Chlorin e6-associated photodynamic therapy enhances abscopal antitumor effects via inhibition of PD-1/PD-L1 immune checkpoint

Abstract

We hypothesized that photodynamic therapy (PDT) with Chlorin e6 (Ce6) enhances antitumor abscopal effects via inhibition of the programmed cell death-1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint. By using syngeneic melanoma and pancreatic tumor mouse models, we studied the Ce6-PDT-induced immune responses in local and distant tumor microenvironments. In addition, the Ce6-PDT's target in the PD-1/PD-L1 interaction was analyzed in MC38-hPD-L1 colon cancer and PD-1 expressing Jurkat T cell coculture. The tumors in the irradiated and non-irradiated sites in the abscopal effective (Abs_eff) group of both mouse models were regressed, proving the abscopal effect. The immunogenic effect in the Abs_eff group was associated with an expansion of T cell and other immune cells infiltration without changes in the CD39⁺ population in either the right or left tumors compared to control group. Furthermore, the abscopal ineffective (Abs_ineff) group demonstrated lesser increase of T cells, decreased immune cell infiltration, and increased CD39-expressing Treg cells without suppression of tumor growth. In the coculture with PD-1-expressing Jurkat T cell, Ce6-PDT efficiently suppressed the PD-1/PD-L1 interactions by increasing the proliferation and cytotoxic activity of CD8⁺ T cells while decreasing CD39-expressing Treg cells in a dose-dependent manner. Likewise, the inhibition of PD-1/PD-L1 interactions was also correlated with the increased production of IL-2 and Granzyme B. Our findings imply that Ce6-PDT is a promising immunotherapy with the potential to improve the abscopal effect.

Figure 1

Abscopal antitumor effects in the melanoma mouse model. (A) Experimental scheme, (B) C57BL/6 mice were inoculated with B16F10 cells, received an intravenous injection of Ce6 (2.5 mg/kg) followed by irradiation using a diode laser (λ = 660 nm) at a rate of 100 J/cm² for 8 min 20 s. (C) Mice were divided into Abs_eff and Abs_ineff groups on the basis of the abscopal effect with Ce6-PDT. (D) Right tumor (RT) volume, (E) left tumor (LT) volume, (F) right and left tumor weight in the control, Abs_eff and Abs_ineff groups respectively. Data are from an experiment representative with n = 3 in the control, n = 3 in the effective, and n = 4 in the ineffective group. *P < 0.05 compared to right control tumor.  ^#P < 0.05 compared to right tumor of the abscopal effective group, ^$P < 0.05 compared to left control tumor, and ^&P < 0.05 compared to left tumor of the abscopal effective group (by one-way ANOVA with Tukey's post hoc test for multiple comparisons).

Figure 2

Abscopal antitumor effects in the pancreatic tumor mouse model. Panc02 cells of 5 × 10⁵ cell density were injected in the right and left flanks of mice. On the 12^th day, Ce6 (2.5 mg/kg) was injected intravenously and after 3 h followed by irradiation using a diode laser (λ = 660 nm) at a rate of 100 J/cm² for 8 min 20 s. (A) The experimental scheme, (B) right tumor volume (RT), (C) left tumor (LT) volume, (D) tumor weight and (E) spleen weight in the control, Abs_eff, and Abs_ineff groups. Data are from an experiment representative with n = 3 in the control, n = 4 in the effective, and n = 4 in the ineffective group. *P < 0.05 compared to right control tumor. #P < 0.05 compared to right tumor of the abscopal effective group, ^$P < 0.05 compared to left control tumor, and ^&P < 0.05 compared to left tumor of the abscopal effective group (by one-way ANOVA with Tukey's post hoc test for multiple comparisons).

Figure 3

Enhanced accumulation and activation of T cells by Ce6-PDT in the melanoma mouse tumors. (A-E) Flow cytometry analysis to count and estimate the intratumoral fraction of (A) CD3⁺, (B) CD45⁺, (C) CD25⁺, (D) CD103⁺, and (E) CD39⁺ T cells, isolated from the irradiated right and non-irradiated left tumors in control, Abs_eff and Abs_ineff group. After 28 days of tumor cell injection, T cells in tumor tissues were isolated from B16F10 tumor-bearing mice. Data are from an experiment representative with n = 3 in the control, n = 3 in the abscopal effective, and n = 4 in the abscopal ineffective group. *p < 0.05 compared to irradiated right tumors in abscopal effective group. #p < 0.05 compared to irradiated right tumors in abscopal ineffective group (by one-way ANOVA with Tukey's post hoc test for multiple comparisons).

