Vaccination with Mage-b DNA induces CD8 T-cell responses at young but not old age in mice with metastatic breast cancer

Abstract

Background: Elderly individuals react less efficiently to vaccines than do adults, mainly because of T-cell unresponsiveness. In this study, we analysed whether tumour-associated antigen (TAA)-specific CD8 T-cell responses could be induced by vaccination in old mice with metastatic breast cancer.

Methods: The effect of pcDNA-3.1- and Listeria-based vaccines, expressing TAA Mage-b, on Mage-b-specific immune responses was tested in spleens and draining lymph nodes (LNs) of mild (4TO7cg) and aggressive (4T1) syngeneic metastatic mouse breast tumour models at young (3 months) and old (20 months) age.

Results: Interferon gamma and interleukin-2 levels increased significantly in draining LNs and spleens of Mage-b-vaccinated mice compared with those in control groups at young but not old age in both mouse tumour models. A significant increase was observed in the number of IFNgamma-producing Mage-b-specific CD8 T cells after Mage-b vaccination in the 4T1 model at young but not old age. This correlated with a reduced protective effect of Mage-b vaccination against metastatic breast cancer at old compared with young age.

Conclusions: The absence of CD8 T-cell responses after Mage-b vaccination and the accompanying reduced protection against metastatic breast cancer in old compared with young mice point towards the need for tailoring cancer vaccination to older age.

Figure 1

Schematic depiction of immunisations in 4TO7cg and 4T1 mice. (A) 4TO7cg mice were twice immunised intramuscularly (im) with pcDNA3.1-Mage-b, pCDNA3.1, or with saline at days 1 and 21. Two weeks after the last immunisation, mice were injected with 10⁵ 4TO7cg tumour cells into a mammary fat pad. Four weeks later, mice were killed and analysed. (B) 4T1 mice were immunised intraperitoneally (ip) with pcDNA3.1-Mage-b+GM-CSF, pCDNA3.1+GM-CSF, or saline+GM-CSF at days 1, 21, and 42. All mice of the 4T1 model received thioglycollate broth (TGB) ip 4 days before each immunisation (grey short arrow). At day 39, mice were injected with 10⁵ 4T1 tumour cells into a mammary fat pad. Four weeks later, they were killed and analysed. (C) BALB/c mice were immunised thrice intraperitoneally with 0.1 × LD₅₀ (10⁷ CFU) of Listeria-based Mage-b_311–660 vaccine, 0.1 × LD₅₀ of LM-LLO vector control, or with saline, using 1-week time intervals. Four days after the second immunisation, mice were injected with 10⁵ 4T1 tumour cells in a mammary fat pad. Two weeks after tumour challenge, mice were killed and analysed. Young mice were 3 months of age, and old mice 20 months.

Figure 2

Expression levels of Mage-b in metastases and primary tumours of the 4T1 model at old age. The Mage-b-specific RT–PCR products of 632 bp were detected by Southern blotting using a DNA probe encoding Mage-b. β-actin was used as RNA control for each sample. The lanes were loaded as follows: lanes 1 and 2, 4T1 primary tumours; lanes 3 and 4, metastases mesenteric lymph nodes (LNs); lanes 5 and 6, metastases liver; lanes 7 and 8, metastases spleen; lanes 9 and 10, metastases liver; lanes11 and 12, metastases diaphragm; and lanes 13 and 14, normal breast. Tumour and normal tissues were randomly analysed from different mice (n=5). Two representative examples of each tissue are shown in this figure.

Figure 3

The effect of Mage-b vaccination on metastases and primary tumours at young and old age in 4TO7cg and 4T1 models. The effect of vaccination with pcDNA3.1-Mage-b was measured on the growth of metastases and primary tumours of the 4TO7cg model (A–D). Young (3 months) and old (20 months) mice were immunised and challenged with 4TO7cg tumour cells as described in Figure 1A. The number of metastases per mouse was determined in young (panel A) and old (panel B) mice. Furthermore, tumour weight was determined in young (panel C) and old (panel D) mice. At young age n=15 mice per group, and at old age n=7 mice per group. Results were averaged per group and subjected to statistical analysis using the Mann–Whitney test (P<0.05 is significant). This experiment was performed once. The effect of Mage-b vaccination was also measured on the growth of metastases and primary tumours of the 4T1 model (E–H). Young and old mice were immunised and challenged with 4T1 tumour cells as described in Figure 1B. The number of metastases per mouse was determined in young (panel E) and old (panel F) mice. Furthermore, tumour weight was determined in young (panel G) and old (panel H) mice. The results shown in this study are the average of three independent experiments, and subjected to statistical analysis (Mann–Whitney P<0.05 is significant). n=5–10 mice per group. Each triangle represents one mouse. In addition, the effect of vaccination with LM-LLO-Mage-b_311–660 was measured on the growth of metastases and primary tumours of the 4T1 model at young and old (I–L) age. Young and old mice were immunised and challenged with 4T1 tumour cells as described in Figure 1C. The number of metastases per mouse was determined in young (panel I) and old (panel J) mice. Moreover, tumour weight was determined in young (panel K) and old (panel L) mice. Results were averaged per group and subjected to statistical analysis (Mann–Whitney P<0.05 is significant). In this experiment, n=5 mice per group, and the experiment was performed once. In all figures, the results in the Mage-b group were compared with those in both control groups; error bars represent s.e.m.

