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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Cancer Immunol Res. 2015 Feb 19;3(6):610–619. doi: 10.1158/2326-6066.CIR-14-0138

PD-1 Restrains Radiotherapy-Induced Abscopal Effect

Sean S Park 3,*, Haidong Dong 1,2,*, Xin Liu 2, Susan M Harrington 2, Christopher J Krco 2, Michael P Grams 3, Aaron S Mansfield 4, Keith M Furutani 3, Kenneth R Olivier 3, Eugene D Kwon 2
PMCID: PMC4827718  NIHMSID: NIHMS666676  PMID: 25701325

Abstract

We investigated the influence of PD-1 expression on the systemic antitumor response (abscopal effect) induced by stereotactic ablative radiotherapy (SABR) in preclinical melanoma and renal cell carcinoma models. We compared the SABR-induced antitumor response in PD-1-expressing wild-type (WT) and PD-1-deficient knockout (KO) mice, and found that PD-1 expression compromises the survival of tumor-bearing mice treated with SABR. None of the PD-1 WT mice survived beyond 25 days, whereas 20% of the PD-1 KO mice survived beyond 40 days. Similarly, PD-1-blocking antibody in WT mice was able to recapitulate SABR-induced antitumor responses observed in PD-1 KO mice and led to increased survival. The combination of SABR plus PD-1 blockade induced near complete regression of the irradiated primary tumor (synergistic effect), as opposed to SABR alone or SABR plus control antibody. The combination of SABR plus PD-1 blockade therapy elicited a 66% reduction in size of non-irradiated, secondary tumors outside the SABR radiation field (abscopal effect). The observed abscopal effect was tumor-specific and was not dependent on tumor histology or host genetic background. The CD11ahigh CD8+ T-cell phenotype identifies a tumor-reactive population, which was associated in frequency and function with a SABR-induced antitumor immune response in PD-1 KO mice. We conclude that SABR induces an abscopal tumor-specific immune response in both the irradiated and non-irradiated tumors, which is potentiated by PD-1 blockade. The combination of SABR and PD-1 blockade has the potential to translate into a potent immunotherapy strategy in the management of metastatic cancer patients.

Keywords: PD-1, abscopal effect, immune blockade, radiotherapy, tumor

Introduction

Ionizing radiation therapy (RT) is a widely used cancer treatment. Among the effects of RT is its potential for inducing necrotic tumor cell death, a prerequisite for eliciting an antitumor immune response. Stereotactic ablative radiotherapy (SABR) optimizes local control by delivering tumor-ablative doses while sparing the surrounding normal tissues. SABR is increasingly viewed as a noninvasive alternative to surgery (1–4) with comparable local control rates. Although a detailed understanding of the effects of SABR on the immune system is incomplete, reports of partial or complete eradication of tumors outside the RT field, defined as the abscopal effect, suggest that SABR is capable of priming and expanding tumor-reactive T cells within the irradiated tumor and among the draining lymph tissues. Presumably these activated, tumor-specific T cells then migrate to and eliminate non-irradiated (henceforth referred to as “secondary”) tumors. Preclinical models have established that the abscopal effect is T-cell dependent (5).

The infrequent incidence of the abscopal effect in patients following SABR may be due to the suboptimal radiation doses or the inhibition of immune responses (induction, expansion, or effector phases) by conventional immunoregulatory mechanisms. Immune checkpoint blockade at the level of immune priming (CTLA-4 blockade) or effector function (B7-H1/PD-1) is being investigated in many clinical trials and is yielding promising results. This study addresses the question of whether a distinct RT regimen – e.g., SABR -- synergizes with an immune checkpoint blockade and elicits a potential systemic curative response.

The combination of anti-CTLA-4 and RT is capable of inducing abscopal effects in melanoma patients (6, 7). A B7-H1 or PD-1 blockade in preclinical RT models increased the rate of tumor regression, thereby recapitulating the results characteristic of PD-1 blockade in conjunction with other therapies in advanced cancer patients. However, the impact of PD-1 blockade on SABR-mediated abscopal effects is largely unexplored.

