Skip to main content
PMC home page
Cancer Immunology, Immunotherapy : CII logoLink to Cancer Immunology, Immunotherapy : CII
. 2007 Sep 8;57(4):549–561. doi: 10.1007/s00262-007-0393-1

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

Sanjay Sharma 1, Ana Lucia Dominguez 1, Dominique B Hoelzinger 1, Joseph Lustgarten 1,✉
PMCID: PMC11031019  PMID: 17828396

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.

Keywords: Aging, Antigen-presenting cells, Toll-receptor ligands, Tregs, Immunization, Tumors

Introduction

Antigen-presenting cells (APCs), such as dendritic cells (DC), play a major role in the initiation and regulation of adaptive immune responses; therefore, the manipulation and/or targeting of DCs has tremendous potential for cancer immunotherapy and antitumor vaccination [9, 24, 36]. The impact of aging on DCs or macrophages (Mϕ) function has been assessed by a variety of means and in numerous assays with mixed results [12, 40]. Though some studies show aging does not affect the number of APCs or their function [5], several others have demonstrated the occurrence of age-related alterations like antigen-presenting capacity [26] or cytokine secretion [38]. It is important to note that when young and old DCs are derived in vitro from the culture of blood-borne monocytes with IL-4 and GM-CSF, there is generally no age-associated difference in the appearance or behavior of the resultant APCs [18]. Thus, it appears that given optimal culture conditions, the generation of Mϕ or DCs in the old may not be impaired. The majority of evaluations assessing the functional capacity of old Mϕ or DCs have been performed utilizing cultured cells. However, it is unclear whether physiological activation of Mϕ or DCs in vivo might take place in the old resulting in the activation of an immune response.

The innate immune response relies on the recognition of the antigen by receptors that recognize specific structures found exclusively in microbial pathogens termed pathogen-associated molecular patterns (PAMPs) [14, 41]. The recognition of PAMPs by APCs is mediated by the Toll-like receptor (TLRs) family [16, 35]. There are more than ten known TLR family members capable of sensing bacterial components such as Poly-I:C (TLR-3), LPS (TLR-4), flagellin (TLR-5), Imiquimod (TLR-7), CpG-ODN (TLR-9) and other microbial products [13, 39]. The TLRs have distinct patterns and locations of cellular expression. A wide variety of TLRs are expressed in immature or mature DCs, macrophages (Mϕ) and monocytes; and these receptors control the activation of those APCs. For example, myeloid DCs express TLR4 and TLR7, whereas plasmacytoid DCs express TLR9 and TLR7 [8]. Furthermore, there is accumulative evidence indicating that several TLR ligands activate different signal pathways inducing the secretion of diverse pro-inflammatory cytokines and chemokines [22, 34]. Now that specific ligands have been identified for most of the TLRs, it is finally possible for immunotherapy to move away from the nonspecific effects of whole bacterial extracts and determine whether the same or even better therapeutic responses may be induced using synthetic TLR ligands. A key question is which of the TLRs induces the most useful therapeutic effects to generate an antitumor response? This question becomes even more critical in elderly patients since in the aged there could be alterations in the function of APCs [38, 26]. Even though many laboratories are evaluating a variety of vaccination strategies to induce antitumor immune responses, none of these laboratories have taken into consideration the effect that aging has on altering the immune system. Additionally, the immune system of the elderly is very different from the young and it is difficult to extrapolate results obtained in the young, for use in the old. We have demonstrated that immunotherapeutic intervention could be effective in young animals, but that the same therapy is not effective in old animals [19, 32]. Therefore, a successful vaccine should be customized in order to be effective in both the young and the old.

In this study, we evaluated whether targeting in vivo APCs with TLR-ligands results in the induction of immune responses and activation of antitumor responses in the old. We compared the antitumor potential of TLR ligands such as Poly-I:C, LPS, flagellin, Imiquimod and CpG-ODN in young and old tumor bearing mice. Our results indicated that only intratumoral (i.t.) injections of CpG-ODN induced the complete rejection of tumors in young and old mice. Intratumoral injections of Poly-I:C also induced the rejection of tumors in the young but not the old. We observed significant differences in the activation of immune responses following CpG-ODN and Poly-I:C injections in the aged. The induction of an antitumor response by CpG-ODN correlates with the activation of a Th1 type pro-inflammatory response resulting in the generation of CD4+, CD8+ T and, NK cell responses, activation of APCs and significant reduction in the number of Tregs in the old. These studies indicate that the selection of an adjuvant (e.g. CpG-ODN) is critical to optimize a vaccination strategy for the young and the old.

Materials and methods

Mice, cell lines and reagents

Young (2–3 months old) and old (18–22 months old) BALB/c or C57BL/6 mice were purchased from the National Institutes on Aging (Bethesda, MD) and housed under specific pathogen-free conditions. TUBO, a cloned cell line generated from a spontaneous mammary gland tumor from a BALB-neuT mouse [28] was provided by Dr. G. Forni (University of Turin, Italy). TUBO cells form tumors in BALB/c mice. The TRAMP-C2 tumor cell line derived from the TRAMP transgenic mouse [11] was obtained from Dr. Norman Greenberg (Fred Hutchinson Cancer Research Center, Seattle, Washington). TRAMP-C2 cells form tumors in C57BL/6 mice. Anti-GITR (DTA-1) was obtained from Dr. Shimon Sakaguchi (Kyoto University, Kyoto, Japan). All cell lines were maintained in complete RPMI medium (RPMI 1640) supplemented with 10% FCS, 2 mM glutamine, 5 x 10−5 M 2-mercapethanol (ME) and 50 μg/ml gentamicin. TLR ligands, CpG-ODN (1826, 5′-TCCATGACGTTCCTGACGTT-3′) and control-CpG (1982, 5′-TCCAGGACTTCTCTCAGGTT-3′) were purchased from Oligo Etc, Imiquimod (3M pharmaceuticals), Poly-I:C and LPS were purchased from Sigma–Aldrich, MO, USA. Flagellin was purified as previously reported [6].

