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The Journal of Immunology, 2006, 177: 2565-2574.
Copyright © 2006 by The American Association of Immunologists

IL-10 Is Required for Optimal CD8 T Cell Memory following Listeria monocytogenes Infection

Kathryn E. Foulds, Masashi J. Rotte and Robert A. Seder1

Cellular Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-10 is an important immunoregulatory cytokine that plays a central role in maintaining a balance between protective immunity against infection and limiting proinflammatory responses to self or cross-reactive Ags. We examined the full effects of IL-10 deficiency on the establishment and quality of T cell memory using murine listeriosis as a model system. IL-10–/– mice had reduced bacterial loads and a shorter duration of primary infection than did wild-type mice. However, the number of Ag-specific T cells in secondary lymphoid and nonlymphoid organs was diminished in IL-10–/– mice, compared with wild-type mice, at the peak of the effector response. Moreover, the frequency and protective capacity of memory T cells also were reduced in IL-10–/– mice when assessed up to 100 days postinfection. Remarkably, this effect was more pronounced for CD8 T cells than CD4 T cells. To address whether differences in the number of bacteria and duration of primary infection could explain these findings, both strains of mice were treated with ampicillin 24 hours after primary infection. Despite there being more comparable bacterial loads during primary infection, IL-10–/– mice still generated fewer memory CD8 T cells and were less protected against secondary infection than were wild-type mice. Finally, the adoptive transfer of purified CD8 T cells from previously infected wild-type mice into naive recipients conferred better protection than the transfer of CD8 T cells from immune IL-10–/– mice. Overall, these data show that IL-10 plays an unexpected role in promoting and/or sustaining CD8 T cell memory following Listeria monocytogenes infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune system is carefully regulated to provide protective cellular immune responses against infection but limit reactivity to self or cross-reactive Ags. In this regard, IL-10 is an important immunoregulatory cytokine having a central role in maintaining such a balance. IL-10 is produced by a variety of cell types and is a potent inhibitor of innate and adaptive cellular immunity (1). Accordingly, IL-10 deficiency improves resistance to infection, as shown by studies using a variety of intracellular pathogens which include Listeria monocytogenes (2, 3, 4, 5, 6), Mycobacterium species (7, 8, 9, 10), and Leishmania major (11, 12). With some infectious agents, such as Toxoplasma gondii (13, 14), Plasmodium chaubudi (15), or some strains of Trypanosoma cruzi (16, 17), there is an increase in the frequency of effector CD4 T cells in IL-10-deficient mice, resulting in immunopathology in the form of a toxic shocklike syndrome from overproduction of Th1 cytokines. Finally, IL-10 has been shown to have a role in maintaining Th1 responses sufficient to mediate protection against the persistent pathogen, L. major (12, 18). Together, these data show that IL-10 can enhance the generation of CD4 T cell effector responses and sustain protective immunity that is mediated by CD4 T cells.

Although the effects of IL-10 on Th1 responses have been well characterized, less is known about the role of IL-10 in regulating CD8 T responses in vivo. Thus, it was of interest to examine the full effects of IL-10 deficiency on the establishment and maintenance of CD8, as well as CD4, T cell responses following an acute infection. We used the mouse model of L. monocytogenes because high levels of both IL-12 (19) and IL-10 (20) are released from macrophages following infection, making murine listeriosis an ideal system for studying the effect of IL-10 deficiency on Th1 and CD8 T cell responses. In addition, previous studies have shown that, although the innate immune response is critical for containing L. monocytogenes early during infection, T cells are required for sterile clearance of the bacteria and immunological memory (21, 22).

There are a number of studies that suggest that IL-10 extends the course of Listeria infection (23, 24, 25) and reduces the cellular immune response (2). For example, IL-10 transgenic mice are highly susceptible to infection with L. monocytogenes, compared with wild-type mice (24). Similarly, adult mice treated with anti-IL-10 or anti-IL-10 receptor Abs show increased resistance to infection during the early stage of infection. In contrast, during the late stage of infection, treated mice have similar or increased bacterial loads (6). One study demonstrated that CD4 and CD8 T cell responses are increased in IL-10-deficient mice following infection with L. monocytogenes, and that these mice have increased protection upon secondary infection (2). However, a subsequent report demonstrated that the number of L. monocytogenes-specific CD8 T cells was reduced in the livers of IL-10 deficient mice, and that these mice did not have significantly different bacterial loads upon secondary infection, compared with wild-type mice (4). Thus, the effect of IL-10 on memory T cell responses following L. monocytogenes infection remains unclear.