In this study, we demonstrate that SABR is a potent inducer of local and systemic (i.e., abscopal effect) antitumor immunity and that PD-1 is a major impediment to antitumor immune responses induced by SABR. Accordingly, PD-1 inhibition (in KO and blocking antibody models) significantly enhances SABR-induced antitumor immunity and results in the suppression of both irradiated and non-irradiated (primary and secondary, respectively) tumor growth. We have established that PD-1 expression is up-regulated in a tumor-reactive CD11ahigh CD8+ T-cell population and contributes to restraining the T-cell effector activity(8). Here, we confirm the correlation between the tumor-reactive CD11ahigh CD8+ T cells and the SABR-induced antitumor response. The combination of SABR and PD-1 blockade may be a promising therapeutic approach for managing metastatic cancer patients.

Materials and Methods

Mice

Six- to eight-week-old BALB/c and C57BL/6 mice were obtained from Taconic Animal Laboratory (Germantown, NY) or Jackson Lab (Bar Harbor, ME) and maintained under pathogen-free conditions in the animal facility at Mayo Clinic Comparative Medicine Department. PD-1 KO C57BL/6 mice were provided by L. Chen (Yale University, New Haven, CT) with the permission of Dr. T. Honjo (Kyoto University). All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Mayo Clinic.

Cell line and reagents

RENCA and 4T1 are BALB/c mouse-derived immunogenic renal cell carcinoma and poorly immunogenic mammary carcinoma cell lines, respectively. RENCA (CRL-2947) and 4T1 (CRL-2947) cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Both cell lines were passaged for less than six months for these studies. ATCC authenticates cell lines by Short Tandem Repeat (STR) analysis. B16-OVA is a C57BL/6 mouse-derived poorly immunogenic melanoma cell line obtained from Dr. Richard G. Vile (Mayo Clinic). B16-OVA cells had undergone quality control analysis by reverse PCR in Dr. Vile’s lab prior to receipt and were passaged for less than six months for the described experiments. All cell lines were proven free of Mycoplasma contamination (Mycoplasma Detection kit; Roche Diagnostics, Chicago, IL). Tumor cells were cultured in DMEM (Invitrogen Corporation, Carlsbad, CA) or RPMI 1640 medium (Cellgro, Hendon, VA) with 10–20% FBS (Life Technologies, Carlsbad, CA), 1 U/ml penicillin, 1 µg/ml streptomycin, and 20 mM HEPES buffer (all from Mediatech, Manassas, VA). Anti-B7-H1 (PD-L1) (Clone MIH5), LAG-3 (Clone C9B7W), and Tim-3 (Clone B8.2C12) monoclonal antibodies (mAb) were used to detect tumor or immune cells for their expression of B7-H1, LAG-3, and Tim-3. Anti-PD-1 hamster mAb G4 was purified as previously described (9). Anti-CTLA-4 hamster mAb (Clone 9H10), anti-CD11a (Clone M17/4) mAb, and appropriate isotype control Ig were purchased from Bio X Cell (West Lebanon, NH).

Tumor-cell challenge and treatment

C57BL/6 and BALB/c mice were injected subcutaneously (s.c.) with 1×105 B16-OVA or 5×105 RENCA cells in the right hindlimb (primary tumor; irradiated) on day 0 and in the left flank (secondary tumor; non-irradiated) on days 3–4. The third party tumor cells (EL4 or 4T1) were injected s.c. in the right flank (secondary tumor; non-irradiated) on days 3–4. Perpendicular tumor diameters were measured using a digital caliper, and tumor sizes were calculated as length × width (mm2). On day 8, when primary tumors were palpable, animals were randomly assigned to treatment groups. Radiotherapy (15 Gy) in a single fraction was administered locally to the primary tumors in the right hindlimb. Briefly, all mice were positioned in a modified 50ml conical plastic tube, which allowed the area of the tumor to be irradiated while keeping the rest of the body outside the RT field. Radiotherapy was delivered using 12 MeV electrons to a field including the tumor with a TrueBeam Linear Accelerator (Varian Medical Systems, Palo Alto, CA) fitted with a 20 × 20 cm2 cone. EBT3 Gafchromic film (Ashland ISP Advanced Materials, Bridgewater, NJ) dosimetry confirmed homogeneity of the prescribed dose within +/− 5%. Superflab bolus (1.0 cm tissue equivalent material) was placed over the tumor, and the tumor received a dose of 15 Gy in one fraction at a rate of 600 cGy/min. PD-1-blocking mAb G4, CTLA-4-blocking mAb (Clone 9H10), or isotype control Ig was administered as an intraperitoneal (i.p.) injection at a dose of 200 µg/mouse (10 mg/kg) alone or as a combination of PD-1- and CTLA-4-blocking mAbs. Tumor growth was evaluated every 2 to 3 days until days 35–40, when all mice were euthanized. In compliance with animal care guidelines, mice were euthanatized when either primary or secondary tumors reached 300 mm2.

