Abstract
In vivo CRISPR screens in CD8+ T cells have previously uncovered targets for cancer immunotherapy; however, a minority of the genome has been individually annotated, suggesting that additional regulators remain to be discovered. Here we assessed 899 genes in CD8+ T cells responding to murine melanoma and identified the E3 ubiquitin ligase STUB1 as a new negative regulator of anti-tumor CD8+ T cell function. We demonstrated that Stub1 knockout CD8+ T cells effectively control tumor growth across multiple murine models. Mechanistically, STUB1 interacts with the adapter protein CHIC2 to regulate cytokine receptor expression in mouse and human CD8+ T cells. Among the regulated cytokine receptors, interleukin-27 receptor α is essential for tumor growth control mediated by Stub1/Chic2 knockout CD8+ T cells. Together, these findings establish the STUB1–CHIC2 complex as a regulator of cytokine receptor expression in CD8+ T cells and provide rationale for inhibiting this pathway to enhance CD8+ T cell-mediated anti-tumor immunity.
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Data availability
The CRISPR-inferred essential gene list is available at https://depmap.org/portal. Screen sequencing data have been deposited on the National Center for Biotechnology Information’s (NCBI’s) Gene Sequence Read Archive with the accession nos SRR28803792 and SRR32938604. RNA-seq data have been deposited on NCBI’s Gene Expression Omnibus with the accession nos GSE265929 and GSE294202. MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset accession nos PXD049402 and PDX062475. Flow cytometry data are available at ImmPort (https://www.immport.org) under the accession no. SDY3084. Source data are provided with this paper.
Code availability
All code and scripts used for data analysis related to the FAUST algorithm are available on GitHub at https://github.com/scyrusm/faust.
Change history
14 August 2025
Since the version of the article initially published, Source Data Fig. 2 has been amended to correct “Control-g1 (1/10)” and “Stub1-g1 (3/10)” to “Control-g1 (2/10)” and “Stub1-g1 (2/10)” for the Fig. 2e data. The updated Source data is now available online.
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Acknowledgements
We thank Y. Sun and Q. Tjokrosurjo for technical support. We thank the Center for Computational and Integrative Biology (CCIB) at Massachusetts General Hospital for the use of the CCIB DNA Core Facility, which performed CRISPR sequencing for indel assessment. We thank the Thermo Fisher Scientific Center for Multiplexed Proteomics at Harvard Medical School (https://tcmp.hms.harvard.edu), which performed the MS analyses. This work was supported by grant no. U19AI133524 from the National Institute of Allergy and Infectious Diseases to A.H.S. and J.G.D. M.W.L. is supported by a National Cancer Institute K99 award (no. 1K99CA290077-01A1). S.C.M. is supported by a Cancer Research Institute Immuno-Informatics Postdoctoral Fellowship (no. CRI5009) and was supported through the American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists. M.W.L. and A.H.S. also received funding for this work from: Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc.; the Blavatnik Biomedical Accelerator at Harvard University; the Quadrangle Fund for Advancing and Seeding Translational Research at Harvard Medical School; and Calico Life Sciences LLC.
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Contributions
Conceptualization: M.W.L. and A.H.S. Methodology: L.E.M., P.P., M.W.L., A.M.L., I.S.L.S., P.K.S.H., N.M.D., E.R., H.X., A.K. and J.G.D. Analysis: L.E.M., P.P., A.M.L., I.S.L.S., P.K.S.H., N.M.D., V.L., E.R., H.X., T.H.N., S.C.M. and M.W.L. Investigation: L.E.M., P.P., V.L., A.M.L., I.S.L.S., P.K.S.H., N.M.D., E.R., H.X., A.K. and M.W.L. Writing—original draft: M.W.L., L.E.M., A.M.L., P.P. and A.H.S. Writing—review and editing: all authors. Supervision: M.W.L. and A.H.S. Funding acquisition: A.H.S. and M.W.L.
