High Accumulation of T Regulatory Cells Prevents the Activation of Immune Responses in Aged Animals¹

Active immunotherapy or vaccination to induce tumor-specific killer and Th cells is potentially the most powerful and broadly applicable immunotherapeutic strategy. The majority of studies to augment the immune response against tumors has been conducted in young murine models and has spent significant efforts in aspects, such as increasing the immunogenicity. However, very little attention has been paid to the competence of the aging population, which shows a decline in immune system function. The age-associated increase in cancer may be due in part to a global decrease in cell-mediated immunity (1, 2). We have demonstrated previously that young animals mount a protective response after immunization with the immunogenic BM-185-enhanced GFP (EGFP)³ tumor cells (3). In contrast, aged mice were not able to mount an immune response against the tumor. These results indicate that there are alterations in the immune system of aged animals, preventing the activation of immune responses against antigenic determinants (1, 2, 3).

Recent evidence has demonstrated that regulatory T (T_reg) cell-mediated immunosuppression is one of the crucial tumor immune evasion mechanisms and contributes to the failure of tumor immunotherapy (4, 5, 6, 7). The T_reg-like cells were first reported >30 years back and named suppressor T cells. However, problems in isolating these cells at that time and failure to characterize the mechanisms responsible for their suppressive activity led these cells to be discredited (8). In the last few years, modern technologies and new experimental approaches have resulted in reidentifying T cell populations with suppressive activities, calling them T_reg cells. Although several different cell types have been attributed with these regulatory capacities, the most characterized groups are the CD4⁺CD25⁺ T cells (5, 6). However, the use of CD25 as a marker for T_reg cells is problematic as CD25 is also expressed on non-T_reg cells in settings of immune activation, such as during an immune response to a pathogen.

The forkhead lineage-specific transcription marker, Foxp3, has been proven to be a specific marker for T_reg cells (9). In mice, CD4⁺CD25⁺Foxp3⁺ T cells are exclusively derived as a T cell lineage in the thymus; however, in the humans, CD4⁺CD25⁺Foxp3⁺ phenotype may represent CD4⁺ T_reg cells derived from the thymus and induced in the periphery (10). Mice and human T_reg cells also differ in that Foxp3 is inducible in human CD4⁺CD25⁻ T cells, but not in mice, and that humans have two Foxp3 isoforms, whereas mice appear to have only one (10). Foxp3 is not expressed on activated T cells, and the T_reg cell population (as more accurately defined by Foxp3 expression) extends beyond the CD4⁺CD25⁺ operational definition. CD4⁺ T_reg cells constitutively express other molecules, including the glucocorticoid-induced TNFR-related family and CTLA-4 (11). The CD4⁺ T_reg cells can secrete several cytokines, including IL-4, IL-10, and TGF-β (7, 8), and can act as immunoregulatory cells that inhibit T cell responses in vitro and in vivo (4, 5). Studies performed in several murine models indicate that CD4⁺ T_reg cells also play an important role in the prevention of autoimmune diseases (11, 12). Presently, the typical T_reg cell population is characterized as CD4⁺CD25⁺Foxp3⁺; however, a CD8⁺CD25⁺Foxp3⁺ population has also been identified recently (13, 14). The T_reg cells are critical for the maintenance of peripheral T cell tolerance, because their depletion leads to organ-specific autoimmune disorder (11, 12).

The mechanism of suppression still remains controversial. Although some studies have shown that T-T cell contact is required for suppression (9, 10), other studies have revealed that suppression is mediated by cytokines IL-10 and TGF-β (7, 8). Furthermore, depletion of CD4⁺CD25⁺ T cells by the administration of anti-CD25 mAb has been shown to suppress the growth of a variety of different syngeneic tumors in mice (15, 16, 17). The observation that the removal of immunoregulatory CD4⁺CD25⁺ T cells can abrogate unresponsiveness to syngeneic tumors in vivo, leading to the spontaneous development of tumor-specific responses, indicates that T_reg cells negatively regulate tumor immunity and that depletion of these cells might be critical for controlling tumor growth, also indicating that the maintenance of self-tolerance against tumor-self Ags could potentially be lifted.