The aim of the present study was to clarify the effect(s) of IL-10 deficiency on the establishment and maintenance of CD4 and CD8 T cell memory. By using recombinant L. monocytogenes-expressing OVA (rLM-OVA),2 we were able to analyze the magnitude and quality of primary and secondary CD4 and CD8 T cell responses using well-defined CD4 and CD8 T cell epitopes within the same mouse strain. Unexpectedly, we found that IL-10 deficiency reduced the number and protective capacity of memory CD8 T cells in both lymphoid and nonlymphoid organs. These data show that IL-10 can have role in promoting and/or sustaining CD8 T cell memory.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6J and homozygous IL-10-deficient B6 (IL-10–/–) female mice (26) were purchased from The Jackson Laboratory, maintained in the Vaccine Research Center Animal Care Facility (Bethesda, MD) under pathogen-free conditions, and used at 6–8 wk of age. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee.

Bacteria

rLM-OVA was a generous gift from Dr. H. Shen (University of Pennsylvania, School of Medicine, Philadelphia, PA). In brief, rLM-OVA was derived from the wild-type strain 10403S and contains a chromosomally integrated Ag cassette encoding truncated OVA (aa 134–387). The OVA sequence was fused to a virulence gene (hly) promoter and signal sequence that control the expression and secretion of OVA (27, 28). The strain was maintained as a –80°C stock in brain-heart infusion (BHI)/50% glycerol. Before each experiment, rLM-OVA was streaked onto BHI agar. A single colony was inoculated into BHI, and the culture was grown overnight at 37°C with aeration.

Infection of mice and determination of bacterial load

Overnight cultures were serially diluted in PBS to the desired dose and injected into the lateral tail vein of mice. Inocula were plated to verify dose. Mice were infected with 5 x 104 CFU (0.1 LD50) for primary infections and 5 x 105 CFU (LD50) for secondary infections unless otherwise noted. In some experiments, ampicillin (Sigma-Aldrich) was given to mice at 2 mg/ml in their drinking water 24 h after primary infection. Bacterial loads were determined by plating 10-fold serial dilutions of spleen and liver homogenates in sterile 1% Triton X-100/PBS on BHI agar.

Intracellular staining and flow cytometric analysis

Spleens from C57BL/6 and IL-10–/– mice were aseptically removed and passed through a nylon mesh screen. RBC were lysed with ACK lysing buffer (Biosource). Splenocytes were resuspended in RPMI 1640 complete medium (10% FCS, 4 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin) and incubated with 2 µg/ml anti-CD28 and 1 µg/ml GolgiStop (BD Pharmingen) with or without 1 µM synthetic peptides. After a 5-h incubation at 37°C with 5% CO2, cells were stained using the Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s instructions. The CD4 T cell epitope from listeriolysin O (LLO)190–201 (NEKYAQAYPNVS), and the CD8 T cell epitope from OVA, OVA257–264 (SIINFEKL), were synthesized by Biosynthesis. All mAb (anti-CD4-PerCP, anti-CD8-PE, anti-IFN-{gamma}-allophycocyanin, anti-CD210-PE) were purchased from BD Pharmingen. Splenocytes were analyzed with a FACSCalibur (BD Biosciences), and data were analyzed using FlowJo (Tree Star).

Adoptive transfer

Splenocytes from L. monocytogenes-infected C57BL/6 and IL-10–/– mice were prepared as described above. Forty to 100 days postinfection, CD4 and CD8 T cells were purified from splenocytes using anti-CD4 and anti-CD8 magnetic beads, respectively, according to the manufacturer’s directions (Miltenyi Biotec) and resuspended in sterile PBS. A total of ~1 x 107 CD4 or CD8 T cells per mouse was injected i.v. into C57BL/6 female mice. The frequency of Ag-specific CD8 memory T cells was 2- to 3-fold higher in wild-type than in IL-10–/– mice. Eighteen hours later, mice were injected with 1 x 105 CFU rLM-OVA. Mice were sacrificed at 48 h postinfection, and bacterial loads were determined as described above.

Statistical analysis

Student’s t tests (two-tailed, 95% confidence intervals) were performed using Prism software (GraphPad). All data from bacteria numbers were log transformed before statistical tests were conducted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Bacterial loads are reduced in IL-10–/– mice during primary L. monocytogenes infection

As the aim of these studies was to assess the role of IL-10 on effector and memory T cell responses, we first determined the effect of IL-10 deficiency on the course of primary L. monocytogenes infection. IL-10–/– and C57BL/6 mice were infected with 5 x 104 CFU rLM-OVA and bacterial loads in the spleens and livers of infected mice were determined 3 and 8 days later. On day 3 postinfection, IL-10–/– mice had ~2 log10 less bacteria in their spleens and ~4 log10 less bacteria in their livers than C57BL/6 mice (Fig. 1A). This early reduction in bacterial loads is likely due to the enhanced innate immune response in the absence of IL-10 (29, 30, 31, 32, 33, 34, 35, 36, 37). By day 8 postinfection, both strains of mice had cleared the bacteria from their spleens; however, the C57BL/6 mice still had ~2.5 x 103 CFU of bacteria in their livers (Fig. 1B). These mice did subsequently clear all of the bacteria in their livers (data not shown). Together, these data indicate that the absence of IL-10 during primary L. monocytogenes infection leads to lower bacterial titers in the spleens and livers of infected mice and a shortened course of infection.


