Down-regulation of the immune response in aging individuals puts this population at a potential risk against infectious agents. In-depth studies conducted in humans and mouse models have demonstrated that with increasing age, the T cell immune response against pathogens is compromised and response to vaccinations is subdued. In the present study, using a mouse model, we demonstrate that older animals exhibit greater susceptibility to Encephalitozoon cuniculi infection, and their ability to evoke an Ag-specific T cell response at the gut mucosal site is reduced. The dampening of T cell immunity was due to the defective priming by the dendritic cells (DC) isolated from the mucosal tissues of aging animals. When primed with DC from younger mice, T cells from older animals were able to exhibit an optimal Ag-specific response. The functional defect in DC from older mice can be attributed to a large extent to reduced IL-15 message in these cells, which can be reversed by addition of exogenous IL-15 to the cultures. IL-15 treatment led to optimal expression of costimulatory molecules (CD80 and CD86) on the surface of older DC and restored their ability to prime a T cell response against the pathogen. To our knowledge, this is the first report which demonstrates the inability of the DC population from aging animals to prime a robust T cell response against an infectious agent. Moreover, the observation that IL-15 treatment can reverse this defect has far-reaching implications in developing strategies to increase vaccination protocols for aging populations.

Age-associated decreases in immune function are thought to contribute greatly to the increased morbidity and mortality seen in elderly populations. Aging of the immune system involves both humoral and cell-mediated immunity. Changes in humoral immunity consist of a decrease in the number of B lymphocytes leading to a poor Ab response (1, 2). Defects in cellular immunity include a decrease in absolute numbers of naive CD3, CD4, and CD8 T cells and alterations in signal transduction through TCR (3). Furthermore, substantial changes in the functional and phenotypic profiles of T cells have been reported in aging humans and rodents (4, 5).

Recent studies have shown that age-related defects in the immune system are not restricted to adaptive immunity but can also be extended to the innate immune response. Neutrophils, macrophages, and NK, important first lines of defense against bacterial and parasitic infections, are impaired with advancing age (6, 7, 8). Moreover, studies with animal models suggest that dendritic cells (DC),3 which serve as important APCs (9), are less able to stimulate T cells, thereby contributing to changes in their functionality during advanced age (10).

Microsporidia are single-celled obligate intracellular parasites that have emerged as an opportunistic infection causing diarrhea and systemic disease in persons with AIDS (11). Increased awareness and improved diagnostics of microsporidia infections has revealed a high occurrence of disease in non-HIV-infected elderly populations (12, 13, 14). One reason microsporidial infections are more frequent in aged human populations could be attributed to down-regulation of immune responses against the pathogen in these individuals.

Most of what is known about the immune response against microsporidia is based on Encephalitozoon cuniculi. The importance of T cells in controlling E. cuniculi infection was demonstrated by adoptive transfer studies using athymic or SCID mice (15, 16). Although both T cell subsets (CD4 and CD8) produce IFN-γ during E. cuniculi infection (17), when challenged via the i.p. route, protective immunity is mediated exclusively by CD8+ T cells (18, 19). Mutant animals lacking CD8+ T cells or perforin gene are highly susceptible to i.p. challenge with the pathogen (19). The situation is somewhat different after oral infection, as depletion of both CD4 and CD8+ T cells is needed to achieve mortality in animals challenged via this route (20). Apparently, IFN-γ production by CD4 or CD8+ T cells is critical for survival, since knockout mice exhibit severe susceptibility to per-oral infection. Although not as susceptible as IFN-γ−/− mice, perforin−/− animals exhibit mortality to per-oral infection, suggesting the importance of host CTL response against the pathogen (21).

Recent observations from our laboratory have demonstrated the importance of DC in priming mucosal T cell responses against this pathogen (21). In the present study, we evaluated the age-related changes in T cell responses against E. cuniculi infection in older mice and the ability of DC to prime immune responses against this pathogen.

C57BL/6 mice of different age groups were purchased from Charles River Breeding Laboratories. SCID mice on the C57BL/6 background, originally purchased from The Jackson Laboratory, were bred in our animal research facility. Animals were housed and bred under Institutional Animal Care and Use Committee-approved conditions at the Animal Research Facility at Louisiana State University Health Sciences Center (New Orleans, LA) and George Washington University (Washington, DC).

A rabbit isolate of E. cuniculi (genotype II), provided by E. Didier (Tulane Regional Primate Research Center, Covington, LA) was maintained by continuous passage in rabbit kidney cells (RK-13), obtained from American Type Culture Collection. The RK-13 cells were maintained in RPMI 1640 (Mediatech) containing 10% FCS (HyClone). Organisms were collected from the culture medium, centrifuged at 325 × g for 10 min, and washed twice with PBS. Mice were orally infected with 2 × 107 spores/mouse.

Quantitation of cytokine mRNA was performed by PCR. Mesenteric lymph node (MLN) lymphocytes and splenocytes from E. cuniculi-infected animals from different age groups were collected on day 14 postinfection (p.i.). RNA was isolated using TRIzol (Invitrogen) according to manufacturer’s instructions. Reverse transcription was performed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and random hexamer primers (Promega). Expression of mRNA for IFN-γ was analyzed by quantitative PCR with the PQRS quantitative method according to published protocol (20). The lymphocytes from uninfected mice were used to establish a baseline value of 1.0, against which the cytokine message level in the test was quantitated.

