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MYC regulates the antitumor immune response through CD47 and PD-L1

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Science  08 Apr 2016:
Vol. 352, Issue 6282, pp. 227-231
DOI: 10.1126/science.aac9935
  • Fig. 1 MYC regulates the expression of CD47 and PD-L1 in murine and human leukemia and lymphomas.

    (A) Flow cytometry median fluorescence intensity (MFI) was used to determine the relative cell surface expression of CD47 (blue), PD-L1 (green), and other immune proteins after MYC inactivation in MYC T-ALL 4188 cells in vitro (n = 3 replicates). (B) Tumors were harvested from primary MYC-driven lymphomas 0 to 4 days after MYC inactivation. mRNA and protein levels were quantified by qPCR and flow cytometry MFI (n = 3 tumors per condition). Representative flow cytometry histograms are shown to the right. (C) CD47 (blue) and PD-L1 (green) protein levels in Jurkat and CCRF-CEM cells were quantified by flow cytometry MFI after MYC inhibition by conditional shRNA knockdown or 10 μM JQ1 treatment (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate SEM.

  • Fig. 2 MYC regulates CD47 and PD-L1 expression in human and mouse tumors and binds to the promoters of the corresponding genes.

    (A and B) mRNA and protein levels of MYC (gray), CD47 (blue), and PD-L1 (green) in human melanoma SKMEL28 and human NSCLC H1299 cells as determined by qPCR and flow cytometry MFI, respectively, 48 hours after MYC inactivation in vitro. MYC was inactivated by 10 μM JQ1 treatment or MYC shRNA knockdown (n = 3 biological and 3 technical replicates for qPCR, n = 3 biological replicates for flow cytometry). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate SEM. (C) ChIP-seq analysis of MYC binding to the promoter sequence of the genes encoding CD47 and PD-L1 in mouse MYC T-ALL cells. Immunoglobulin G (IgG) was used as a negative control. ChIP-seq traces were generated from GSE44672 (34). Exons are represented as vertical bars, the untranslated region is represented by a black line, and arrows indicate the direction of transcription.

  • Fig. 3 Constitutive expression of CD47 and PD-L1 in mouse MYC T-ALL 4188 cells prevents recruitment of immune effectors after MYC inactivation.

    (A) Quantification of CD4+ T cells in transplanted control (gray) or constitutive CD47- or PD-L1–expressing (colored) tumors before 2, 4, 11, or 21 days after MYC inactivation. Control, CD47-expressing, or PD-L1–expressing MYC T-ALL 4188 tumor cells were transplanted into FVB RAG1−/− mice 1 week after reconstitution with fLuc+ CD4+ T cells. Administration of Dox to inactivate MYC in established tumors occurred on day 0. (Left): Representative bioluminescence images of tumor-bearing RAG1−/− animals. MSCV, murine stem cell virus. (Right) Average bioluminescence signal of the T cells (n = 5 tumors per group). (B) Quantification of F4/80+ or CD69+ cells in transplanted control (gray) or constitutive CD47- or PD-L1–expressing (colored) tumors before or 4 days after MYC inactivation, by immunohistochemistry using markers for macrophages (F4/80) and activated T cells (CD69). Tumor cells were transplanted into wild-type (WT) FVB hosts. Administration of Dox to inactivate MYC in established tumors occurred on day 0. The y axis denotes the number of positively-staining cells per field. For representative images, see fig. S13. Data represent mean ± SEM derived from measurements of three independent tumors and three measurements per tumor. *P < 0.05; ***P < 0.001.

  • Fig. 4 Down-regulation of CD47 or PD-L1 is required for tumor regression, shutdown of angiogenesis, and induction of senescence upon MYC inactivation.

    (A) Survival after MYC inactivation of syngeneic FVB/N mice that had been transplanted with either MSCV control (gray), CD47-expressing (blue), or PD-L1–expressing (green) fLuc+ MYC T-ALL cells. MYC was inactivated when tumors reached 1.5 cm3 (day 0) (n = 5 tumors for control, n = 10 tumors for CD47, and n = 5 tumors for PD-L1). (B) MYC expression before (day 0) or after MYC inactivation (day 4). (C) Bioluminescence imaging measurement of tumor burden before and after MYC inactivation in control (gray), CD47-expressing (blue), and PD-L1–expressing (green) tumors. Data for three representative animals per group are shown. (D) Minimal residual disease (remaining tumor cells) after MYC inactivation was measured by bioluminescence imaging. (E) Angiogenesis was measured 0 and 4 days after MYC inactivation in control, CD47-expressing, and PD-L1–expressing tumors growing in WT FVB hosts by immunofluorescence for CD31. For representative images, see fig. S15. (F) Control, CD47-expressing, and PD-L1–expressing tumors [as described in (E)] were analyzed by immunostaining for senescence-associated β-gal. The y axis denotes the number of positively-staining microvessels (E) or cells (F) per field. For representative images, see fig. S15B. Data represent mean ± SEM derived from measurements of three independent tumors and three measurements per tumor. *P < 0.05; **P < 0.01; ***P < 0.001.

Supplementary Materials

  • MYC regulates the antitumor immune response through CD47 and PD-L1

    Stephanie C. Casey, Ling Tong, Yulin Li, Rachel Do, Susanne Walz, Kelly N. Fitzgerald, Arvin M. Gouw, Virginie Baylot, Ines Gütgemann, Martin Eilers, Dean W. Felsher

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S17
    • Table S1
    • Full Reference List
    Correction (1 April 2016): In the original version of the author list, Arvin M. Gouw's middle initial was omitted, and Ines Gütgemann's surname was misspelled. Those errors have been corrected here.
    The original version is accessible here.

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