Figure 1
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  		FIGURE 1. Assessment of bacterial loads in wild-type and IL-10–/– mice following L. monocytogenes infection. Bacterial loads in the spleens and livers of IL-10–/– and C57BL/6 mice were enumerated 3 (A) and 8 (B) days postinfection with 5 x 104 CFU rLM-OVA. Solid lines represent the geometric mean. The limit of detection is depicted as a dotted line. Data are from one of two independent experiments with similar results.

 
IL-10–/– mice have decreased numbers of effector T cells following L. monocytogenes infection

Since IL-10 has been shown to inhibit the activation and effector function of T cells (38, 39, 40, 41, 42, 43, 44), we hypothesized that more effector T cells would be generated in the absence of IL-10. Thus, the frequencies of LLO190–201-specific CD4 and OVA257–264-specific CD8 T cells in the spleens and livers of infected IL-10–/– and C57BL/6 mice were assessed 8 days postinfection, at the peak of the effector T cell response, by intracellular staining for IFN-{gamma}. Remarkably, the frequencies of LLO-specific IFN-{gamma}+ CD4 T cells in the spleens and livers of IL-10–/– mice were decreased nearly 2-fold, compared with C57BL/6 mice (Fig. 2A). In contrast, the frequencies of OVA-specific IFN-{gamma}+ CD8 T cells were similar in the spleens of both strains of mice, and ~2-fold higher in the livers of IL-10–/– mice (Fig. 2B). Similar results were obtained in a separate experiment using tetramer staining to identify the OVA-specific CD8 T cells (data not shown). We also compared the activation states of T cells between the two strains of mice at this time point using a panel of activation and memory markers. Ag-specific T cells from both strains of mice had similar levels of the IL-2 receptor {alpha}-chain (CD25), whereas CD8 T cells from C57BL/6 mice had a modest enhancement in the expression of the IL-2/IL-15 receptor beta-chain (CD122), compared with CD8 T cells from IL-10–/– mice (data not shown). There were no marked differences in the levels of CCR7, CD62L, or the IL-7 receptor {alpha}-chain on Ag-specific cells between the two strains of mice for either T cell subset (data not shown).


Figure 2
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  		FIGURE 2. Primary CD4 and CD8 T cell responses to L. monocytogenes in IL-10–/– and C57BL/6 mice. IL-10–/– and C57BL/6 mice were infected with 5 x 104 CFU rLM-OVA, and lymphocytes were prepared from the spleens and livers of infected mice 8 days postinfection. Frequencies of LLO190–201-specific CD4 T cells (A) as a percentage of total CD4 T cells and OVA257–264-specfic CD8 T cells (B) as a percentage of total CD8 T cells were determined by intracellular staining for IFN-{gamma}. Absolute numbers of LLO190–201-specific CD4 T cells (C) and OVA257–264-specfic CD8 T cells (D) were calculated based on the frequency of IFN-{gamma}+ T cells, the total number of CD4 or CD8 T cells, and the total number of splenocytes or liver lymphocytes recovered from each mouse. Bars represent the mean + SEM of four mice from one of two independent experiments with similar results.

 
The absolute numbers of Ag-specific IFN-{gamma}+ CD4 and CD8 T cells also were determined on day 8 postinfection, because IL-10–/– mice had substantially smaller spleens and fewer liver lymphocytes than C57BL/6 mice. Since naive IL-10–/– and C57BL/6 mice have similar numbers of splenocytes and liver lymphocytes, this difference in spleen sizes on day 8 postinfection is likely due to the lower bacterial burdens and more rapid clearance of the infection in IL-10–/– mice. As with the frequencies of LLO-specific IFN-{gamma}+ CD4 T cells, the numbers of LLO-specific IFN-{gamma}+ CD4 T cells were lower in the spleens and livers of IL-10–/– mice, compared with C57BL/6 mice (Fig. 2C). For CD8 T cells, the absolute numbers of OVA-specific IFN-{gamma}+ CD8 T cells in the spleens of IL-10–/– and C57BL/6 mice were comparable: however, there were 2-fold less OVA-specific IFN-{gamma}+ CD8 T cells in the livers of IL-10–/–, compared with C57BL/6 mice (Fig. 2D). Thus, while the frequency of OVA-specific IFN-{gamma}+ CD8 T cells was higher in the livers of IL-10–/– than in C57BL/6 mice, there were actually fewer numbers of Ag-specific IFN-{gamma}+ CD8 T cells. This was due to the presence of nearly four times as many total CD8 T cells in the livers of C57BL/6, compared with IL-10–/– mice. The decrease in the number of total CD8 T cells in the liver of IL-10–/– mice may have been due to impaired recruitment of these cells in the absence of IL-10. This is consistent with a previous report that IL-10 induces the recruitment and proliferation of CD8 T cells, but not CD4 T cells (45). In summary, our results show that a smaller number of effector CD4 and CD8 T cells are present in the spleens and livers of IL-10–/– mice than in C57BL/6 mice at the peak of the primary T cell response.