Several studies have shown that there is an increase in the number of CD4⁺CD25^high T cells in the elderly (18, 19, 20, 21). Some of these studies have demonstrated that there is a correlation between a higher number of CD4⁺CD25^high T cells and T_reg suppression activity (18, 19), whereas other studies have shown no correlation (20, 21). Although some of these studies analyzed the expression of Foxp3 in CD4⁺CD25^high T cells by PCR, they did not analyze whether these CD4⁺CD25^high T cells were Foxp3 positive by functional Foxp3 protein intracellular flow cytometer staining (22). Currently, there are no studies demonstrating the expression of Foxp3 in CD4⁺/CD8⁺ T cells in the elderly or old animals or analyzing whether these populations influence the activation an immune response in the aged. We evaluated whether there are differences in the number of T_reg cells between young and old animals and whether T_reg cells influence the activation of antitumor immune responses in the aged. Our results indicated that old mice contained the double amount of CD4⁺CD25⁺Foxp3⁺ and CD8⁺CD25⁺Foxp3⁺ populations in spleen and lymph nodes when compared with spleens and lymph nodes from young mice. Furthermore, depletion of CD25⁺ cells with anti-CD25 mAb in old mice resulted in the rejection of BM-185-EGFP tumor cells and the generation of a protective memory response against BM-185 wild-type (w.t.) cells, and restores antitumor T cell cytotoxic activity. We have demonstrated previously that the addition of costimulatory molecules could restore the antitumor response against BM-185-EGFP tumors in old animals (3). TLR ligands such as LPS, flagellin, CpG-oligodeoxynucleotide (ODN), imiquimod, poly(I:C), and others are strong coactivator molecules capable of activating antitumor responses. We tested whether these ligands were capable of restoring an antitumor immune response in old mice. Our results indicate that only CpG-ODN vaccination induced the rejection of BM-185-EGFP tumors in old mice. We observed that after CpG-ODN treatment, the numbers of T_reg cells in old animals were reduced to the same levels as young mice. These results indicate that the accumulated T_reg cells in the old inhibit or prevent the activation of antitumor responses, and the depletion or reduction of T_reg cells might be critical to optimally activate an immune response in the aged.

Young (2- to 3-mo-old) and old (18- to 22-mo-old) BALB/c mice were purchased from the National Institute of Aging and housed under specific pathogen-free conditions. The tumor cell lines, BM-185 w.t., and BM-185 expressing EGFP (BM-185-EGFP) and BM-185-EGFP-CD80 were provided by D. Kohn (University of Southern California, Los Angeles, CA). P815 cells were purchased from the American Type Culture Collection. Anti-CD25 (PC61) mAb was purified by passage of culture supernatant through a protein G matrix affinity column (Sigma-Aldrich). All cell lines were maintained in complete RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 5 × 10⁻⁵ M 2-ME, and 50 μg/ml gentamicin.

The prevalence of T_reg populations in young and old animals was determined by analysis of CD4⁺CD25⁺Foxp3⁺ and CD8⁺CD25⁺Foxp3⁺ T_reg cells in the spleen and lymph nodes. Spleen and lymph nodes from young and old animals were first surface stained with anti-CD4 (FITC) or anti-CD8 (FITC) and anti-CD25 (allophycocyanin) Abs (eBioscience). Intracellular Foxp3 (PE) staining was performed using a commercially available kit (eBioscience) following the manufacturer’s protocol.

To evaluate the effect of depleting CD25⁺ T cells, old mice were injected on days −7, −4, and −1 with 100 μg/injection of anti-CD25 mAb. Young and old animals were injected s.c. with 10⁵ BM-185-EGFP cells on day 0, and mice were examined twice per week for tumor development and survival. To evaluate whether old mice developed a memory response after treatment with the anti-CD25 mAb, animals were challenged 60 days later with an s.c. injection of 10⁵ BM-185-w.t. cells. Survival analysis used the Breslow modification of the Kaplan-Meier test.