Figure 3

The effect of Mage-b vaccination on metastases and primary tumours at young and old age in 4TO7cg and 4T1 models. The effect of vaccination with pcDNA3.1-Mage-b was measured on the growth of metastases and primary tumours of the 4TO7cg model (A–D). Young (3 months) and old (20 months) mice were immunised and challenged with 4TO7cg tumour cells as described in Figure 1A. The number of metastases per mouse was determined in young (panel A) and old (panel B) mice. Furthermore, tumour weight was determined in young (panel C) and old (panel D) mice. At young age n=15 mice per group, and at old age n=7 mice per group. Results were averaged per group and subjected to statistical analysis using the Mann–Whitney test (P<0.05 is significant). This experiment was performed once. The effect of Mage-b vaccination was also measured on the growth of metastases and primary tumours of the 4T1 model (E–H). Young and old mice were immunised and challenged with 4T1 tumour cells as described in Figure 1B. The number of metastases per mouse was determined in young (panel E) and old (panel F) mice. Furthermore, tumour weight was determined in young (panel G) and old (panel H) mice. The results shown in this study are the average of three independent experiments, and subjected to statistical analysis (Mann–Whitney P<0.05 is significant). n=5–10 mice per group. Each triangle represents one mouse. In addition, the effect of vaccination with LM-LLO-Mage-b_311–660 was measured on the growth of metastases and primary tumours of the 4T1 model at young and old (I–L) age. Young and old mice were immunised and challenged with 4T1 tumour cells as described in Figure 1C. The number of metastases per mouse was determined in young (panel I) and old (panel J) mice. Moreover, tumour weight was determined in young (panel K) and old (panel L) mice. Results were averaged per group and subjected to statistical analysis (Mann–Whitney P<0.05 is significant). In this experiment, n=5 mice per group, and the experiment was performed once. In all figures, the results in the Mage-b group were compared with those in both control groups; error bars represent s.e.m.

Figure 4

Mage-b-induced immune responses (in vitro) at young and old age in the 4TO7cg and 4T1 model. Mage-b-induced immune responses were determined at young and old age. Mice were vaccinated (pcDNA3.1-Mage-b) and challenged with 4TO7cg or 4T1 tumour cells, as shown in Figure 1A or B, respectively. Cells from spleens were transfected with Mage-b DNA, and draining LNs were re-stimulated with syngeneic 64pT breast tumour cells, highly expressing Mage-b. The production of IL-2 by cells in the spleen (A and C) and of IFNγ by cells in the draining lymph nodes (LNs) (B and D) of vaccinated and control mice was determined by quantitative ELISA. Controls such as nonstimulated cells from spleens or draining LNs were negative (data not shown). All experiments were performed in triplicate and subjected to statistical analysis using the Tukey–Kramer Multiple Comparison test (P<0.05 is significant). The cytokine levels produced in the Mage-b group were compared with those produced in the saline and vector control groups. At young age n=10 mice per group, and at old age n=15. Mice were also vaccinated with LM-LLO-Mage-b_311–660 and challenged with 4T1 tumour cells, as shown in Figure 1C. Cells from spleens were re-stimulated with bone marrow cells transfected with pcDNA3.1-Mage-b and pCMV1-GM-CSF plasmid DNA. Two days later, Mage-b-specific immune responses were analysed by ELISPOT (E). Involvement of Mage-b-specific CD8 T cells was determined by negative depletion, using magnetic beads with anti-CD8 antibodies. Controls such as BM cells transfected with pcDNA3.1-Mage-b or with pCMV-GM-CSF, and nontransfected BM cells were negative (data not shown). All experiments were performed in triplicate. n=5 mice per group, and were used once. Results were averaged and subjected to statistical analysis using the Mann–Whitney test (P<0.05 is significant). The number of IFNγ-producing cells in the Mage group was compared with that in the saline and vector control groups. In all figures, cells from spleens or draining LNs were pooled; error bars represent the s.e.m.

Figure 5

Mage-b-specific immune responses (in vivo) at young and old age in the 4T1 model. Young and old mice were vaccinated, as shown in Figure 1B. CD8 T cells, CD4 T cells, macrophages, NK cells, and B cells were analysed by fluorescence-activated cell sorter (FACS) for the production of intracellular IFNγ without re-stimulation in vitro. Spleen cells of 15–20 mice in each group were pooled. Of each sample, 10 000 spleen cells were analysed by FACS. The percentage of IFNγ-producing cells was determined for each cell type.