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The Journal of Immunology, 2008, 180, 825 -832
Copyright © 2008 by The American Association of Immunologists, Inc.
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Essential Role of CD8+CD122+ Regulatory T Cells in the Recovery from Experimental Autoimmune Encephalomyelitis1

Young-Ho Lee*, Yoshiyuki Ishida{dagger}, Muhaimin Rifa’i*,{ddagger},§, Zhe Shi*, Ken-ichi Isobe* and Haruhiko Suzuki2,*

* Department of Immunology, Nagoya University Graduate School of Medicine, {dagger} Radioisotope Research Center, Medical Branch of Nagoya University, Nagoya, and {ddagger} Japan Society for the Promotion of Science, Tokyo, Japan; and § Brawijaya University, East-Java, Indonesia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Experimental autoimmune encephalomyelitis (EAE) is one of the best-documented animal models of autoimmune disease. We examined the role of CD8+CD122+ regulatory T cells, which we previously identified as naturally occurring regulatory T cells that effectively regulate CD8+ T cells, in EAE. Depletion of CD8+CD122+ regulatory T cells by in vivo administration of anti-CD122 mAb resulted in persistent EAE symptoms. Transfer of CD8+CD122+ regulatory T cells into EAE mice at the peak EAE score clearly improved symptoms, indicating an important role of CD8+CD122+ regulatory T cells in the recovery phase of EAE. This was further confirmed by an increase and a decrease in the number of infiltrating T cells in the CNS and T cell cytokine production in mice that were depleted of or complemented with CD8+CD122+ cells. Furthermore, transfer of preactivated CD8+CD122+ regulatory T cells resulted in diminished EAE symptoms, especially in the recovery phase of EAE. These results elucidate the essential role of CD8+CD122+ regulatory T cells in the recovery phase of EAE and suggest the preventive effect of preactivated CD8+CD122+ regulatory T cells for EAE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune system has evolutionally developed in mammals as a self-defense mechanism that fights against microorganisms invading the body. This system is necessary for the maintenance of life but at the same time, self-attack of the immunity is sometimes harmful. Autoimmune diseases are typical examples of bad effects caused by immune attacks. Therefore, the immune system has developed certain mechanisms to prevent such attacks, including special regulatory T cells that suppress immune reactions. Although the regulatory cells that potentially suppress immune reactions consist of many different types of cells, there is no doubt that a subset of T cells has regulatory activity.

Experimental autoimmune encephalomyelitis (EAE)3is an inflammatory demyelinating disease of the CNS that is primarily mediated by CD4+ Th1 cells specific for autoantigens in the CNS (1). Numerous studies have demonstrated the role of CD4+ T cells in EAE, including the observation that CD4+ T cells are critical for the induction of EAE (2). In contrast to conventional CD4+ T cells that commonly work as effector cells of immune pathways, CD4+CD25+ regulatory T cells play a critical role in the regulation of autoimmune diseases including EAE (3). Compared with CD4+ T cells, the role of CD8+ T cells in EAE is less clear. Early research into the roles of CD8+ T cells in EAE suggested a regulatory/suppressor function (4, 5). However, more recent studies have reported the ability of myelin Ag-specific CD8+ T cells to function as potent effectors of CNS autoimmunity instead of as suppressors (6, 7).

Although many different types of regulatory T cells have been postulated, the importance of naturally occurring CD4+CD25+ regulatory T cells has exclusively been evaluated. Nevertheless, several types of CD8+ regulatory T cells have been reported (8, 9). We found that a subset of CD8+ cells had very important regulatory activity in immune homeostasis and that the CD8+CD122+ subset contains naturally occurring regulatory T cells. These CD8+CD122+ regulatory T cells directly control CD8+ cells and have regulatory effects in vitro. CD8+CD122+ regulatory T cells produce IL-10 and suppress IFN-{gamma} production and proliferation of CD8+ T cells (10, 11).