Young and old BALB/c mice were immunized with an s.c. injection of 10⁵ BM-185-EGFP cells. Two weeks later, spleens from primed animals were removed and spleen cells were restimulated in vitro with BM-185-EGFP-CD80 cells. After 5 days, CTLs were assayed for lytic activity. The BM-185-w.t., BM-185-EGFP, P815, and P815 pulsed with the H2-K^d-HYLSTQSAL peptide (a K^d-EGFP-derived immunodominant epitope) cells were incubated with 150 μCi of ⁵¹Cr sodium chromate for 1 h at 37°C. Cells were washed three times and resuspended in complete RPMI 1640 medium. For the cytotoxic assay, ⁵¹Cr-labeled target cells (10⁴) were incubated with varying concentrations of effector cells in a final volume of 200 μl in U-bottom 96-well microtiter plates. Supernatants were recovered after 5 h of incubation at 37°C, and the percentage of lysis was determined by the following formula: percentage of specific lysis = 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release).

TLR ligands, CpG-ODN (1826) and control-ODN, were purchased from Oligos Etc.; imiquimod (a soluble form of this compound was obtained directly from 3M Pharmaceuticals), poly(I:C), and LPS were purchased from Sigma-Aldrich. Flagellin was purified, as previously reported (23). Old animals were injected s.c. with 10⁵ BM-185-EGFP cells on day 0. On day 5 after tumor inoculation, animals were randomly divided into groups of five mice/group. Animals received 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 three times per week (30 μg/injection) for 3 wk. Evaluation of memory responses and survival was performed, as described above.

Spleen cells (10⁶) from old mice were plated in 24-well plates and not treated or treated with CpG-ODN, LPS, poly(I:C), imiquimod, and flagellin at 1 μg/ml. After 24 h, supernatants were collected and levels of IL-6 were determined by the Luminex-100 flow cytometry assay.

Statistical significance of data was determined by Student’s t test to evaluate the p value.

Spleen and lymph nodes from naive young and old BALB/c mice were evaluated for the presence of CD4⁺CD25⁺FoxP3⁺ cells. As shown in Fig. 1, old mice contained a significantly larger population of CD4⁺Foxp3⁺ T cells in their spleen (ranging from 5 to 9%) (p < 0.04) and lymph nodes (ranging from 10 to 14%) (p < 0.02) when compared with spleens (ranging from 4 to 6%) and lymph nodes (ranging from 6 to 8%) from young mice. Further analysis of CD4⁺ populations in terms of comparative expression of Foxp3 and CD25 demonstrates that a significantly larger population of CD4⁺ cells also coexpresses Foxp3 and CD25 in spleens (21%) (p < 0.01) and lymph nodes (27%) (p < 0.01) when compared with spleens (14%) and lymph nodes (12%) from young mice (Fig. 2). It has been demonstrated recently that T_reg cells are not exclusively confined to CD4⁺ T cells; CD8⁺ T cells also express Foxp3 and can act as immunoregulatory cells inhibiting T cell responses (13, 14). We next evaluated whether there were differences in the number of CD8⁺CD25⁺FoxP3⁺ cells accumulated in spleens and lymph nodes in young and old mice. Our results indicated that spleens (4.9%) and lymph nodes (5.3%) from old mice contained a higher accumulation (p < 0.05) of CD8⁺CD25⁺FoxP3⁺ cells in comparison with young spleens (2.6%) and lymph nodes (2.5%) (Fig. 3).

Compared with young animals, the aged have a higher prevalence of CD25⁺Foxp3⁺ regulatory cells (Figs. 2 and 3). Based on these results, we hypothesize that elimination of CD25⁺ cells could improve the antitumor immune response of old mice against BM-185-EGFP. Old mice were treated with anti-CD25 mAb on days −7, −3, and −1, and animals were inoculated with 10⁵ BM-185-EGFP cells on day 0. We included young mice inoculated with BM-185-EGFP cells as a positive control. Our results indicated that 100% of old mice treated with anti-CD25 mAb developed an antitumor response capable of rejecting the BM-185-EGFP cells in the same manner as young mice (Fig. 4). Old mice without anti-CD25 mAb treatment formed tumors (Fig. 4). We next evaluated whether old mice depleted of CD25⁺ cells developed a protective memory response against the BM-185 w.t. cells. As shown in Fig. 4, challenging the animals on day 60 posttumor implantation, 100% of old animals originally injected with the BM-185-EGFP cells plus anti-CD25 mAb rejected the BM-185-w.t. cells. As expected, young mice inoculated with BM-185-EGFP also developed a protective memory response after the challenge. Our results show that aged animals accumulate T_reg cells and inhibit specific immunity, and their depletion might be a simple means to supplement antitumor immunity in the aged hosts.

