Antitumor Efficacy of Radiation plus Immunotherapy Depends upon Dendritic Cell Activation of Effector CD8+ T Cells

Abstract

Tumor cells dying after cytotoxic therapy are a potential source of antigen for T-cell priming. Antigen-presenting cells (APC) can cross-present MHC I-restricted peptides after the uptake of dying cells. Depending on the nature of the surrounding environmental signals, APCs then orchestrate a spectrum of responses ranging from immune activation to inhibition. Previously, we had demonstrated that combining radiation with either agonistic monoclonal antibody (mAb) to CD40 or a systemically administered TLR7 agonist could enhance CD8 T-cell-dependent protection against syngeneic murine lymphoma models. However, it remains unknown how individual APC populations affect this antitumor immune response. Using APC depletion models, we now show that dendritic cells (DC), but not macrophages or B cells, were responsible for the generation of long-term immunologic protection following combination therapy with radiotherapy and either agonistic CD40 mAb or systemic TLR7 agonist therapy. Novel immunotherapeutic approaches that augment antigen uptake and presentation by DCs may further enhance the generation of therapeutic antitumor immune responses, leading to improved outcomes after radiotherapy. Cancer Immunol Res; 4(7); 621-30. ©2016 AACR.

Figure 1. Combination therapy with RT and CD40 mAb led to the generation of durable antitumor immune responses capable of preventing disease recurrence in murine lymphoma models.

A, Survival curve of EG7 tumor bearing mice following treatment with a single 10 Gy dose of RT either alone or in combination with CD40 mAb. * P < 0.05 compared to control mice (log-rank; Mantel-Cox test). B and C, survival curve following contralateral rechallenge of LTS mice with EG7 (B) and tumor growth curve for rechallenge with EL4 (C) cells. D and E, representative dot blot of IFNγ production (D) and frequency of IFNγ⁺ CD8⁺ T cells (E) isolated from either tumor naïve or LTS mice originally treated with RT and CD40 mAb following coculture with 25 Gy irradiated EL4 cells for 5 days, followed by priming with 25 Gy irradiated EL4 cells. ** P < 0.0l (Mann-Whitney test). F, representative density plots of peripheral blood confirming lymphocyte depletion. G, Survival curve of mice bearing established BCL₁ tumors following treatment with a single dose of 5 Gy RT in combination with CD40 mAb. Lymphocytes (CD4⁺ or CD8⁺) were depleted 1 day prior to therapy with depletion maintained for up to 2 weeks. ***, P > 0.00l, *, P > 0.0l, *, P > 0.05, Mann-Whitney test. Experimental groups contained at least 5 mice and are representative of at least 2 independent experiments.

Figure 2. Depletion of B cells does not impact the efficacy of combination therapy with RT and CD40 mAb in models of T cell lymphoma.

A and B, Survival curve of EL4 (A) and tumor volumes of EG7 (B) bearing mice following combined therapy (10 Gy RT in combination with CD40 mAb). Not significant (n/s), P > 0.05 compared to control mice (log-rank; Mantel-Cox test). n/s, P < 0.05, Mann-Whitney test. Depletion of B cells achieved by i.p. administration of CD20 mAb 1 day prior to therapy. C, representative dot plots of peripheral blood from control or CD20 mAb-treated mice to confirm depletion. Experimental groups contained at least 5 mice and are representative of 2 independent experiments.

Figure 3. Macrophages are dispensable for efficacy following combination therapy with RT and CD40 mAb.

A and B) BCL₁ lymphoma cells labeled with PKH-26 were injected i.p in to BALB/c mice and treated with 5 Gy RT. Phagocytosis of irradiated tumor cells by peritoneal MØs was assessed after 24 h by fluorescence microscopy (A) or flow cytometry (B). MØs were labeled with alexa fluor 488-phalloidin or F4/80-FITC respectively. C and D, Survival curves of BCL₁ (C) and EL4 (D) tumor bearing mice following treatment with a single 5 Gy (C) or 10 Gy (D) dose of RT in combination with CD40 mAb. Mice received either PBS or clodronate encapsulated liposomes (PBS-lip and Clod-lip, respectively) on day -1 day +2 and +4 with respect to therapy. n/s, P > 0.05 compared to controls (log-rank; Mantel-Cox test). Experimental groups contained at least 5 mice and are representative of at least 2 independent experiments.

Figure 4. Dendritic cell depletion abrogates the therapeutic effect of combination therapy.

A, Splenic CD11c⁺ DC were isolated and co-incubated with PKH-26–labeled irradiated BCL1 lymphoma cells at a ratio of 20:1. Uptake was assessed by flow cytometry after 3 h. ***, P < 0.001 vs. control). B and C, CD11c-DTR chimeric mice received DT (100 ng i.p) and spleen cells analysed for presence of CD11c⁺ cells after 24 hours in BALB/c mice by flow cytometry (B) or by immunohistochemistry in C57B1/6 mice (C). D and E, mice were inoculated with either BCL₁ (D) or EL4 (E) tumor cells and received combined therapy (RT; either 5 Gy or 10 Gy respectively, in combination with CD40 mAb). For DC depletion, animals received 100 ng DT i.p. 3 times per week for up to 2 weeks i.p (D). F, Incidence of metastatic disease present in the lymph nodes in mice following treatment outlined in (E). * P < 0.05 compared to combination therapy (log-rank; Mantel-Cox test). Experimental groups contained at least 5 mice and are representative of at least 2 independent experiments.

Figure 5. Dendritic cells are critical for the therapeutic effect of combination therapy with RT and a systemically administered TLR7 agonist.

A and B, Growth (A) and survival (B) curves of EL4 tumor bearing mice following combination therapy with 10 Gy RT and R848. Frequency of systemic disease in cohort expressed as fraction relative to population size (A). Depletion of B cells was achieved by i.p. administration of CD20 mAb 1 day prior to therapy. For DC depletion, animals received 100 ng DT i.p. 3 times per week for up to 2 weeks i.p. n/s, P > 0.05 compared to controls (log-rank; Mantel-Cox test). C, Mice received either PBS or clodronate encapsulated liposomes (PBS-lip and Clod-lip, respectively) on day -1 day +2 and +4 with respect to therapy with 10 Gy RT and R848. Experimental groups contained at least 5 mice and are representative of at least 2 independent experiments.