In this study, we tried to elucidate the role of CD8+CD122+ regulatory T cells in EAE, a model of autoimmune disease in which CD8+ T cells are involved in the development of the disease. We demonstrate that the depletion of CD122+ cells increases the duration of EAE symptoms in mice and transfer of CD8+CD122+ regulatory T cells into EAE-suffering mice dramatically diminishes EAE symptoms, indicating that CD8+CD122+ regulatory T cells play an essential role in the recovery from EAE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, Ag, and Ab

Female C57BL/6 mice (6–8 wk old) were purchased from SLC. RAG-2–/– mice were originally obtained from the Central Institute for Experimental Animals (Kanagawa, Japan) and were maintained in our animal facility. enhanced GFP (EGFP)-transgenic mice originally derived from The Jackson Laboratory were maintained in our animal facility. The myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (MEVGWYRSPFSRVVHLY RNGK) was synthesized by Sigma Genosys. Anti-CD122 mAb (TM-β1) was provided by Dr. H. Shiku (Mie University, Tsu, Japan). FITC-conjugated anti-mouse CD8 (53-6.7), FITC-conjugated anti-mouse CD25 (PC61.5), PE-conjugated anti-mouse CD4 (GK1.5), PE-conjugated anti-mouse CD44 (IM7), PE-conjugated anti-mouse CD62L (MEL-14), biotin-conjugated anti-mouse CD122 (5H4), biotin-conjugated anti-mouse IFN-{gamma} (XMG1.2), biotin-conjugated anti-mouse IL-10 (JES5-2A5), biotin-conjugated anti-mouse NK1.1 (PK136), and FITC- or biotin-conjugated anti-mouse CD45.1 (A20) Abs and the streptavidin-PE-Cy5 conjugate were purchased from eBioscience.

In vivo depletion

Depletion of CD122+ cells was achieved either by i.v. injection of 250 µg of anti-CD122 mAb (TM-β1) on days –4, 10, and 24 (MOG immunization: day 0) or by injection of 25 µg of anti-CD122 mAb on day –4. In the initial experiment to simply deplete CD122+ cells (see Fig. 2), three injections of 250 µg of mAb were performed; however, for the following experiment to transfer CD8+CD122+ cells into CD122+ cell-depleted mice, 25 µg of anti-CD122 mAb was only injected on day –4. For depletion of NK1.1+ cells, 250 µg of anti-NK1.1 mAb (PK136) was injected on days –4, 10, and 24.

EAE induction

EAE was induced by immunizing female C57BL/6 mice with 200 µg of MOG35–55 peptide emulsified in CFA containing 4 mg/ml heat-killed Mycobacterium tuberculosis H37RA (Difco) on day 0 intradermally in the tail base. Mice also received 200 ng of pertussis toxin (List Biological Laboratories) i.p. on days 0 and 2. Disease severity was monitored according to the following scale: 0, no overt signs of disease; 1, limp tail or hind limb weakness but not both; 2, limp tail and hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; and 5, death by EAE. To determine the effect of anti-CD122 mAb treatment on the development of EAE, 6- to 8-wk-old female C57BL/6 mice were treated with anti-CD122 mAb (TM-β1) and anti-NK1.1 mAb (PK136) on days –4, 10, and 24 of immunization with MOG35–55.

Cell culture and cytokine detection

A single-cell suspension of splenic tissue taken from simple EAE mice on day 23 were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C with 5% CO2. Splenocytes were cultured with MOG35–55 (30 µg/ml) at a density of 6 x 106/ml for 72 h. For nonspecific stimulation, the single-cell suspension was cultured on an anti-CD3-coated plate (BD BioCoat; BD Biosciences) or cultured with anti-CD3/CD28-coated microbeads (T cell expander; Dynal) with 50 ng/ml rIL-2 in a density of 2 x 106/ml (2 x 105 cells/well) for 24 h. Supernatants from cell cultures were taken and ELISA was performed according to the manufacturer’s instructions (R&D Systems). For intracellular cytokine detection, cells were analyzed according to the manufacturer’s protocol for Cytofix/Cytoperm (BD Biosciences).