We have demonstrated previously that injection of BM-185-EGFP cells induces T cell responses against EGFP⁺ tumors and specific CD8 T cell responses against the EGFP-H2-K^d-restricted peptide (HYLSTQSAL) in young animals, but not in old animals (3, 24). We evaluated whether depletion of CD25⁺ cells could restore the activation of EGFP-specific responses in old mice. Young and old mice were treated or not treated with anti-CD25 mAb and immunized with BM-185-EGFP cells. Two weeks later, animals were sacrificed and T cell cytotoxic activity was evaluated. As shown in Fig. 5, young mice (Fig. 5,A) demonstrated a robust cytotoxic response against BM-185-EGFP and P815 cells pulsed with EGFP-H2-K^d peptide; in contrast, old mice could not activate a cytotoxic T cell response (Fig. 5,B). However, old mice treated with anti-CD25 mAb generate a cytotoxic T cell response against BM-185-EGFP and P815 cells pulsed with EGFP-H2-K^d peptide similar to those observed in young mice (Fig. 5 C). Taken together, these results suggested that CD25⁺ T_reg cells negatively modulate the immune response in the old mice, and depletion of CD25⁺ cells could be used as strategy to restore the immune response in old animals.

We have demonstrated previously that costimulatory molecules, such as B7.1, anti-OX40, or anti-4-1BB mAb, could restore the immune responses in old mice. It has been demonstrated that TLR ligands are potent activators of immune responses and significantly up-regulate the costimulatory molecules on APCs. It is not clear which of these adjuvants would be the most effective to induce an immune response in old mice. To this end, we compared the antitumor immune responses in old tumor-bearing mice after injecting poly(I:C) (TLR-3), LPS (TLR-4), flagellin (TLR-5), imiquimod (TLR-7), and CpG-ODN (TLR-9). Old mice were implanted s.c. with. 10⁵ BM-185-EGFP cells on day 0. On day 5, 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 three times per week (30 μg/injection) for 3 wk. Our results demonstrate that only injections of CpG-ODN (p < 0.001) restored the immune responses in old mice, resulting in the complete rejection of tumors (Fig. 6). Treatment with poly(I:C), imiquimod, LPS, or flagellin did not have any effect in controlling the tumor growth. No antitumor effect was observed in old mice injected with control-ODN, indicating that the immune response stimulated by the CpG-ODN was specific against the tumor.

We evaluated whether vaccination with CpG-ODN influenced the number of CD4⁺Foxp3⁺ T_reg cells in old mice. As shown in Fig. 7,A, treatment with CpG-ODN resulted in a significant reduction (∼40%) of CD4⁺Foxp3⁺ T_reg cells in the spleens of old animals as compared with aged controls (p < 0.05). Furthermore, the levels of T_reg cells in old controls after CpG-ODN treatment were comparable with the level of the young controls. We also evaluated whether CpG-ODN restored the cytotoxic activity against EGFP Ags in old mice. As expected, old mice immunized with BM-185-EGFP cell did not induce an immune response (Fig. 7,B), whereas immunization with BM-185-EGFP plus CpG-ODN restored the CTL responses against BM-185-EGFP and P815 cells pulsed with EGFP-H2-K^d peptide (Fig. 7 C). These results indicate that CpG-ODN vaccination influences the levels of circulating CD4⁺Foxp3⁺ T_reg cells in old mice by reducing their numbers to the same level as young animals, which correlate with the restoration of immune responses in the aged.

We wanted to evaluate the effect of CpG-ODN on T_reg cells. We treated in vitro CD4⁺CD25⁺ T cells with CpG-ODN, and our initial results indicate that CpG-ODN does disrupt the suppressive activity of these cells (data not shown). These results are in agreement with the data of Caramalho et al. (25), which demonstrated that T_reg cells do not express TLR-9. It has been demonstrated that IL-6 is capable of reversing the suppressive activity of CD4⁺ T_reg cells (26). We evaluated IL-6 secretion by splenocytes following incubation with CpG-ODN, LPS, poly(I:C), imiquimod, and flagellin. Treatment with CpG-ODN significantly increased the levels of IL-6 (p < 0.001) (Fig. 8) as compared with controls or other groups treated with other TLR ligands, correlating it with the reduced T_reg population in CpG-ODN-treated old mice.