In vivo tumor studies

TUBO (1 × 106) or TRAMP-C2 (5 × 106) were implanted subcutaneously (s.c.) in young or old BALB/c or C57BL/6 mice, respectively. Tumors were allowed to grow for 10 days before treatment was initiated. On day 10 after tumor challenge (tumor size ∼200 mm3), animals were randomly divided into groups of 5–8 mice/group. Animals received s.c. injections on the opposite site from where the tumor was implanted or i.t. injections of CpG-ODN, Poly-I:C, Flagellin, Imiquimod, LPS and CpG-control. Animals were injected with the various ligands (10, 30 and 100 μg/injection) three times a week for 3 weeks. For the i.t. injections animals were injected with a final volume of 50 μl/injection. Animals injected with PBS or control-ODN served as controls. The 30 μg/injection dose was found to be an optimum dose because at higher doses toxicity or side effects were observed (data not shown). To evaluate whether young and old BALB/c mice developed a memory response after treatment with the different TLR-ligands, animals were challenged 60 days later with a lethal dose of 106 TUBO cells. Tumor growth was monitored every 5 days and growth rates were determined by caliper measurements in two diameters. Tumor volume was expressed as: (minor diameter)2 × major diameter/2. Statistical analysis was determined by Student’s t-test. Survival analysis used the Breslow modification of the Kaplan–Meier test.

Depletion studies

Anti-CD4 (GK1.5) and anti-CD8 (56–6.37) mAbs were used for in vivo depletion of T cell subsets. Hybridoma cell lines were purchased from the American Type Culture Collection and supernatants were passed through a protein G matrix (Sigma–Aldrich, St. Louis, MO) to purify the Abs. Anti-asialo GM1 (Wako Pure Chemical Industries, VA, USA) was used to deplete NK cells. Animals were injected i.p. with 300 μg of anti-CD4 and anti-CD8 mAb or 50 μg of anti-asialo GM1 twice per week, starting 1 week before inoculation of the tumor cells and continuing for the duration of the experiment. Depletion of T and NK cells was confirmed by FACS analysis of the lymph nodes and spleen (data not shown). To deplete Tregs, animals were injected with anti-GITR mAb i.p. once a week for 3 weeks on days 9, 16 and 23 (300 μg/injection).

Flow cytometry analysis

To analyze the immune-infiltrates at the tumor site, animals were implanted with TUBO tumors (106) and the tumor was allowed to grow until its size was ∼400 mm3. Tumors were injected i.t. three times a week with CpG-ODN or Poly-I:C (30 μg/injection) for 1 week. The next day following the last injection, animals were killed and tumors were excised, homogenized on fine nylon mesh in RPMI media to form single cell suspensions. Tumor infiltrating cells were isolated by Histopaque 1077 (Sigma). Tumor samples were stained with labeled anti-CD4, anti-CD8, anti-DX5 mAb to evaluate the presence of NK cells, anti-CD11b/anti-B7.1 to evaluate the presence of macrophges/monocytes and anti-CD11c/anti-B7.1 to evaluate the presence of DCs. Percentages of specific populations were obtained by FACS analysis. The expression of TLR-3 and TLR-9 on CD11b+ and CD11c+ cells was analyzed by intracellular staining. Spleen or lymph nodes from young and old mice were non-treated or treated with CpG-ODN and Poly-I:C and stained with anti-CD11b, anti-CD11c, anti-TLR-3 and anti-TLR-9 (eBiosciences, CA, USA) following the manufacturer’s protocol.

Analysis of CD4+ Foxp3+ Tregs

The prevalence of Treg populations in the tumor microenvironment, spleen and lymph nodes (LN) of young and old animals following CpG-ODN or Poly-I:C treatment or non treatment was determined by the analysis of CD4+Foxp3+. The same experimental protocol was followed as described in Analysis of immune-infiltrates section. Tumor samples were first stained with anti-CD4 (FITC) antibodies (eBioscience). Intracellular Foxp3 staining was performed using a commercially available kit (eBioscience) following the manufacturer’s protocol. The same staining protocol was followed for analysis of Tregs in spleen and LN.

Multiplex analysis

For determining soluble factors within the tumor microenvironment, a tumor extract was prepared in T-per extraction buffer (Pierce) supplemented with protease inhibitor cocktail (Pierce). About 10 ml of buffer was used per 1 g of tumor sample. The tumor was homogenized on a fine nylon mesh to get a single cell suspension and centrifuged at 10,000 rpm for 30 min at 4°C. Supernatant was kept frozen at −80°C until analyzed. Levels of cytokines and chemokines were assayed using multiplex luminescent beads (Biosource, MA, USA) according to the manufacturer’s instructions and analyzed with a Luminex 100 plate reader (Luminex Corp., TX, USA). A cytokine mixture and aliquots of three experimental samples were run on each plate to assess interassay variability. Fluorescence intensity was transformed into cytokine concentration using StatLIA software (Brendan Scientific, CA, USA). The lower limit of detection was 1.5 pg/ml for each cytokine. Cytokines that were monitored: IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12p40, IFN-γ, TNF-α, CCL2, CCL3 and CCL5. For detection of TGF-β in tumor samples ELISA assay was used following the manufacturer’s instructions (R&D Systems Inc., MN, USA).

Statistical analyses

Statistical significance of data was determined by Student’s t-test to evaluate the P value. The relationship between two parameters was tested using regression analysis and P < 0.05 was considered significant.