Flow cytometry analysis of IFNγ-producing CD8 T cells

For the in vitro tumor-cell challenge, 5–10×105 tumor-infiltrating lymphocytes (TIL), which were isolated from tumor tissues or lymphoid organs, were cultured with OVA peptide (1 µl/ml) for 4–5 hours in the presence of 1 µl/ml of Brefadin A (Sigma), washed and incubated with rat anti-mouse CD16/CD32 mAb (2.4G2) to block nonspecific binding, and then stained with CD8-PE-Cy5 and IFNγ-FITC or control antibodies according to the manufacturer’s instructions (BD Pharmingen). Tumor antigen-specific CD8 T cells were identified by staining with OVA-tetramer (Beckman Coulter) and TRP-2 pentamer (ProImmune). Cells were analyzed using FACScan flow cytometer and FlowJo version X.10 (Tree Star, Ashland, OR) software.

Statistical analysis

All statistical analyses were performed using GraphPad Prism software 5.0 (GraphPad Software, Inc., San Diego, CA). A two-sided, unpaired or paired Student T test was used to assess statistical differences in experimental groups. A p value <0.05 was considered statistically significant.

Results

In the absence of PD-1 expression, the SABR-induced abscopal effect is enhanced

To examine to what extent PD-1 may influence the abscopal effect induced by SABR, B16-OVA melanoma cells were injected into the right hindlimb (primary; irradiated) and left flank (secondary; non-irradiated) of wild-type (WT) and PD-1-deficient (PD-1 KO) C57BL/6 mice. Eight days following tumor-cell injection tumors in the right hindlimb (primary tumors) were administered a single dose of 15 Gy. The secondary tumors (left flank) were kept out of the radiation field. The results in Figure 1A show that SABR resulted in a five-fold reduction (p<0.05, n=5) in primary tumor size 24 days post SABR in the PD-1 KO mice, as compared with that of the WT mice. Importantly, non-irradiated secondary tumors in PD-1 KO mice exhibited a significant reduction in growth (i.e., an abscopal effect; Figure 1B, p<0.05), as compared with that of the secondary tumors in WT mice. As shown in Figure 1C, none of the WT mice survived beyond 25 days, whereas 60% of PD-1 KO mice were alive at day 25 and 20% survived beyond 40 days. From these observations, we conclude that PD-1 expression suppresses the SABR-induced antitumor response and that the abscopal effect is enhanced in the absence of PD-1, thereby improving survival.

Figure 1. Enhanced antitumor abscopal effect in PD-1-deficient mice induced by radiation therapy.

Figure 1

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Both primary and secondary B16-OVA tumors (2 × 105) were injected into wild-type (WT) or PD-1-deficient (KO) mice (5 mice per group). Tumor growth was measured following RT. Data show mean size ± SD of five mice per group of primary tumors (A) and secondary tumors (B). *P<0.05 compared with WT mice. (C) The survival of WT mice was shorter than that of PD-1 KO mice (p<0.05, n=5) following RT.

PD-1 blockade recapitulates SABR-induced abscopal effect observed in PD-1 KO mice

In Figure 1, we observed that SABR induced an abscopal effect in the absence of PD-1 and conferred increased survival by suppressing the growth of secondary non-irradiated tumors. Here, we addressed whether PD-1 blockade in WT mice, when combined with SABR, would recapitulate the abscopal effect induced by SABR in the KO model. B16-OVA tumor cells were injected into the right hindlimb (primary tumor) and left flank (secondary tumor) of WT mice, and SABR was administered 8 days post-injection. On days 7, 9, 11, 14, and 16 following tumor-cell inoculation, either PD-1-blocking or isotype-matched control antibody (200 µg) was administered.

Without PD-1 blockade, SABR treatment resulted in the retardation of tumor growth only at the site of irradiation (primary tumor). PD-1 blockade alone modestly suppressed primary and secondary tumor growth. However, the combination of SABR and PD-1 blockade resulted in a near complete response of the primary tumor when compared with SABR plus control Ig or PD-1 blockade alone (Figure 2A). Therefore, a synergistic effect between SABR and PD-1 blockade was noted in the primary tumor.

Figure 2. PD-1 blockade enhanced anti-tumor abscopal effects induced by radiation therapy.

