c-Rel is a myeloid checkpoint for cancer immunotherapy (original) (raw)

Data availability

Microarray and RNA-seq data that support the findings of this study have been deposited in the ArrayExpress under accession nos. E-MTAB-8674 and E-MTAB-8714. Source data for Fig. 1 and Figs. 4–7 are provided with this paper and are available online. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank H.-C. Liou (Cornell University), W. Weinberg (US FDA) and J. Zakrzewski (Cornell University) for providing the breeding pair of the _Rel_−/− mice. We thank D. Gabrilovich, J. Goldsmith, P. Fang, L. Wan, M. Lin, D. Zhang, L. Guan, J. Devergiilis and J. Sun for valuable discussions, technical support and reagents. We thank the University of Pennsylvania Pancreatic Islet Cell Biology Core and D.P. Beiting from the University of Pennsylvania School of Veterinary Medicine for technical support. This work was supported in part by grants from the National Institutes of Health (nos. R01-AI152195, R01-AI099216, R01-AI121166, R01-AI143676 and R01-AI136945 to Y.H.C.); X.L. was partially supported by grant no. NIH-T32-DK007780.

Author information

Author notes

  1. These authors contributed equally: Ting Li, Xinyuan Li.

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
    Ting Li, Xinyuan Li, Ali Zamani, Wei Wang, Chin-Nien Lee, Mingyue Li, George Luo, Emily Eiler, Honghong Sun & Youhai H. Chen
  2. Department of Microbiology and Immunology, College of Physicians & Surgeons, Columbia University, New York, NY, USA
    Sankar Ghosh
  3. Mount Sinai Center for Therapeutics Discovery, Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Jian Jin
  4. Department of Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Jian Jin
  5. Department of Biomedical Sciences, Research Division of Immunology, Cedars-Sinai Medical Center, Los Angeles, CA, USA
    Ramachandran Murali
  6. Shandong Eye Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China
    Qingguo Ruan & Weiyun Shi

Authors

  1. Ting Li
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  2. Xinyuan Li
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  3. Ali Zamani
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  4. Wei Wang
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  11. Jian Jin
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  12. Ramachandran Murali
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Contributions

T.L. and X.L. designed and executed the experiments and wrote the manuscript. A.Z. designed and performed some of the MDSC suppression experiments. H.S., M.L., W.W., C.-N.L., G.L., E.E., Q.R. and W.S. helped complete certain molecular, cellular or animal experiments. S.G. provided the Rel gene floxed mice. J.J. and R.M. designed and optimized the c-Rel inhibitor. Y.H.C. conceived and supervised the study and wrote the manuscript.

Corresponding author

Correspondence toYouhai H. Chen.

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Competing interests

Y.H.C. and R.M. are inventors of the following patent that describes the c-Rel inhibitor used in this study: Chen Y.H., Murali R. & Sun J. Rel inhibitors and methods of use thereof (USA patent no. US8609730B2). Y.H.C. is a member of the advisory board of Amshenn Pharmaceutical Company and Binde Company.

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Extended data

Extended Data Fig. 1 Global and myeloid Rel gene deletion blocks tumor growth and reduces MDSCs in mice.