Results

Evaluation of TLR ligands for the induction of antitumor responses in young and old mice

It has been demonstrated that TLR-ligands are able to enhance antitumor responses in several tumor models [2, 29]. However, there are no reports evaluating whether direct injections of TLR-ligands are able to induce an antitumor response in the old. We compared the effect of different TLR-ligands in inducing an antitumor response in young and old mice. Young (2 months old) and old (18 months old) BALB/c mice were implanted s.c. with TUBO cells on day zero and on day 10, animals started treatment with s.c. injections of Poly I:C, LPS, flagellin, Imiquimod, CpG-ODN and control-ODN (as a control) in the opposite flank from where the tumor was injected. Under these conditions no antitumor response could be observed in any of the treated groups in both young or in old animals (data not shown). No significant differences in tumor growth were observed between treated and non-treated young and old mice. These results indicated that administration of various TLR-ligands away from the tumor did not result in the activation of a systemic antitumor response in young or aged animals. Next, we hypothesized that by targeting APCs at the tumor site, it would result in the modulation of the immune responses within the tumor resulting in the activation of an antitumor response. We evaluated whether in situ vaccination at the tumor site would generate an antitumor response. Our results indicated that i.t. injections of CpG-ODN induced an antitumor response in BALB/c young (Fig. 1a) and old (Fig. 1b) mice resulting in tumor rejection. An important observation is that i.t. injections of Poly-I:C also induced the rejection of tumors in BALB/c young mice (Fig. 1a) but not in old mice (Fig. 1b). To make sure that these differences in the induction of antitumor responses generated by TLR-ligands between young and old mice was not due to a specific characteristic of the tumor model, we tested the effect of the different TLR-ligands in young and old C57BL/6 mice implanted with TRAMP-C2 tumors. We observed that only i.t. injections of CpG-ODN significantly delayed the tumor growth in old mice (Fig. 1d) while i.t. injections of CpG-ODN and Poly-I:C delayed the tumor growth in young animals (Fig. 1c) when compared to control animals. No antitumor effect was observed in young or old mice injected i.t. with control-ODN indicating that the immune response stimulated by the CpG-ODN was selective. Treatment with i.t. injections of LPS, Imiquimod or flagellin did not have any effect in controlling the tumor growth in BALB/c and C57BL/6 young or old mice. These results indicated that not all TLR-ligands are effective for inducing an antitumor response and those TLR-ligands capable of inducing an immune response might react differently in young and old mice. We also performed dose escalation experiments immunizing with 10, 30 and 100 μg/injection of TLR-ligands. Intratumoral injections with 10 μg/injection of CpG-ODN showed a delay in tumor growth (see more details in below) while other TLR-ligands demonstrate no antitumor effect. Repetitive injections of TLR-ligands at 100 μg/injection showed toxic effects in which several animals died. Data shown in Fig. 1 was performed with 30 μg/injection that show no toxic effects.

Fig. 1.

Fig. 1

Open in a new tab

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)

CpG-ODN induces a protective memory response in young and old mice

We next evaluated whether young and old BALB/c animals that rejected the tumor after vaccinations with CpG-ODN would induce a protective memory response against TUBO cells. Young and old mice were challenged with 106 TUBO cells 60 days after injection of the primary tumor. As shown in Fig. 2a, 100% of young and old mice that received CpG-ODN rejected the TUBO cells indicating that a protective memory response was established. Young animals that rejected the tumor after treatment with Poly-I:C also rejected the tumor after the challenge, showing the induction of protective memory immunity. These results suggest that targeting APCs within the tumor with CpG-ODN could result in the generation of a systemic immune response capable of activating a protective memory response in both young and old mice. We confirmed that the immune responses induced by CpG-ODN are dependent on CD4+ and CD8+ T cells and NK cells in old mice since depletion of the cells abrogated the antitumor immune responses (Fig. 2b).

Fig. 2.

Fig. 2

Open in a new tab

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

Evaluation of immune infiltrates within the tumor after CpG-ODN and Poly I:C treatment in young and old mice

Our results indicate that young mice respond to CpG-ODN and Poly I;C and old mice respond only to CpG-ODN. Critical for the generation of an antitumor response is the influx of immune-infiltrates to the tumor site. First, we evaluated whether the APC population within the tumor microenvironment was influenced by the i.t. injections of CpG and Poly I:C in young and old mice. We evaluated DCs and macrophage tumor infiltration in terms of CD11c and CD11b expression, respectively. Our results indicated that the expression of CD11b and CD11c were significantly higher (P < 0.05) in CpG-ODN treated young (Fig. 3a) and old mice (Fig. 3b) when compared to control tumors. Treatment with Poly I:C only induced the expression of CD11c in young animals (Fig. 3a) but not of CD11b (Fig. 3c) in young animals or CD11c or CD11b in the old (Fig. 3b,d). Analysis of the levels of B7.1 or class II on CD11c+ cells indicated that there cellular markers were up-regulated after in CpG-ODN treated mice when compared to control animals (data not shown). We also enumerated the immune-infiltrates within the tumor microenvironment after treatment with CpG and Poly-I:C. As summarized in Table 1, we did not observe a drastic difference in the number of immune-infiltrates evaluated between young and old control (non-treated) mice. However, following CpG-ODN treatment, we observed a 3–4 fold increase in the number of CD4, CD8 T cells, NK cells, macrophages/monocytes and DC in both young and old mice. These results indicate that in situ immunizations with CpG-ODN modulate the tumor microenvironment resulting in the attraction, accumulation and activation of immune-infiltrates at the tumor site. The accumulation of immune-infiltrates positively correlated with the antitumor response since treatment with Poly I:C increased the number of immune-infiltrates in the young but not in the old mice.

Fig. 3.

Fig. 3

Open in a new tab

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

Table 1.

