Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice

Abstract

High-dose ionizing irradiation (IR) results in direct tumor cell death and augments tumor-specific immunity, which enhances tumor control both locally and distantly. Unfortunately, local relapses often occur following IR treatment, indicating that IR-induced responses are inadequate to maintain antitumor immunity. Therapeutic blockade of the T cell negative regulator programmed death-ligand 1 (PD-L1, also called B7-H1) can enhance T cell effector function when PD-L1 is expressed in chronically inflamed tissues and tumors. Here, we demonstrate that PD-L1 was upregulated in the tumor microenvironment after IR. Administration of anti-PD-L1 enhanced the efficacy of IR through a cytotoxic T cell-dependent mechanism. Concomitant with IR-mediated tumor regression, we observed that IR and anti-PD-L1 synergistically reduced the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which suppress T cells and alter the tumor immune microenvironment. Furthermore, activation of cytotoxic T cells with combination therapy mediated the reduction of MDSCs in tumors through the cytotoxic actions of TNF. Our data provide evidence for a close interaction between IR, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and radiotherapy.

Figure 1. The profile of PD-L1 and PD-1 expression in tumor microenvironments is altered after IR.

BALB/c mice were injected s.c. into the flank with 1 × 10⁶ TUBO cells. On day 14, mice were locally treated with one 12-Gy dose of IR. Three days after IR, tumors were removed and digested into single-cell suspensions, which were blocked with anti-FcR mAbs and then subjected to surface staining. PD-L1 expression on myeloid cells and tumor cells (A) and PD-1 expression on T cells (B). Representative data are shown from three (A and B) experiments conducted using 3 mice per group.

Figure 2. IR and PD-L1 blockade synergistically amplify the antitumor effect.

(A) Combination of anti–PD-L1 (αPD-L1) and IR significantly enhanced the inhibition of TUBO tumor growth. BALB/c mice were inoculated s.c. on day 0 with 1 × 10⁶ TUBO cells. Tumors locally received one 12-Gy dose on day 14 and/or 200 μg anti–PD-L1 (clone 10F.9G2) or isotype control i.p. every three days for a total of four times. **P < 0.01; ***P < 0.001. (B) Combination therapy greatly delayed MC38 tumor growth compared with single treatments. C57BL/6 mice were injected s.c. on day 0 with 1 × 10⁶ MC38 cells. Tumors received 20 Gy on day 8, and antibodies were started on day 8 and administered as described in A. *P < 0.05; ***P < 0.001. (C) Tumor-free mice that underwent combination therapy were resistant to the tumor rechallenge. Thirty days after tumor eradication, the mice treated as in A were rechallenged with 2 × 10⁶ TUBO cells on the opposite flank. (D) Systemic effect of combination treatment greatly reduced the growth of secondary tumors. TUBO tumors on the right flank were treated with 12 Gy or anti–PD-L1 alone, or with 12 Gy plus anti–PD-L1, as described in A. Tumors on the left flank were measured and monitored. Representative data are shown from three (A) or two (B–D) experiments conducted with 6 to 8 (A and D), 5 (B), or 4 (C) mice per group.

Figure 3. CD8⁺ T cells are required for the efficacy of IR and anti–PD-L1 combination treatment.

(A) Tumor regression by combination treatment with anti–PD-L1 and IR was mediated by CD8⁺ T cells. Tumors received 12 Gy and mice were treated with anti–PD-L1, as described in Figure 2A. Starting from 1 day before IR, 250 μg of depletion antibodies against CD8⁺ T cells (clone 2.43) was injected i.p. every 3 days for a total of four times. *P < 0.05. (B) Combination therapy greatly enhanced the antigen-specific response of CD8⁺ T cells. TUBO tumors received 12 Gy, and mice were treated with anti–PD-L1 as described in Figure 2A. Nine days after IR, the draining LNs were removed and subjected to ELISPOT assays. **P < 0.01; ***P < 0.001. Representative data are shown from two (A) and three (B) experiments conducted with 5 to 6 (A) or 4 (B) mice per group.

Figure 4. IR and PD-L1 blockade induce the reduction of MDSCs.

Tumors received 12 Gy, and mice were treated with anti–PD-L1 as described in Figure 2A. Three days or 10 days after IR, tumors were removed to obtain cell suspensions for surface staining. (A) Flow cytometric analysis of MDSCs (CD11b⁺Gr1⁺) gated on CD45⁺ cells in tumors 10 days after IR. (B) Quantitative data of the percentage of MDSCs (CD11b⁺Gr1⁺), macrophages (CD11b⁺F4/80⁺), CD8⁺ T cells, and CD4⁺ T cells relative to CD45⁺ cells on day 10 (left) and on day 3 (right) after IR. *P < 0.05; **P < 0.01; ***P < 0.001. Representative data are shown from two (A and B) experiments conducted with 5 mice per group.

Figure 5. CD8⁺ T cells mediate the reduction of MDSCs in IR and anti–PD-L1 combination treatment.

TUBO tumor–bearing mice were treated with IR and antibodies as described in Figure 2A and Figure 3A. Ten days after IR, the tumors were removed. (A) Immunofluorescence staining of frozen tumor sections. Top row, untreated tumor; Bottom row, tumor treated with IR and anti–PD-L1. Scale bars: 100 μm; original magnification, ×4. Inset scale bars: 5 μm; original magnification, ×100. (B) Quantification of distance from CD11b⁺Gr1⁺ cells to the closest CD8⁺ T cell in a high-power field (×40). Fifteen to eighteen high-power fields were counted for each section. **P < 0.01. Sections were obtained from three tumors per group. The quantification performed on the individual tumor from each group is shown in each histogram. (C) Representative dot plots of MDSCs gated on a CD45⁺ cell population. (D) The reduction of the proportion of MDSCs with combination therapy was rescued after the depletion of CD8⁺ T cells. **P < 0.01. Representative data are shown from two (A–D) experiments conducted with 3 (A and B) or 4 (C and D) mice per group.

Figure 6. CD8⁺ T cells induce the apoptosis of MDSCs through TNF-α following combination therapy.

(A and B) Isolated CD8⁺ T cells derived from naive BALB/c mice were stimulated with anti-CD3 and anti-CD28 for 6 hours. Then, Gr1⁺ cells purified from the spleen of TUBO-bearing mice were added for an additional 21 hours’ coincubation. Representative dot plots (A) and the percentage (B) of annexin V⁺ in MDSCs are shown. Cells were gated on a CD11b⁺Ly-6C⁺ cell population. **P < 0.01; ***P < 0.001. (C) Percentage of annexin V⁺ in MDSCs after treatment with increasing concentrations of TNF. Gr1⁺ cells purified from the spleen of TUBO-bearing mice were treated with different concentrations of TNF or IFN-γ for 21 hours. (D) The inhibition of tumor growth with combination therapy was abrogated after the treatment of TNFR-hIgG. TUBO tumor–bearing mice were treated with IR and antibodies as described in Figure 2A. 500 μg TNFR-hIgG (etanercept) was administered every 4 days for a total of three times starting on the first day of IR. *P < 0.05. (E) Depletion of MDSCs augmented the efficacy of IR treatment. C57BL/6 mice were injected s.c. on day 0 with 1 × 10⁶ MC38 cells. Tumors received 20 Gy on day 9, and 300 μg of depletion antibody against MDSCs (clone 1A8) was administered every 2 days for a total of four times starting 1 day prior to IR. **P < 0.01. Representative data are shown from two (A–E) experiments conducted with 4 (D) or 5 (E) mice per group. (F) Schematic of proposed mechanism for tumor destruction induced by IR and PD-L1 blockade.