Local irradiation of murine melanoma affects the development of tumour-specific immunity

Abstract

Radiation therapy affects the immune system. In addition to killing radiosensitive immune cells, it can induce functional changes in those cells that survive. Our recent studies showed that the exposure of dendritic cells (DCs) to radiation in vitro influences their ability to present tumour antigen in vivo. Here we show that local radiation therapy of B16 melanoma tumours inhibits the development of systemic immunity to the melanoma antigen MART-1. This inhibition could not be overcome by intratumoral injection of DCs expressing human MART-1 after radiation therapy, suggesting that a form of immune suppression might have developed. On the other hand, injection of MART-expressing DCs prior to tumour irradiation was able to prevent inhibition from developing. These results suggest that local radiation therapy may block the generation of immunity under some circumstances and that strategies may be required to prevent this and allow radiation-induced cell death to translate fully into the development of systemic immunity.

Figure 1

Radiation decreases immunity generated by adenovirus encoding MART-1 cDNA transduced dendritic cells (AdVMART-1/DC). C57BL/6 mice were treated with 5 × 10⁵ irradiated (10 Gy) or non-irradiated AdVMART-1/DC. A second immunization was performed 10 days later with 5 × 10⁵ irradiated or non-irradiated AdVMART-1/DC. One week after the last immunization, MART-1-specific (a) interferon-γ (IFN-γ) and (b) interleukin-4 (IL-4) responses were assessed using enzyme-linked immunospot (ELISPOT) assays. Splenocytes were restimulated for 48 hr with EL4(MART-1) cells (black bars) or EL4 cells (white bars), or were not restimulated (control; grey bars). Mice immunized with AdVMART-1/DC that had been irradiated showed a reduced number of IFN-γ- and IL-4-expressing cells compared to those injected with non-irradiated dendritic cells. Furthermore, irradiated AdVMART-1/DC, whether given before or after AdVMART-1/DC, decreased their ability to generate responses. Results are shown as mean ± 1 standard error of the mean (SEM) of triplicate data of one representative of three independent experiments [*P< 0·05 compared with the EL4(MART-1)-restimulated group; **P< 0·0001 as compared with the AdVMART-1/DC-injected group].

Figure 2

Radiation suppresses B16 anti-tumour immunity. C57BL/6 mice were implanted with viable B16 cells and irradiated with 0 or 10 Gy when the tumour size reached approximately 5–6 mm in diameter. One week after the treatment, the number of splenic interferon-γ (IFN-γ)-producing lymphocytes was detected using enzyme-linked immunospot (ELISPOT) assays 48 hr after restimulation with EL4(MART-1) cells (black bars) or EL4 cells (white bars), or without restimulation (control; grey bars). Three mice were used in each treatment group. Mice without any treatments served as controls. The results shown are the mean ± 1 standard error of the mean (SEM) of triplicate data from one representative of three experiments (*P< 0·05 compared with the EL4(MART-1)-restimulated group).

Figure 3

Flow cytometric analyses of cell populations in B16 tumour-bearing mice. C57BL/6 mice with 5–6-mm-diameter tumours were irradiated with 0 or 10 Gy. Cells were harvested from tumour (Fig. 3a) and spleens (Fig. 3b) and, either 2 or 4 days after the radiation treatment, were stained with antibodies to various immunecell subset markers and analyzed by flow cytometry. Mice without tumours are used as a control in Fig. 3b. Data in Fig. 3a,b represent the subpopulation in total tumour cells and in gated splenic lymphocytes respectively. Figure 3(c) shows CD4⁺ CD25⁺ Foxp3+ T-regulatory cells as a fraction of CD4⁺ cells in both tumour lymphocytes and splenic lymphocytes. Results are shown as mean ± 1 standard error of the mean (SEM) of three to five experiments. (*P< 0·05 as compared with the 0 Gy group in Fig. 3a,c and with the control group in Fig. 3b).

Figure 4

Intratumoral injection of adenovirus encoding MART-1 cDNA transduced dendritic cells (AdVMART-1/DCs) before, but not after, radiation therapy prevents radiation-induced immune suppression. C57BL/6 mice bearing 5–6-mm-diameter B16 tumours were irradiated (0 or 10 Gy) 1 day before or after the intratumoral delivery of AdVMART-1/DCs (5 × 10⁵). Six groups of mice were used in this study: (i) without tumours or treatments; (ii) with tumours and no treatments; (iii) with B16 tumours irradiated with 10 Gy; (iv) with tumours injected with AdVMART-1/DCs; (v) with tumours and AdVMART-1/DCs injected 1 day prior to local tumour irradiation (10 Gy); and (vi) with tumours irradiated (10 Gy) followed 1 day later by AdVMART-1/DC injection. One week after the first treatment, interferon-γ (IFN-γ)-producing lymphocytes were detected using enzyme-linked immunospot (ELISPOT) assays after 48 hr of restimulation with EL4(MART-1) cells (black bars) or EL4 cells (white bars), or with no restimulation (control; grey bars). Three mice were used in each treatment group. The results shown are the mean ± 1 standard error of the mean (SEM) of triplicate data from one representative experiment of two carried out (*P< 0·05 as compared with the EL4(MART-1)-restimulated group).

Figure 5

In vivo migration of irradiated or non-irradiated dendritic cells (DCs). Lipopolysaccharide (LPS)-treated and untreated DCs were labeled with the green fluorescent dye, PKH2, irradiated (0 or 10 Gy) and injected subcutaneously (s.c.) into the mouse foodpad. Mice without DC injection were used as controls. Fluorescent cells within the lymph node (LN) population were detected by flow cytometry 48 hr after injection of DC. Three mice were used in each treatment group. Results shown are the mean ± 1 standard error of the mean (SEM) of three experiments (*P< 0·05 compared with the 0 Gy DC group).