Analysis of immune-infiltrates within the tumor

Control CpG-ODN Poly I:C
Young Old Young Old Young Old
CD4 1.8 ± 0.3a 0.9 ± 0.6 5.4 ± 1.4 3.8 ± 1.4 4.7 ± 0.9 1.3 ± 0.5
CD8 0.9 ± 0.4 0.6 ± 0.3 3.7 ± 0.8 2.7 ± 0.8 3.1 ± 0.7 1.0 ± 0.4
NK 1.3 ± 0.5 1.5 ± 0.7 4.2 ± 0.6 5.1 ± 0.8 3.8 ± 1.1 1.9 ± 0.4
Mac/Mon 2.7 ± 0.8 3.1 ± 1.1 8.6 ± 1.7 9.3 ± 2.1 7.1 ± 1.2 4.3 ± 0.9
DC 2.3 ± 0.7 1.8 ± 0.9 7.7 ± 2.1 6.2 ± 1.5 6.6 ± 2.0 3.1 ± 1.1
Open in a new tab

Animals were inoculated with 106 TUBO cells on day 0. Tumor was allowed to grow until tumor size was approximately ∼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 analyzed for the presence of CD4, CD8 NK Macrophages (Mac/Mon) and dendritic cells (DC). Controls are animals implanted with tumors and treated with PBS

A significant (P < 0.05, Student’s t-test) difference in the number of immune-infiltrates observed in the tumor microenvironment was found between control mice and mice that were treated with CpG-ODN (or Poly I:C for young animals). One of two experiments, three animals/group/experiment

aPercentages of specific populations were obtained by FACS analysis

Evaluation of TLR-3 and TLR-9 on APCs from young and old animals

To evaluate whether the difference between young and old animals in responding to CpG and Poly-I:C was due to the differential expression of TLR-9 or TLR-3. Spleen cells from young and old mice were non-treated or treated with CpG-ODN or Poly-I:C and stained with anti-CD11b, anti-CD11c, anti-TLR-3 and anti-TLR-9. Our results indicate that the levels of expression of TLR-3 and TLR-9 between young and old CD11b+ and CD11c+ cells was very similar without major differences in non-treated or treated samples (Fig. 4). Similar results were found with young and old lymph nodes (data not shown). These results indicate that lack of stimulation of Poly-I:C in the old is not due to the lack of expression of TLR-3 but probably to an unknown deficiency.

Fig. 4.

Fig. 4

Open in a new tab

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

Analysis of tumor infiltrating CD4+ Foxp3+ Tregs following treatment with CpG and Poly-I:C in young and old mice

There is extensive evidence indicating that CD4+Foxp3+ Tregs can control and suppress immune responses. We analyzed the level of CD4+Foxp3+ Tregs within the tumor in young and old mice over time. We observed that the number of tumor infiltrating CD4+Foxp3+ Tregs increased over time in young and old mice (Fig. 5a). Our data also indicated that the number of CD4+Foxp3+ Tregs found within the tumor was higher in old mice when compared to young animals. We next evaluated whether i.t. injections of CpG-ODN and Poly-I:C influenced the number of CD4+Foxp3+ Tregs within the tumor in young and old mice. As shown in Fig. 5b,c following treatment with CpG-ODN the numbers of CD4+Foxp3+ Tregs within the tumor were drastically reduced (reduction of 80%) in young and old animals. Treatment with Poly-I:C also reduced the numbers of CD4+Foxp3+ Tregs within the tumor in young animals (reduction of 75%). However, we only observed a 20% reduction in the numbers of CD4+Foxp3+ Tregs in old animals following Poly-I:C injections.

Fig. 5.

Fig. 5

Open in a new tab

Analysis of CD4+Foxp3+ Tregs in the tumor microenvironment following treatment with CpG and Poly-I:C in young and old mice. a The accumulation of CD4+Foxp3+ Tregs 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 Tregs between young and old mice. b Representative experiment on single animals analyzing the levels of CD4+Foxp3+ Tregs 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+ Tregs within the tumor after treatment with CpG-ODN or Poly-I:C. **P < 0.01 in the number of Tregs found between control mice and young and old animals treated with CpG-ODN (or Poly I:C for young animals)

The levels of Tregs correlate with the induction of an antitumor response

Our data indicate that after CpG-ODN treatment the numbers of Tregs were drastically reduced within the tumor, however, these results do not indicate whether low levels of Tregs correlate with the induction of an antitumor response. First, we evaluated whether treatment with CpG-ODN or Poly-I:C (10 or 30 μg/injection) affect the number of Tregs in spleen or LN. Young and old mice were implanted with tumors and when tumors reached a size of ∼400 mm3 , the mice were treated with i.t. 10 or 30 μg/injection of CpG-ODN and Poly-I:C. Following implantation of the tumor cells we observed an increase in the number of CD4+Foxp3+ T cells in spleen and LN from both young and old mice (Fig. 6). Animals injected with 30 μg/injection of CpG-ODN showed a ∼30–35% reduction in the number of CD4+Foxp3+ T cells in spleen and LN to similar numbers as animals without tumors (Fig. 6). Young or old animals treated with 10 μg/injection of CpG-ODN demonstrate a minimal reduction in the number of CD4+Foxp3+ T cells. As expected Poly-I:C treatment did not have an effect in reducing the levels of CD4+Foxp3+ T cells in old mice, while young mice treated with 30 μg/injection of Poly-I:C showed ∼25% reduction in the number of CD4+Foxp3+ T cells in LN and spleens. These results indicate that CpG-ODN vaccination influences the levels of CD4+Foxp3+ T cells by reducing their numbers. Therefore a relationship exists between the reduction of CD4+Foxp3+ T cells and the activation of an immune response with the use of CpG-ODN.

Fig. 6.