A CTL assay was performed according to a standard procedure in our laboratory (22). Briefly, mouse peritoneal macrophages were harvested 2 days after i.p. inoculation with 1 ml of thioglycolate. The macrophages were washed three times in PBS and dispensed at a concentration of 5 × 104 cells/well in U-bottom 96-well plates. After overnight incubation, the cells were infected with 2 × 105 spores of E. cuniculi per well for 48 h. The cells were washed extensively with PBS to remove any extracellular parasites. Macrophages were labeled with 51Cr (0.5μCi/well) for 2 h at 37°C. Macrophages were washed five times with PBS and incubated with MLN and spleen cells at various E:T ratios in a final volume of 200 μl of culture medium. The microtiter plates were centrifuged at 200 × g for 3 min and incubated at 37°C for 4 h. Samples (100 μl) were removed and assayed for released cpm by scintillation counting. The percentage of lysis was calculated as follow: ((mean cpm of test sample − mean cpm of spontaneous release)/(mean cpm of maximal release − mean cpm of spontaneous release))/100.

Ag-specific proliferation of T cell population was determined by a thymidine incorporation assay according to a standard protocol in our laboratory (22). Briefly, MLN and spleen lymphocytes were isolated and cultured in 96-well flat-bottom plates in RPMI 1640 at a concentration of 2 × 105 cells/well. The cells were stimulated with E. cuniculi spores (5 × 103 spores/well). After a 72-h incubation at 37°C in 5% CO2, thymidine (0.5μCi/well; Amersham) was added to the wells. Cells were harvested on a glass filter using an automated multiple sample harvester (Brandel M12), dried, and incorporation of radioactive thymidine was determined by liquid scintillation (Beckman Coulter).

C57BL/6 mice of different age groups (8 wk and 9 mo old) were infected per orally as mentioned above. Animals were sacrificed at day 14 p.i, MLN and spleen were collected, and T cells from the tissues were isolated by magnetic purification using TCRβ Ab according to the manufacturer’s instructions (Stem Cell Technology). Sorted T cells (92–96% pure) were adoptively transferred (5 × 106 cells/mouse) to SCID mice via the i.v. route. Two days after transfer, recipient mice were orally challenged with 2 × 107E. cuniculi spores/mouse. Morbidity and mortality were monitored daily until termination of the experiment.

MLN and splenic DC were isolated from naive C57BL/6 mice of different age groups according to previously published protocol (23). Briefly, mice were sacrificed, MLN harvested, and DC isolated by enzymatic digestion (DNase I and collagenase D) followed by mechanic disruption. Cells were then labeled with anti-CD11c biotin-conjugated Ab (eBioscience) followed by positive magnetic selection as described above. The cells were further purified on a cell sorter (FACSAria; BD Biosciences) after incubation with streptavidin-conjugated to PE-Cy5.5 (eBioscience).

Sorted DC were plated at 20,000 cells/well and incubated overnight with five E. cuniculi spores per DC. In some of the experiments, IL-15 (50–100 ng/ml; R&D Systems) was added to the wells. The next day, plated DC were extensively washed and purified T cells were added to the culture. T cells were isolated from MLN of naive C57BL/6 mice (8 wk old or 9 mo old) by positive magnetic selection as mentioned above. After a 72-h incubation, Ag-specific proliferation was measured by thymidine incorporation. In separate studies, the cultures were incubated for 96 h and cytotoxic activity against E. cuniculi-infected macrophages was determined by radioisotope release according to the protocol described above.

MLN DC from young and older mice were isolated as described above and plated at 20,000 cells/well with 1 × 105E. cuniculi spores/well in the presence or absence of recombinant murine IL-15 (50 ng/ml). After overnight incubation, the supernatants were harvested and assayed for IL-12p40 production using a commercially available ELISA kit (Biolegend) according to manufacturer’s instructions.

IL-15 message was measured by real-time PCR using the standard protocol of our laboratory (23). Briefly, cell cultures were set up as described above and MLN DC were harvested after a 48-h incubation at 37°C. RNA was isolated with a RNeasy kit (Qiagen) according to the manufacturer’s instructions, followed by reverse transcription with SuperScript III first-strand synthesis system (Invitrogen). Real-time PCR was performed on a MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) using 200 nM of primers (5mIL-15, 5′-CATCCATCTCGTACTTGTGTT-3′; 3mIL15, 5′-CATCTATCCAGTTGGCCTCTTTT-3′; 5mβ-actin, 5′-AGAGGGAAATCGTGCGTGAC-3′; and 3mβ-actin, 5′-CAATTAGTGATGACCTGGCCGT-3′) and SYBR GreenER qPCR Supermix (Invitrogen). Samples were subjected to 50 cycles of 15 s at 95°C and 2 min at 60°C followed by melt curve analysis. Noninfected DC were used to establish a baseline value of 1.0, against which the cytokine message level was quantitated.

Purified DC (40,000 cells/well) were incubated overnight in the presence of E. cuniculi (five spores per DC). Eighteen hours later, cells were harvested and labeled with anti- CD40, CD80, CD86, and MHC class II Abs conjugated to PE (eBioscience). DC were then analyzed with a FACSAria (BD Biosciences).

