Abstract
T cell-based immunotherapies have revolutionized cancer treatment, yet durable responses remain elusive. Here we show that PCIF1, an RNA N6 2′-O-dimethyladenosine (m6Am) methyltransferase, negatively regulates CD8+ T cell antitumor responses. Whole-body or T cell-specific Pcif1 knockout (KO) reduced tumor growth in mice. Single-cell RNA sequencing shows an increase in the number of tumor-infiltrating cytotoxic CD8+ T cells in Pcif1-deficient mice. Mechanistically, proteomic and m6Am-sequencing analyses pinpoint that Pcif1 KO elevates m6Am-modified targets, specifically ferroptosis suppressor genes (Fth1, Slc3a2), and the T cell activation gene Cd69, imparting resistance to ferroptosis and enhancing CD8+ T cell activation. Of note, Pcif1-deficient mice had enhanced responses to anti-PD-1 immunotherapy, and Pcif1 KO chimeric antigen receptor T cells improved tumor control. Clinically, cancer patients with low PCIF1 expression in T cells have enhanced responses to immunotherapies. These findings suggest that PCIF1 suppresses CD8+ T cell activation and targeting PCIF1 is a promising strategy to boost antitumor immunity.
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Data availability
scRNA-seq data have been deposited in the Gene Expression Omnibus under accession code GSE254353. m6Am-seq data have been deposited in the Gene Expression Omnibus under accession code GSE254597. The TMT, PRM and IP–MS proteomics data have been deposited in the ProteomeXchange Consortium via the iProX partner repository under the dataset identifier PXD048902. Data in Fig. 6a,b were derived from https://resilience.ccr.cancer.gov/ ref. 46. Further information and reagents are available from the corresponding author J.Z. upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank SpecAlly Life Technology for analysis of proteomic data. We also thank the staff at the core facility of the Medical Research Institute at Wuhan University for technical support. This work was supported by grants from the National Key Research and Development Program of China (2023YFC3402100 and 2022YFC3401500 to J.Z., 2021YFC2302400 to M.Z., 2023YFC3402200 to C.Y.), the National Natural Science Foundation of China (82341023 to G.C., 31970732 and 82273062 to J.Z., 22425071 and 92153303 to C.Y., 82203319 to M.G., 82100193 to H.Z.), the Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University (ZNJC202312 to J.Z.), the Fundamental Research Funds for the Central Universities (2042022dx0003 to J.Z.), Knowledge Innovation Program of Wuhan-Basic Research (2022020801010105 to J.Z.), the Natural Science Foundation of Hubei Province of China (2022CFA008 to J.Z., 2022CFB029 to H.Z.) and the Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZLCX20212300 to G.C.).
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Contributions
B.X., M.Z., K.L., Z.Z. and Y.L. performed most of the experiments with help from M.G., X.W., X. Xiao, Y.S., C.H., J.S., H.F., X. Xing, G.X. and Y.Y. J.Z., C.Y., H.Z., G.C., B.X., M.Z., Z.Z. and Y.L. designed the experiments. M.Z., K.L. and C.Y. performed the m6Am sequencing and analyzed the data. Z.Z. and H.Z. performed the anti-CD19 CAR T cell assays. J.Z., C.Y., H.Z. and G.C. guided and supervised the project. J.Z. and B.X. wrote the paper. All authors commented on the paper.
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Nature Immunology thanks Joseph Fraietta and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Nick Bernard, in collaboration with the Nature Immunology team.Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Pcif1 KO mice exhibit normal phenotypes and retarded tumor growth.
