ScRNA-seq and bulk RNA-seq data reported in this paper are accessible at the Gene Expression Omnibus under accession numbers GSE210704 and GSE212179, respectively. MS data have been deposited in an international public repository (MassIVE proteomics repository at https://massive.ucsd.edu/) under dataset accession number MSV000087205. There are no restrictions for data availability. Source data are provided with this paper.
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We thank members of the Wen laboratory for discussion; Y. Wan for critical feedback; M. Croft for the OT-II mice; Y. Fu for the MC38 cell line; Y. Yu from the University of Delaware Mass Spectrometry Core Facility for the assistance of mass spectrometry assay; P. Yan and X. Chen from the Genomics Shared Resource at the Ohio State University for the assistance of bulk RNA-seq and scRNA-seq assay. This work was supported by National Institutes of Health grants R01GM135234 and R01AI162779 (H.W.), R01AI077283 and R01CA262089 (Z.L.), R01GM131399 (Q.M.), R01DE026728 (Y.L.), R01AI134972 (K.J.O.) and The Ohio State University Comprehensive Cancer Center Intramural Research Program (A.M., D.M.J., K.H. and H.W.).
The authors declare no competing interests.
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Extended Data Fig. 1 Susd2−/− mice show no change in global immune cell populations at steady state.
a, Cartoon of the strategy to generate Susd2−/− mice with a CRISPR/Cas9-mediated genome engineering strategy. The sequences of two guide RNA and the primers used for genotyping were shown. b, Genotyping results for WT or Susd2−/− alleles. c-j, Flow cytometry analysis of T cells (CD3+) and natural killer cells (NK1.1+) (c), CD4+ and CD8+ T cells (d), regulatory T cells in naïve status (CD4+CD25+) (f), macrophage (CD11b+F4/80+) (g), conventional dendritic cells (CD11b+CD11c+) (h), monocytes (CD11b+Ly6C+) (i), neutrophils (CD11b+Ly6G+) (j), and histogram of B cells (CD19+) (e) in spleen from wild-type and Susd2−/− mice were. c-j, WT, n = 4 mice, Susd2−/−, n = 5 mice. b-j, data are representative of three independent experiments. Statistical significance was determined by two-tailed, unpaired Student’s t-test, there is no significant difference between WT and Susd2−/− in c-j (P > 0.05). All data are mean ± SD.
Extended Data Fig. 2 Susd2 deficiency does not affect intratumoral myeloid cells, NK cells and CD4+ T cells.
a-g, Flow cytometry analysis of CD11b+F4/80+ macrophages (a), CD11b+CD11c+ dendritic cells (b), CD11b+Ly6C+ monocytes (c), CD11b+Ly6G+ neutrophils (d), NK1.1+ NK cells (e), IFN-γ+NK1.1+ NK cells (f), IFN-γ+CD4+, GzmB+CD4+ and TNF+CD4+ T cells (g) in MC38 tumor isolated from WT or Susd2−/− mice at Day 18. h,i, Spectral flow cytometry analysis of intratumor CD8+ T cells from WT and Susd2−/− mice at day18 post MC38 tumor inoculation. UMAP of individual marker expression patterns (h) and frequencies of individual clusters of WT and Susd2−/− samples (i), Boxes represent median and 25th to 75th percentiles, whiskers are minimum to maximum values excluding outliers (two-sided Wilcoxon’s rank-sum P value). j-l, Flow cytometry analysis of PD-1+CD8+ T cells and LAG-3+CD8+ T cells in MC38 (j), EG7 (k) or B16-OVA (l) tumors isolated from WT or Susd2−/− mice at day 18 post tumor inoculation. a–d,g,j-l, n = 5, e,f,i, n = 8. n, number of mice per group. a–d,g,j,k,l, data are representative of three independent experiments, e,f,i, data are representative of two independent experiments. a-g, j-l, statistical significance was determined by two-tailed, unpaired Student’s t-test, there is no significant difference between WT and Susd2−/− group (P > 0.05). All data are mean ± SD.
a, Flow cytometry analysis of IFN-γ+CD8+ T cells, GzmB+CD8+ T cells and TNF+CD8+ T cells in different OVA257–264 dosage stimulated splenocytes isolated from WT or Susd2−/− OT-I mice. b-e, CD8+ T cells were isolated from total splenocytes of either WT or Susd2−/− OT-I mice left untreated or stimulated with OVA257–264 for 3 days and were subjected to RNA-seq assay. The volcano plot of RNA-seq data demonstrates differential gene expression between WT and Susd2−/− CD8+ T cells at Day 0 (b) and Day 3 (d). A heat map of the top thirty genes representing genes differentially expressed between WT and Susd2−/− CD8+ T cells at Day 0 (c) and Day 3 (e). f, Intracellular accumulation of IFN-γ in CD8+ T cells isolated from WT or Susd2−/− OT-I mice that were co-cultured with either WT or Susd2−/− bone marrow-derived dendritic cells (BMDCs) that have been pulsed with OVA257–264. a,f, n = 3, b-e, n = 4. n, number of mice per group. a,f, data are representative of four independent experiments. b-e, data are representative of two independent experiments. b,d statistical significance was calculated using two-sided Wilcoxon’s rank-sum test and adjusted with Bonferroni’s correction. Statistical significance was determined by two-way ANOVA followed by Sidak’s multiple comparisons test(a,f) with P values noted in the figure. All data are mean ± SD.