DC from naive C57BL/6 mice were purified, plated (5 × 104 cells/well), and pulsed with irradiated E. cuniculi (five spores per DC). After a 24-h incubation, the cells were harvested, washed, and adoptively transferred to 9-mo-old mice (5 × 105 DC/mouse) via the i.v. route Two weeks after transfer, the recipients were sacrificed, tissues (MLN and spleen) were harvested, and the CTL assay was performed.

Statistical analysis of the data was performed using a two-sampled Student’s t test (24).

Previous studies from our laboratory have demonstrated that young (7- to 8-wk-old) mice are able to survive oral E. cuniculi infection without any signs of morbidity (20). To assess the ability of different aged mice to resist E. cuniculi infection, C57BL/6 mice of different age groups (4, 9, and 12 mo old) were infected with 2 × 107E. cuniculi spores via the oral route. As shown in Fig. 1, although 4- and 9-mo-old infected mice survived, 12-mo-old infected animals succumbed to infection. Infected 12-mo-old mice became lethargic, lost weight, and died starting as early as 28 days p.i., with all of the animals from this age group succumbing to the infection by day 42 p.i. Conversely, 4- and 9-mo-old mice showed no signs of mortality or morbidity. These results demonstrate that with an increase in age, older animals lose their ability to survive E. cuniculi challenge.

FIGURE 1.

Older mice cannot survive oral E. cuniculi infection. C57BL/6 mice of different age groups (4, 9, and 12 mo) were orally infected with 2 × 107E. cuniculi spores (six mice per group). Animals were monitored daily for mortality and morbidity until the end of the experiment. The experiment was repeated twice with similar results.

FIGURE 1.

Older mice cannot survive oral E. cuniculi infection. C57BL/6 mice of different age groups (4, 9, and 12 mo) were orally infected with 2 × 107E. cuniculi spores (six mice per group). Animals were monitored daily for mortality and morbidity until the end of the experiment. The experiment was repeated twice with similar results.

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Since cellular immunity has been shown to play a critical role in the protective immunity against E. cuniculi (18, 19), we studied the induction of Ag-specific T cell responses against this pathogen in older animals. At 2 wk p.i, cell suspensions (spleen and MLN) from infected tissues were prepared and proliferative and cytotoxic assays were performed. As shown in Fig. 2, A and B, MLN and spleen cells from infected mice of the younger age group (8 wk and 4 mo old) exhibited significant proliferation (p < 0.001) in response to antigenic restimulation. Although 9-mo-old mice survived the infection (Fig. 1), the splenocytes from these mice exhibited a significant reduction in Ag-specific proliferation (p = 0.02; Fig. 2,B) and response was reduced to background levels in MLN (p ≤ 0.001; Fig. 2,A). This defect was more pronounced in 12-mo-old infected animals where both spleen and MLN cells failed to proliferate in response to E. cuniculi restimulation (p < 0.001; Fig. 2, A and B). Spleen and MLN cells from all of the groups with the exception of the 12-mo-old animals exhibited a normal mitogenic response (data not shown).

FIGURE 2.

Defective mucosal immune response after E. cuniculi infection. C57BL/6 mice (8 wk, 4 mo, 9 mo, and 12 mo old) were infected per orally with 2 × 107 spores. Two weeks p.i., mice were sacrificed (three animals per group) and MLN and spleen cell suspensions were prepared. Proliferative response of MLN lymphocytes (A) and splenocytes (B) was measured by thymidine incorporation after a 72-h incubation with E. cuniculi spores. In a separate experiment, MLN (C) and spleen cell suspensions (D) were prepared and incubated with 51Cr-labeled uninfected or infected macrophages at various E:T ratios. After a 4-h incubation, the cytolytic activity was determined by radioisotope release into culture supernatants. MLN (E) and splenic (F) lymphocytes were isolated 14 days after E. cuniculi infection (three mice per group). mRNA was prepared and assayed for IFN-γ message by RT-PCR. Data are representative of two separate experiments.

FIGURE 2.

Defective mucosal immune response after E. cuniculi infection. C57BL/6 mice (8 wk, 4 mo, 9 mo, and 12 mo old) were infected per orally with 2 × 107 spores. Two weeks p.i., mice were sacrificed (three animals per group) and MLN and spleen cell suspensions were prepared. Proliferative response of MLN lymphocytes (A) and splenocytes (B) was measured by thymidine incorporation after a 72-h incubation with E. cuniculi spores. In a separate experiment, MLN (C) and spleen cell suspensions (D) were prepared and incubated with 51Cr-labeled uninfected or infected macrophages at various E:T ratios. After a 4-h incubation, the cytolytic activity was determined by radioisotope release into culture supernatants. MLN (E) and splenic (F) lymphocytes were isolated 14 days after E. cuniculi infection (three mice per group). mRNA was prepared and assayed for IFN-γ message by RT-PCR. Data are representative of two separate experiments.