Isolation of CD8+ or CD8+CD122+ T cells

Single-cell suspensions of splenic and lymph node tissue were prepared and CD8+ T cells were positively selected with MACS magnetic beads coated with anti-CD8a Ab and a LS magnetic bead column (Miltenyi Biotec). To isolate CD8+CD122+ or CD8+CD122– T cells, purified CD8+ T cell populations were incubated with anti-CD8-FITC (clone 53-6.7) and anti-CD122-biotin Ab (clone 5H4) and then stained with streptavidin-PE Cy5. CD8+CD122+ and CD8+CD122– T cells were sorted out with a FACSVantage cell sorter (BD Biosciences).

Histology

Spinal cords for histological analysis were obtained from mice 23 days after immunization and frozen blocks were made in 4% carboxyl methyl cellulose. They were stored at –80°C until cryosectioning. Specimens of 5-µm thickness were prepared with a Leica CW3050-4 cryostat. Tissue sections were stained either by conventional H&E or by FITC-conjugated anti-CD4, PE-conjugated anti-CD8, and FITC-conjugated anti-GFP Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Depletion of CD122+ cells by in vivo administration of anti-CD122 mAb

We first tried to deplete CD122+ cells in mice and to induce EAE in mice. Mice were sacrificed and analyzed on day 4 after administration of anti-CD122 mAb (TM-β1). The percentage of CD122+ cells in the CD8+ population significantly decreased in mice treated with anti-CD122 mAb (Fig. 1A). At the same time, CD8–CD122+ cells were also depleted. The majority of these CD8–CD122+ cells were found to be NK cells by another analysis (data not shown). Therefore, we set another control group of mice treated with anti-NK1.1 mAb (PK136) to deplete NK1.1+ NK cells (Fig. 1B) and leave CD8+CD122+ T cells intact (Fig. 1A, right panel). In contrast to the clear depletion of CD8+CD122+ cells, CD4+CD25+ cells were not affected by the anti-CD122 mAb treatment (Fig. 1C).


Figure 1
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  		FIGURE 1. CD8+CD122+ cells are depleted by in vivo administration of anti-CD122 mAb but not anti-NK1.1 mAb. A, C57BL/6 mice were sacrificed on day 4 after i.v. injection of 250 µg of anti-CD122 mAb or anti-NK1.1 mAb. Splenic cells were analyzed for the expression of CD8 and CD122. Percentages of CD8+CD122+ cells in CD8+ cells are shown in each panel. Note that both CD8+CD122+ cells and CD8–CD122+ cells decreased in anti-CD122-treated mice. Treatment with anti-NK1.1 mAb resulted in a decrease of CD8–CD122+ cells but not of CD8+CD122+ cells, suggesting that the majority of CD8–CD122+ cells were NK cells. B, Splenic cells were analyzed for the expression of CD8 and NK1.1. Percentages of CD8–NK1.1+ cells in total CD8– cells are shown in each panel. C, Splenic cells were analyzed for the expression of CD4 and CD25. Percentages of CD4+CD25+ cells in CD4+ cells are shown in each panel. A–C, Representative results of at least three experiments are shown. D, CD8+CD122+ cells obtained from C57BL/6CD45.1 mice were transferred into RAG-2–/– mice of C57BL/6 genetic background. Seven days later, mice were either treated or not treated with anti-CD122 mAb, and then splenic cells were analyzed on day 4. Transferred cells (CD45.1+) were observed in untreated mice but almost disappeared in anti-CD122-treated mice.

 
It has been reported that CD4+CD25+ regulatory T cells are not really depleted but CD25 molecules on the cell surface are down-regulated when mice are treated with anti-CD25 mAb in vivo (12). To exclude the possibility of down-regulation of CD122 molecules, we purified CD8+CD122+ cells from C57BL/6CD45.1 mice by cell sorting and transferred them into RAG-2–/– mice. On day 7 after cell transfer, mice were inoculated with anti-CD122 mAb, and splenic cells were analyzed 4 days after the mAb treatment. CD45.1+ cells mostly disappeared in anti-CD122-treated mice (Fig. 1D), indicating that disappearance of CD122+ cells in anti-CD122-treated mice reflected the actual depletion of CD122+ cells not down-regulation of the CD122 molecule.