The capacity of a host to mount an effective immune response can be limited by the pre-existence of counterregulatory elements. Thus, controlling regulatory mechanisms may represent a powerful strategy for controlling chronic infection or enhancing the efficiency of vaccines. This is of particular significance to the aging population, which has a diminished immune system. Recent evidence has demonstrated that T_reg cell-mediated immunosuppression is one of the crucial tumor immune evasion mechanisms, contributes to the failure of tumor immunotherapy, and controls the activation of primary and memory T cell responses. One subset is represented by naturally occurring CD4⁺ T_reg cells, generated in the thymus, which constitutively express CD25 (6, 7). Other CD4⁺ T_reg populations include cells secreting high levels of TGF-β1, which can be induced by oral Ag administration or stimulation of CD4⁺CD25⁻ T cells in the presence of TGF-β1. Additionally, T_reg class 1 cells secreting IFN-γ and IL-10 can be induced following in vitro stimulation in the presence of exogenous IL-10 (8). These T_reg subsets are most likely generated in the periphery, which enables the development of peripheral tolerance to self Ags (27).

Foxp3 expression is confined only to CD25^high cells in humans, whereas expression of Foxp3 is distributed throughout the CD25-staining population in mice (10). There is cumulative evidence indicating that CD4⁺CD25⁺Foxp3⁺ T cells are capable of suppressing the activation and expansion of other T cells and NK cells (9, 10). Furthermore, the ectopic expression of Foxp3 conferred a suppressive function on peripheral CD4⁺CD25⁻ T cells (9). This demonstrates that Foxp3 is a key player in CD4⁺CD25⁺ suppressor/T_reg cells. The prevalence of higher CD4⁺CD25^high T cells with immunosuppressive activity has been described in the elderly (18, 19, 20). However, these studies did not evaluate the expression of Foxp3; therefore, it is difficult to predict how many of those cells are truly T_reg cells. Additionally, the function of CD4⁺CD25^high T cells does not always correlate with the function of CD4⁺Foxp3⁺ T_reg cells (24, 27). Recently, it was shown that CD4⁺CD25⁺Foxp3⁺ T cells of aged mice are comparable with those of young animals in showing potent immunosuppressive activities (28). However, the relation of CD25⁺Foxp3⁺ T_reg activity with regard to inducing antitumor immunity in old animals is not completely understood. This is the first study that shows quantitative data of T_reg cells in old hosts in terms of Foxp3 in context to host antitumor responses.

Our results demonstrate that there is accumulation of CD25⁺Foxp3⁺ T_reg cells in the spleen and lymph nodes of aged animals when compared with young animals. Our results show that depletion of CD25⁺ cells could restore the immune responses, resulting in the rejection of BM-185-EGFP tumor cells in old mice. Furthermore, depletion of CD25⁺ cells also induces the generation of a protective memory response against BM-185 w.t. cells in the same manner as young mice. Additionally, we observed that old mice did not have the ability to prime a cytotoxic immune response against EGFP Ags following immunization with BM-185-EGFP cells. However, old mice treated with anti-CD25 mAb generate a cytotoxic T cell response against EGFP⁺ targets similar to those observed in young mice. Recently, Bienvenu et al. (29) showed that CD8⁺CD25⁺ T cells could also inhibit the responses of CD25⁻ T cells. Our results demonstrate that there is also accumulation of CD8⁺CD25⁺Foxp3⁺ T_reg cells in old animals as compared with young mice. Therefore, both CD4⁺ and CD8⁺ T_reg cells could contribute to the immune suppression induced by T_reg cells in old animals, and for the optimal stimulation of an immune response, it might be necessary to make sure to eliminate both CD4⁺ and CD8⁺ T_reg cells in the elderly. Taken together, these results suggested that CD25-positive T_reg cells negatively modulate the immune response in the old mice, and depletion of CD25⁺ cells could be used as a strategy to restore the immune response in old animals.