T cell memory to L. monocytogenes is decreased in the absence of IL-10

We next determined the effect of IL-10 deficiency on the generation of T cell memory to L. monocytogenes. In addition, a more thorough characterization of the quality of the responses was done to include the frequency of total IFN-{gamma}+ memory T cells comprised of IFN-{gamma} single-positive and IFN-{gamma}/IL-2 double-positive T cells. Of note, the frequencies of IL-2 single-positive T cells were very low and therefore are not shown. There was a modest decrease in the frequency of total LLO-specific IFN-{gamma}+ memory CD4 T cells in the spleens and a ~2-fold reduction in the livers of IL-10–/– mice in comparison to C57BL/6 mice when assessed 40 days postinfection (Fig. 3, A and C). There was also a 2- to 3-fold decrease in the frequencies of total OVA-specific IFN-{gamma}+ memory CD8 T cells in the spleens and livers of IL-10–/– mice, compared with C57BL/6 mice (Fig. 3, B and D). Similar results were obtained in a separate experiment using tetramer staining to identify the OVA-specific CD8 T cells (data not shown). At 40 days postinfection, the spleen sizes and total cell numbers were comparable between the IL-10–/– and C57BL/6 mice commensurate with the fact that the infection had resolved in both strains. Therefore, the frequencies of IFN-{gamma}+ T cells corresponded to the absolute numbers of IFN-{gamma}+ T cells. Such differences were also evident up to 100 days postinfection and were most notable for CD8 T cells.We also compared the activation states of T cells from the two strains of mice at 40 days postinfection It was notable that Ag-specific CD8 T cells from C57BL/6 mice had higher levels of both CD25 and CD122 expression than those from IL-10–/– mice (data not shown). There were no substantial differences in the levels of CCR7, CD62L, or the IL-7 receptor {alpha}-chain on Ag-specific cells between the two strains of mice for either T cell subset (data not shown). Taken together, these data show that fewer effector (Fig. 2, C and D) and memory CD4 and CD8 T cells are generated in the absence of IL-10.


Figure 3
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  		FIGURE 3. Frequencies of total IFN-{gamma}+, IFN-{gamma}+/IL-2–, and IFN-{gamma}+/IL-2+ memory T cells following primary infection. A and B, Frequencies of LLO190–201-specific CD4 T cells and OVA257–264-specific CD8 T cells, respectively, in the spleens and livers of rLM-OVA infected mice were determined by intracellular staining for IFN-{gamma} and IL-2 40 days postinfection with rLM-OVA. Bars represent the mean + SEM of 4 mice from one of 4 independent experiments with similar results. C and D, Cells were gated on the CD4+ or CD8+ population, respectively. Numbers show the percentage of CD4 or CD8 T cells that are IFN-{gamma}+/IL-2– or IFN-{gamma}+/IL-2+. Flow cytometry profiles are from one representative mouse from groups of four from one of four independent experiments with similar results.

 
Memory T cell responses generated in the absence of IL-10 are decreased in ampicillin-treated mice

The reduced memory T cell responses to L. monocytogenes infection in IL-10–/– mice may have been due to the reduced bacterial loads and shorter duration of infection following primary infection (Fig. 1). To address the role of Ag load and persistence, IL-10–/– and C57BL/6 mice were treated with ampicillin 24 h after infection. At the time of ampicillin treatment, 1 day postinfection, the bacterial loads in the spleens, but not livers, of IL-10–/– and C57BL/6 mice were slightly lower (Fig. 4A). Twenty-four hours after treatment with ampicillin, which was 2 days postinfection, the bacterial loads in the spleens and livers of both strains of mice were comparable (Fig. 4B).


Figure 4
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  		FIGURE 4. IL-10–/– and C57BL/6 mice have comparable bacterial loads in their spleens and livers after treatment with ampicillin. A, Number of bacteria in the spleens and livers of IL-10–/– and C57BL/6 mice 1 day postinfection with 5 x 104 CFU rLM-OVA. B, Bacterial loads in the spleens and livers of IL-10–/– and C57BL/6 mice were enumerated 2 days postinfection with (+amp) or without (-amp) ampicillin treatment 24 h after infection. Solid lines represent the geometric mean. The limit of detection is depicted as a dotted line. Data are from one of three independent experiments with similar results.