Depletion of CD122+ cells increases the duration of EAE symptoms induced by MOG peptide

Anti-CD122 or anti-NK1.1 mAb (250 µg) was i.v. injected into a C57BL/6 mouse on days –4, 10, and 24 of immunization with MOG peptide. EAE was induced as described in Materials and Methods. Mice were monitored for signs of EAE until 40 days after immunization. Mice that did not receive the mAb (simple EAE mice) showed the typical course of EAE symptoms that appeared ~10–11 days after MOG immunization, reached their peak around days 15 and 16, and then gradually declined. In clear contrast, anti-CD122-treated EAE mice showed continuing EAE symptoms with high clinical scores from day 16 until day 40, although the day of onset and maximum score were not different between anti-CD122-treated EAE and simple EAE mice (Fig. 2A). In the case of NK1.1-depleted EAE mice, EAE symptoms did not differ from those of simple EAE mice (Fig. 2A).


Figure 2
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  		FIGURE 2. Mice depleted of CD122+ cells show prolonged symptoms of EAE. A, C57BL/6 mice were repeatedly treated with mAb, and EAE was induced by immunizing mice with MOG33–55 peptide. The EAE score was monitored by their neurological symptoms. Data were obtained from eight mice in each group, and their mean values and their SDs as error bars are shown. The difference between anti-CD122-treated mice and untreated mice was statistically significant through the clinical course after peak symptoms occurred (day 16 after immunization with MOG). B, On day 29 after immunization with MOG33–55 peptide, splenic cells were analyzed for their population of CD8+CD122+ cells. Data are representative of three mice in each group. Percentages of CD8+CD122+ cells among the total CD8+ cell population are shown in each panel.

 
Flow cytometric analysis of splenic cells obtained from mice on day 29 after immunization revealed that anti-CD122-treated EAE mice showed a decreased number of CD8+CD122+ cells compared with simple EAE mice but anti-NK1.1-treated EAE mice had a similar number of CD8+CD122+ cells to simple EAE mice (Fig. 2B).

Transfer of CD8+CD122+ regulatory T cells obtained from naive C57BL/6 mice effectively suppresses EAE symptoms

Next, we tried to examine the direct effect of CD8+CD122+ regulatory T cells on EAE by transferring CD8+CD122+ cells into mice with EAE. However, when we transferred CD8+CD122+ cells from naive C57BL/6 mice into EAE mice that had received 250 µg of anti-CD122 mAb, the transferred cells quickly disappeared because of the remaining anti-CD122 mAb (data not shown). To decide the appropriate amount of anti-CD122 mAb for the CD8+CD122+ cell transfer at 21 days after the mAb injection, we injected various amounts of anti-CD122 mAb and counted the number of CD8+CD122+ cells (Fig. 3A). Four days after the mAb injection, we analyzed the splenic cells by flow cytometry and found that 25 µg of anti-CD122 mAb was enough to deplete the CD8+CD122+ population. With this amount, the percentage of CD25+ cells in the CD4+ population was unchanged (Fig. 3A). Next, 10, 25, or 50 µg of anti-CD122 mAb was injected into C57BL/6 mice and the recovery of CD122+ cells was evaluated. By 25 µg of anti-CD122 mAb administration, the percentage of CD8+CD122+ cells in the CD8+ population recovered to approximately one-half of the initial percentage on 21 days after the mAb treatment (Fig. 3B). From the results of these experiments, 25 µg of anti-CD122 mAb per mouse was used for experiments involving transfer of CD8+CD122+ cells.