Fig. 6

Open in a new tab

Optimal treatment with CpG-ODN reduces the levels of Tregs. 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+ Tregs 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 Tregs 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

Analysis of cytokines following treatment with CpG and Poly-I:C in young and old mice

Cytokines and chemokines within tumors can contribute to the progression of tumors to a more aggressive metastatic phenotype or play a role in tumor therapy [17, 37]. The levels of several cytokines and chemokines within the tumor microenvironment were analyzed in non-treated, CpG-ODN and Poly-I:C treated young and old animals by Multipex assay. As shown in Table 2, we observed that injections of CpG-ODN induced a strong pro-inflammatory response in which the levels of IL-1b, IL-6, IL12, INF-γ, TNF-α,( CCL2, CCL3 and CCL5 were significantly higher (P < 0.01) in young and old tumors. Treatment with Poly-I:C also augmented the level of these cytokines and chemokines in young animals. In contrast, old animals treated with Poly-I:C showed no significant enhancement in the levels of cytokines and chemokines secreted within the tumor when compared to control animals. Following treatment with CpG-ODN or Poly I:C there were no significant changes in the level of IL-2, IL-4, IL-10 or TGF-β in young or old animals.

Table 2.

Evaluation of cytokine and chemokines levels within the tumor after treatment with CpG-ODN and Poly I:C in young and old mice

Cytokine/chemokinea Young Old
Controlb CpG-ODN Poly I:C Control a CpG-ODN Poly I:C
IL1b 76 ± 6.3a 346 ± 19.7 135 ± 16.5 19 ± 2.5 114 ± 9.3 34 ± 3.2
IL2 20 ± 3.2 28 ± 4.2 21 ± 1.5 39 ± 5.3 44 ± 2.2 40 ± 3.2
IL4 1.9 ± 0.3 2.3 ± 0.4 3.6 ± 0.7 2.3 ± 0.3 2.8 ± 0.6 2.0 ± 0.4
IL6 9.7 ± 1.3 157 ± 7.3 78 ± 6.2 8.7 ± 0.7 178 ± 11.3 34 ± 2.1
IL10 136 ± 17 114 ± 12 128 ± 21 177 ± 18 168 ± 28 151 ± 14
IL12 67 ± 12 2341 ± 143 1654 ± 176 36 ± 9 1387 ± 198 97 ± 18
INF-γ 6.9 ± 0.6 27.5 ± 2.7 19.4 ± 1.8 5.3 ± 0.6 24.9 ± 3.2 8.3 ± 0.9
TNF-α 11.7 ± 1.2 108 ± 7.5 45 ± 2.6 14 ± 1.8 96.3 ± 5.6 19 ± 1.8
TGFβ 14 ± 2.8 11 ± 3.6 12.6 ± 1.9 19 ± 3.7 15.7 ± 2.5 16.9 ± 4.1
CCL2 211 ± 13.2 1134 ± 53.8 819 ± 29.3 139 ± 7.6 830 ± 38.7 301 ± 11.8
CCL3 512 ± 21.4 3044 ± 90.4 1432 ± 63.1 476 ± 28.6 2398 ± 115.6 628 ± 35.4
CCL5 789 ± 65.2 6077 ± 321.5 7629 ± 384.4 318 ± 20.6 1956 ± 87.4 939 ± 33.7
Open in a new tab

A significant (P < 0.01, Student’s t-test) difference in the level of cytokines and chemokines found in the tumor microenvironment between control mice and mice that were treated with CpG-ODN

One of three experiments, three animals/group/experiment

apg/ml

bNot treated mice

Effect of CpG-ODN vaccination and Treg depletion

The above experiments suggest that due to the lack of further reduction in the number of CD4+Foxp3+ T cells animals treated with 10 μg/injection of CpG-ODN could not generate an effective antitumor response. Next, we evaluated whether suboptimal treatment with CpG-ODN (10 μg/injection) in combination with inhibition of Treg suppression would induce an antitumor response. As shown in Fig. 6, treatment with CpG-ODN (10 μg/injection) alone or anti-GITR mAb alone delayed the tumor growth but was not sufficient for tumor eradication. However, when CpG-ODN (10 μg/injection) and anti-GITR was combined complete tumor rejection was observed in both young (Fig. 7a) and old (Fig. 7b) mice. Taken together, these results suggest that there is a direct correlation between the reduction or inhibition of Tregs and the induction of a competent antitumor response for tumor eradication.

Fig. 7.

Fig. 7

Open in a new tab

Suboptimal CpG-ODN treatment and Treg 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

Discussion

The evidence regarding APC function in the aged is not clear. Some studies have shown that APC function is unaltered in the aged [23], while other reports indicate APCs from aged mice are defective [31]. Therefore, it is unclear whether alterations in APC function are a factor that contributes to the lack of an effective tumor immune response in the aged. Currently there are no studies indicating whether targeting APCs in vivo with adjuvants would result in the induction of an antitumor response in aged mice. The current study was designed to evaluate whether it was possible to stimulate an antitumor response by targeting APCs with TLR-ligands in aged mice.