 
To assess whether memory T cell responses were similar between IL-10–/– and C57BL/6 mice after ampicillin treatment, the frequencies of IFN-{gamma}+ CD4 and CD8 memory T cells from spleens and livers were assessed 100 days postinfection In general, the frequencies of IFN-{gamma}+ T cells from ampicillin-treated mice were decreased, compared with those from untreated mice (Fig. 5, A–C), except for the frequencies of IFN-{gamma}+ CD8 T cells in the livers of C57BL/6 mice, which were nearly the same (Fig. 5D). In both the spleens and livers of ampicillin-treated mice, the frequencies of CD4 T cells were comparable in IL-10–/– and C57BL/6 mice (Fig. 5, A and B). However, there were substantially fewer IFN-{gamma}+ CD8 T cells in IL-10–/– than C57BL/6 mice in both the spleens and livers of ampicillin-treated mice (Fig. 5, C and D). Thus, even when the number of bacteria and duration of infection were comparable after 1 day of infection, the number of memory CD8 T cells generated in the absence of IL-10 was lower than in the presence of IL-10.


Figure 5
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  		FIGURE 5. IL-10–/– mice treated with ampicillin have decreased memory T cell responses. IL-10–/– and C57BL/6 mice were infected with 5 x 104 CFU rLM-OVA; 24 h later, a group of mice was treated with ampicillin (+amp), and another group was left untreated (–amp). Frequencies of LLO190–201-specific CD4 T cells (A and B) and OVA257–264-specfic CD8 T cells (C and D) in the spleens and livers were determined by intracellular staining for IFN-{gamma} and IL-2 100 days postinfection Bars represent the mean + SEM of four mice from one of three independent experiments with similar results.

 
IL-10–/– mice have increased bacterial loads upon secondary infection

To determine whether the lower magnitude of CD4 and CD8 memory T cells in IL-10–/– mice corresponded to reduced protection following secondary challenge, infected IL-10–/– and C57BL/6 mice (~40 days postinfection) were rechallenged with a lethal dose of rLM-OVA (5 x 105 CFU), and the bacterial loads were determined 1 day postreinfection. As shown in Fig. 6A, both IL-10–/– and C57BL/6 mice had ~2- to 3-log reduction in bacterial load in their spleens, compared with naive C57BL/6 mice. However, previously infected IL-10–/– mice had more bacteria in their spleens than did C57BL/6 mice on day 1 postinfection (Fig. 6). Thus, since immunity to L. monoyctogenes reinfection is mediated almost exclusively by T cells (22, 46), these data suggest that the decreased numbers of memory T cells generated in the absence of IL-10 provide less protection upon reinfection.


Figure 6
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  		FIGURE 6. IL-10–/– mice have diminished protection upon secondary challenge with L. monocytogenes. A, Mice that were infected with 5 x 104 CFU rLM-OVA were challenged 40 days later with 5 x 105 CFU rLM-OVA. Bacterial loads in the spleens and livers of IL-10–/– and C57BL/6 mice were determined 1 day postinfection I, immune; N, naive. B, Same as in A except that a group of mice was treated with ampicillin (+amp) 24 h after primary infection and another group was left untreated (–amp). Solid lines represent the geometric mean. The limit of detection is depicted as a dotted line. Data are from two to three independent experiments with similar results.

 
We next assessed whether ampicillin treatment during primary infection altered protective immunity following secondary challenge. Consistent with the decreased frequencies of memory T cells in ampicillin-treated IL-10–/– mice (Fig. 5), there was ~4 log10 more bacteria in the spleens of such mice 1 day after reinfection (+amp, Fig. 6B). Thus, memory T cells generated in the absence of IL-10 were lower in frequency and conferred less protection in the spleen, compared with wild-type mice, even when the bacterial loads and duration of infection were comparable 24 h after primary infection. Finally, it is notable that there was no detectable difference in bacterial loads in the livers of IL-10–/– and C57BL/6 ampicillin-treated mice despite the differences in the frequency of epitope-specific CD8 T cells. Since CD4 T cells play a more significant role in protection in the liver than the spleen (47) and CD4 and CD8 T cells are believed to act synergistically to clear Listeria (48), the frequency of memory CD4 T cells in the ampicillin-treated mice may have been too low to help confer protection at day 1 postinfection

IL-10–/– mice have decreased T cell responses following secondary infection

To determine whether IL-10 deficiency has an effect on the magnitude and quality of responding T cells upon secondary infection, the frequency and cytokine profile of responding CD4 and CD8 T cells was assessed 7 days postreinfection. The frequency of total IFN-{gamma} producing LLO-specific CD4 T cells in the spleens of IL-10–/– and C57BL/6 mice were comparable at the peak of the secondary response (Fig. 7, A and C). However, there were nearly 2-fold less IFN-{gamma}+/IL-2+ LLO-specific CD4 T cells in the spleens of IL-10–/– than C57BL/6 mice. Moreover, there was an ~5-fold decrease in the frequency of IFN-{gamma}+/IL-2– CD4 T cells and an ~10-fold reduction in the frequency of IFN-{gamma}+/IL-2+ CD4 T cells in the livers of IL-10–/–, compared with C57BL/6 mice. As spleen sizes and lymphocyte numbers were similar in the IL-10–/– and C57BL/6 mice 7 days postreinfection, the frequencies of IFN-{gamma}+ T cells corresponded directly to the absolute numbers of IFN-{gamma}+ T cells.