Figure 3
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  		FIGURE 3. Recovery of CD8+CD122+ cells depends on the dose of injected anti-CD122 mAb. A, C57BL/6 mice were treated with i.v. injections of various amounts of anti-CD122 mAb, and splenic cells were analyzed by flow cytometry on day 4 after mAb injection. Percentages of CD122+ cells in the CD8+ population and CD25+ cells in the CD4+ population are shown. Data are mean values obtained from three experiments. B, Ten, 25, or 50 µg of anti-CD122 mAb was injected into C57BL/6 mice, and the recovery of CD8+CD122+ cells was evaluated until 32 days after mAb injection. Percentages of CD122+ cells in CD8+ population are shown as the mean value ± SD obtained from three mice.

 
CD8+CD122+ cells were depleted by inoculating anti-CD122 mAb (25 µg/mouse) 4 days before immunization with 100 µg of MOG35–55 peptide. CD8+CD122+ cells and CD8+CD122– cells were isolated from the spleens and lymph nodes of naive C57BL/6CD45.1 mice by cell sorting and 1 x 106 cells were transferred into the CD122+ cell-depleted mice on day 18. Transfer of CD8+CD122+ cells quickly decreased EAE symptoms, whereas mice that received either CD8+CD122– cells or no cells showed continuing EAE symptoms with a high clinical score (Fig. 4A). CD8+CD122+ cell-transferred EAE mice were sacrificed and splenic cells were analyzed on day 23 (5 days after cell transfer). At this point, most donor cells (CD45.1+) had their original phenotype of CD122+CD62L+CD44+, whereas host cells showed a mostly naive phenotype of CD122–CD62L+CD44– (Fig. 4B).


Figure 4
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  		FIGURE 4. Transfer of CD8+CD122+ cells into CD122+ cell-depleted (dep.) mice significantly reduces EAE symptoms. A, C57BL/6 mice were treated or not treated with 25 µg/mouse of anti-CD122 mAb 4 days before MOG-peptide inoculation, and EAE was induced by immunization with MOG33–55 peptide. On day 18 after MOG immunization, 1 x 106 of either CD8+CD122+ cells or CD8+CD122– cells obtained from C57BL/6CD45.1 mice were transferred into EAE mice. EAE scores are shown as the mean value plus SDs obtained from at least four mice in each group. Statistically significant (p < 0.05) values of mice transferred with CD8+CD122+ cells compared with those without cell transfer are marked with asterisks. B, On day 23 after immunization with MOG peptide, mice that had received CD8+CD122+ cells derived from C57BL/6CD45.1 mice were sacrificed and the status of T cells in spleen was analyzed. Left panels, Expression of CD122, CD62L, and CD44 in host-type (CD45.1–) CD8+ cells; right panels show that in donor-type (CD45.1+) CD8+ cells.

 
CD8+CD122+ regulatory T cells suppress the infiltration of T cells into the CNS

On day 23 after immunization, histological examination including immunohistochemical analysis using anti-CD4 and anti-CD8 Abs, was performed on spinal cord specimens of simple EAE. Mononuclear cells infiltrating into the spinal cord were evident (Fig. 5, A and C), as well as CD4+ and CD8+ cells (Fig. 5, B and D). In the case of anti-CD122-treated mice, more CD4+ and CD8+ T cells were found in the spinal cord (Fig. 5, E and F) than in the spinal cord of simple EAE mice. In contrast, fewer CD4+ T cells were observed in the spinal cord of CD8+CD122+ cell-transferred EAE mice than in anti-CD122-treated mice (Fig. 5, G and H). Transferred CD8+CD122+ cells were mostly localized in the lymphatic organs such as the spleen (Fig. 4B) and lymph nodes (data not shown). Only a few cells were detected inside the CNS (Fig. 5, I and J), suggesting that CD8+CD122+ cells more likely play roles in the peripheral lymphatic organs rather than in the specified local area of the immune reaction.