a, A schematic representation of the process for creating whole-body Pcif1 KO mice through the crossbreeding of Pcif1flox/flox mice with CAG-Cre transgenic mice. b, The mRNA level of Pcif1 was analyzed by real-time PCR in Pcif1 KO or WT mice. n = 4 mice/each group. c, The protein level in tissues of both WT (n = 3) and Pcif1 KO (n = 4) mice. d-f, The representative images show the body size (d), tissue weight/body weight ratio (e) (n = 6), and body weight (f) (n = 14) of WT or Pcif1 KO mice. g,h, The assessments of liver function (g), and routine blood tests (h) between Pcif1 KO mice and their WT littermates. n = 8 mice/each group. i-k, Flow cytometry analysis of thymocytes, peripheral T cells isolated from spleen and lymph nodes of Pcif1 KO and WT mice. i,j, Percentages of CD4+ and CD8+ T cells in spleen and lymph nodes of Pcif1 KO and WT mice. k, Percentages of CD4−CD8− double-negative (DN), CD4+CD8+ double-positive (DP), CD4+ single-positive (CD4SP) and CD8+ single-positive (CD8SP) cells out of the total number of thymocytes from Pcif1 KO and WT mice. n = 4 mice/each group. l-n, Tumor growth of LLC (l), B16-F10 (m) or MC38 cells (n) was assessed in WT or Pcif1 KO mice. Tumor volumes of mice were measured every two days. Each symbol represents an individual mouse. Two-tailed unpaired Student’s t-test (b, e-k) was performed. Data are shown as the mean ± SEM.
Extended Data Fig. 2 scRNA-seq result analyses of LLC tumors in WT and Pcif1 KO mice.
a, Uniform Manifold Approximation and Projection (UMAP) showing 20 clusters of total cells from LLC tumors carried subcutaneously in WT or Pcif1 KO mice (n = 2 mice/each group). b, Heat map from scRNA-seq displaying normalized expression of selected genes in each cluster. c-i, Dot plot showing the expression of representative genes for each cell type (c). And the UMAP displays six clusters of total cells (d) and marker genes (e-i). j, UMAP plot showing secondary clusters of lymphoid cells. k, Heat map from scRNA-seq displaying normalized expression of selected genes in each cluster. l-r, Dot plot (l) and UMAP plots (m-r) showing the expression of selected marker genes for each cell type.
Extended Data Fig. 3 Analysis of NK and TAM in scRNA-seq results from LLC tumors.
a-e, Volcano plots of differentially expressed genes between WT and Pcif1 KO intratumoral NK cells (a) and violin plots showing Gzmc (b), Gzmd (c), Gzme (d), and B2m (e) expression in WT and Pcif1 KO intratumoral NK cells. f, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the significantly changed genes in WT and Pcif1 KO intratumoral NK cells. g,h, UMAP plot showing secondary clusters of myeloid cells (g). And a heat map from scRNA-seq displaying normalized expression of selected genes in each cluster (h). i-q, Dot plot (i) and UMAP (j) of intratumoral myeloid cells, marker genes (k-p) and quantitation of each cell type (q). Two-sided wald-test with Benjamini-Hochberg correction (a) or one-sided hypergeometric test (f) were performed.
Extended Data Fig. 4 Analyses of tumor growth and tumor-infiltrating CD8+ T cells.
a, Tumor growth of LLC cells was assessed in WT or Pcif1 KO mice with indicated treatments. Tumor volumes of mice were measured every two days. b, The representative FACS gating strategies for analyzing tumor-infiltrating immune cells. This gating strategy is applied to analyzing all tumor-infiltrating leukocytes. c-f, Flow cytometry analysis showing the percentage of stem-like PD-1+Tcf-1+CD8+ T cells(c), effector memory CD44+CD8+ T cells (Tem) (d), tissue-resident memory CD69+CD103+CD8+ T cells (Trm) (e), and exhausted PD-1+ Tim3+CD8+ T cells (Tex) (f) in LLC subcutaneous tumor isolated from WT or Pcif1 KO mice 23 days after tumor inoculation. n = 5 mice/each group. Each symbol represents an individual mouse. Two-tailed unpaired Student’s t-test (c-f) was performed. Data are shown as the mean ± SEM.
Extended Data Fig. 5 Pcif1 cKO mice exhibit normal phenotypes and retarded tumor growth.