Figure 4

Flow cytometric analysis of the immune cell in the irradiated and non-irradiated tumor of melanoma mouse tumors. Percentages of (A) CD11c⁺, (B) F4/80⁺, (C) NK1.1⁺ (D) CD86⁺, and (E) CD 206⁺ in irradiated and non-irradiated tumor in control, Abs_eff group, and Abs_eff group. Data are from an experiment representative with n = 3 in the control, n = 3 in the effective, and n = 4 in the ineffective group. *p < 0.05 compared to irradiated right tumors in abscopal effective group. #p < 0.05 compared to irradiated right tumors in abscopal ineffective group (by one-way ANOVA with Tukey's post hoc test for multiple comparisons).

Figure 5

Enhanced accumulation and activation of T cells by Ce6-PDT in the pancreatic mouse tumors. (A-E) Flow cytometry analysis to count and estimate the intratumoral fraction of (A) CD3⁺, (B) CD4⁺, (C) CD8⁺, (D) CD25⁺ (E) CD103⁺, and (F) CD39⁺ T cells, isolated from the irradiated right and non-irradiated left tumor in Abs_eff group and Abs_ineff group. After 28 days of tumor cell injection, T cells within tumors were isolated from Panc02 pancreatic cancer bearing mice. Data are from an experiment representative of three mice in the effective and four mice in the ineffective group. *P < 0.05 compared to right tumor. #P < 0.05 compared to left tumor (by one-way ANOVA with Tukey's post hoc test for multiple comparisons).

Figure 6

Flow cytometric analysis of the immune cells in the irradiated and non-irradiated tumor of pancreatic mouse tumors. Percentages of (A) CD11c⁺, (B) F4/80⁺, (C) NK1.1⁺ (D) CD86⁺ (E) CD 206⁺ (F) CD68⁺, and (E) CD163⁺ in (A) 24 and (B) 48 h from the irradiated and non-irradiated pancreatic tumor in the Abs_eff group and Abs_ineff group. Data are from an experiment representative of three mice in the effective and four mice in the ineffective group. *P < 0.05 compared to right tumor #P < 0.05 compared to left tumor (by one-way ANOVA with Tukey's post hoc test for multiple comparisons).

Figure 7

Effect of Ce6-PDT on IL-2 cytokine release in Jurkat T cells and MC38 colon cancer cells coculture model. Cell culture media was analyzed to measure IL-2 level by cytokine ELISA in (A) 24 and (B) 48 h. The concentration of Ce6 only is 4 μΜ. Data are presented as mean ± S.E. of three representative independent experiments. *P < 0.05 compared to vehicle-treated control. (by one-way ANOVA with Tukey's post hoc test for multiple comparisons).

Figure 8

Inhibition of PD-1/PD-L1 checkpoint by Ce6-PDT and increased granzyme B production. Cell culture media was analyzed to measure granzyme level by cytokine ELISA in (A) 24 and (B) 48 h. The concentration of Ce6 only is 4 μΜ. Data are presented as mean ± S.E. of three representative independent experiments. *P < 0.05 compared to vehicle-treated control. (by one-way ANOVA with Tukey's post hoc test for multiple comparisons).

Figure 9

Augmentation of cytotoxic T cells and deduction of CD39⁺ Treg cells through the blockage of PD-1/PD-L1 interaction by Ce6-PDT. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37 °C for 20 h prior to the addition of PD-1 effector cells. Cells were treated with an increasing concentrations of Ce6 (1–4 µM) followed by PDT. The concentration of Ce6 only is 4 μΜ. PD-1/PD-L1 immune check-point blockage by Ce6-PDT was analyzed to measure the CD8⁺ T cells and CD39⁺ T cells (A), (C) in 24 h and (B), (D) in 48 h of coculture respectively. Data are presented as the means only.