Figure 7
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  		FIGURE 7. T cell responses following secondary infection are decreased in IL-10–/– mice. Mice that were infected with 5 x 104 CFU rLM-OVA were challenged 40 days later with 5 x 105 CFU rLM-OVA. A and B, Frequencies of LLO190–201-specific CD4 T cells and OVA257–264-specfic CD8 T cells, respectively, in the spleens and livers of rLM-OVA-infected mice were determined by intracellular staining for IFN-{gamma} and IL-2 seven days after secondary infection. Bars represent the mean + SEM of four mice from one of two independent experiments with similar results. C and D, Cells were gated on the CD4+ or CD8+ population, respectively. Numbers show the percentage of CD4 or CD8 T cells that are IFN-{gamma}+/IL-2– or IFN-{gamma}+/IL-2+. Flow cytometry profiles are from one representative mouse from groups of four from one of two independent experiments with similar results.

 
The frequencies of total IFN-{gamma} and IFN-{gamma}+/IL-2+ producing OVA-specific CD8 T cells also were lower in the spleens and livers of IL-10–/– than C57BL/6 mice (Fig. 7, B and D). Most notably, there was a 2-fold reduction in the frequency of total IFN-{gamma}+ OVA-specific CD8 T cells and a nearly 4-fold reduction in the frequency of IFN-{gamma}+/IL-2+ OVA-specific CD8 T cells in the livers of IL-10–/–, compared with C57BL/6 mice. Together, these data show that the recall response by memory T cells generated in the absence of IL-10 is smaller upon secondary infection with a lower frequency of IFN-{gamma}+ T cells that also produce IL-2. Finally, it is notable that the dramatic increase in the secondary responses in wild-type mice occurred despite the fact that the bacterial loads were lower in the spleens and livers of these mice 1 day postreinfection (Fig. 6).

CD8 T cells from immune IL-10–/– mice have reduced protective capacity following transfer to wild-type hosts

To directly show that memory T cells primed in the absence or presence of IL-10 could differentially mediate protection, such cells were adoptively transferred 55 days after primary infection into naive C57BL/6 mice and challenged 1 day later with rLM-OVA. Under these experimental conditions, any differences in protection following secondary infection could be attributed to the transferred T cells only since the host mice were the same. CD8 T cells were purified from spleens following primary infection and transferred to naive wild-type mice at a 1:1 donor-to-host ratio. Although the same number of total CD8 T cells from immune wild-type and IL-10–/– were transferred (~1 x 107 cells), there were ~3-fold more OVA-specific CD8 T cells in wild-type than IL-10–/– immune mice (Fig. 3, B and D). Mice that received immune CD8 T cells from wild-type or IL-10–/– mice had fewer bacteria in their spleens and livers than naive mice that received no cells (Fig. 8A). However, mice that received CD8 T cells from IL-10–/– mice had almost 100-fold more bacteria in their spleens and nearly 10-fold more bacteria in their livers than mice that received CD8 T cells from C57BL/6 mice. The enhanced protection conferred by CD8 T cells from immune wild-type mice is consistent with the increased numbers of Ag-specific cells generated following primary infection. Next, to equalize the number of Ag-specific memory CD8 T cells transferred, 1 x 107 CD8 T cells from IL-10–/– mice and 3-fold less cells from wild-type mice were transferred to naive wild-type mice and challenged 1 day later. There was no protection conferred by the transfer of immune CD8 T cells from either strain of mice (data not shown). Thus, the number of transferred cells was below the critical number of cells required to mediate protection. As a result, whether the observed differences in the quality of memory CD8 T cells between the two strains of mice also may have contributed to the difference in protection remains undetermined.


Figure 8
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  		FIGURE 8. Memory T cells from IL-10–/– mice are less protective than memory T cells from C57BL/6 mice following adoptive transfer. IL-10–/– and C57BL/6 mice were infected with 5 x 104 CFU rLM-OVA and at least 40 days later, CD8 (A) or CD4 (B) T cells were purified from previously infected splenocytes and equal numbers of cells were adoptively transferred into naive C57BL/6 mice. Eighteen hours later, mice were injected with 1 x 105 CFU rLM-OVA. Bacterial numbers in the spleens and livers were determined 2 days postinfection Solid lines represent the geometric mean. The limit of detection is depicted as a dotted line. Data are from one of two independent experiments with similar results.