Figure 2

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The primary tumors were treated with local RT at 15 Gy on day 8 after tumor injection. Anti-PD-1 mAb (Clone G4) or control Ig (200 ug/mouse) were injected intraperitoneally on day 7 and every other day thereafter for a total of 5 doses. Tumor growth was measured in primary (A) and secondary (B) tumors and was shown as mean size ± SD of five mice per group. * p<0.05 compared with mice treated with anti-PD-1 mAb alone (n=5). (C) The survival of mice treated with anti-PD-1 mAb and RT was longer than that of mice treated with anti-PD-1 mAb alone (p<0.05, n=5) or without treatment. One of three independent experiments is shown.

To address whether PD-1 blockade would enhance the SABR-induced abscopal effect, secondary tumor growth (outside of the radiation field) was assessed. Whereas neither PD-1 blockade alone or SABR plus control Ig treatment had any effect on the secondary tumor outgrowth, the combination of SABR and PD-1 blockade was effective in eliciting a 66% reduction in the secondary tumor size (100 mm versus 300 mm; Figure 2B, P<0.05) 20 days post treatment. PD-1 blockade alone or SABR plus control Ig had minimal effect on survival, as compared with mice treated solely with control Ig (day 22 survival versus day 19; Figure 2C). In contrast, the incorporation of PD-1 blockade in conjunction with SABR elicited a significant increase (p<0.05) in survival (20% of mice alive at day 33; Figure 2C). Together, results shown in figures 1 and 2 demonstrate that SABR is capable of inducing antitumor immunity, which is restrained by PD-1. Furthermore, PD-1 inhibition by KO or blockade potentiates the SABR-induced abscopal effect and leads to increased survival. Since CTLA-4 also has been reported to promote abscopal effects (10), we next tested whether the combined PD-1 and CTLA-4 blockade would lead to synergistic effects. SABR with PD-1 or CTLA-4 blockade alone produced minimal reduction of primary and secondary tumor growths in our melanoma animal model with larger tumor burden. However, SABR and the combined PD-1 and CTLA-4 blockade significantly reduced the growth of secondary tumors (Supplemental Figure 1). These results suggest that the combined checkpoint inhibition (PD-1 and CTLA-4 blockade) has synergistic effects in enhancing SABR-induced abscopal effects and warrants clinical investigation in patients with a large metastatic tumor burden.

Combination SABR and PD-1 blockade therapy is tumor antigen-specific and is not dependent on tumor histology or host genetic background

To demonstrate that the applicability of SABR and PD-1 combination therapy is not limited to a specific tumor histology or host genetic factors and that the combination therapy is tumor antigen-specific, mouse renal carcinoma (RENCA) as primary and secondary tumors and breast cancer (4T1) as the third (experimental) tumor histology were injected into Balb/c mice that had a different genetic background from C57BL/6 mice (Figure 3A). Primary RENCA tumor (grown on the right hindlimb) was administered a single 15 Gy. Tumor growth was measured at 1) the primary irradiated RENCA tumor (right hindlimb); 2) the secondary non-irradiated RENCA tumor (left flank); and 3) the non-irradiated experimental 4T1 tumor (right flank). Due to the institutional mouse tumor-burden guidelines, survival endpoints were not a component of this experiment. In both the primary irradiated and secondary non-irradiated RENCA tumor sites, SABR in combination with PD-1 blockade elicited either complete or nearly complete remission of RENCA tumors (Figures 3B and 3C). However, the growth of 4T1 tumors was not impacted by SABR, PD-1 blockade, or the combined therapy (Figure 3D). These data support the hypothesis that PD-1 restrains tumor-specific antitumor immunity in mice treated with SABR and PD-1 blockade. We conclude that the SABR-induced antitumor abscopal effect is potentiated by PD-1 blockade and that it is tumor-specific and independent of tumor histology or host genetic background.

Figure 3. PD-1 blockade enhanced tumor-specific antitumor abscopal effects induced by radiation therapy.