a, A schematic representation of the process for creating conditional knockout Pcif1 mice (Pcif1 cKO) through the crossbreeding of Pcif1flox/flox mice with Cd4-Cre transgenic mice. b,c, The mRNA and protein level of Pcif1 in CD8 T cells of both WT and Pcif1 cKO mice. n = 3 mice/each group. d-f, The representative images show the body size (d), tissue weight/body weight ratio (e) (n = 6), and body weight (f) (n = 14) of WT or Pcif1 cKO mice. g,h, The assessments of liver function (g) and routine blood tests (h) between Pcif1 cKO mice and their WT littermates. n = 6 mice/each group. i-k, Flow cytometry analysis of thymocytes, peripheral T cells isolated from spleen and lymph nodes of Pcif1 cKO and WT mice. i, j, Percentages of CD4+ and CD8+ T cells in spleen (i) and lymph nodes (j) of Pcif1 cKO and WT mice. k, Percentages of CD4−CD8− double-negative (DN), CD4+CD8+ double-positive (DP), CD4+ single-positive (CD4SP) and CD8+ single-positive (CD8SP) cells out of the total number of thymocytes from Pcif1 cKO and WT mice. n = 4 mice/each group. l, Tumor growth of LLC cells was assessed in WT or Pcif1 cKO mice. Tumor volumes of mice were measured every two days. m-p, WT or Pcif1 cKO mice were injected subcutaneously with 105 B16-F10 cells. Tumor growth (m,o), tumor weight (n), and survival (p) were monitored. Each symbol represents an individual mouse. Two-tailed unpaired Student’s t-test (b,e-k,n), two-way ANOVA followed by Sidak’s multiple comparisons test (m), or Kaplan-Meier survival analysis and log-rank (Mantel-Cox) test survival analysis (p) were performed. Data are shown as the mean ± SEM.
Extended Data Fig. 6 Pcif1 KO enhances CD8+ T cell activation.
a, CD69+ percentages and MFI were quantified in WT or Pcif1 KO CD4+ T cells stimulated with anti-CD3/CD28 for 5 hours. n = 3 mice/each group. b, WB were showed the protein levels of CD69 in WT or Pcif1 KO CD8+ T cells stimulated with anti-CD3/CD28 for 5 hours. c-h, The percentages and MFI of cytokines in WT or Pcif1 KO CD8+ T cells were assessed after stimulation with anti-CD3/CD28 for 48 hours. n = 4 mice/each group. i,j, The secreted cytokines in the culture media of WT and Pcif1 KO CD8+ T cells were quantified using ELISAs. n = 3 mice/each group. k,l, The Ki67+ percentages were quantified in WT or Pcif1 KO CD8+ (k) or CD4+ (l) T cells stimulated with anti-CD3/CD28 for 72 hours. n = 4 mice/each group. m,n, EdU staining (m) and CellTrace dilution (n) in WT and Pcif1 KO CD8+ T cells. n = 4 mice/each group. o, WB showing the expression of p-Lck(Y394), Lck, p-Lat(Y220), and Lat in splenic naïve CD8+ T cells derived from the mice with subcutaneous LLC tumors. Total Lck, Lat and Vinculin proteins were used as loading controls. Right: Quantification of p-Lck(Y394)/Lck or p-Lat(Y220)/Lat. n = 3 mice/each group. p-r, MFI, RT-qPCR, and ELISA analysis of cytokines in Pcif1 KO CD8+ T cells retrovirally transduced with GFP, Pcif1 WT-GFP, or Pcif1 N552A-GFP restimulated with 2 μg/mL anti-CD3/CD28 for 48 hours. n = 3 mice/each group. s, Flow cytometry analysis of retrovirus-mediated expression of GFP, Pcif1-WT-GFP and Pcif1-N552A-GFP in Pcif1 KO CD8+ T cells. t-y, The percentages, MFI, RT-qPCR and ELISA analysis of cytokines in Pcif1 KO CD8+ T cells retrovirally transduced with GFP, Pcif1 WT-GFP, or Pcif1 N552A-GFP restimulated with 2 μg/mL anti-CD3/CD28 for 72 hours. n = 3 mice/each group. Each symbol represents an individual mouse. Two-tailed unpaired Student’s t-test (c-j,o) or two-way ANOVA followed by Sidak’s multiple comparisons test (a,k-n), two-way ANOVA followed by Tukey’s multiple comparisons test (p-r,t-y) were performed. Data are shown as the mean ± SEM.
