Antitumor effects of anti-CD40/CpG immunotherapy combined with gemcitabine or 5-fluorouracil chemotherapy in the B16 melanoma model

Abstract

Our previous studies demonstrated that anti-CD40 mAb (anti-CD40) can synergize with CpG oligodeoxynucleotides (CpG) to mediate antitumor effects by activating myeloid cells, such as macrophages in tumor-bearing mice. Separate teams have shown that chemotherapy with gemcitabine (GEM) or 5-fluorouracil (5-FU) can reduce tumor-induced myeloid-derived suppressor cells (MDSC) in mice. In this study we asked if the same chemotherapy regimens with GEM or 5-FU will enhance the antitumor effect of anti-CD40 and CpG. Using the model of B16 melanoma growing intraperitoneally in syngeneic C57BL/6 mice, we show that these GEM or 5-FU treatment regimens reduced MDSC in the peritoneal cavity of tumor-bearing mice. Treatment of mice with GEM or 5-FU did not significantly affect the antitumor function of macrophages as assessed in vitro. In vivo, treatment with these GEM or 5-FU regimens followed by anti-CD40/CpG resulted in antitumor effects similar to those of anti-CD40/CpG in the absence of GEM or 5-FU. Likewise, reduction of MDSC by in vivo anti-Gr-1 mAb treatment did not significantly affect anti-CD40/CpG antitumor responses. Together, the results show that the GEM or 5-FU chemotherapy regimens did not substantially affect the antitumor effects induced by anti-CD40/CpG immunotherapy.

Keywords: 5-Fluorouracil; Anti-CD40; CpG; Gemcitabine; Immunotherapy.

Figure 1

Effect of GEM or 5-FU on i.p. B16 melanoma-induced MDSC. A, B: C57BL/6 mice were injected i.p. with 10⁵ B16 cells on day 0. TBM (n= 6 per group) received either 120 mg/kg GEM (TBM GEM) or PBS (TBM) i.p. on day 11. On day 14, PEC were collected from TBM, TBM GEM and naïve mice (n=3–4 mice per group), stained with FITC-conjugated anti-CD45, PE-conjugated anti-Gr-1 and APC-conjugated anti-CD11b, and subjected to flow cytometry. Graphs A and B represent the results of one out of three similar experiments. C, D: B16 TBM (n=3 or 4 per group) received either 50 mg/kg of 5-FU (TBM 5-FU) or DMSO control (TBM) i.p. on day 7 and 14. PECs were collected on day 16 from TBM, TBM 5-FU and naïve mice (n=4), and subjected to flow cytometry using a similar protocol as A, B. Graphs C and D represent the results of one out of two similar experiments. The percentage (A, C) and absolute number (B, D) of CD45⁺ CD11b⁺Gr-1⁺ cells out of total PECs were calculated. The data are shown as Mean ± SD. * P<0.05, ** P < 0.01, *** P<0.001, NS: Non-significant.

Figure 2

Effect of GEM or 5-FU on Mφ function. A, B: Two groups of naïve C57BL/6 mice (n=2 per group) were injected with either 50 mg/kg of 5-FU (5-FU) or DMSO (Control) i.p., and PEC were collected 5 days later. Total PEC (2x10⁵/well) and B16 tumor cells (10⁴/well) were placed in 96-well plates with medium or stimulated with IFN-γ (10 U/ml) and LPS (1 ng/ml). C: C57BL/6 mice were injected with B16 cells i.p. on day 0, and injected with either 50 mg/kg of 5-FU (TBM 5-FU) or DMSO (TBM, control) i.p. on days 5 and 10. PECs from naïve, TBM and TBM 5-FU (n=3–4 per group) were collected on day 14. Total PEC were stimulated with IFN-γ (10 U/ml) and LPS (1 ng/ml). The results of one out of two similar experiments are shown. D: B16 i.p. TBM were injected with 120 mg/kg of GEM (TBM GEM) or PBS (TBM) i.p. on day 11. Total PEC were collected on day 14 and placed in 96-well plates with medium or IFN-γ (10 U/ml) and LPS (1 ng/ml). All plates were incubated for 48 hours. D shows a combined graph of three similar experiments (8–9 mice per group). Counts of B16 cells were measured based on thymidine incorporation (A), and NO activity was determined by nitrite level in the supernatants (B, C, D). The data are shown as Mean ± SD. # Counts <150. NS: Non-significant.