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To further assess the development of Ag-specific responses against E. cuniculi infection in older mice, the cytolytic activity of spleen and MLN cells against parasite- infected macrophages was determined. As shown in Fig. 2,C, MLN from older mice (9 and 12 mo of age) failed to lyse E. cuniculi-infected targets. The cytolytic response in MLN from older animals was reduced to the background lytic level at all E:T ratios tested (Fig. 2,C). In comparison, splenic cells from 9-mo-old mice at an E:T ratio of 40:1 were able to lyse 18–20% target cells (Fig. 2 D). Similar to our earlier observations (20), both spleen and MLN cells from younger animals (8 wk and 4 mo old) exhibited a strong cytotoxic response against parasite-infected targets.

Because IFN-γ has been reported to play an important role in immune protection against E. cuniculi infection (25), the level of cytokine message in the tissues of older animals was analyzed. Tissues (spleen and MLN) from mice orally infected with 2 × 107 spores of E. cuniculi were harvested 14 days p.i. and assayed for IFN-γ mRNA levels by semiquantitative PCR. In agreement with our previous findings (20), IFN-γ expression in both spleen and MLN of 8-wk-old mice increased after E. cuniculi infection (Fig. 2, E and F) and a similar pattern was observed in the tissues from 4-mo-old infected animals. Although spleen from 9-mo-old infected mice exhibited an increase in IFN-γ mRNA, the message levels in the MLN of these mice did not rise above the background levels. However, at 12 mo of age, mice infected with E. cuniculi failed to up-regulate IFN-γ message in both the MLN and spleen (Fig. 2, E and F). Based on these findings, it can be strongly suggested that age-related immunosuppressive responses to E. cuniculi infection occur by 9 mo and seem to be more pronounced in the MLN than spleen.

Subsequently, adoptive transfer studies were performed to establish the defect in the mucosal immune response of the older mice. T cells from mice of different age groups (8 wk and 9 mo old) were isolated 2 wk after oral infection and adoptively transferred to naive SCID animals. As shown in Fig. 3, recipient mice injected with MLN T cells from older mice (9 mo old) were unable to withstand E. cuniculi infection and all of the animals (six of six) died by day 43 after challenge. Interestingly, splenic T cells isolated from the same donors were able to protect SCID animals and recipients survived until termination of the experiment (Fig. 3). As expected, SCID mice treated with T cells from either spleen or MLN of younger (8-wk-old) mice were able to protect the recipients against E. cuniculi infection. These results further emphasize that age-related suppression of immune response against E. cuniculi infection occurs earlier in the gut mucosa as compared with the systemic immune compartment.

FIGURE 3.

MLN T cells from older mice fail to protect SCID recipients: MLN lymphocytes and splenocytes from 8-wk-old and 9-mo-old mice (four mice per group) were isolated and adoptively transferred to SCID mice (5 × 106 cells/mouse). Two days after transfer, recipients were challenged per orally with 2 × 107E. cuniculi spores. Morbidity and mortality were monitored daily until the end of the experiment. There were six animals per group and the experiment was performed twice with similar results.

FIGURE 3.

MLN T cells from older mice fail to protect SCID recipients: MLN lymphocytes and splenocytes from 8-wk-old and 9-mo-old mice (four mice per group) were isolated and adoptively transferred to SCID mice (5 × 106 cells/mouse). Two days after transfer, recipients were challenged per orally with 2 × 107E. cuniculi spores. Morbidity and mortality were monitored daily until the end of the experiment. There were six animals per group and the experiment was performed twice with similar results.

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As DC are known to play a crucial role in the priming of T cells (9), we decided to evaluate the ability of these cells to induce T cell responses against E. cuniculi in older animals. Since a suboptimal immune T cell response was detected in the MLN of 9-mo-old mice, further studies were conducted with these animals. DC from the MLN of young (8-wk-old) or older (9-mo-old) mice were isolated and cultured overnight with E. cuniculi spores. Twenty-four hours later, affinity-purified T cells (TCRβ+) from 8-wk-old mice were added to the culture. After 72 h, the proliferative response of T cells was measured by a [3H]thymidine incorporation assay. As shown in Fig. 4,A, although DC isolated from young mice induced a strong, dose-dependent T cell proliferative response, T cells primed with cells from older animals showed minimal proliferation. To establish that the suboptimal proliferation is due to a defect in the ability of DC derived from older mice to prime T cells, TCRβ+ T cells were isolated from mice of different age groups (8 wk old and 9 mo old) and incubated with E. cuniculi-pulsed DC from 8-wk-old mice. As shown in Fig. 4 B, T cells from both young and older mice showed comparable proliferative responses when primed with DC isolated from younger mice.

FIGURE 4.

A, MLN DC from older mice are unable to prime young T cells in vitro. MLN DC from 8-wk-old and 9-mo-old mice were isolated (four mice per group), plated at different concentrations, and stimulated overnight with E. cuniculi spores. The next day, T cells isolated from naive 8-wk-old mice (three mice per group) were added to the culture. After a 72-h incubation, proliferative response was measured by thymidine incorporation. B, T cells from young and older mice respond equally well to in vitro priming. MLN DC from 8-wk-old mice (four mice) were isolated, plated at different concentrations, and stimulated with E. cuniculi. After overnight incubation, T cells isolated from MLN of naive 8-wk-old or 9-mo-old mice (three mice per group) were added to the culture. After a 72-h incubation, proliferative response was measured by thymidine incorporation. C, Defective IL-12 production by MLN DC from older mice. Culture supernatants were assayed for IL-12 production by ELISA. Results are representative of two separate experiments.