Extended Data Fig. 7 Proteomic and m6Am-seq analysis identifies Pcif1 downstream targets.
a, Metabolic labeling analysis to measure total protein synthesis in both WT and Pcif1 KO CD8+ T cells. b, c, GSEA shows enrichment of ferroptosis (b) and T cell-mediated cytotoxicity (c) pathways in Pcif1 KO CD8+ T cells. NES: normalized enrichment score. d, m6Am levels of naïve CD8+ T cells were quantified by LC-MS/MS. n = 2 biological replicates with 3 technical replicates each. e, m6Am-seq workflow. Total RNA was fragmented and immunoprecipitated with a cap-m7G antibody (m7G-IP), followed by [FTO (+)] or [FTO (−)] treatment, then m6A-IP. Sequencing profiles were normalized and compared, identifying ‘Demethylase-sensitive peaks’ as m6Am. f, Correlation analysis of m6Am enrichments between replicates. g, Metaplot showing m6Am overlapped with CAGE-annotated TSSs. h, Relative mRNA levels of the top 10 genes in activated WT and Pcif1 KO CD8+ T cells. n = 3 mice/each group. i, Genome browser views of Fth1, Cd69 and Slc3a2 using m6Am-seq: The 5′-UTR methylation peak decreased in ‘FTO (+)’ vs. ‘FTO (-)’ samples, indicating a demethylase-sensitive m6Am (upper panel). High-SRD adenosine residues were defined as m6Am sites, overlapping with FANTOM TSS (lower). j, WB quantification of Fth1, Cd69, and Slc3a2 in splenic activated WT or Pcif1 KO CD8+ T cells. n = 3 mice/each group. k-m, RNA expression of target genes at specific time points. n = 3 mice/each group. n-p, RNA decay assay showing Fth1, Slc3a2, Cd69 mRNA level in activated CD8+ T cells post-actinomycin D treatment. n = 3 mice/each group. q, WB analysis of Ctbp2 knockdown on target genes expression in WT and Pcif1 KO CD8+ T cells. r, GO analysis of PCIF1-interacting proteins (log2 FC > 2) highlights top 10 enriched pathways. s, 7-AAD staining of activated CD8+ T cells, treated for 24 hours with 0.1 μM RSL3 or 20 μM Erastin. n = 3 mice/each group. Each symbol represents an individual mouse. One-sided permutation test (b,c), one-sided hypergeometric test, without adjustment (r), two-way ANOVA followed by Sidak’s multiple comparisons test (k-p,s) or two-tailed unpaired Student’s t-test (d,i,j) were performed. Data are shown as the mean ± SEM.
Extended Data Fig. 8 Pcif1-deficient CD3+ CAR T cells show improved anti-tumor efficacy.
a, Tumor growth of LLC cells was assessed in WT or Pcif1 cKO mice with indicated treatments. Tumor volumes of mice were measured every two days. n = 9 mice/each group. b, Overview of generation of CAR T cell production and a schematic treatment plan for C57BL/6 J mice bearing subcutaneous LLC-hCD19 or MC38-hCD19 cells. c-e, WT C57BL/6 J mice were injected subcutaneously with 1 × 105 LLC-hCD19 cells on day 0 and treated with 1 × 106 WT or Pcif1 KO anti-CD19 CD3+ CAR T cells on day 7. Tumor growth (c,e) and tumor weight (d) were monitored. n = 5 mice/each group. f-h, Flow cytometry analysis of IFNγ (f), TNF (g), and Gzmb (h) -expressing intratumoral anti-CD19 CD8+ CAR T cells in LLC-hCD19 tumors. n = 5 mice/each group. i-k, WT C57BL/6 J mice were injected subcutaneously with 1 × 105 MC38-hCD19 cells on day 0 and treated with 1 × 106 WT or Pcif1 KO anti-CD19 CD3+ CAR T cells on day 7. Tumor growth (i,k) and tumor weight (j) were monitored. n = 5 mice/each group. l-m, Flow cytometry analysis of IFNγ (l), TNF (m), and Gzmb (n) -expressing intratumoral anti-CD19 CD8+ CAR T cells in MC38-hCD19 tumors. n = 5 mice/each group. o,p, Tumor burden of RAJI cells was assessed in NCG mice with indicated treatments (o) and quantified on representative days (p) in different treatment groups. n = 5 mice/each group. Each symbol represents an individual mouse. Two-way ANOVA followed by Tukey’s multiple comparisons test (c,i) one-way ANOVA by Tukey’s multiple comparisons test (d,j), two-tailed unpaired Student’s t test (f-h,l-n) were performed. Data are shown as the mean ± SEM.