Extended Data Fig. 4 Susd2 deficiency does not affect effector function of CD4+ T cells or inhibitory function of Treg cells.
a, Flow cytometry analysis of IFN-γ+CD4+ T cells, GzmB+CD4+ T cells and TNF+CD4+ T cells in OVA323–339 stimulated splenocytes isolated from WT or Susd2−/− OT-II mice. b, Flow cytometry analysis of intranuclear level of Foxp3 in spleen CD4+ T cells from WT or Susd2−/− mice. c, Cell proliferation of naïve CD4+ T cells upon stimulation with CD3-CD28 antibody in the absence or presence of WT or Susd2−/− Treg cells at the indicated cell: cell ratio was measured by the staining of carboxyfluorescein diacetate succinimidyl ester (CFSE), followed by FACS analysis. a–c, n = 3. n, number of mice per group. Data are representative of four independent experiments. Statistical significance was determined by two-tailed, unpaired Student’s t-test, there is no significant difference between WT and Susd2−/− group in a–c (P > 0.05). All data are mean ± SD.
a, Sanger sequencing result of pCMV3×Flag-IL2RA vector. b, Immunoblotting of Flag-IL2Rα in 293 T cells transfected with pCMV3×Flag-IL2RA vector. Data are representative of three independent experiments.
a, Transcript of Susd2 in mouse CD8+ T cells stimulated with CD3-CD28 antibody, IL-2, IL-7 or IL-15 for 0, 1, 2 and 3 days. b-d, Immunoblotting of STAT5 in OVA257–264-primed WT or Susd2−/− OT-I T cells which were rested overnight, and then stimulated with IL-2 (100 U/ml), IL-7 (5 ng/ml) or IL-15 (10 ng/ml) for 0, 30, 60, 120 and 240 minutes. e, Flow cytometry analysis of p-STAT5 in WT or Susd2−/− Treg cells stimulated with IL-2 (100 U/ml) for 0, 30, 60 and 120 minutes. f, CD8+ T cells isolated from WT or Susd2−/− OT-I mice were co-cultured with WT or Susd2−/− bone marrow-derived dendritic cells (BMDCs) that have been pulsed with OVA257–264. Intracellular accumulation of IFN-γ in WT or Susd2−/− CD8+ T cells in the absence or presence of blocking antibodies against IL-2, IL-2Rα, or IL-2Rβ were measured by FACS analysis. g,h, Tumor growth in WT and Susd2−/− mice bearing B16-F10 tumor cells which were injected with IL-2/AbCD25 complex (g) or IL-2/AbCD122 complex(h). i-k, Flow cytometry analysis of intracellular accumulation of IFN-γ, GzmB and TNF-expressing intratumoral CD8+ T cells. a,e,f, n = 3; g-k, n = 5. n, number of mice per group. a-h, data are representative of three independent experiments; i-k, data are representative of two independent experiments. Statistical significance was determined by two-way ANOVA followed by Sidak’s multiple comparisons test (e-g) or one-way ANOVA followed by Tukey’s test (a,i-k) with P values noted in the figure. All data are mean ± SD.
a, Validation of EL4 thymoma cell line expressing human CD19 (EL4-hCD19) with the deletion of its intracellular domain. b, Percentages of CD8+ T cells retrovirally transduced with a chimeric antigen receptor (CAR) containing a portion of hCD19 single chain variable fragment (ScFv) fused with signaling domains of mouse CD28 and mouse CD3ζ sequence (with first and third ITAMs of the CD3ζ molecule inactivated) before and after cell sorting were assessed by the staining with anti-Thy1.1 antibody. c, Transcript of Susd2 in CAR T cells that have been electroporated with scrambled gRNA(sgRNA) or Susd2 gRNA-Cas9 nucleoprotein (RNP) complex, sgRNA versus Susd2 gRNA (P = 0.0010). c, n = 3. a–c, data are representative of two independent experiments. Statistical significance was determined by two-tailed, unpaired Student’s t-test (c) with P values noted in the figure. The data represent mean ± SD.
Extended Data Fig. 8 A model for an inhibitory role of SUSD2 in effector CD8+ T cell antitumor immunity by modulating IL-2R signaling.
The present study has identified SUSD2 as a negative regulator of IL-2-mediated effector CD8+ T cell functions and antitumor immunity. Both SUSD2 and IL-2Rα chain (IL-2Rα) contain the sushi domain (SD). Genetic ablation of SUSD2 (Susd2−/−) leads to elevated IFN-γ, GzmB and TNF production in effector CD8+ T cells and improved tumor growth control in multiple syngeneic tumor models. Mechanistically, SD-dependent interaction between SUSD2 and IL-2Rα competitively inhibits IL-2-IL-2Rα binding, leading to an attenuated IL-2R signaling. Therefore, SUSD2 represents a promising therapeutic target of tumor immunotherapy. Green and red arrows indicate promoting and inhibiting effect, respectively.
Supplementary Table 1. Marker transcripts of the 18 single-cell clusters. Supplementary Table 2 Marker transcripts of the four CD8+ T cell clusters. Supplementary Table 3 DEGs between WT and Susd2 KO at Day 0. Supplementary Table 4 DEGs between WT and Susd2 KO at day 3. Supplementary Table 5 The list of Susd2-interacting proteins detected by anti-V5 immunoprecipitates coupled with MS analysis. Supplementary Table 6 Primer sequences for molecular cloning. Supplementary Table 7 Primer sequences for site-directed mutagenesis. Supplementary Table 8 Primer sequences for RT–PCR
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Zhao, B., Gong, W., Ma, A. et al. SUSD2 suppresses CD8+ T cell antitumor immunity by targeting IL-2 receptor signaling. Nat Immunol (2022). https://doi.org/10.1038/s41590-022-01326-8
Received: 02 December 2021
Accepted: 08 September 2022
Published: 20 October 2022