a, Tumor growth in WT and _Rel_−/− mice (n = 5 mice/group) injected s.c. with B16F0 tumor cells (***, P < 0.0001). b, Tumor weight of WT and _Rel_−/− mice (n = 10 mice/group) treated in Fig. 1a. (*, P = 0.0188). c, Percentages of CD11b+Gr-1+ cells in the blood of WT and _Rel_−/− mice treated with anti-Gr-1 or IgG isotype control (n = 3 mice/group), 7 days post B16F10 tumor cell inoculation (**, P = 0.0037; ***, P < 0.0001). d, Percentages of CD11b+Gr-1+ cells in the tumor of WT mice treated with anti-Gr-1 or IgG isotype control (n = 3 mice/group), 7 days post B16F10 tumor cell inoculation (***, P = 0.0008). e, Percentages of CD4+CD25+ cells in the spleen of WT and _Rel_−/− mice treated with anti-CD25 or IgG isotype control (n = 3 mice/group), 14 days post B16F10 tumor cell inoculation (***, P < 0.0001). f, Tumor size on Day 14 of WT and _Rel_−/− mice treated as in Fig. 1c–e. n = 16 for the WT + IgG group, n = 15 for the _Rel_−/−+IgG group, n = 11 for the WT + anti-Gr1 group, n = 10 for the _Rel_−/−+anti-Gr1 group, n = 9 for the WT + anti-CD25 group, and n = 9 for the _Rel_−/−+anti-CD25 group (*, P = 0.0303; **, P < 0.01; ***, P = 0.0001). g, c-Rel expression in Gr-1+ and Gr-1- splenocytes of LyzM-Cre (Cre) and LyzM-Cre _Rel_F/F (_Rel_F/F) tumor-bearing mice as determined by Western blot. Representative blots from biologically independent experiments were shown and the bar graph shows the relative quantities of the c-Rel protein in the corresponding group shown below (n = 3 mice for each group; *, P = 0.0101). h,i, Percentages of CD11b+Ly6G+ (h) and CD11b+Ly6C+ (i) leukocytes in the tumor of WT and _Rel_−/− mice treated in Fig. 1a (n = 5 mice/group). **, P = 0.0011 for panel h; **, P = 0.0087 for panel i. j, Percentages of CD8+CD25+ leukocytes in the tumor of LyzM-Cre and LyzM-Cre _Rel_F/F mice treated in Fig. 1f (n = 8 mice/group; ***, P = 0.0005). k-n, Percentages of the indicated leukocyte subsets in the spleen of WT and _Rel_−/− mice treated in Fig. 1a. (n = 4 mice/group in the panels k and l; n = 5 mice/group in the panels m and n; *, P = 0.037). Statistical significance was determined by two-tailed Mann-Whitney _U-_test (a), two-tailed unpaired _t-_test (b-f, h-j, m), or one-way ANOVA with Tukey post-hoc test (g). For all panels, data are presented as means ± s.e.m.

Source data

Extended Data Fig. 2 Percentages of immune cell subsets in tumor-bearing and naïve LyzM-Cre and LyzM-Cre _Rel_F/F mice.

a, Percentages of CD11b+Gr-1+ cells in the spleen of mice treated in Fig. 1f. n = 6 mice/group. b-d, Percentages of CD4+ cells in the spleen (b, n = 6/group), blood (c, n = 6/group), and tumor (d, n = 8/group) of mice treated in Fig. 1f. e-g, Percentages of CD4+CD25+ cells in the spleen (e, n = 6/group), blood (f, n = 6 for the LyzM-Cre group and n = 5 for the LyzM-Cre _Rel_F/F group), and tumor (g, n = 3 for the LyzM-Cre group and n = 6 for the LyzM-Cre _Rel_F/F group) of mice treated in Fig. 1f. h-j, Percentages of CD8+ cells in the spleen (h, n = 6/group), blood (i, n = 6/group), and tumor (j, n = 8/group) of mice treated in Fig. 1f. k-n, Percentages of the indicated leukocyte subsets in the tumor (n = 3 for the LyzM-Cre group and n = 6 for the LyzM-Cre _Rel_F/F group) of mice treated in Fig. 1f (k, l), and the spleen (n = 3 for the LyzM-Cre group and n = 5 for the LyzM-Cre _Rel_F/F group) of naïve mice (m, n). (***, P = 0.0002) Statistical significance was determined by two-tailed unpaired _t-_test (k). For all panels, data are presented as means ± s.e.m.

Source data

Extended Data Fig. 3 Reduced suppressive function, reactive oxygen species (ROS) production, and cell growth in _Rel_−/− MDSCs.