Extended Data Fig. 9 Pcif1-deficient CD8+ CAR T cells show enhanced anti-tumor efficacy.
a, Tumor growth of MC38-hCD19 cells was assessed in C57BL/6 J mice with 1 × 106 CD8+/CD4+ CAR T cells treatment. n = 5 mice/each group. b,c, Tumor growth and tumor cell death of MC38-hCD19 cells were assessed in C57BL/6 J mice with 2 × 105 CD8+ CAR T cells treatment. n = 5 mice/each group. d, In vitro cytotoxic activity of anti-CD19 CD8+ CAR T cells against MC38-hCD19 cells at various effector-to-target (E:T) ratios after 24 h co-culture. n = 3 mice/each group. e, The target protein levels in anti-CD19 CD8+ sh_CAR T cells of both WT and Pcif1 KO mice. f,g, Cellular Fe2+ assay of anti-CD19 CD8+ sh_CAR T cells detected with flow cytometry. n = 6 mice/each group. h,i, Representative examples and quantification of Oxidized BODIPY-FITC staining in anti-CD19 CD8+ sh_CAR T cells detected via flow cytometry. n = 6 mice/each group. j, Tumor growth of MC38-hCD19 cells was assessed in C57BL/6 J mice with 1 × 106 anti-CD19 CD8+ sh_CAR T cells treatment. n = 6 mice/each group. Each symbol represents an individual mouse. Two-tailed unpaired Student’s t test (c), two-way ANOVA followed by Tukey’s multiple comparisons test (d), one-way ANOVA by Tukey’s multiple comparisons test (g,i) were performed. Data are shown as the mean ± SEM.
Extended Data Fig. 10 The schematic of PCIF1 suppressing CD8+ T cell anti-tumor immunity.
Based on our results, PCIF1 inhibits CD8+ T cell anti-tumor activity through two parallel pathways. First, PCIF1 catalyzes m6Am modifications on FTH1 and SLC3A2 mRNAs, leading to the suppression of their protein expression, potentially promoting ferroptosis in CD8+ T cells. Second, PCIF1-mediated m6Am modification on CD69 mRNAs reduces its protein translation, thereby inhibiting CD8+ T cell activation, as evidenced by decreased effector cytokine production. When PCIF1 is deficient in CD8+ T cells, anti-tumor activity is enhanced by inhibiting ferroptosis through the upregulation of FTH1 and SLC3A2, and by promoting CD8+ T cell activation via increased CD69 expression.
Supplementary information
Supplementary Tables 1–6
Supplementary Table 1: The m6Am peaks on mRNAs within activated WT mice CD8+ T cells were detected through m6Am-seq. Supplementary Table 2: The identified m6Am sites on mRNAs within activated WT mouse CD8+ T cells were detected through m6Am-seq. Supplementary Table 3: The potential m6Am sites on mRNAs within activated WT mouse CD8+ T cells were detected through m6Am-seq. Supplementary Table 4: The list of PCIF1-interacting proteins within activated WT mouse CD8+ T cells was identified by IP–MS. Supplementary Table 5: Clinical characteristics of patients with OSCC undergoing immunotherapy. Supplementary Table 6: Primer sequences used in the main text.
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Statistical source data.
Source Data Extended Data Fig. 8
Statistical source data.
Source Data Extended Data Fig. 9
Statistical source data.
Source Data Extended Data Fig. 1
Unprocessed western blots.
Source Data Extended Data Fig. 5
Unprocessed western blots.
Source Data Extended Data Fig. 6
Unprocessed western blots.
Source Data Extended Data Fig. 7
Unprocessed western blots.
Source Data Extended Data Fig. 9
Unprocessed western blots.
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Xiang, B., Zhang, M., Li, K. et al. The epitranscriptional factor PCIF1 orchestrates CD8+ T cell ferroptosis and activation to control antitumor immunity. Nat Immunol (2025). https://doi.org/10.1038/s41590-024-02047-w
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DOI: https://doi.org/10.1038/s41590-024-02047-w