Figure 3

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(A) A diagram of tumor models treated with RT and/or anti-PD-1 mAb. The primary RENCA tumor cells (5×105) were injected into the right hindlimb of Balb/c mice. The secondary RENCA tumor cells (2.5×105) and a different tumor cell line 4T1 (5 ×104) were injected into the left or right flanks, respectively, four days after primary tumor-cell injection in the same mice. The primary tumors were treated with 15 Gy on day 8 after tumor-cell injection. Anti-PD-1 mAb (Clone G4) or control Ig (200 ug/mouse) were injected intraperitoneally on day 7 and every other day thereafter for a total of 5 doses. Tumor growth was measured in the primary (B) and secondary sites. (C) RENCA tumors and was shown as mean size ± SD of five mice per group. * p<0.05, **p<0.01 compared with mice treated with anti-PD-1 mAb alone. (D) The growth of 4T1 tumors did not respond to RT targeting RENCA tumors and anti-PD-1 treatment. One of two independent experiments is shown.

Tumor-reactive CD11ahigh CD8+ T cells from primary and secondary tumor sites express PD-1

Our observations are consistent with the ability of SABR to induce an in situ antitumor response at the irradiated site, which then traffics to secondary tumor sites outside the radiation field. The observation of PD-1 blockade augmenting SABR-induced antitumor immunity is consistent with PD-1 functioning as an immune checkpoint inhibitory molecule. Since CD11a expression is required in the rejection of tumors (11), we previously established that CD11ahigh CD8+ T cells are a tumor-reactive population (8). Since both melanoma and RENCA tumor lines used in our experiments express B7-H1 (PD-L1; a ligand for PD-1) (12), the expression of PD-1 by CD11ahigh CD8+ T cells from primary and secondary tumors was examined (Figure 4). B16-OVA cells were injected into the right hindlimb and the left flank of C57BL/6 mice. A single SABR fraction of 15 Gy was administered to the right hindlimb on day eight post-injection. Seven days post-SABR, CD11ahigh CD8+ T cells were identified from irradiated (right hindlimb), non-irradiated (left flank), and control tumor-bearing mice which did not receive RT. Levels of PD-1 (represented by percentages of positive) expressed by CD11ahigh CD8+ T cells are higher in the primary tumor site as compared with those of the secondary tumor site (p<0.05 on day 15). In contrast to mice that received SABR, CD11ahigh CD8+ T cells in the tumor tissues of non-irradiated mice expressed only modest levels of PD-1 (Figure 4A, p<0.01 on day 15). To confirm whether these PD-1+ CD8 T cells are indeed tumor antigen-reactive effector T cells, we measured their intracellular IFNγ production following a brief re-stimulation with surrogate tumor-antigen peptide (OVA peptide) ex vivo. Figure 4B shows that 33–61% of PD-1+ CD8 T cells produced IFNγ at the primary and secondary tumor sites, suggesting that PD-1+ CD11ahigh CD8+ T cells are effector T cells within the tumors. The expression of PD-1 ligand (PD-L1; B7-H1) increased in tumor cells after SABR in both primary and secondary tumors, but it did not change in that of the non-tumor host (leukocyte) cells (Figure 4C). In addition to PD-1, we measured the expression of other immune checkpoint molecules expressed by CD8 T cells. The results presented in Figure 4D show that tumor-reactive CD11ahigh CD8+ T cells from both primary and secondary tumors express high levels of LAG-3, but not Tim-3, and SABR did not alter the LAG-3 expression by CD8+ T cells in either of the tumor sites. In contrast, CD11alow CD8+ T cells did not express either LAG-3 or Tim-3 before or after SABR at both primary and secondary tumor sites. These results suggest that RT does not induce the expression of LAG-3 regulatory molecules within tumors.

Figure 4. Immune checkpoint molecule expression in primary and secondary tumors following radiation therapy.

Figure 4

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On day 8 after injection of both primary and secondary B16-OVA tumor cells (2 × 105), primary tumors of the right hindlimb were irradiated with a single dose of 15 Gy. In the following weeks, tumor-infiltrating lymphocytes (TIL) were isolated and analyzed for PD-1 expression (A) and CTL function (B) of CD11ahigh CD8+ T cells. Data show percentages of PD-1+ in total CD11ahigh CD8+ TILs of three to five mice per group. * p<0.05, **p<0.01 compared with that in mice without RT I (A). Arrow indicates the day of radiation therapy (day 8). The production of IFNγ was analyzed by intracellular staining for IFNγ after a brief re-stimulation with OVA peptide ex vivo for 5 hours. Numbers in brackets show the percentages of IFNγ-producing cells per PD-1+ population. (C) B7-H1 (PD-L1) expression by CD45- (tumor cells) and CD45+ (leukocytes) cells within both tumor sites. (D) LAG-3 and Tim-3 expression by CD11ahigh or CD11alow CD8+ TILs within both tumor sites.