Corresponding authors
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Competing interests
A.H.S. has patents or is pending royalties on the PD-1 pathway from Roche and Novartis. M.W.L., L.E.M., I.S.L.S., A.M.L., P.K.S.H., N.M.D. and A.H.S. have a patent on methods for modulating STUB1 for the treatment of cancer. A.H.S. is on the advisory boards for Elpiscience, Monopteros, Corner Therapeutics, Bioentre, Alixia, GlaxoSmithKline, Janssen, Amgen, AltruBio, ImmVue and MabQuest, and receives research funding from Calico Life Sciences LLC and Taiwan Bio. J.G.D. consults for Microsoft Research, Abata Therapeutics, Servier, Maze Therapeutics, BioNTech, Sangamo and Pfizer, consults for and has equity in Tango Therapeutics, serves as a paid scientific advisor to the Laboratory for Genomics Research, is funded in part by GlaxoSmithKline and receives funding support from the Functional Genomics Consortium: AbbVie, Bristol Myers Squibb, Janssen and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc. His interests were reviewed and are managed by the Broad Institute in accordance with its conflict-of-interest policies. The other authors declare no competing interests.
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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.
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Extended data
Extended Data Fig. 1 Additional data related to Fig. 1.
(a) Gating for the naive screen. (b-c) Tumor growth curves for the (b) naive screen (n = 6 groups of 8-10 mice) or (c) activated screen (n = 4 groups of 10 mice). (d-e) Number of recovered transferred T cells in the (d) naive screen or (e) activated screen (Naive screen: n = 6 groups of 8-10 mice and n = 3 input samples, Activated screen: n = 4 groups of 10 mice and n = 2 input samples). (f-g) Number of recovered gRNAs in the (f) naive screen or (g) activated screen. The dotted orange line indicates the maximum gRNAs recoverable (Naive screen: n = 6 groups of 8-10 mice and n = 3 input samples, Activated screen: n = 4 groups of 10 mice and n = 2 input samples). (h-i) Comparison of the log2-normalized fold change in gRNA abundance between the tdLN and input (left) or the tumor and input (right) between replicates in the naive (h) or activated (i) screens. Intraclass correlation coefficient (ICC2) values are listed (n = 6 for the naive screen and n = 4 for the activated screen). (j-l) Screen results for tumor normalized to tdLN. Volcano plots of the median CLES at the gRNA level and q-value at the gRNA-UMI level in the naive screen (j) or activated screen (k). Comparison of the median CLES for the naive and activated screens (l) (Naive screen: n = 6 groups of 8-10 mice, performed twice; Activated screen: n = 4 groups of 10 mice, performed once; Both screens: one-sided FAUST test on gRNA-UMI counts with liberal setting and Benjamini-Hochberg correction). Spearman’s correlation was used to test the significance of correlation. Data represent: mean and s.e.m. (b,c) or mean and s.d. (d-g).
Extended Data Fig. 2 Additional data related to Fig. 2.
(a) Percentage of Stub1 insertion deletions (indels) in Control-g1-, Stub1-g1-, or Stub1-g2-nucleofected naive CD8+ T cells. (b) Quantification (as in Fig. 2c) of the log2-normalized fold change at day 10 in the tdLN relative to input for the indicated populations (n = 6-8 replicate animals per group, performed twice, one-sided one-way ANOVA with Tukey’s multiple comparisons test within each model, the double-headed bar indicates each pairwise comparison). (c) Quantification of the log2-normalized fold change in the spleen at day 4 relative to input for Stub1-g1-nucleofected naive OT-1 CD8+ T cells in B16-OVA-challenged mice (n = 8 replicate animals per group, performed twice, two-sided ratio paired t-test). (d) Survival curve for LLC-OVA-challenged mice in Fig. 2g (n = 10 mice per group, performed twice, two-sided Mantel-Cox test performed on indicated comparisons). (e) Percentage of indels in Stub1 for the indicated bone marrow chimeras (BMC) in Fig. 2i. (f) Changes in mouse weight throughout BMC study in Fig. 2i (n = 10 mice per group, performed twice, one-sided two-way ANOVA with Tukey’s multiple comparisons test, the double-headed bar indicates each pairwise comparison, selected comparisons are plotted). (g) Survival curve for mice in Fig. 2i (n = 10 mice per group, performed twice, two-sided Mantel-Cox test performed on indicated comparisons). (h) Tumor growth curves for BMCs challenged with B16-OVA tumors (n = 9-10 mice per group, performed twice, one-sided one-way ANOVA with Tukey’s multiple comparisons test). (i) Survival curve for mice in (h) (n = 9-10 mice per group, performed twice, two-sided Mantel-Cox test performed on indicated comparisons). (j) Survival curve for mice in Fig. 2j (n = 9-10 mice per group, performed twice, two-sided Mantel-Cox test performed on indicated comparisons). Data represent: mean and s.d. (b,c,e,f) or mean and s.e.m. (h).