 
Finally, the protective capacity of CD4 T cells primed in the absence or presence of IL-10 was assessed following adoptive transfer into naive C57BL/6 mice. There were ~2-fold more LLO-specific CD4 T cells transferred from wild-type than IL-10–/– immune mice, because these mice had ~2 times as many memory CD4 T cells in their spleens as shown in Fig. 3. Although there was little decrease in the bacterial loads in the spleens of recipient mice that received transferred CD4 T cells, there was a substantial decrease in the livers of these mice in comparison to naive mice (Fig. 8B). However, there was little difference in the protection conferred by transferred CD4 T cells from immune IL-10–/– or C57BL/6 mice, consistent with the modest differences in the frequencies of memory CD4 T cells seen in such mice. Overall, these results are in agreement with a prior report that adoptively transferred CD8 T cells are more efficient than CD4 T cells at transferring protection in the spleen while CD4 T cells are at least as protective as CD8 T cells in the liver (47).

The IL-10 receptor is expressed on CD8 T cells from IL-10–/– and C57BL/6 mice following L. monocytogenes infection

The results above, that IL-10–/– mice had reduced CD8 T cell memory and protection despite having comparable Ag loads after treatment with ampicillin, suggested that that IL-10 might be having a direct effect on T cells. Thus, surface expression of the IL-10 receptor was assessed on Ag-specific CD4 and CD8 T cells from IL-10–/– and C57BL/6 mice at 8 and 42 days postinfection (Fig. 9). Although we found no differences between the two strains of mice, nearly 60% of Ag-specific CD8 T cells in the spleen expressed the IL-10 receptor on day 8 postinfection, compared with 20% of Ag-specific CD4 T cells (Fig. 9, A and C). Similar differences in IL-10 receptor expression between CD4 and CD8 T cells existed in the livers. By day 42, only ~5% of resting Ag-specific memory T cells expressed the IL-10 receptor, with no significant differences between CD4 and CD8 T cells (Fig. 9, B and C). Thus, it appears that the IL-10 receptor is up-regulated on activated CD8 T cells during the primary response, allowing for the possibility that IL-10 has a direct enhancing effect on CD8 T cells during Listeria infection. These data are consistent with a recent report that IL-10 acts directly on CD8 T cells during priming to increase the number of responding CD8 T cells (49).


Figure 9
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  		FIGURE 9. Surface expression of the IL-10 receptor on Ag-specific T cells. IL-10–/– and C57BL/6 mice were infected with 5 x 104 CFU rLM-OVA and lymphocytes were prepared from the spleens and livers of infected mice 8 and 42 days postinfection Ag-specific T cells were identified by intracellular staining for IFN-{gamma} following stimulation with LLO190–201 and OVA257–264. Frequencies of Ag-specific T cells that expressed IL-10r were determined by surface staining 8 days (A and C) and 42 (B and C) days postinfection. In C, numbers are the percentage of CD4 or CD8 T cells express IL-10r. Flow cytometry profiles are from one representative mouse from groups of four.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, the effects of IL-10 deficiency on primary and secondary CD4 and CD8 T cell responses in both lymphoid and nonlymphoid organs following L. monocytogenes infection were characterized. Unexpectedly, the absence of IL-10 actually reduced the frequency of epitope-specific memory T cells in IL-10-deficient mice, compared with wild-type mice. This was particularly true for CD8 T cells. Increased bacterial loads in IL-10 deficient mice, compared with wild-type mice, following secondary infection indicated that the decrease in memory T cell responses was functionally important.

Our findings are only partially in agreement with a previously published report that described increased innate and acquired immunity to L. monocytogenes infection in IL-10 deficient mice (2). The studies reported in this study used IL-10–/– mice that had been back-crossed >10 generations, a higher dose of Listeria, and characterization of epitope-specific CD8 T cell responses by FACS analysis, rather than by culture and assessment of responses by ELISA. We also compared protection 1 day postinfection when there were still bacteria in spleens rather than 2 days postinfection when there were bacteria only in livers. Notably, we only observed statistically significant differences in protection in the spleens, and not the livers, of these mice.

The beneficial effect of IL-10 on CD8 T cell memory may occur either by direct and/or indirect mechanisms. Consistent with a direct role, IL-10 has been shown to increase the proliferation (49, 53, 54, 55, 56, 57), recruitment (45), and cytotoxicity (58, 59) of CD8 T cells in vitro. More recently, the presence of IL-10 was shown to increase CD8 T cell numbers following priming with peptide-pulsed IL-10R2–/– dendritic cells in vitro, demonstrating a direct enhancing effect of IL-10 on CD8 T cells (49). We also showed in this study that the IL-10 receptor was up-regulated on activated Ag-specific CD8 T cells. Thus, it is possible that the increased CD8 T cell response in the presence of IL-10 seen in vivo in our study may be the result of IL-10 acting directly on CD8 T cells to alter their proliferation and differentiation. Experiments with IL-10R–/– mice are currently under way to determine whether this is the case.