FIGURE 4.

A, MLN DC from older mice are unable to prime young T cells in vitro. MLN DC from 8-wk-old and 9-mo-old mice were isolated (four mice per group), plated at different concentrations, and stimulated overnight with E. cuniculi spores. The next day, T cells isolated from naive 8-wk-old mice (three mice per group) were added to the culture. After a 72-h incubation, proliferative response was measured by thymidine incorporation. B, T cells from young and older mice respond equally well to in vitro priming. MLN DC from 8-wk-old mice (four mice) were isolated, plated at different concentrations, and stimulated with E. cuniculi. After overnight incubation, T cells isolated from MLN of naive 8-wk-old or 9-mo-old mice (three mice per group) were added to the culture. After a 72-h incubation, proliferative response was measured by thymidine incorporation. C, Defective IL-12 production by MLN DC from older mice. Culture supernatants were assayed for IL-12 production by ELISA. Results are representative of two separate experiments.

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IL-12 production by DC is important for the initiation of the Th1 immune response (26). Given that the role of IL-12 in the immune response against E. cuniculi has been reported (25), we analyzed the ability of DC from MLN to produce IL-12 in response to E. cuniculi stimulation. DC from young and older mice were isolated and pulsed overnight with E. cuniculi spores. The following day, the supernatants were harvested and levels of IL-12 were determined by ELISA. As expected, MLN DC from younger mice produced significant amounts of IL-12 when stimulated with E. cuniculi spores (Fig. 4 C). Conversely, DC from older mice exhibited a defect in IL-12 production and yielded significantly less cytokine (p = 0.0015) as compared with the cells isolated from younger animals.

Next, we assayed the ability of splenic DC from this age group to prime Ag-specific responses. Splenic DC isolated from young (8-wk-old) and older (9-mo-old) animals were pulsed with E. cuniculi spores overnight and their ability to prime T cells to proliferate was determined by a [3H]thymidine incorporation assay as mentioned above. Interestingly, unlike MLN DC, splenic DC from 9-mo-old mice were able to prime T cell responses comparable to DC from younger mice (Fig. 5,A). Similar to the results obtained with proliferation assays the defect in IL-12 release was not observed in splenic DC from older mice. When pulsed overnight with E. cuniculi spores, they produced IL-12 levels comparable to the splenic DC isolated from younger mice (Fig. 5 B).

FIGURE 5.

Splenic DC from older mice are able to prime splenocytes in vitro. Splenic DC from 8-wk-old and 9-mo-old mice were isolated, plated (20,000 DC/well) and stimulated with E. cuniculi spores. The next day, splenic T cells isolated from naive 8-wk-old mice were added to the culture. A, After a 72-h incubation, proliferative response was measured by thymidine incorporation. B, Culture supernatants were assayed for IL-12 production by ELISA. Results are representative of two separate experiments.

FIGURE 5.

Splenic DC from older mice are able to prime splenocytes in vitro. Splenic DC from 8-wk-old and 9-mo-old mice were isolated, plated (20,000 DC/well) and stimulated with E. cuniculi spores. The next day, splenic T cells isolated from naive 8-wk-old mice were added to the culture. A, After a 72-h incubation, proliferative response was measured by thymidine incorporation. B, Culture supernatants were assayed for IL-12 production by ELISA. Results are representative of two separate experiments.

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These findings demonstrate that MLN DC from 9-mo-old mice are unable to prime an immune response against E. cuniculi infection as manifested by their inability to secrete IL-12 and induce T cell proliferation in vitro. Conversely, splenic DC from these animals have intact function, exhibit optimal levels of IL-12, and induce robust T cell proliferation.

To fully determine the ability of DC from older mice to prime the E. cuniculi-specific immune response in vivo, adoptive transfer studies were performed. DC isolated from MLN or spleen from young (8-wk-old) and older (9-mo-old) mice were pulsed in vitro with E. cuniculi spores and subsequently transferred to naive mice at 9 mo of age. Two weeks after transfer, the recipient animals were sacrificed, tissues (spleen and MLN) were isolated, and the CTL assay performed. As shown in Fig. 6, A and B, the MLN DC from older animals failed to induce a cytotoxic response in the mucosal or splenic compartment of recipient animals at all of the E:T ratios tested. Conversely, the recipients injected with splenic DC from the same donors were able to exhibit cytotoxic activity against E. cuniculi-infected targets in both mucosal and splenic sites (Fig. 6, A and B). This response was comparable to the one observed in the recipients treated with DC isolated from younger animals (Fig. 6, A and B). These results demonstrate that, compared with spleen, age-related defects in DC start earlier in the mucosal compartment.

FIGURE 6.

Adoptive transfer of MLN DC form older mice fail to prime a CTL response in vivo. DC from MLN and spleen from 8-wk-old and 9-mo-old mice were isolated, plated (5 × 104 cells per well), and pulsed with irradiated E. cuniculi spores (2.5 × 105/well). After overnight incubation, cultures were harvested and cells were adoptively transferred to 9-mo-old mice (5 × 105/mouse) via the i.v. route. Fourteen days later, recipient mice were sacrificed, MLN (A) and splenic (B) lymphocytes were prepared and assayed for cytotoxicity. Experiment was performed twice with similar results and data are representative of one experiment.