Extended Data Fig. 3 Additional data related to Fig. 3.
(a-b) Western Blots (a) and protein quantification (b) of STUB1 and β-ACTIN from naive or activated CD8+ T cells (n = 2 biological replicates per group). (c) Stub1 expression normalized to 18S rRNA from naive CD8+ T cells and CD44+ intratumoral CD8+ T cells. (n = 3-4 biological replicates per group, two-sided Student’s unpaired t-test). (d-e) Control-g1- and Stub1-g1-nucleofected naive OT-1 T cells were transferred into mice subsequently challenged with B16-OVA tumors. The percentages of SLAMF6+ TIM3−, CX3CR1+ TIM3+, and CX3CR1− TIM3+ cells were quantified in the (d) tdLN and (e) tumor (n = 5-6 mice per group, multiple two-sided Student’s unpaired t-tests with FDR correction, q-value < 0.025 is significant). (f-i) RNA sequencing of control and Stub1 KO OT-1 T cell isolated from B16-OVA tumors (n = 2-3 biological replicates per group). (f) Principal component analysis of intratumoral OT-1 T cells. (g) Differential genes in Stub1 KO vs. control cells. Dotted lines indicate fold change of –1 or 1 (x-axis) and adjusted p-value of 0.05 (y-axis), (two-sided DESeq2 adjusted for multiple comparisons). (h-i) Enriched signatures in the Stub1 KO cells (h) or control cells (i). Gene ratios are plotted for each bar (hypergeometric test adjusted for multiple comparisons). (j) Percentage of killing of SIINFEKL-pulsed B16 tumor cells by Control-g1- or Stub1-g1-nucleofected OT-1s (n = 3 biological replicates per group, two-sided Student’s unpaired t-test). (k-l) Schematic (k) and tumor growth curves (l) following transfer of WT Control-g1-, WT Stub1-g1-, or PERFORIN KO Stub1-g1-nucleofected naive OT-1 CD8+ T cells into mice that were subsequently challenged with B16-OVA tumor cells (n = 9-10 mice per group, one-sided one-way ANOVA with Tukey’s multiple comparisons test). Data represent: mean and s.d. (b-e,j) or mean and s.e.m. (l). All experiments were performed twice (except f-i, performed once).
Extended Data Fig. 4 Additional data related to Fig. 4.
(a) qPCR of Chic2 from naive CD8+ T cells and CD44+ CD8+ T cells isolated from B16-OVA tumors. Chic2 expression was normalized to 18S rRNA expression. All values were normalized to the average expression of Chic2 in naive CD8+ T cells (n = 3-4 biological replicates per group, performed twice, two-sided Student’s unpaired t-test). (b) Percentage of indels in Chic2-g1-nucleofected naive CD8+ T cells. (c-g) Control-g1-, Stub1-g1-, or Chic2-g1-nucleofected naive OT-1 CD8+ T cells were adoptively transferred into mice that were subsequently challenged with B16-OVA tumor cells. (c-e) Quantification of the percentages of tumor-infiltrating OT-1 T cells expressing (c) CX3CR1, (d) GRANZYME B and PERFORIN, and (e) indicated coinhibitory receptors. (f-g) Quantification of the percentages of OT-1 T cells in the tdLN expressing (f) TOX and (g) T-BET (n = 10 mice per group, performed twice, one-sided one-way ANOVA with Tukey’s multiple comparisons test for each marker/population). (h) Survival curves for Fig. 4j (n = 10 mice per group, performed twice, two-sided Mantel-Cox test performed on indicated comparisons). Data represent: mean and s.d. (a,c-g).
Extended Data Fig. 5 Additional data related to Fig. 5.
(a) Representative histograms (left) and quantification (right) of IL6ST surface expression in activated Control-g1-, Stub1-g1-, or Chic2-g1-nucleofected CD8+ T cells (n = 3, performed twice, one-sided one-way ANOVA with Tukey’s multiple comparisons test). (b-c) Control-g1-, Stub1-g1-, or Chic2-g1-nucleofected naive OT-1 CD8+ T cells were adoptively transferred into mice that were subsequently challenged with B16-OVA tumor cells. Quantification of the MFI of FOXP3 (b) and PD-L1 (c) for tumor-infiltrating OT-1 T cells expressing these markers (n = 10 mice per group, performed twice, one-sided one-way ANOVA with Tukey’s multiple comparisons test). (d) Western Blots of IFNγR1, IL-27Rα, and β-ACTIN (loading control) from Control-g2-nucleofected, Stub1 KO, or Chic2 KO EL4 cells (performed twice). Data represent: mean and s.d. (a-c).
