Mechanisms involved in radiation enhancement of intratumoral dendritic cell therapy

Abstract

We have previously reported that local tumor irradiation, without inducing cell death, can augment the therapeutic efficacy of intratumoral (IT) dendritic cell (DC) vaccination. This study examined potential mechanisms underlying radiation enhancement of IT DC therapy in this setting. Even though ionizing radiation did not mediate tumor cell killing, bone marrow-derived DCs acquired in vitro tumor antigens from irradiated D5 murine melanoma cells more efficiently than from untreated cells. This radiation-enhanced loading of DCs did not induce DC maturation, but was associated with improved cross-priming of T cells both in vitro and in vivo. Furthermore, in vivo pulsing of DCs with irradiated versus untreated tumor cells resulted in superior presentation of tumor antigens to T cells. In addition, tumor irradiation facilitated homing of IT administered DCs to the draining lymph node, possibly by down-regulating CCL21 expression within the tumor mass. Studies of the tumor microenvironment in irradiated versus untreated tumors did not reveal significant inflammatory changes. Moreover, radiation did not promote accumulation of CD4 or CD8 effector T cells within solid tumors. Our results indicate that, without inducing cytotoxicity, tumor irradiation can enhance the ability of DCs to capture tumor antigens, migrate to the draining lymph node, and present processed antigens to T cells. These findings may prove useful in designing future strategies for human cancer immunotherapy.

FIGURE 1

A, Radiation does not induce apoptosis or necrosis in D5 cell cultures. Immediately and 18 hours after a single radiation dose of 60 Gy, D5 cells were analyzed for apoptotic and necrotic cells using the Annexin V-FITC/PI FACS assay. Untreated cells served as a negative control. The lower left quadrant represents viable cells, the lower right quadrant depicts cells in early apoptosis, and the upper right quadrant shows necrotic and late apoptotic cells. B, DCs acquire antigens from irradiated D5 cells more efficiently than from untreated tumor cells. DCs were cultured with CFSE-stained irradiated versus untreated D5 cells for 18 hours. After pulsing, cells were labeled with R-PE-conjugated anti-MHC class II mAb and analyzed by flow cytometry. Bottom dot plots were gated on FL2 positive cells. Some of the control groups, namely CFSE-labeled irradiated and untreated D5 cells, and unpulsed-DCs are shown. The numbers inside the dot plots shown in A and B represent the percentage of cells within the corresponding quadrant. Experiments were repeated at least three times with similar results.

FIGURE 2

Tumor irradiation improves T cell cross-priming in vitro and in vivo. A and B, D5-G6 TDLN cells were stimulated in vitro by irradiated versus non-irradiated tumor cells pulsed-DCs (RP-DC versus URP-DC, respectively) at various ratios. The incidence of IFN-γ-secreting cells (A) and the amount of IFN-γ released to the supernatant (B) were determined via ELISPOT and ELISA assays, respectively. In control samples containing D5-G6 TDLN cells alone, RP-DCs alone, URP-DCs alone, and D5-G6 TDLN cells co-cultured with unpulsed-DCs no IFNγ spots or IFNγ secretion were detected. (A), Data are reported as the average number of spots per 1×10⁵ responders ± SE of triplicate samples. (B), Data are reported as the average concentration of IFNγ (pg/ml) per 1×10⁶ responders per 48 h ± SE of triplicate samples. *, P<0.05. C and D, DCs pulsed with irradiated tumor cells (RP-DCs) are more effective than DCs pulsed with non-irradiated tumor cells (URP-DCs) in eliciting tumor-specific IFN-γ secretion of splenocytes (C) and mediating lung metastases regression (D). Mice were inoculated i.v. with D5 tumor cells on day 0. RP-DCs were administered i.d. on days 3, 7, and 11. Control groups received either no treatment (PBS), irradiated D5 cells (RT-D5), unpulsed-DCs (UP-DCs), tumor lysate pulsed-DCs (TP-DCs) or DCs pulsed with non-irradiated tumor cells (URP-DCs). Spleens and lungs were harvested 15 days after tumor inoculation. C, Splenocytes from treated and control mice were cultured with or without specific or irrelevant irradiated tumor cells in an IFN-γ ELISPOT assay. Data are reported as the average number of spots per 5 × 10⁴ responders ± SE of triplicate samples. *, P<0.01 versus all other groups. D, Pulmonary metastases were enumerated. Data are reported as the mean number of metastases ± SE of six mice per group. * P<0.001 versus PBS, RT-D5, UP-DC and URP-DC. Experiments were repeated at least two times with similar results.