FIGURE 6.

Adoptive transfer of MLN DC form older mice fail to prime a CTL response in vivo. DC from MLN and spleen from 8-wk-old and 9-mo-old mice were isolated, plated (5 × 104 cells per well), and pulsed with irradiated E. cuniculi spores (2.5 × 105/well). After overnight incubation, cultures were harvested and cells were adoptively transferred to 9-mo-old mice (5 × 105/mouse) via the i.v. route. Fourteen days later, recipient mice were sacrificed, MLN (A) and splenic (B) lymphocytes were prepared and assayed for cytotoxicity. Experiment was performed twice with similar results and data are representative of one experiment.

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Previous studies from our laboratory have reported the essential role of IL-15 in the priming of T cell responses by DC (27). Moreover, a recent study by Ohteki et al. (28) has shown that DC are the primary source of IL-15. We analyzed IL-15 message expression in MLN DC from 8-wk and 9-mo-old mice after in vitro activation with E. cuniculi. Forty-eight hours after E. cuniculi stimulation, MLN DC isolated from 8-wk-old mice exhibited a 4.5-fold increase in IL-15 expression while those from older mice showed only a 2-fold increase over their respective nonstimulated controls (p = 0.047; Fig. 7,A). To determine whether IL-15 treatment would restore DC function of older mice, cultures of purified DC pulsed with E. cuniculi spores were treated overnight with IL-15. Twenty-four hours later, cells were washed and purified T cells were added to the cultures. Proliferation was measured after 4 days of incubation by a thymidine incorporation assay. As shown in Fig. 7 B, IL-15 treatment significantly improved the proliferation of T cells cultured with DC from older mice (p = 0.048), suggesting that this cytokine can reverse the inability of older DC to prime T cell responses.

FIGURE 7.

IL-15 improves T cell priming by older DC and their costimulatory molecule expression. A, MLN DC from older mice are unable to up-regulate the IL-15 message after E. cuniculi stimulation. MLN DC from 8-wk-old and 9-mo-old mice were isolated and plated (20,000 DC/well). Cells were incubated for 48 h with E. cuniculi spores and analyzed for IL-15 mRNA expression normalized to β-actin mRNA levels. Relative expression was measured by using the mean from each group and the formula relative transcript level = 2 − ΔCt × 1000. B, IL-15 treatment restores the ability of older DC to prime young T cells in vitro. MLN DC from 8-wk-old and 9-mo-old mice were isolated and plated (20,000 DC/well). DC were incubated overnight with E. cuniculi spores and exogenous IL-15 (100 and 50 ng/ml). The next day, cell cultures were washed and T cells isolated from MLN of naive 8-wk-old mice were added. After a 72-h incubation, the proliferative response was measured by thymidine incorporation. C, Treatment with IL-15 restores the ability of MLN DC from older mice to prime a CTL response. MLN DC from 8-wk and 9-mo-old mice (10–15 mice/group) were isolated, plated (5 × 104 DC/well), and stimulated overnight with E. cuniculi (2.5 × 105 spores/well). The following day, purified T cells from naive 8-wk-old mice (three mice per group) were added to the culture and, subsequently, after a 4-day incubation, a CTL assay was performed. D, IL-15 increases costimulatory molecule expression by older DC. MLN DC from 8-wk and 9-mo-old mice were isolated, plated at 40,000 DC/well, and incubated overnight with E. cuniculi spores. The next day, cells were harvested and labeled for MHC class II (I–iv), CD80 (v–vii), CD86 (ix–xii), and CD40 (xiii–xvi). Results are presented as mean ± SD of triplicates and experiment was performed twice with similar results.

FIGURE 7.

IL-15 improves T cell priming by older DC and their costimulatory molecule expression. A, MLN DC from older mice are unable to up-regulate the IL-15 message after E. cuniculi stimulation. MLN DC from 8-wk-old and 9-mo-old mice were isolated and plated (20,000 DC/well). Cells were incubated for 48 h with E. cuniculi spores and analyzed for IL-15 mRNA expression normalized to β-actin mRNA levels. Relative expression was measured by using the mean from each group and the formula relative transcript level = 2 − ΔCt × 1000. B, IL-15 treatment restores the ability of older DC to prime young T cells in vitro. MLN DC from 8-wk-old and 9-mo-old mice were isolated and plated (20,000 DC/well). DC were incubated overnight with E. cuniculi spores and exogenous IL-15 (100 and 50 ng/ml). The next day, cell cultures were washed and T cells isolated from MLN of naive 8-wk-old mice were added. After a 72-h incubation, the proliferative response was measured by thymidine incorporation. C, Treatment with IL-15 restores the ability of MLN DC from older mice to prime a CTL response. MLN DC from 8-wk and 9-mo-old mice (10–15 mice/group) were isolated, plated (5 × 104 DC/well), and stimulated overnight with E. cuniculi (2.5 × 105 spores/well). The following day, purified T cells from naive 8-wk-old mice (three mice per group) were added to the culture and, subsequently, after a 4-day incubation, a CTL assay was performed. D, IL-15 increases costimulatory molecule expression by older DC. MLN DC from 8-wk and 9-mo-old mice were isolated, plated at 40,000 DC/well, and incubated overnight with E. cuniculi spores. The next day, cells were harvested and labeled for MHC class II (I–iv), CD80 (v–vii), CD86 (ix–xii), and CD40 (xiii–xvi). Results are presented as mean ± SD of triplicates and experiment was performed twice with similar results.