The possibility also exists that IL-10 can influence CD8 T cell memory indirectly by altering the amount and duration of bacterial burden during primary infection. Although the amount and duration of infection have been shown previously to have little effect on the magnitude of the CD8 T cell effector response following L. monocytogenes infection (60), a recent report demonstrated that if the duration of infection is limited to 24–48 h, the ensuing memory population is significantly diminished (61). The reduced numbers of memory T cells generated following a short duration of infection could be increased if the dose of bacteria was increased 10-fold. In the study reported here, when we attempted to control for differences in bacterial load and duration between IL-10 deficient and wild-type mice during primary infection by treatment with ampicillin, we still observed a reduction in the number and protective capacity of memory CD8 T cells in IL-10 deficient mice. These data suggest that the differences in Ag load may not be the cause of the differences in CD8 T cell memory between the two strains of mice. However, it is possible that the slightly larger Ag load (~0.5 log) seen within the first day of infection in the spleens of wild-type, compared with IL-10–/– mice, before ampicillin treatment may explain the increase in T cell memory in such mice. Even if this was the case, it would suggest that IL-10, by inhibiting the innate immune response, increases the amount of bacteria and duration of infection, leading to increased numbers of memory CD8 T cells.

A final consideration for the mechanism by which IL-10 promotes the formation or maintenance of CD8 T cell memory relates to the decreased production of proinflammatory cytokines such as IL-12 and TNF-{alpha} in the presence of IL-10 following infection. Although IL-12 has been shown to have a role in enhancing CD8 T cell effector responses, it remains possible that it, or other proinflammatory cytokines that are down-regulated by IL-10, may have a deleterious effect on memory, at least in this model. Thus, in this case, IL-10 would enhance CD8 T cell memory by decreasing the production of proinflammatory cytokines that have a negative effect on the generation of memory CD8 T cells.

The finding that IL-10 may actually be important for sustaining memory and protection has been previously shown in the mouse model of L. major infection (12, 18). However, the roles of IL-10 in promoting T cell memory in the L. major and L. monocytogenes models of infection are very different. L. major is a chronic infection taking up to 3 mo to resolve and requires Th1 cells to mediate protection. Treatment of L. major-infected mice with Ab to the IL-10 receptor 6 mo after primary infection results in sterilizing immunity and loss of protection against rechallenge. Thus, in this model, IL-10 mediates its effects indirectly by preventing the elimination of Ag that is required to sustain CD4 effector/memory T cells. By contrast, L. monocytogenes infection is cleared much faster, within 7–10 days after infection, and CD8 T cells are more critical for protection. The data presented in this study suggest that innate production of IL-10 early during the course of infection (20) promotes the generation of increased numbers of memory CD8 T cells. Combined, these studies imply that IL-10 can play a positive role in regulating both the maintenance of CD4 effector/memory T cells for an infection that persists and the induction of CD8 effector/memory T cells for an acute infection.

In conclusion, we have directly demonstrated that IL-10 has a beneficial effect on the generation of CD8 T cell memory in response to infection with a pathogen. These data, in conjunction with the role that IL-10 has in sustaining CD4 T cell-mediated immunity against a persistent infection such as L. major, underscore the complicated nature of IL-10 as an immunoregulatory cytokine. Perhaps the presence of IL-10 is beneficial for T cell memory following vaccination with a live vector such as Listeria because it could help to prolong the duration of Ag presentation, recruit CD8 T cells to the sites of infection, and act directly on CD8 T cells to enhance their proliferation. The possibility exists that IL-10 may have different effects with other live or nonlive vectors. For example, IL-10 could have a beneficial role in the generation and maintenance of T cell memory following vaccination with a live vector and an inhibitory role with a nonlive vector. Since treatment with anti-IL-10 or anti-IL-10 receptor Abs has been proposed as a method to increase T cell responses to vaccines, careful analysis of the effects of such treatments on the generation of memory cells will need to be performed.


   Acknowledgments
 
We thank members of the Seder laboratory (Bethesda, MD) for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 Address correspondence and reprint requests to Dr. Robert A. Seder, Cellular Immunology Section, Vaccine Research Center, National Institutes of Health, 40 Convent Drive, Room 3512, Bethesda, MD 20892. E-mail address: rseder{at}mail.nih.gov Back

2 Abbreviations used in this paper used: rLM-OVA, recombinant Listeria monocytogenes-expressing OVA; BHI, brain-heart infusion; LLO, listeriolysin O. Back

Received for publication January 3, 2006. Accepted for publication May 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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J. Immunol., October 1, 2007; 179(7): 4520 - 4528.
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J.-B. Lee, K.-A Lee, and J. Chang
Phenotypic changes induced by IL-12 priming regulate effector and memory CD8 T cell differentiation
Int. Immunol., September 6, 2007; (2007) dxm072v1.
[Abstract] [Full Text] [PDF]

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