Aging is accompanied by numerous functional and phenotypic changes in the immune system; moreover, increases in autoimmunity, infections, and occurrence of cancer have been reported in aged people (1, 2). We have demonstrated previously that transfecting the BM185-EGFP tumors with the CD80 molecule allowed the old mice to reject the tumors (3). However, under these conditions, old animals do not develop memory responses. But when EGFP-CD80 tumors were given in combination with anti-OX40 and anti-4-1BB mAb, old mice developed long-term memory responses capable of rejecting a challenge against w.t. tumors (3, 30). These data indicate that the aged immune repertoire can be exploited for the induction of tumor immunity, and that it is possible to convert aged animals from nonresponder to responder status with the inclusion of additional costimulation (3, 30). Activation of the innate immune system through TLR ligand had been demonstrated to be a good strategy to activate antitumor immune responses. Our result demonstrated that only immunizations with CpG-ODN restored the immune responses in old mice, resulting in the complete rejection of tumors and generation of memory responses. Treatment with LPS, imiquimod, flagellin, or poly(I:C) did not have any effect on controlling the tumor growth. These results have clinical implications, indicating that signaling via different TLR ligands could induce qualitatively or quantitatively different types of responses in old APCs, which ultimately could trigger an antitumor response (S. Sharma and J. Lustgarten, manuscript in preparation). Our responses with CpG-ODN were also comparable with observations of Maletto et al. (31) and Alignani et al. (32), who have shown that CpG-ODN can function as efficient adjuvants in both young and old in a peptide vaccination setting. Analysis of T_reg cells in CpG-ODN-immunized old animals revealed that numbers and levels of T_reg cells are significantly reduced to the level found in the young animals. Furthermore, we observed that cytotoxic T cell responses were restored after CpG-ODN immunization in old mice. Therefore, there is a relationship between CpG-ODN treatment, reduction in the level of T_reg cells, and the induction of T cell responses. Vaccinations with CpG-ODN induce potent antitumor responses by stimulating a proinflammatory response (33, 34). It was demonstrated recently that IL-6 and perhaps other unidentified soluble factors secreted by DCs following stimulation with CpG-ODN reversed the suppressive activity of CD4⁺ T_reg cells (26). We evaluated IL-6 secretion by spleen cells following incubation with CpG-ODN and other TLR ligands. Our results indicate that CpG-ODN is the only TLR ligand that significantly increased the levels of IL-6 (p < 0.001), correlating it with down-regulation of T_reg cells and the activation of immune responses. We have observed that after CpG-ODN treatment, the levels of IL-6 within the tumor microenvironment are also increased by >20-fold (S. Sharma and J. Lustgarten, manuscript in preparation). This report shows for the first time that one of the effects of immunizing with CpG-ODN results in the inhibition of T_reg cells.

In summary, this study has shown for the first time that there are higher pools of CD4⁺CD25⁺FoxP3⁺ and CD8⁺ CD25⁺FoxP3⁺ cells accumulating in spleens and lymph nodes from old mice when compared with young animals. Furthermore, the existence of these suppressor populations leads to an inhibition of an immune activation because depletion of CD25⁺ with anti-CD25 mAb induced an antitumor response in old animals, inducing the rejection of BM-185-EGFP tumors, restoring the cytotoxic T cell response, and generating a protective memory response against w.t. tumors. Immunization of old animals with CpG-ODN inhibits the accumulation of T_reg cells and restores the antitumor immune responses against BM185-EGFP tumors. During aging, thymic output may change the pattern of T cells, and T_reg homeostasis may also be affected. An imbalance of T_reg homeostasis would then predispose the aged to immune dysfunction, resulting in a higher risk of immune-mediated diseases, cancer, or infections. The reason for the accumulation of T_reg cells in old mice is not yet understood. It has been demonstrated that there is shift from a Th1 to a Th2 response in aged mice (35), with an increased production of IL-10 (36), which may account in part for the high accumulation of T_reg cells in these animals. Further studies are needed to completely understand the reason that T_reg cells accumulate or expand in the old and how these populations relate to other immune-regulatory effects, such as cytokines, homeostasis, and APCs, and determine their specificity.

The authors have no financial conflict of interest.