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Next, we determined whether IL-15 can restore the ability of MLN DC from older mice to prime a CTL response against E. cuniculi. MLN DC from mice of different age groups (8 wk and 9 mo old) were pulsed overnight with E. cuniculi spores and, after a 24-h incubation, purified T cells isolated from younger animals (8 wk old) were added to the cultures. Four days later, the cultures were harvested, incubated with radiolabeled infected macrophages, and lytic activity was determined by 51Cr release. As shown in Fig. 7,C, unlike DC from 8-wk-old mice, cells isolated from animals of 9 mo of age failed to induce an in vitro CTL response. Addition of exogenous rIL-15 to DC cultures from older mice restored their ability to prime cytotoxic responses (Fig. 7 C). However, IL-15 treatment was not able to bring the IL-12 production of older MLN DC to those observed with cells from younger mice (data not shown).

To further determine the mechanism of IL-15-mediated up-regulation of T cell priming by older DC, the cells were analyzed for MHC class II expression, which is pivotal for Ag presentation to CD4+ T cells and CD80 and CD86, costimulatory molecules important for T cell stimulation (29). Although no difference in MHC class II expression between E. cuniculi-primed DC from older and young mice was observed (Fig. 7,C, i–iv), significantly lower levels of both CD80 and CD86 expression (p = 0.0007 and p = 0.02, respectively) was noted in the DC from older animals (Fig. 5,C, v–xii). In vitro IL-15 treatment restored the expression of CD80 and CD86 molecules on DC from older animals. However, no difference in the expression of the CD40 molecule in response to E. cuniculi infection between DC isolated from young or older animals was observed (Fig. 7, xiii–xvi). Also, addition of exogenous IL-15 to the cultures had no effect on the expression of this molecule.

Our results demonstrate that with increasing age, the ability of animals to launch an optimal immune response against E. cuniculi infection is reduced, which may prove to be a risk for their survival. Down-regulation of the immune response in the older animals is manifested by the inability of T cells from the MLN of these mice, to exhibit Ag-specific proliferation or cytotoxic response against pathogen-infected targets. Furthermore, our data for the first time demonstrate that generation of ineffective Ag-specific T cell responses in the MLN of older animals is due to the suboptimal priming by DC in this tissue. When primed with DC from younger animals, T cells from older mice exhibit a normal proliferative and cytotoxic response. The defect in the DC population from older animals appears to be restricted to the cells from MLN as their ability to secrete IL-12 and prime T cell responses against E. cuniculi infection is severely compromised. In comparison, the splenic DC isolated from the older animals produce normal IL-12 levels and induce an optimal T cell response to this pathogen. Moreover, although transfer of DC isolated from the tissues (MLN and spleen) of younger mice led to the generation of Ag-specific cytotoxic activity in the recipient animals, treatment with MLN DC from older animals failed to generate a detectable CTL response. Similar to the cells isolated from young animals, DC from the splenic compartment of older donors were able to induce cytotoxic responses in the tissues of recipient mice. Down-regulation of the DC response against E. cuniculi in older animals appears to be to a large extent due to reduced IL-15 message levels in MLN, which leads to decreased expression of costimulatory molecules like CD80 and CD86 on these cells. However, treatment with IL-15 reverses this down-regulation, leading to optimal expression of these molecules and restores their ability to prime a T cell response against the pathogen.

Age-related changes in the immune response are known to render individuals more susceptible to infection and decreases their ability to generate responses against tumors (30, 31, 32). The immune dysfunction due to aging which contributes to morbidity and mortality is not restricted only to the very elderly, but in all likelihood can be observed during middle age (33). Vaccination of the elderly population with the influenza virus fails to generate a strong effector response in these individuals (34, 35). Although, both primary and secondary Ab responses to vaccinations are subdued, the highest degree of impairment is seen in the T cell arm of the immune system (35). A substantial decrease in the number of naive lymphocytes as a result of thymic output and expansion of oligoclonally and functionally incompetent memory lymphocytes as a result of age-associated changes have been reported (36). In a murine model, it has been demonstrated that the frequency and number of lymphocyte precursors are considerably reduced by 7 mo of age (37). Although the total number of T cells in secondary lymphoid organs and the CD4:CD8 ratio are relatively unaffected by aging, there is a considerable decrease in effector response of both T cell subsets (38, 39). Also, naive CD4 T cells from aged animals have been reported to secrete less IL-2, leading to a decrease in the expression of CD25. These cells are unable to exhibit optimal proliferation to antigenic stimulation and cannot undergo complete differentiation to Th type 1 or type 2 effector cells (40).

Although there is an abundance of information related to observed defects in effector T cell responses in the aging population, the status of the innate immune response in these individuals has not been studied. Most of the above-mentioned reports have emphasized the defect in development of effector T cell response in these individuals. In the present study, we demonstrate that the age-related T cell defect in the older animals can be restored if DC from younger mice are involved in the priming of Ag-specific T cell responses (both in vitro and in vivo). When cultured with E. cuniculi-pulsed DC from younger animals, effector function of the T cells from older mice was restored. Moreover, transfer of DC from younger animals led to induction of Ag-specific CTL responses in the tissues of older animals. In addition, although transfer of MLN T cells from older mice failed to protect SCID animals from E. cuniculi challenge, 100% survival was observed among the groups treated with immune T cells isolated from tissues of younger mice. This study strongly suggests that poor priming by DC from the mucosal site is an important factor in T cell defect during early aging.

