bottom). b Dot plot showing the top marker genes of each cell type. c UMAP plots (top) and PRDM1 expression (bottom) in malignant cells from two patients. d UMAP plot and violin plot showing distinct T cell subclusters and their marker genes. e Dot plot depicting the relative frequency of each T cell subpopulation between patient 1 and patient 2. f The top 10 GO terms of upregulated genes in PRDM1-high tumor cells from patient 2.
Fig. 8 Single-cell analysis of intra-tumoral cell populations. a In total, 8 cell types were identified and shown using UMAP. The colors were coded by cell types (top) and patients (bottom). b Dot plot showing the top marker genes of each cell type. c UMAP plots (top) and PRDM1 expression (bottom) in malignant cells from two patients. d UMAP plot and violin plot showing distinct T cell subclusters and their marker genes. e Dot plot depicting the relative frequency of each T cell subpopulation between patient 1 and patient 2. f The top 10 GO terms of upregulated genes in PRDM1-high tumor cells from patient 2.CC BY · PubMed Central

Verification

Claims the paper makes

112 Own result · 29 Own interpretation · 7 Own method · 4 Partly supported · 2 Supported so far · 2 No basis given · 1 Supported · 1 No full text of the sources · accuracy not yet measured

  • Probably not a substantial claim. 13
  • It may state two separate claims as one. 19
  • The quote only partly states this claim. 1
30 of 30

  • The paper relies on this without support.

    The mechanisms underlying PD-L1 regulation are not fully elucidated.

    “However, the mechanisms underlying PD-L1 regulation are not fully elucidated.”

  • The paper relies on this without support.

    The role of the PRDM family in regulating anti-tumor immunity and immune molecules in HCC cells remains largely unknown.

    “However, the role of the PRDM family in regulating anti-tumor immunity and immune molecules in HCC cells remains largely unknown.”

  • The paper relies on this without support.

    SPI1 is a transcription factor for PD-L1.

    “SPI1, a transcription factor for PD-L1, was the most likely candidate based on fold change (Fig. 3b).”

  • The paper relies on this without support.

    T cell exhaustion due to PD-L1/PD1 inhibitory receptor signaling impairs T cell proliferation and effector functions.

    “As T cell exhaustion due to PD-L1/PD1 inhibitory receptor signaling impairs T cell proliferation and effector functions, the relationship between tumoral BLIMP1 protein expression and exhausted T cells in HCC specimens was examined by profiling the content and function of CD8+ T cells in tumor-infiltrating T lymphocytes (TILs) using flow cytometry in cohort 1”

  • The paper relies on this without support.

    The ubiquitin-proteasome system enhances proteasomal degradation of its target proteins.

    “The ubiquitin-proteasome system (UPS) enhances proteasomal degradation of its target proteins.”

  • The paper relies on this without support.

    Deubiquitinating enzymes control ubiquitin-dependent pathways by separating protein-ubiquitin bonds.

    “Deubiquitinating enzymes (DUBs) control ubiquitin-dependent pathways by separating protein-ubiquitin bonds.”

  • The paper relies on this without support.

    Only a fraction of patients with HCC respond to PD-(L)1 mAb therapy.

    “However, only a fraction of patients with HCC respond to PD-(L)1 mAb therapy.”

  • The paper relies on this without support.

    PD-(L)1 mAb has been proven to be beneficial for patients with various advanced malignancies.

    “PD-(L)1 mAb has been proven to be beneficial for patients with various advanced malignancies.”
8 of 8

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

2 of 2

Citations

Cites

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Cited by

Sorted by citations

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Related

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Metadata

Authors
Li, Q., Zhang, L., You, W., Xu, J., Dai, J., Hua, D., Zhang, R., Yao, F., Zhou, S., Huang, W., Dai, Y., Zhang, Y., Baheti, T., Qian, X., Pu, L., Xu, J., Xia, Y., Zhang, C., Tang, J., & Wang, X.
Affiliations
  • Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
  • Southeast University (CN)
  • Jiangsu Province Hospital (CN)
  • Nanjing Medical University (CN)
Corresponding author
  • Xu, Jiali
  • Xia, Yongxiang
  • Zhang, Chuanyong
  • Tang, Jinhai
  • Wang, Xuehao
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2022-12-12
Volume
13
Issue
1
Pages
7677-7677
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-022-35469-x
PMC
PMC9744896
OpenAlex
W4311824088
Semantic Scholar
8cc67529818e6c3235226e246230d27027fa9130
Cited by
120
References
42
Citations by year
2026: 27 · 2025: 39 · 2024: 47 · 2023: 7
FWCI
10.60
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (82103440, 82272791, 31930020, 82203659)
  • China Postdoctoral Science Foundation
  • Government of Jiangsu Province
  • Natural Science Foundation of Jiangsu Province (BK20220731, 31930020)
  • Nanjing Medical University
Topics
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Health Sciences › Medicine › Hepatology › Hepatocellular Carcinoma Treatment and Prognosis
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
Keywords
Blimp-1, hepatocellular carcinoma, tumor immune evasion, PD-L1, PD-1, USP22, PU.1, deubiquitination, CD8+ T cell exhaustion, anti-PD-1 therapy, antitumor immunity, transcriptional regulation
MeSH terms
Positive Regulatory Domain I-Binding Factor 1, Animals, Carcinoma, Hepatocellular, Liver Neoplasms, CD8-Positive T-Lymphocytes, Mice, Immune Evasion, B7-H1 Antigen, Programmed Cell Death 1 Receptor

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-10-07, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-10-07 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Cited sources are checked with a language model’s help (gpt-6-sol).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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