Figure 3

Sorting CD11b⁺Gr-1⁺ Cells from 5-FU treated mice. C57BL/6 mice were injected i.p. with 10⁵ B16 cells on day 0. TBM received either 50 mg/kg of 5-FU (n=9 per group, TBM 5-FU) or DMSO control (n=5 per group, TBM) i.p. on days 5 and 11. PEC were collected on day 14 from all TBM and two naïve mice. A, B: PEC were stained with FITC-conjugated anti-B220, PE-conjugated anti-Gr-1 and APC-conjugated anti-CD11b. CD11b⁺Gr-1⁺ cells from TBM 5-FU and TBM, as well as B220⁺ cells from TBM were sorted. In 96-well plates, adherent naïve PEC or sorted PEC (10⁵/well) were incubated with B16 cells (10⁴/well) in medium or IFN-γ (10 U/ml) and LPS (1 ng/ml) for 48 hours. Counts of B16 cells (A) inhibited by PEC were measured based on thymidine incorporation in tumor cells, and NO activity was determined by nitrite level (B) in the supernatants. Results in panel A and B are representative of two independent experiments. C: Spleen cells pooled from two naïve mice were placed 2x10⁵/well with sorted PEC (1x10⁵/well) in medium or anti-CD3 (0.5μg/ml)/anti-CD28 (5μg/ml). PEC were obtained from TBM treated with 5-FU (TBM-5-FU) or without treatment (TBM). Counts of spleen cells were measured by thymidine incorporation assay 48 hours later. Panel C represents the combined results from two similar experiments. The data are shown as Mean ± SD. * P < 0.05, ** P < 0.01. # counts < 150 or nitrite level < 1μM. NS: Non-significant. NT: Not tested.

Figure 4

Antitumor effect of GEM, 5-FU or Gr-1⁺ cell depletion in combination with anti-CD40/CpG in vivo. C57BL/6 mice were injected s.c. (A, B, D) or i.p. (C) with 10⁵ B16 cells on day 0. A: TBM ( n=4 or 5 per group) had no treatment (control) or were treated with GEM i.p. on days 7 and 14, anti-CD40/CpG i.p. on days 8/11 and 15/18, or a combination of GEM and anti-CD40/CpG. B: TBM (n=6 or 7 per group) had no treatment or were treated with 5-FU i.p. on day 6, anti-CD40/CpG i.p. on days 7/10 and 14/17, or a combination of 5-FU and anti-CD40/CpG. C: TBM (n=11 per group) had no treatment or were treated with 5-FU i.p. on days 5 and 15, anti-CD40/CpG i.p. on day 9/12, or a combination of 5-FU and anti-CD40/CpG. The graph represents the combination of two similar experiments. D: C57BL/6 mice were injected s.c. with 10⁵ B16 cells on day 0. TBM (n=6 or 7 per group) had no treatment or were treated with 0.2mg of anti-Gr-1 i.t. on days 7, 10, 14 and 17, anti-CD40/CpG i.p. on days 7/10 and 14/17, or a combination of anti-Gr-1 and anti-CD40/CpG. Panels A, C and D represent single experiments, while B represents two similar experiments. Means ± SE of tumors volumes (A, B, D) are presented. Control mice received DMSO, PBS, or Rat IgG. * P < 0.05, ** P < 0.01 and *** P < 0.001 for control group versus treatment groups. There was no statistically significant difference between the anti-CD40/CpG and combined treatment groups. Arrows indicate treatment schedule.