CpG-ODN but not other TLR-ligands restore the antitumor responses in old mice: the implications for vaccinations in the aged

Abstract

Aim: There is accumulative evidence indicating that targeting antigen presenting cells (APCs) with different types of adjuvants could result in the induction of antitumor immune responses. It has been hypothesized that APCs function may be altered in the elderly contributing to a decline in the immune function. We evaluated whether targeting APCs following injection with Poly I:C, LPS, flagellin, imiquimod and CpG-ODN would induce an antitumor response in the old.

Materials and methods: The immune and antitumor responses induce Poly I:C, LPS, flagellin, imiquimod and CpG-ODN were compared in young (2 month old) and old (18 months) mice.

Results: Our results indicated that only intratumoral (i.t.) injections of CpG-ODN completely rejected the tumor in both young and old mice. Injections of Poly I:C also induced the rejection of tumors in the young but not in the old. Furthermore, i.t. injections of CpG-ODN promoted the development of protective memory responses in the young and the old. Analysis of the immune responses in the old indicated that CpG-ODN but not Poly-I:C induces: a pro-inflammatory Th1 type response; accumulation and activation of CD4+, CD8+ T and, NK cell responses; activation of APCs; and reduction in the number of Tregs. The activation of these immune-parameters positively correlates with the induction of an antitumor response.

Conclusions: These studies indicate that there are differences in the level of stimulation with TLR-ligands between young and old APCs and that the aged immune responses can be rescued and exploited for the induction of tumor immunity by targeting APCs with specific TLR-ligands. These results have important clinical implications for developing immunization strategies containing TLR-ligands that will be effective in both the young and old.

Fig. 1

Analysis of the antitumor effect of TLR-ligands in young and old mice. Young (a, c) and old (b, d) BALB/c or C57BL/6 mice were implanted with 10⁶ TUBO (a, b) or TRAMP-C2 (c, d) cells on day zero. On day 10 animals received i.t. injections of CpG-ODN, Poly-I:C, Flagellin, Imiquimod, LPS and CpG-control (30 μg/injection) three times a week for 3 weeks. Animals were monitored for the development of tumors. Data are the means of two independent experiments ±SEM with 5–8 animals per group per experiment. *P < 0.001 difference was found between control mice and mice injected with CpG-ODN (or Poly I:C for young animals)

Fig. 2

CpG-ODN induces a protective memory response in young and old mice. a Young and old BALB/c mice were implanted with 10⁶ TUBO cells on day zero. On day 10 animals received i.t. injections of CpG-ODN (30 μg/injection) three times a week for 3 weeks. Young mice also received i.t. injections of Poly-I:C. About 60 days later animals were challenged with a lethal dose (10⁶) of TUBO cells Control group are naïve animals inoculated with 10⁶ TUBO cells at the time of challenge. Survival was monitored and percent of survival was determined. Five to seven animals were included per group. Data are representative of two experiments. b Old BALB/c animals were depleted of CD4 and CD8 T cells and NK cells. Animals were s.c. implanted with 10⁶ TUBO cells on day zero. On day ten, animals started i.t. injections with CpG-ODN as described above. Animals were monitored for survival. Six animals were included per group. Data are representative of two experiments

Fig. 3

Analysis of infiltrating DC and macrophage activation following treatment with CpG and Poly I:C in young and old animals. Young (a, c) and old (b, d) BALB/c mice were implanted with 10⁶ TUBO cells on day zero. Tumor was allowed to grow until tumor size was ∼400 mm³. Tumors were then treated with i.t. injections of CpG-ODN or Poly I:C (30 μg/injection) three times a week for 1 week. Next day following the last injection, animals were killed and tumors were excised and stained with anti-CD11c (a and b) and anti-CD11b (c and d) mAb. The results shown are representative of two independent experiments

Fig. 4

Analysis of TLR-3 and TLR-9 expression in young and old CD11b⁺ and CD11c⁺ cells. Spleen cells from young and old mice were, non-treated (control) or treated with CpG-ODN and Poly-I:C (1 μg/ml). After 48 h cultures were collected surface stained with anti-CD11b or anti-anti-CD11c and then intracellular stained for TLR-3 and TLR-9. The results shown are representative of three independent experiments

Fig. 5

Analysis of CD4⁺Foxp3⁺ T_regs in the tumor microenvironment following treatment with CpG and Poly-I:C in young and old mice. a The accumulation of CD4⁺Foxp3⁺ T_regs within the tumor over time was evaluated in young and old animals. Young and old Balb/c mice were implanted with 10⁶ TUBO cells on day zero. Animals were killed on day 7, 14, 21 and 28. Tumors were extracted at the determined times and stained against CD4 and Foxp3 as described in Material and Methods. *P < 0.05 in the number of T_regs between young and old mice. b Representative experiment on single animals analyzing the levels of CD4⁺Foxp3⁺ T_regs within the tumor after treatment with CpG-ODN or Poly-I:C in young and old animals. Young and old Balb/c mice with established tumors were treated with CpG-ODN and Poly-I:C as described in Fig. 3. c Accumulative data of all animals analyzed for the levels of CD4⁺Foxp3⁺ T_regs within the tumor after treatment with CpG-ODN or Poly-I:C. **P < 0.01 in the number of T_regs found between control mice and young and old animals treated with CpG-ODN (or Poly I:C for young animals)

Fig. 6

Optimal treatment with CpG-ODN reduces the levels of T_regs. Young (a, c) and old (b, d) mice were implanted s.c. with 10⁶ TUBO cells. Tumor was allowed to grow until tumor size was ∼400 mm³. Tumors were then treated with i.t. injections (10 or 30 μg/injection) of CpG-ODN or Poly-I:C (CpG-10, CpG-30, Poly-10, or Poly-30) three times a week for 3 weeks. Next day following the last injection, animals were sacrificed and the levels of CD4⁺Foxp3⁺ T_regs in spleen (a, b) and LN (c, d) were evaluated. The group of animals without tumors (naïve) or with tumor but not treated (tumor) served as controls. *P < 0.01 in the number of T_regs found between mice with tumors and non-treated and young and old animals treated with 30 μg/injection of CpG-ODN. Data are representative of two experiments, three animals per group

Fig. 7

Suboptimal CpG-ODN treatment and T_reg inhibition induces complete tumor rejection. Young (a) and old (b) Balb/c mice were implanted with 10⁶ TUBO cells on day zero. On day 10 animals were treated with; i.t. injections of CpG-ODN (10 μg/injection) three times a week for 3 weeks; i.p injections of anti-GITR on day 9, 16 and 23 (300 μg/injection); and the combination of CpG-ODN and anti-GITR. Data are the means of two independent experiments ±SEM with five animals per group per experiment. *P < 0.001 difference was found between control mice and animals injected with CpG-ODN plus anti-GITR