Extended Data Fig. 6 Additional data related to Fig. 6.
(a) Pairwise structure alignment of crystal structures of the STUB1 TPR domain (human-8EHZ and 7TB1) and full-length STUB1 (mouse-2C2L). (b) Western Blots of IL-27Rα, phosphorylated p62 (positive control), β-ACTIN (loading control), and IFNγR1 following treatment of EL4 cells with vehicle or MG132 (performed twice). (c) Western Blots of V5, IL-27Rα, and IFNγR1 following V5 immunoprecipitation (left) and corresponding whole cell lysates with β-ACTIN loading control (right) of EL4 cells transduced with Chic2-V5-tagged cDNAs (performed twice).
Extended Data Fig. 7 Additional data related to Fig. 7.
(a) Percentage of indels in Ifngr1 and Il27ra in naive CD8+ T cells. (b) Schematic of the IL-27 neutralization experiment. (c) Quantification of the number of transferred cells in the tumors of isotype or αIL-27p28-treated mice that received a 50:50 mix of Control-g1- and Stub1-g1-nucleofected naive OT-1 T cells (n = 10 replicate animals per group, performed twice, two-sided Student’s paired t-test for comparisons within the isotype or αIL-27p28 group and two-sided Student’s unpaired t-test for comparisons between isotype and αIL-27p28 groups). (d) Representative histograms (left) of IL-27Rα expression on Control-g1-, Stub1-g1-, Chic2-g1-, or Stub1-g1/Chic2-g1-nucleofected OT-1 CD8+ T cells from B16-OVA tumors and IL-27Rα MFI of IL-27Rα-expressing cells (right) (n = 10 mice per group, performed twice, one-sided one-way ANOVA with Tukey’s multiple comparisons test, the triple-headed bar indicates all pairwise comparisons). (e) Quantification of phosphorylated STAT1 (left) and phosphorylated STAT3 (right) following IL-27 stimulation of activated CD8+ T cells in vitro (n = 1-2 mice per group, performed four times, graph represents pooled data from the four experiments, mixed-effects analysis, p-value is for analysis of the curves). (f-h) (f) Quantification of the percentage of IL-27Rα-expressing cells and IL-27Rα MFI. (g) Representative histogram of CXCR3 expression, quantification of the percentage of CXCR3-expressing cells, and CXCR3 MFI. (h) Quantification of the percentage of CXCR3-expressing cells and CXCR3 MFI on IL-27Rα+ cells (n = 8 replicate animals per group, performed twice, two-sided Student’s paired t-test). (i) Schematic of competitive experiment to analyze Stub1/Il27ra double KO CD8+ T cells in the tdLN. (j) Quantification of the percentage of cells expressing CXCR3 and the CXCR3 MFI on CXCR3-expressing cells (n = 7 replicate animals per group, performed twice, two-sided Student’s paired t-test). Data represent: mean and s.d. (c,d,f-h,j) or mean and s.e.m. (e).
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2 and Note 1.
Supplementary Table 1
Counts from RNA-seq of naive CD8+ T cells and CD8+ T cells isolated from the tdLN and tumor of mice bearing B16-OVA tumors.
Supplementary Table 2
Naive and activated T cell CRISPR screen gRNA list, UMI list, raw gRNA/gRNA-UMI counts and gene scoring.
Supplementary Table 3
Raw counts and DEseq2 output from RNA-seq of control and Stub1 KO CD8+ T cells from B16-OVA tumors.
Supplementary Table 4
Ubiquitin MS raw data, normalized values and fold changes for primary CD8+ T cells.
Supplementary Table 5
Proteome MS raw data, normalized values and fold changes for primary CD8+ T cells.
Supplementary Table 6
Proteome MS raw data, normalized values and fold changes for EL4 cells.
Supplementary Table 7
Reagents and resources.
Supplementary Data
Protein Data Bank validation reports for 7TB1, 8EHZ and 2C2L.
Source data
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LaFleur, M.W., Milling, L.E., Prathima, P. et al. A STUB1–CHIC2 complex inhibits CD8+ T cells to restrain tumor immunity. Nat Immunol 26, 1476–1487 (2025). https://doi.org/10.1038/s41590-025-02231-6
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DOI: https://doi.org/10.1038/s41590-025-02231-6