TLR ligands are potent inducers of APC activity and have shown enhanced immune responses acting as adjuvants. Considering the potential deficiencies in aged APCs, it would be important to evaluate whether TLR ligands could overcome those deficiencies. We screened various TLR ligands, such as Poly I:C, Imiquimod, Flagellin, CpG-ODN, and LPS for stimulating anti-tumor responses in vivo in old hosts. Our results indicated that CpG-ODN was the only TLR-ligand capable of inducing an immune response in both the young and the old. Interestingly, Poly I:C could also induce an anti-tumor response in young animals but not in old ones. Renshaw et al. [27] demonstrated that the expression of TLRs in splenic and thioglycollate-elicited macrophages is reduced in aged animals when compared to young animals. This decline in TLR expression on aged macrophages is associated with lower secretions of cytokines following stimulation with different TLR-ligands when compared to young macrophages. We analyzed by intracellular staining the expression of TLR-3 and TLR-9 from CD11c+ and CD11b+ cells isolated from young and old mice non-stimulated or stimulated with CpG-ODN and Poly-I:C. Our results indicated non-obvious differences in the expression of TLR-3 or TLR-9 between young and old APCs. These results suggest that the lack of stimulation by Poly-I:C in aged animals is probably due to a deficiency in the TLR-3 signal transduction events in old APCs. At this point we cannot correlate the results of Renshaw et al. with our results after stimulating an immune response with CpG-ODN in old mice. It is difficult to envision all the events that happened in vivo following i.t. injection of CpG-ODN or how DCs or other APCs are influenced or affected by adjuvants or cytokines. Additionally, the complexity of TLR signaling may be dramatically different in vitro as compared to in vivo. It has been demonstrated that there is a different pattern of expression of TLRs among APCs and expression of TLR on APCs varies with the state of activation and anatomical localization. The expression of TLRs on DCs or other APCs could be influenced by the environment of the host. For example, Kadowaki et al. [15] showed that the expression of TLR-9 could be up-regulated in DCs by cytokines. We have demonstrated that after CpG-ODN treatment there is a strong Th1 pro-inflammatory response where IL-6, IL-12 TNF-α INF-γ and other cytokines and chemokines are up regulated. Therefore, it can be speculated that the secretion of these cytokines and chemokines might influence the expression of TLRs on APCs or the signaling pathways [3, 21].

An effective antitumor response could very well be composed of multiple components. Our results showed a significant CD4, CD8 T cell, NK cell and APC infiltration, secretion of pro-inflammatory cytokines/chemokines and reduction of Tregs at the tumor site. These multiple factors directly correlate with the antitumor outcome in old animals, showing that there are probably various immune pathways involved in tumor rejection. The infiltration and activation of APCs is a critical step for the induction of an antitumor response. TLR ligands directly or indirectly, through cytokines can activate APCs. Our results indicate that following CpG-ODN treatment young and old CD11b+ and CD11c+ cells were activated as reflected by the up-regulation of cellular markers such class II and B7.1 (data not shown) and secretion of cytokines and chemokines. Additionally after CpG-ODN treatment the infiltration of DCs and macrophages was similar between young and old mice and was consistent with the antitumor response. Treatment with Poly-I:C also induced the activation and infiltration of APCs in young animals but not in the old which correlated with the therapeutic response. These results indicate that the effector-function differs between young and old APCs and that these cells react differently to specific adjuvants. We do not yet understand the differences between CpG-ODN and Poly-I:C in stimulating young and old APCs in vivo. We are currently conducting microarray analysis of CD11c+ and CD11b+ cells isolated from young and old mice non-stimulated or stimulated to assess the differences in response to TLR-ligands. Hopefully, these studies will help us to elucidate possible defects in the effector-function of aged-APCs.

Previous studies from our laboratory revealed that there is a higher accumulation of Tregs in old mice when compared to young animals [33]. The accumulation of Tregs in old mice inhibits immune responses against nominal antigens since reduction of Tregs to the same level as young animals restore the immune responses in the old [33]. In these studies, we observed that the number of Tregs keeps accumulating over time at the tumor site and that there is higher accumulation of Tregs in tumors of old mice when compared to those in young mice. These results are in agreement with our recent findings demonstrating that the number of CD4+Foxp3+ Tregs are highly elevated in old mice when compared to young mice. We also observed that following i.t. injections of CpG-ODN the number of Tregs within the tumors were drastically reduced. Young and old mice treated with 30 μg/injection of CpG-ODN showed a strong reduction in the number of CD4+Foxp3+ T cells in LN and spleen almost to the same levels as naïve young mice and these animals were able to reject the tumor, while animals treated 10 μg/injection of CpG-ODN only delayed the tumor growth with a minimal reduction in the number of Tregs. Our data indicated that following i.t. injections of CpG-ODN (30 μg/injection) the levels of IL-6, IL12, INF-γ, TNF-α and chemokines within the tumor were significantly elevated. In contrast, treatment with i.t. injections of CpG-ODN at 10 μg/injection the levels of cytokines and chemokines were not as high as those induced by 30 μg/injection (data not shown). Only when Tregs were suppressed with anti-GITR mAb in combination with immunizations using 10 μg/injection of CpG-ODN an antitumor response capable of rejecting the tumors was observed. We do not yet understand the reason for the high accumulation of Tregs in old mice. It has been demonstrated that there is shift from a Th1 to a Th2 response in aged mice [30, 7] with an increased production of IL-10 [4] and TGF-β [10] which may account in part for the high accumulation of Tregs in these animals. Our results suggest that there is a relationship between the optimal treatment with CpG-ODN, induction of a Th1 pro-inflammatory response, reduction in the number of Tregs and activation of an antitumor immune response. New preliminary data emerging from our laboratory indicate that IL-6, Eotaxin-3 and perhaps other factors but not CpG-ODN inhibit de novo conversion of non-regulatory T cells to regulatory T cells (data not shown, Hoelzinger et al. in preparation). We believe that the cytokines secreted after CpG-ODN treatment (30 μg/injection) influence the homeostasis of Tregs in old tumor bearing hosts by preventing de novo conversion of Tregs and inhibiting their suppressing activity [25]. As such, these results indicate that by regulating the levels of Tregs in the old it is possible to restore the immune responses resulting in the activation of antitumor responses and development of protective memory responses. These results have important clinical implications for the optimization of immunization strategies in the old. It might be critical to utilize immunization strategies where APCs are properly activated and capable of activating a Th1 type response that subsequently these cytokines influence the levels or affect the effector-function of Tregs. Alternatively, it might be necessary to use methods where vaccination protocols are combined with depletion or inhibition of Tregs. Although our results argue that it is important to reduce the levels of Tregs in order to generate an antitumor response, it is important to note that Treg inhibition alone with anti-GITR was not sufficient for tumor rejection. The activation of innate and adaptive components of the immune response in combination with Treg inhibition was critical to optimally generate an antitumor response in the old.

