125 I RPI upregulated the expression of PD-1/PD-L1 in the TME
To investigate PD-1/PD-L1 axis involvement in immunosuppression following 125I RPI treatment, tumor tissues were collected on days 6 and 12 after 125I RPI treatment. The expression of PD-L1 on tumor cells and PD-1 on CD8 + tumor infiltrating lymphocytes (TILs) was analyzed by flow cytometry. Compared with the control group, the expression of PD-L1 was upregulated in the 125I RPI group on day 6 (P = 0.0053), and further upregulated on day 12 (Fig. 1A and 1B; when compared with expression at day 6 and the control; P = 0.0057 and P < 0.0001, respectively). On day 6 after 125I RPI, the expression of PD-1 on CD8 + TILs was not significantly different from that in the control group (P = 0.1351), but was upregulated on day 12 (Fig. 1C and 1D; P = 0.0001). This shows that following 125I RPI implantation, as time increased, the number of exhausted T cells increased, and the immune response may have been inhibited by PD-1/PD-L1. Therefore, we speculate that PD-1/PD-L1 blockade therapy may improve the efficacy of 125I RPI.
Combination 125I RPI and anti-PD-1 therapy inhibits tumor growth and induces the abscopal effect
To test whether combination 125I RPI and anti-PD-1 therapy inhibited tumor growth and induced the abscopal effect, the mice were divided into the following groups 10 days after tumor inoculation: PBS group, 125I RPI group, α-PD-1 group, and 125I RPI + α-PD-1 group. On day 10, 125I particles were implanted, and anti-PD-1 antibodies were injected on days 10, 12, 14, 16, and 18. Anti-PD-1 therapy alone had little effect on tumor volume, 125I RPI delayed tumor growth, and combined therapy significantly delayed tumor growth (P = 0.007, α-PD-1 vs. 125I RPI + α-PD-1 = 2734.70 ± 548.16 mm3 vs. 1186.16 ± 218.14 mm3 on day 22; P = 0.0139, 125I RPI vs. 125I RPI + α-PD-1 = 1921.32 ± 307.35 mm3 vs. 1186.16 ± 218.14 mm3 on day 22; Fig. 2A and 2B). In addition, combination therapy inhibited the growth of secondary tumors, whereas monotherapy did not (P = 0.0410, anti-PD-1 vs. 125I RPI + α-PD-1 = 2503.2764 ± 473.71 mm3 vs. 1624.67 ± 155.10 mm3 on day 22; P = 0.0494, 125I RPI vs. 125I RPI + α-PD-1 = 2621.33 ± 563.22 mm3 vs. 1624.67 ± 155.10 mm3 on day 22; Fig. 2C). As shown in Fig. 2D, the median survival time of the PBS, 125I RPI, α-PD-1, and 125I RPI + α-PD-1 groups were 20, 22, 26, and 34 days, respectively. Compared with 125I RPI and α-PD-1, combination therapy resulted in significantly longer survival (P = 0.0026 and P = 0.0019, respectively; Fig. 2D). These results suggest that anti-PD-1 therapy can improve the efficacy of 125I RPI and combination therapy can induce the abscopal effect and improve the prognosis in mice.
Combination Therapy Increases Proliferation And Activation Of Cd8 + tils
To determine whether combination therapy could activate anti-tumor immunity, we analyzed T lymphocytes in the TME. Mice treated with 125I RPI or anti-PD-1 had a slightly increased proportion of CD8 + TILs (P = 0.4103 and P = 0.6318, respectively), but those treated with combination therapy had significantly increased numbers of CD8 + TILs (P < 0.05 compared with all other groups; Fig. 3A and 3B). There was no difference in the proportion of CD4 + TILs among the groups (P = 0.1539, 125I RPI + α-PD-1 vs. PBS; Fig. 3A and 3B). To evaluate the activity of CD8 + TILs, we analyzed the ability of CD8 + TILs to produce IFN-γ and TNF-α (Fig. 3C). There were approximately five-fold more IFN-γ + CD8 + TILs in the 125I RPI + α-PD-1 group than in the PBS group (Fig. 3D; P = 0.0013), and six-fold more TNF-α + CD8 + TILs than in the PBS group (Fig. 3D; P = 0.0002). These results showed that combined therapy could activate anti-tumor immunity and increase the number and activity of T cells in the TME. We also showed that the immune activation effect of 125I RPI is achieved through PD-1 blockade.
Combined therapy reduces the number of Tregs in the TME
Tregs can promote tumor growth by inhibiting T cell activation(25). Studies have found that RT can upregulate the expression of Tregs in the TME(13), but anti-PD-1 treatment can reduce the expression of Tregs(26). However, the effect of 125I RPI and 125I RPI combined with anti-PD-1 treatment on Tregs is unknown. To this end, we analyzed the proportion of Tregs and showed that 125I RPI did not affect the proportion of Tregs (P = 0.2442). The proportion of Tregs decreased after anti-PD-1 treatment (P = 0.0051), but the decrease was more significant after the combined treatment (Fig. 4A and 4B; P = 0.0227). We further analyzed the ratio of CD8 + T/Tregs, which was significantly higher in the 125I RPI + α-PD-1 group than in the other groups (P < 0.05 compared with all other groups; Fig. 4B). These results suggest that combination therapy could reshape T cell immunity and make the tumor immune microenvironment into one of immune activation.
The schedule is a critical determinant affecting the efficacy of combination therapy
The sequence of RT and anti-PD-1 treatment affects the efficacy of the combined therapy. Previous studies showed that concurrent therapy is better than sequential therapy(12, 27–29). To determine whether administration of 125I RPI or α-PD-1 mAb at different times affects the efficacy of combination therapy, we examined three different combination schedules: 125I RPI followed after three days by α-PD-1 mAb (schedule A); concurrent administration of α-PD-1 mAb and 125I RPI (schedule B); and α-PD-1 mAb followed after three days by 125I RPI (schedule C) (Fig. 5A). The treatment start time of the three schedules was on day 10 after injection of the tumor cells. The median survival time for schedules B and C were 34 and 33 days, respectively, while schedule A reduced the survival time compared to 125 I RPI only (P = 0.0128, 22 days vs. 26 days; Fig. 5B). In schedule A, α-PD-1 mAb did not control tumor growth, demonstrating that when 125I RPI was used at a later stage, the tumor burden was already too high. This could explain why schedule A was less efficient. These results suggest that initiating 125I RPI earlier to delay tumor progression represents a more effective combination schedule.