Research ArticleCancer Immunotherapy

Blockade of TNFR2 signaling enhances the immunotherapeutic effect of CpG ODN in a mouse model of colon cancer

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Sci. Signal.  02 Jan 2018:
Vol. 11, Issue 511, eaan0790
DOI: 10.1126/scisignal.aan0790
  • Fig. 1 The in vitro and in vivo effects of an antibody recognizing TNFR2 (M861) on Treg cells.

    (A to C) Magnetic-activated cell sorting–purified CD4+ T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The cells were cultured with interleukin-2 (IL-2; 10 ng/ml) alone or with tumor necrosis factor (TNF; 20 ng/ml) and M861 (10 μg/ml), as indicated, for 72 hours. Proliferation of regulatory T (Treg) cells shown by CFSE dilution (A), the proportion of Treg cells in CD4+ cell cultures (B), and the surface abundance of TNF receptor type II (TNFR2) on Treg cells (C) were analyzed with fluorescence-activated cell sorting (FACS), gating for Foxp3+ staining (A and C) or not gated (total cells; B). (D) Wild-type Balb/c mice were injected intraperitoneally with phosphate-buffered saline (PBS), lipopolysaccharide (LPS; 200 μg), M861 (100 μg), mouse immunoglobulin G (muIgG), or a combination thereof, as indicated, for 24 hours. The abundance of TNFR2 on the surface of Treg cells in the spleen was analyzed by FACS, gating for CD4+Foxp3+ staining. FACS plots are representative of three independent experiments. Data are means ± SEM of n = 5 mice. Number in each FACS plot indicates the percentage of gated cells. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test (A to D).

  • Fig. 2 M861 in combination with CpG ODN potently inhibits the development of mouse CT26 colon tumors.

    (A) Schematic of the experimental protocol. Balb/c mice were inoculated in the right flank with CT26 tumor cells (2 × 105 cells in 0.2 ml of PBS). When tumor reached 5 to 6 mm in diameter (day 0), mice were then treated with PBS, CpG oligodeoxynucleotide (ODN) plus control (ctrl) IgG, M861 plus control ODN, or CpG ODN plus M861. (B) Mean growth curves of CT26 tumors in mice treated as described in (A). Data are means ± SEM of 15 mice. (C) Survival curves of the CT26 tumor-bearing mice treated as described in (A). (D to G) CT26 tumor growth curves in each individual CT26 tumor-bearing mouse treated with PBS (D), CpG ODN plus control IgG (E), M861 plus control ODN (F), or M861 plus CpG ODN (G). Data are summary of results pooled from three independent experiments (n = 15 mice). The tumor-free mice were reinoculated with CT26 tumor cells into the right flank and 4T1 tumor cells into the left flank 8 weeks after the mice became tumor-free. As a control, normal mice were also inoculated with CT26 tumor cells into the right flank and 4T1 tumor cells into the left flank in the same manner. Data (n = 8 mice) are the percentage of tumor incidence on (H) normal mice and (I) surviving mice on day 26 after (re-)challenge. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way analysis of variance (ANOVA) test (B), log-rank test (C), or Student’s t test (H and I).

  • Fig. 3 Effects of M861 in combination with CpG ODN on tumor-infiltrating TNFR2+ Treg cells and IFN-γ+ CD8 CTLs.

    (A) Representative FACS analysis of Treg cells in CD4+ cells from Balb/c mice inoculated with CT26 tumor cells (as described in Fig. 2), treated as indicated when tumor diameter reached 10 mm, and sacrificed for tumor tissue isolation 1 day after the final treatment. The number indicates the percentage of Treg cells in CD4+ cells. (B) Representative FACS analysis of TNFR2+ cells in Treg cells from mice described in (A). The number indicates the percentage of TNFR2+ cells in CD4+Foxp3+ cells. (C) Representative FACS analysis of IFN-γ+ cells in CD8+ cells from mice described in (A). The number indicates the percentage of interferon-γ–positive (IFN-γ+) cells in CD8+ cells. (D to G) Summary of the proportion of Treg cells in intratumoral CD4+ cells (D), the mean fluorescence intensity (MFI) of TNFR2 on CD4+Foxp3+ cells (E), the proportion of TNFR2+ cells in CD4+Foxp3+ cells (F), and the proportion of IFN-γ+ cells in CD8+ cells (G), each from mice described in (A). Flow analysis was gated on live CD45+CD3+ cells. Data were quantified from, or are representative of, at least three independent experiments; n = 5 mice (A, B, and D to F) or 3 mice (C and G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t test.

  • Fig. 4 Effect of TNFR2 antagonistic antibody and CD25 antagonistic antibody on mouse 4T1 breast cancer model.

    (A) Schematic of the experimental protocol. TR75-54.7 and/or PC61 were intraperitoneally injected into Balb/c mice 3 days before inoculation of 4T1 tumor cells (1 × 105 cells in 0.1 ml of PBS). (B) Growth kinetics of 4T1 tumors in mice. Data are means ± SEM of five mice. (C) Survival curves of the 4T1 tumor-bearing mice. (D) Median survival of the 4T1 tumor-bearing mice. *P < 0.05 by one-way ANOVA test (B) or log-rank test (C).

Supplementary Materials

  • Supplementary Materials for:

    Blockade of TNFR2 signaling enhances the immunotherapeutic effect of CpG ODN in a mouse model of colon cancer

    Yingjie Nie, Jiang He, Hidekazu Shirota, Anna L. Trivett, De Yang, Dennis M. Klinman, Joost J. Oppenheim,* Xin Chen*

    *Corresponding author. Email: xchen{at}umac.mo (X.C.); oppenhej{at}mail.nih.gov (J.J.O.)

    This PDF file includes:

    • Fig. S1. M861 does not induce the death of Treg cells in LPS-treated mice.
    • Fig. S2. M861 does not inhibit the proliferation of TNFR2-expressing CT26 tumor cells.

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    Citation: Y. Nie, J. He, H. Shirota, A. L. Trivett, D. Yang, D. M. Klinman, J. J. Oppenheim, X. Chen, Blockade of TNFR2 signaling enhances the immunotherapeutic effect of CpG ODN in a mouse model of colon cancer. Sci. Signal. 11, eaan0790 (2018).

    © 2018 American Association for the Advancement of Science

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