A direct correlation between the number of tumor-reactive CD11ahigh CD8+ T cells within both primary and secondary tumors and the extent of tumor growth delay was also noted in PD-1 KO mice (Figure 5A). The tumor-antigen specificity of CD11ahigh CD8+ T cells within both tumor sites was identified by antigen peptide tetramer or pentamer staining. As shown in Figure 5B, CD11ahigh but not CD11alow CD8+ T cells isolated from both primary (irradiated) and secondary (non-irradiated) tumors demonstrated tumor-antigen (OVA) specificity by binding to OVA tetramer, and to a lesser degree to TRP-2 pentamer, suggesting that the CD11ahigh CD8+ T cell population indicates the presence of tumor-specific CD8 T cells. As shown in Figure 5C, CD11ahigh CD8+ T cells isolated from tumor sites produced IFNγ upon OVA peptide stimulation but not with control peptide stimulation. These new data collectively suggest that CD11ahigh CD8+ T cells inside tumors are tumor-specific functional effector cells and that their function is enhanced by SABR in PD-1 KO mice.

Figure 5. Increased tumor-infiltrating effector CD8 T cells in PD-1-deficient mice induced by radiation therapy.

Figure 5

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One week after RT, lymphocytes containing tumor-reactive CD11ahigh CD8+ T cells were identified within both primary and secondary B16-OVA tumors, as well as within draining lymph nodes and spleen tissue from wild-type (WT) or PD-1-deficient (KO) mice. CTL function was analyzed by their intracellular production of IFNγ. (A) Increased frequency of CD11ahigh CD8+ T cells in tumor tissues in PD-1 KO mice. **P<0.01 compared with WT mice (n=3). (B) Representative dot blot graph shows the binding of OVA tetramer or TRP-2 pentamer to CD11ahigh CD8+ T cells, isolated from WT and PD-1 KO mice treated with or without SABR. (C) Representative histogram shows that CD11ahigh CD8+ T cells, isolated from WT and PD-1 KO mice treated with or without SABR, produced IFNγ upon brief ex vivo re-stimulation with OVA peptide.

We next determined to what degree CD8+ or CD4+ T cells contribute to the SABR-enhanced abscopal effects in the absence of PD-1 by depleting CD8+ or CD4+ T cells in wild-type and PD-1 KO mice treated with SABR. As shown in Figure 6, the depletion of CD8+ T cells, but not the depletion of CD4+ T cells, abolished the synergistic and abscopal effects in the primary and secondary tumors in PD-1 KO mice. Although the high expression of CD11a by CD8 T cells was used as a surrogate marker to identify tumor-reactive T cells, the blockade of CD11a did not affect the SABR-induced abscopal effects in PD-1 KO mice (Figure 6), suggesting that the disruption of CD11a alone may not affect CD8 T cell-mediated antitumor effects. In conclusion, our data suggest that PD-1 is a T-cell regulator that restrains the immune-mediated abscopal effect induced by RT.

Figure 6. CD8 T cells mediated an enhanced antitumor abscopal effect in PD-1-deficient mice induced by radiation therapy.

Figure 6

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Both primary and secondary B16-OVA tumors (2 × 105) were injected into wild-type (WT) or PD-1-deficient (KO) mice (5 mice per group). Depletion of CD8 or CD4 T cells and blockade of CD11a were performed by injection of depleting or blocking antibodies starting at 2 days prior to RT and during the subsequent two weeks. Tumor growth was measured following RT. Data show the average size of primary and secondary tumors of five mice per group.

Discussion

In this study, we demonstrate that SABR-induced antitumor immunity is tumor antigen-specific and that combination SABR and anti-PD-1 therapy is not restricted to tumor histology or host genetic factors. We show that PD-1 restrains the immune-mediated abscopal effect induced by local RT in a preclinical model. PD-1 blockade or deficiency can synergize with RT to induce tumor-specific CD8+ T-cell immunity and result in a clinical response in the secondary tumors outside of the radiation field.

The demonstration of RT-induced T-cell priming within the tumor by the sensitization of the tumor stroma (13) or at tumor-draining lymph nodes via activation of antigen-presenting dendritic cells (14), as well as the accumulation of effector CD8+ T cells inside the tumor microenvironment (15), support the hypothesis that RT can elicit and enhance both the priming and effector phases of antitumor T-cell response. Consequently, if sufficient effector CD8+ cytotoxic lymphocytes are generated, they can migrate to and infiltrate distant metastatic tumors. Our CD8+ T cell-depletion data demonstrated that CD8+ T cells mediated the enhanced antitumor abscopal effects in the absence of PD-1 (Figure 6). Thus, the generation of antitumor effector CD8 T cells appears to be a key mechanism underlying the abscopal effect (16, 17).