In conclusion, we report here for the first time a critical evaluation of the role of TLR ligands as they relate to generating an antitumor response and also assessing whether this response is a function of age. We demonstrated that not all TLR-ligands are able to induce an antitumor response and that there are differences among the various TLR-ligands in their capacity to induce an antitumor response in old mice. This information is very important for the selection of adjuvants in order to induce or enhance an immune response in the elderly. Our results are in agreement with previous studies indicating that CpG-ODN could enhance vaccination formulations in the aged [20, 1]. These studies provide some possible mechanisms on how CpG-ODN enhances the immune responses in the old. Another important observation from our studies is that in order to generate an effective antitumor response, it is critical to deliver the CpG-ODN at the tumor site. However, the drawback of this strategy is that not all tumors will be physically available for performing i.t. injections and it will be difficult to target metastatic lesions. We have developed a system whereby CpG-ODN is linked to antibodies, making it possible to deliver the CpG-ODN anywhere in the body (Sharma et al. in preparation). Overall, our data indicate that CpG-ODN is capable of restoring the antitumor immune responses in the old. These studies should provide the framework for the design of much more effective strategies for stimulating antitumor responses with the use of TLR-ligands in the old.

Acknowledgements

This work was supported by Grant CA78579 and AG028751 (to J. L.) from the National Institutes of Health.