a,b, Representative flow cytometry plots from the MDSC-T cell suppression assay for Fig. 2a (a) and Fig. 2b (b). c, Growth of bone marrow-derived MDSCs from WT (n = 4 mice/group) and _Rel_−/− (n = 9 mice/group) mice. (*, P = 0.03299). d, ROS production by bone marrow-derived MDSCs from WT and _Rel_−/− mice (n = 6 mice/group; ***, P < 0.0001). e, Percentages of CD11b+Ly6G+ and CD11b+Ly6C+ cells in bone marrow-derived MDSCs from WT and _Rel_−/− mice. Data representative of three independent experiments. f, Tumor growth in WT mice injected s.c. with B16F10 tumor cells plus _Rel_−/− MDSCs infected with control (n = 5 mice) or _Rel_-expressing retroviruses (n = 6 mice) (**, P = 0.0041). g, Percentages of CD44high cells in intratumor CD8+ cells of WT mice treated as in panel f. (n = 4 mice/group; *, P = 0.0187). h, The degree of suppression, at the indicated Effector:T cell ratio, of CD8+ T cell proliferation by bone marrow-derived MDSCs and BMDMs from naïve mice (n = 3 mice/group). The concentrations of anti-CD3 and anti-CD28 used (125 ng/mL each) were half of those in Fig. 2b (***, P < 0.0001). i, Representative flow cytometry plots for the MDSC-T cell suppression assay for Panel h. Statistical significance was determined by two-tailed unpaired _t-_test (c-e, g), two-tailed Mann-Whitney _U-_test (f), or two-way ANOVA with Tukey post-hoc test (h). Data are presented as means ± s.e.m. (c,d,f,h).

Source data

Extended Data Fig. 4 Rel gene deletion in MDSCs leads to upregulated expression of inflammatory genes and decreased Cebpb downstream genes.

a,b, Results from Ingenuity Pathway analysis of the RNA-seq data of bone marrow-derived WT and _Rel_−/− MDSCs, showing upregulated ‘inflammatory response’ genes (a, red) and downregulated Cebpb downstream genes (b, green) in _Rel_−/− MDSCs. Statistical significance was determined by calculated a right-tailed Fisher’s Exact Test. All data are pooled from 3 independent experiments.

Extended Data Fig. 5 Rel gene deletion in macrophages results in decreased expression of inflammatory genes.

BMDMs from WT and _Rel_−/− mice were treated with vehicle or LPS (100 ng/mL) for 1 hour and gene microarray analysis was performed for ~30,000 murine genes. Ingenuity Pathway Analysis was performed to identify c-Rel-regulated genes that were downstream of LPS response. Blue genes were decreased, and red genes were increased in _Rel_−/− cells as compared to WT cells, after LPS treatment.

Extended Data Fig. 6 The inhibitor is c-Rel-specific and blocks MDSC suppressive functions.

a, Relative numbers of WT and _Rel_−/− human Jurkat T cells treated with the c-Rel inhibitor (2.5 μM) or vehicle for the indicated times (n = 3 biologically independent cultures/group). b,c, Relative numbers of EL4 (b, n = 3 biologically independent cultures/group) and B16F10 (c, n = 3 biologically independent cultures/group) cells treated with or without the c-Rel inhibitor (5 μM) (**, P = 0.00767). d,e, Relative mRNA levels of IL-2 in WT, _Rel_−/− human Jurkat T cells (d, n = 3 biologically independent cultures/group; **, P = 0.0047), and normal human PBMCs (e, n = 3 biologically independent cultures/group; *, P = 0.0241) that were treated with or without c-Rel inhibitor (1 μM). For stimulation, plate-coated anti-mouse CD3 (250 ng/ml) plus soluble anti-mouse CD28 (250 ng/ml), or PMA (10 ng/ml) plus ionomycin (1 µM) were added to the culture for 4 hours, as indicated. f, Preferential inhibition of c-Rel binding to DNA by the c-Rel inhibitor. Western blot for the indicated NF-κB proteins after the NF-κB oligonucleotide pull-down of the nuclear extracts of WT and _Rel_−/− splenocytes treated with the c-Rel inhibitor (5 µM) or vehicle control (Ctr). g, Body weight change of mice that were injected i.p. with vehicle control only (n = 7 mice), or injected s.c. with B16F10 cells and i.p. with the c-Rel inhibitor (n = 3 mice) or vehicle control (n = 4 mice) as indicated. h,i, Representative flow cytometry plots of the MDSC-T cell suppression assay for Fig. 6e (h) and Fig. 6f (i). Data are presented as means ± s.e.m (a-e, g, and i). n = 3 mice/group (d,e,j). Statistical significance was determined by two-tailed unpaired _t-_test (b-e).

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Li, T., Li, X., Zamani, A. et al. c-Rel is a myeloid checkpoint for cancer immunotherapy.Nat Cancer 1, 507–517 (2020). https://doi.org/10.1038/s43018-020-0061-3

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