The abscopal effect is not frequently seen with RT alone, despite the observation of the RT-induced generation of tumor-specific T cells in both pre-clinical models and in patients (15, 18). However, local RT to the primary tumor is necessary to elicit the abscopal effect, since the anti-PD-1 blockade by itself was not effective in the suppression of secondary tumors (Figures 2 and 3). The low objective response rate is further supported by the modest response rates of 13–24% in the clinical anti-PD-1 and anti-PD-L1 studies (19, 20). This observation is consistent with the hypothesis that RT induces in situ priming of antitumor effector T cells, which is dependent on immunogenic tumor cell death and the induction of danger signals (14, 21). The effector T cells generated from RT-induced tumor cell death may not be sufficient to control a distant tumor due to the up-regulation of immune checkpoint molecules such as CTLA-4 and PD-1 (Figure 4A), as well as PD-1 ligand (B7-H1), by tumor cells (Figure 4C). Therefore, the addition of checkpoint blockade immunotherapy is likely to enhance the antitumor immunity mediated by the effector T cells, especially at the sites remote from irradiation. As a proof of this concept, the blockade of CTLA-4 has been shown to enhance RT-induced abscopal effects in melanoma and lung cancer patients (6, 7, 10). Our findings further demonstrate that the PD-1 blockade or deficiency promotes the immune-mediated abscopal effect induced by SABR in multiple mouse tumor models with different genetic backgrounds and tumor histologies.

RT can be delivered using various regimens (standard, hypofractionated, SABR, etc.) in the clinics, and the RT dose and fractionation for the optimal induction of antitumor immunity has not been validated. Formenti and colleagues suggested that 8 Gy × 3 hypofractionation best induces antitumor immunity in their preclinical model (17). However, this is a sub-curative dose, and we chose a 15 Gy × 1 regimen in our preclinical model because this regimen was previously used in animal models without apparent toxicity, was more readily translatable to the clinical regimen of SABR, and was superior to a 3 Gy × 5 fractionated regimen in immune induction (Dong and Park, unpublished observation). SABR has been demonstrated to improve local control and disease-free survival in multiple prospective clinical studies in patients with oligometastatic cancers (22–24) and may have an important role in a select group of patients with limited metastases. Our preclinical data indicate that a single 15 Gy fraction was not a curative dose but was effective in tumor growth delay at the primary irradiated tumor site. It has been demonstrated that the RT-induced tumor growth delay (sub-curative RT dose) results from a dynamic balance between tumor proliferation and CD8+ T cell-mediated tumor-cell apoptosis (25). Our finding supports this previous report, as the combination of RT and PD-1 blockade or inhibition led to an improvement in tumor growth delay at the primary tumor site (synergism), with 50% of animals achieving a complete response when compared with RT alone.

While RT alone led to the primary tumor growth delay, it was not able to induce tumor regression of the distant secondary tumor that was outside of the RT field. RT can induce the expression of B7-H1 (PD-1 ligand) in the irradiated tumor tissues (25), and B7-H1 functions as a critical barrier to antitumor immunity following RT. We report herein that TILs expressed high levels of PD-1 at both irradiated primary and non-irradiated secondary tumor sites (Figure 4A) and that the tumor growth delay was enhanced at both irradiated and non-irradiated sites when PD-1 blockade or deficiency was combined with RT. Furthermore, the re-stimulation of TILs with OVA antigens confirmed that these TILs were indeed antitumor-specific effector cells that were present not only in the primary tumor site but also in the secondary tumor site (Figure 4B), which supports the immune-mediated mechanism of the abscopal effect observed in Figures 1–3. This observation suggests that PD-1/PD-L1 interaction may be a key checkpoint mechanism of TIL down-regulation in the tumor microenvironment at both the primary and secondary tumor sites.