References

  • 1.Alignani DO, et al. Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice. J Leukoc Biol. 2005;77:898–905. doi: 10.1189/jlb.0604330. [DOI] [PubMed] [Google Scholar]
  • 2.Baines J, Celis E. Immune-mediated tumor regression induced by CpG-containing oligodeoxynucleotides. Clin Cancer Res. 2003;9(7):2693–2700. [PubMed] [Google Scholar]
  • 3.Bhattacharyya S, et al. Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the P13K/Akt pathway and of IkappaB kinase activity. Blood. 2004;104(4):1100–1109. doi: 10.1182/blood-2003-12-4302. [DOI] [PubMed] [Google Scholar]
  • 4.Caslte SC, et al. Age-related impaired proliferation of peripheral blood mononuclear cells is associated with an increase in both IL-10 and IL-12. Exp Gerontol. 1999;34(2):243–252. doi: 10.1016/S0531-5565(98)00064-3. [DOI] [PubMed] [Google Scholar]
  • 5.Castle SC, et al. Antigen presenting cell function is enhanced in healthy elderly. Mech Ageing Dev. 1999;107:137–145. doi: 10.1016/S0047-6374(98)00141-9. [DOI] [PubMed] [Google Scholar]
  • 6.Cuadros C, et al. Flagellin fusion proteins as adjuvants or vaccines induce specific immune responses in aged animals. J Immunol. 2004;72:2810–2816. doi: 10.1128/IAI.72.5.2810-2816.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Deng Y, et al. Age-related impaired type 1 T cell responses to influenza: reduced activation ex vivo, decreased expansion in CTL culture in vitro, and blunted response to influenza vaccination in vivo in the elderly. J Immunol. 2004;172(6):3437–3446. doi: 10.4049/jimmunol.172.6.3437. [DOI] [PubMed] [Google Scholar]
  • 8.Edwards AD, et al. Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8 alpha+ DC correlates with unresponsiveness to imidazoquinolines. Eur J Immunol. 2003;33(4):827–833. doi: 10.1002/eji.200323797. [DOI] [PubMed] [Google Scholar]
  • 9.Esche C, Shurin MR, Lotze MT. The use of dendritic cells for cancer vaccination. Curr Opin Mol Ther. 1999;1(1):72–81. [PubMed] [Google Scholar]
  • 10.Forsey RJ, et al. Plasma cytokine profiles in elderly humans. Mech Ageing Dev. 2003;124(4):487–493. doi: 10.1016/S0047-6374(03)00025-3. [DOI] [PubMed] [Google Scholar]
  • 11.Foster BA, et al. Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Cancer Res. 1997;57(16):3325–3330. [PubMed] [Google Scholar]
  • 12.Grewe M. Chronological ageing and photoageing of dendritic cells. Clin Exp Dermatol. 2001;26:608–612. doi: 10.1046/j.1365-2230.2001.00898.x. [DOI] [PubMed] [Google Scholar]
  • 13.Hawlisch H, Kohl J. Complement and Toll-like receptors: key regulators of adaptive immune responses. Mol Immunol. 2006;43:13–21. doi: 10.1016/j.molimm.2005.06.028. [DOI] [PubMed] [Google Scholar]
  • 14.Janeway CA, Jr, et al. Cross-linking and conformational change in T-cell receptors: role in activation and in repertoire selection. Cold Spring Harb Symp Quant Ciol. 1989;54(Pt 2):657–666. doi: 10.1101/sqb.1989.054.01.077. [DOI] [PubMed] [Google Scholar]
  • 15.Kadowaki N, et al. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med. 2001;194:863–869. doi: 10.1084/jem.194.6.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kaisho T, Akira S. Regulation of dendritic cell function through Toll-like receptors. Curr Mol Med. 2003;3:373–385. doi: 10.2174/1566524033479726. [DOI] [PubMed] [Google Scholar]
  • 17.Lin W, Karin M. A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest. 2007;117:1175–1183. doi: 10.1172/JCI31537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lung TL, et al. Unimpaired dendrititc cells can be derived from monocytes in old age and can mobilize residual function in senescent T cells. Vaccine. 2000;18:1606–1612. doi: 10.1016/S0264-410X(99)00494-6. [DOI] [PubMed] [Google Scholar]
  • 19.Lustgarten J, Dominguez AL, Thoman M. Aged mice develop protective antitumor immune responses with appropriate costimulation. J Immunol. 2004;173:4510–4515. doi: 10.4049/jimmunol.173.7.4510. [DOI] [PubMed] [Google Scholar]
  • 20.Maletto B, et al. CpG-DNA stimulates cellular and humoral immunity and promotes Th1 differentiation in aged BALB/c mice. J Leukoc Biol. 2002;72(3):447–454. [PubMed] [Google Scholar]
  • 21.Martin M, et al. Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide. J Immunol. 2003;171(2):717–725. doi: 10.4049/jimmunol.171.2.717. [DOI] [PubMed] [Google Scholar]
  • 22.Matsushima H, et al. TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. J Immunol. 2004;173(1):531–541. doi: 10.4049/jimmunol.173.1.531. [DOI] [PubMed] [Google Scholar]
  • 23.Norian LA, Allen PM. No intrinsic deficiencies in CD8+ T cell-mediated antitumor immunity with aging. J Immunol. 2004;173:835–844. doi: 10.4049/jimmunol.173.2.835. [DOI] [PubMed] [Google Scholar]
  • 24.Osada T, et al. Dendritic cell-based immunotherapy. Rev Immunol. 2006;25:377–413. doi: 10.1080/08830180600992456. [DOI] [PubMed] [Google Scholar]
  • 25.Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science. 2003;299:1033–1036. doi: 10.1126/science.1078231. [DOI] [PubMed] [Google Scholar]
  • 26.Plowden JM, et al. Impaired antigen-induced CD8+ T cell clonal expansion in aging is due to defects in antigen presenting cell function. Cell Immunol. 2004;229:86–92. doi: 10.1016/j.cellimm.2004.07.001. [DOI] [PubMed] [Google Scholar]
  • 27.Renshaw M, et al. Cutting edge: impaired Toll-like receptor expression and function in aging. J Immunol. 2002;169:4697–4701. doi: 10.4049/jimmunol.169.9.4697. [DOI] [PubMed] [Google Scholar]
  • 28.Rovero S, et al. DNA vaccination against rat her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. Infect Immun. 2000;72:2810–2816. doi: 10.4049/jimmunol.165.9.5133. [DOI] [PubMed] [Google Scholar]
  • 29.Salem ML, et al. Defining the antigen-specific T-cell response to vaccination and poly(I:C)/TLR3 signaling: evidence of enhanced primary and memory CD8 T-cell responses and antitumor immunity. J Immunother. 2005;28(3):220–228. doi: 10.1097/01.cji.0000156828.75196.0d. [DOI] [PubMed] [Google Scholar]
  • 30.Sandmand M, et al. Is ageing associated with a shift in the balance between Type 1 and Type 2 cytokines in humans? Clin Exp Immunol. 2002;127(1):107–114. doi: 10.1046/j.1365-2249.2002.01736.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Saurwein-Teissl M, Romani N, Grubeck-Loebenstein B. Dendritic cells in old age neglected by gerontology? Mech Ageing Dev. 2000;121:123–130. doi: 10.1016/S0047-6374(00)00203-7. [DOI] [PubMed] [Google Scholar]
  • 32.Sharma S, Dominguez AL, Lustgarten J. Aging affect the anti-tumor potential of dendritic cell vaccination, but it can be overcome by co-stimulation with anti-OX40 or anti-4-1BB. Exp Gerontol. 2006;41:78–84. doi: 10.1016/j.exger.2005.10.002. [DOI] [PubMed] [Google Scholar]
  • 33.Sharma S, Dominguez AL, Lustgarten J. High accumulation of T regulatory cells prevents the activation of immune responses in aged animals. J Immunol. 2006;177(12):8348–8355. doi: 10.4049/jimmunol.177.12.8348. [DOI] [PubMed] [Google Scholar]
  • 34.Sun J, et al. TLR ligands can activate dendritic cells to provide a MyD88-dependent negative signal for Th2 cell development. J Immunol. 2005;174(2):742–751. doi: 10.4049/jimmunol.174.2.742. [DOI] [PubMed] [Google Scholar]
  • 35.Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol. 2003;21:335–376. doi: 10.1146/annurev.immunol.21.120601.141126. [DOI] [PubMed] [Google Scholar]
  • 36.Tamir A, et al (2007) Induction of tumor-specific T-cell responses by vaccination with tumor lysate-loaded dendritic cells in colorectal cancer patients with carcinoembryonic-antigen positive tumors. Cancer Immunol Immunother (in press). doi:10.1007/s00262-007-0299-y [DOI] [PMC free article] [PubMed]
  • 37.Tan T, Coussens L. Humoral immunity, inflammation and cancer. Curr Opin Immunol. 2007;19(2):209–216. doi: 10.1016/j.coi.2007.01.001. [DOI] [PubMed] [Google Scholar]
  • 38.Tortorella C, et al. APC-dependent impairment of T cell proliferation in aging: role of CD28- and IL-12/IL-15-mediated signaling. Mech Ageing Dev. 2002;123:1389–1402. doi: 10.1016/S0047-6374(02)00079-9. [DOI] [PubMed] [Google Scholar]
  • 39.Trinchieri G, Sher A. Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol. 2007;7(3):179–190. doi: 10.1038/nri2038. [DOI] [PubMed] [Google Scholar]
  • 40.Vargas A, et al. Age-dependent changes in thymic macrophages and dendritic cells. Microsc Res Tech. 2003;62:501–507. doi: 10.1002/jemt.10411. [DOI] [PubMed] [Google Scholar]
  • 41.Xu D, Liu H, Komai-Koma M. Direct and indirect role of Toll-like receptors in T cell mediated immunity. Cell Mol Immunol. 2004;1(4):239–246. [PubMed] [Google Scholar]

Articles from Cancer Immunology, Immunotherapy : CII are provided here courtesy of Springer

RESOURCES