As stated above, although T cell responses in aged animals has been well studied (39, 41, 42), the information regarding the effect of aging on DC function is very limited and not well defined. In one of the recent reports, it has been demonstrated that aging has no effect on the in vitro generation of monocyte-derived DC and cells isolated from both young or old individuals were capable of stimulating the T cell response (43). However, studies by Maletto et al. (44) have shown that immune dysfunction in aged animals infected with Trypanosoma cruzi is due to a defect in APC function (44). Similarly, poor APC function has also been linked to a suboptimal immune response against pneumococcal vaccines in old mice (45). A study involving monocyte-derived DC from healthy volunteers demonstrated that, although infection with influenza virus was able to evoke an immune response comparable to cells isolated from young individuals, respiratory scyncytial virus-infected cells from old volunteers triggered a significantly reduced response (46). However, GM-CSF as well as M-CSF production is reduced with age and this could explain the difference between in vitro and in vivo observations (47, 48).

Although the effect of aging on DC function remains somewhat enigmatic, available information suggests that down-regulation of DC function can be an important cause for a suboptimal immune response against certain diseases in the elderly population. Our studies demonstrate that oral E. cuniculi infection can be one such situation where older animals are unable to evoke a robust T cell response against the pathogen due to poor DC priming. This defect was especially apparent in the mucosal sites because cells isolated from the MLN failed to prime an effector T cell response against the pathogen. Since IL-15, which is primarily produced by DC, has recently been shown to play a major role in the induction of the CD8 T cell response (49), we decided to focus our attention on this cytokine. Our current observations, for the first time, demonstrate the inability of MLN DC from aging animals to express optimal IL-15 message. Interestingly, in vitro treatment of DC with IL-15 restored their ability to prime T cell immunity against this pathogen, suggesting that the age-related immune defect in the older mice may be significantly linked to this cytokine. Recently, the importance of IL-15 in the activation of the DC response has been shown by several laboratories (50, 51). In one of these studies, in vivo and in vitro exposure of splenic DC to IL-15 led to up-regulation of costimulatory molecules, marked increase in the IFN-γ release by these cells, and enhanced their ability to stimulate Ag- specific CD8+ T cell proliferation (51). Similarly, our laboratory has reported that a lack of IL-15 impairs the DC response, which results in poor CD4+ T cell immunity against T. gondii infection (27). Moreover, recent studies have shown that IL-15 is involved in the cross-talk between different DC subpopulations, which is essential for the activation of these cells (52). In the current study, an increase in IL-12 production after IL-15 treatment was not observed, suggesting that IL-15-mediated restoration of E. cuniculi-specific T cell priming by DC may be independent of IL-12 production. These findings are similar to an earlier report by Paryath et al. (53) in which IL-15 stimulation failed to increase pathogen-related IL-12 production by PBMC from HIV-infected individuals.

The mechanism involved in the restoration of DC function by IL-15 appeared to be dependent on the ability of the cytokine to up-regulate surface costimulatory molecules like CD80 and CD86. Both of these molecules, which are known to play an important role in T cell priming (54), were down-regulated in E. cuniculi-pulsed DC from older mice. However, IL-15 treatment of DC from older animals increased their expression to the levels observed in the cells from younger mice. Recent studies have shown that transduction of the IL-15 gene causes a significant increase in the expression of CD83, CD86, and CD40 molecules on these cells (51, 55). Although down-regulation in CD80 and CD86 expression on the DC of older mice was observed, the expression of MHC class II and the CD40 molecule on these cells was comparable to cells from younger animals. Our observations are in agreement with recent findings by Sharma et al. (56), who demonstrated that adequate costimulation can generate normal CD8+ T cell responses against tumors in old mice treated with DC vaccines. The novelty of our findings is that we convincingly demonstrate for the first time that DC isolated from mucosal sites of older mice are functionally defective against an oral pathogen. These observations strongly suggest that the decreased ability of the aging population to combat oral infections may begin with a defect in the DC capacity to prime Ag-specific response, especially in the gut mucosa.

Since conventional vaccines are unable to protect the elderly population, new strategies need to be developed to protect these individuals against infectious agents. Our data demonstrate that mucosal DC from older animals have a defect in IL-15 induction and addition of this cytokine can, to a great extent, restore the function of these cells. When treated with IL-15, mucosal DC from older mice are able to generate CTL responses against E. cuniculi infection, which, as reported earlier by our laboratory (19), is critical for protective immune response against the pathogen. These findings have strong implications in developing therapeutic agents that will enable the elderly population to combat those intracellular pathogens against which CTL immunity is essential for host protection.

We thank Teresa Hawley for help with flow cytometry.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants AI 071778 and AI 043693 awarded to I.A.K.

3

Abbreviations used in this paper: DC, dendritic cell; MLN, mesenteric lymph node; p.i., postinfection.

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