In our study, RT in the form of SABR combined with PD-1 KO or blockade dramatically improved the control of both primary and secondary tumors. In contrast, Dewan and colleagues reported that CTLA-4 blockade required fractional irradiation to achieve secondary tumor regression (26). CTLA-4 and PD-1 regulate T-cell response at the priming and effector phases, respectively (27). The mechanisms underlying the way in which single-dose and fractionated radiation differ in their ability to synergize with PD-1 or CTLA-4 blockade are not clear. Nevertheless, our results show that the combination of PD-1 and CTLA-4 blockade has synergistic effects in promoting antitumor RT-induced abscopal effects in models with a larger tumor burden (Supplemental Figure 1). Future studies are warranted to determine the optimal radiation fractionation and timing in combination with checkpoint blockade therapy.

Using a similar B16 mouse melanoma model, Lugade and colleagues have shown that a single dose of 15 Gy irradiation resulted in the priming of tumor-specific T cells (15). Given the tendency toward RT-resistance both in vitro and in vivo, B16 tumor models are adequate for us to address the extrinsic host factor tumor growth in control mice during RT. We have compared the accumulation of tumor-antigen specific CD8+ T cells within B16-OVA tumors between a single dose of SABR and fractional RT (3 Gy × 5). We found that the numbers of OT-1 CD8 T cells (with TCR specific for OVA antigen) were significantly higher in mice treated with SABR as compared with fractional RT (Dong and Park, unpublished observation). This is consistent with the fact that single-dose radiotherapy promotes cross-priming of T cells within primary tumors (14) but that the magnitude of the elicited T-cell responses is restrained by PD-1 expression following irradiation (28). It is conceivable that up-regulation of PD-1 in effector CD8+ T cells could result in insufficient control of secondary tumors outside the radiation field. Accordingly, a blockade of PD-1 would protect the effector CD8+ T cells from the immunosuppressive effects of PD-1 to achieve antitumor immunity.

Antibodies targeting immune checkpoint molecules on T cells to enhance antitumor immunity have demonstrated promising effects in the control of advanced/metastatic cancers in recent clinical studies (19, 20). PD-1 blockade was shown to be active as a single treatment in melanoma, renal, and lung carcinomas; however, only a small portion of patients achieved a complete response (19). Previous pre-clinical studies have shown that RT synergizes with anti-PD-1 or anti-B7-H1 to produce tumor regression in several tumor models (25, 28, 29). While the combination RT and PD-1 blockade has not been reported in the clinics, a case report showed an abscopal effect when RT was added to an ongoing anti-CLTA-4 therapy in one melanoma patient (6). Our studies are consistent with clinical predictions of PD-1 blockade, and they support the concept that local RT can synergize with checkpoint blockade therapy to promote antitumor immunity.

In conclusion, our data could be of significant clinical interest because they suggest that the combination of anti-PD-1 blockade and local radiotherapy can lead to the systemic control of tumors that are refractory to treatment with PD-1 blockade alone. Another potential advantage is that a single SABR and short-term PD-1 blockade may focus the antitumor T-cell response in the targeted tissues and be less likely to elicit an autoimmune reaction in other organs.

Supplementary Material

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2

Acknowledgments

Financial Support: NIH R01 CA134345 (Kwon, Dong), Fraternal Order of Eagles Cancer Research Fund (Dong). NIH/NIAID R01 AI095239 (Dong). RSNA Research Scholar (Park)

The authors thank Drs. L. Chen (Yale University, New Haven, CT USA) and T. Honjo (Kyoto University, Japan) for the PD-1 knockout mice.

Footnotes

Delivered as an oral presentation at ASTRO’s 56th Annual Meeting (September 14 – 17, 2014) in San Francisco, CA

Disclosure of Potential Conflicts of Interest: No conflicts of interest by authors.

Authors’ Contributions:

Conception and design: H. Dong, E.D. Kwon, K.R. Olivier, and S.S. Park

Development of methodology: H. Dong, E.D. Kwon, C.J. Krco, and S.S. Park

Acquisition of data: H. Dong, X. Liu, and S.H. Harrington

Analysis and interpretation of data: H. Dong, E.D. Kwon, C.J. Krco, A.S. Mansfield, and S.S. Park

Writing, review and/or revision of manuscript: H. Dong, C.J. Krco, A.S. Mansfield, and S.S. Park

Administrative, technical, or material support: H. Dong, X. Liu, S.H. Harrington, C.J. Krco,, M.P. Grams, K.M. Furutani, and S.S. Park

Study supervision: H. Dong, S.H. Harrington, and S.S. Park

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Supplementary Materials

1
NIHMS666676-supplement-1.pptx (62.3KB, pptx)
2
NIHMS666676-supplement-2.docx (15KB, docx)

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