FIGURE 3

A and B, Lymph node cells draining D5 tumors subjected to combined treatment with radiation plus DCs, but not to monotherapies, undergo effective in vivo priming to tumor antigens. Unpulsed-DCs were injected into irradiated D5 tumors. Control groups of mice received either no treatment (NT), DC only, or radiation only (RT). After 2 days, TDLN cells were harvested and analyzed for IFN-γ, GM-CSF, and IL-2 secretion in response to either D5 cells (A) or anti-CD3 mAb activation (B). Data are reported as the average concentration of cytokine (pg/ml) per 5 × 10⁵ responders per 24 h ± SE of triplicate samples. *, P<0.001 versus all other groups. C, Tumor irradiation enhances DC migration to the draining lymph node. CFSE-labeled unpulsed-DCs were injected into irradiated versus untreated s.c. D5 tumors either 1, 3, or 7 days after radiation. Tumors and TDLNs were harvested 24, 48, and 72 hours after each i.t. injection. Fluorescein-labeled cells were detected using an anti-FITC, horseradish peroxidase-conjugated antibody. Positive cells stained purple in tumor sections, and brown in lymph node sections. Representative fields from two independent experiments are shown [original magnification, X400 (left and right) or X100 (middle)]. Upper left, Tumor section stained with an isotype matched control antibody. Lower left, Tumor section stained with anti-FITC antibody. Upper middle and right, Section of a lymph node draining an untreated tumor. Lower middle and right, Section of a lymph node draining an irradiated tumor. D Tumor irradiation decreases CCL21 gene expression within the tumor mass by one-half compared with untreated tumors (upper left). But CCL21 gene expression within the tumor beds (upper right), and the tumor draining lymph nodes (lower left) does not change significantly between irradiated and untreated tumors. As a result, CCL21 concentration gradient between the tumor and the draining lymph node is increased (lower right). Expression of CCL21 mRNA within irradiated and untreated s.c. D5 tumors, tumor beds, and TDLNs was measured by quantitative real time RT-PCR. CCL21 mRNA expression was first normalized to the expression of HPGRT, and was then normalized to the average expression of CCL21 mRNA in untreated tumors (upper left), in untreated tumor beds (upper left), in untreated TDLNs (lower left) or in corresponding TDLNs (lower right). RNA was extracted from five tumors, three tumor beds, and three TDLNs per group, and gene expression was examined in individual samples. * p < 0.05. In upper right and lower left panels p is non-significant.

FIGURE 4

Characterization of the immune infiltrate within irradiated versus untreated s.c. D5 tumors. A, Mice were inoculated s.c. in bilateral flanks with D5 tumor cells on day 0. Right flank tumors only were locally irradiated on days 7 to 11. Tumors were harvested and weighed on days 12, 19, and 26. Data are reported as the mean tumor weight in grams ± SE of five mice per group. This experiment was repeated two times with similar results. *, P<0.04. B, Twelve day tumors described in A were mechanically disaggregated to single cell suspensions, stained for CD45 and CD11c (left) or CD4 (right) and analyzed by flow cytometry. The absolute number of cells of interest × 10⁶ per gram tumor for each individual tumor was calculated using polystyrene microbeads. Bilateral tumors harvested from the same mouse are represented by the same X axis value. Data represents cumulative results of two experiments. P=0.0006 (left), P=0.0027 (right). C, Tumors described in B were stained for CD45 and CD11c, CD4, CD8, NK1.1, CD14 or Ly6G and analyzed by flow cytometry. Data are reported as the mean percentage (SE) of nine tumors from two independent experiments. *, P<0.03.

FIGURE 5

A (left), Adoptively transferred CD4⁺ effector cells localize randomly between dual s.c. solid tumors. CD45.2 mice were inoculated s.c. in bilateral flanks with D5 cells on day 0. CD45.1 effector cells (2×10⁷) were injected i.v. on day 14. Tumors were harvested 48 hours after adoptive transfer, mechanically disaggregated to single cell suspensions, stained for CD45.1 and CD4 and analyzed by FACS. Each data point represents an individual tumor, and bilateral tumors harvested from the same mouse are represented by the same X axis value. P=0.3361, not significant. (right), CD4⁺ effector cells do not localize preferentially to irradiated s.c. tumors. In additional mice in the study detailed above, right tumors only were locally irradiated on days 7 to 11. Tumors were harvested 24, 48, and 72 hours after adoptive transfer and processed as described above. Data are reported as the mean number of cells × 10³ per gram of tumor ± SE of five tumors per group. *, P=0.034. B, C, and D, CD8⁺ effector cells do not localize preferentially to irradiated s.c. tumors. CD45.1 mice were inoculated s.c. in bilateral flanks with B16-OVA cells on day 0. Right tumors only were locally irradiated on days 7 to 11. CFSE-labeled CD45.2 CD8⁺ effector cells (1×10⁷) derived from OT-1 mice were injected i.v. on day 11, after the last dose of radiation was administered. Twenty four, 48, and 72 hours after adoptive transfer, tumors were harvested, weighed, and mechanically disaggregated to single cell suspensions. Samples were stained for CD45.2 and CD8 and analyzed by FACS. B (left) Tumor irradiation inhibits B16-OVA tumor growth. Data are reported as the mean tumor weight ± SE of at least five tumors per group. *, P<0.05. (right), Twenty four hours after adoptive transfer, non-irradiated tumors contained significantly more host CD8⁺ cells per gram of tumor compared with irradiated tumors. Each data point represents an individual tumor, and bilateral tumors harvested from the same mouse are represented by the same X axis value. P<0.001. C (left), Data are reported as the mean number of adoptively transferred CD8⁺ cells × 10⁶ per gram of tumor ± SE of at least five tumors per group. (right), Transferred CD8⁺ effector cells detected within irradiated versus non-irradiated tumors proliferated to the same extent. The geometrical mean of CFSE intensity of CD45.2⁺CD8⁺ effector cells within irradiated and non-irradiated tumors was recorded at 24, 48, and 72 hours after infusion. Data are reported as the mean of the geometrical mean of CFSE intensity ± SE of at least five tumors per group. D, B16-OVA tumors harvested 24, 48 and 72 hours after adoptive transfer were fixed in formalin and embedded in paraffin. Fluorescein-labeled cells were detected using an anti-FITC, horseradish peroxidase-conjugated polyclonal antibody. Representative fields from non-irradiated (left) and irradiated (right) tumors are shown (magnification, X400).