Figure 5
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  		FIGURE 5. Increased infiltration of CD4+ and CD8+ cells into the CNS of CD122+ cell-depleted EAE mice is improved by CD8+CD122+ cell transfer. C57BL/6 mice were treated or not treated with anti-CD122 mAb and EAE was induced. On day 18 after immunization, CD8+CD122+ cells obtained from normal C57BL/6 mice or from EGFP-transgenic mice were transferred into EAE mice. On day 23 after immunization with MOG35–55 peptide, mice were sacrificed and their spinal cords were harvested from L1 to L2 and analyzed by tissue sections either stained with H&E (A, C, E, and G) or anti-CD4-FITC and anti-CD8-PE (B, D, F, and H). A and B, Spinal cords of C57BL/6 mouse without induction of EAE. C and D, Spinal cord of an EAE-induced C57BL/6 mouse. Infiltrated mononuclear cells are indicated by arrows. E and F, Spinal cord of anti-CD122-treated EAE mouse. Note that infiltrated mononuclear cells are increased. G and H, Spinal cord of a mouse rescued from EAE by inoculation with CD8+CD122+ regulatory T cells. Note that infiltrated mononuclear cells are decreased compared with anti-CD122-treated EAE mice. Scale bars, 100 µm. I and J, Spinal cord of a mouse rescued from EAE by inoculation with CD8+CD122+ cells obtained from EGFP-transgenic mice. Tissue section was stained with anti-GFP-FITC (I) or anti-CD8-PE (J). Note that PE-positive cells (CD8+) are rich but GFP-positive (transferred) cells are rare. Scale bars, 50 µm.

 
IFN-{gamma} production of splenic T cells is increased in CD8+CD122+-depleted EAE mice

To investigate the mechanism of CD8+CD122+ regulatory T cells in suppressing EAE symptom, we took splenic cells obtained from C57BL/6 mice without EAE induction (naive), simple EAE mice, anti-CD122-treated EAE mice, and CD8+CD122+-transferred EAE mice. Splenic cells were cultured under the stimulation of MOG35–55 peptide for 72 h and cytokine levels in culture supernatant were examined by ELISA. Twenty-nine days after MOG immunization, production of IFN-{gamma} was increased in splenic cells of anti-CD122-treated EAE mice compared with that of simple EAE mice (Fig. 6A). IFN-{gamma} production was also high in splenic cells of mice rescued by CD8+CD122+ cell transfer.


Figure 6
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  		FIGURE 6. IFN-{gamma} production is increased in CD122+ cell-depleted mice in the recovery phase from EAE. A, C57BL/6 mice were treated or not treated with 25 µg/mouse of anti-CD122 mAb and immunized with MOG33–55 peptide to induce EAE. CD8+CD122+ cells were transferred into anti-CD122-treated EAE mice on day 18 after MOG immunization. On day 29 after immunization, splenic cells were obtained and were cultured under stimulation with MOG33–55 peptide for 3 days. After the culture, the supernatant was analyzed by ELISA for IFN-{gamma}. Data are shown as mean value and SD obtained from three mice. B, Splenic cells were obtained on day 14 after immunization and separated into CD4+ cells and CD8+ cells. Cells were stimulated by MOG peptide and the production of IFN-{gamma} was measured by ELISA. The p value obtained using Student’s t test is shown. C, Splenic cells obtained on day 14 after immunization were cultured under stimulation with plate-bound anti-CD3 Ab. IFN-{gamma} in culture supernatant was measured by ELISA. The p value obtained using Student’s t test is shown. D, Splenic cells were obtained at days 0, 11, and 14 of EAE induction. Cells were cultured as in C, and IFN-{gamma} production was examined by ELISA. The p value obtained using Student’s t test is shown. E, Cells were prepared as in B or C and production of IL-17 was measured by ELISA.

 
We further proceeded to examine IFN-{gamma} production in EAE-induced mice. When IFN-{gamma} production from purified CD4+ cells was measured on day 14 after MOG immunization, cells obtained from anti-CD122-treated EAE mice produced more IFN-{gamma} than those obtained from simple EAE mice (Fig. 6B). This increased production of IFN-{gamma} was similarly observed in cells nonspecifically stimulated with plate-bound anti-CD3 Ab, instead of MOG peptide (Fig. 6C). Stimulation with anti-CD3 Ab allows for examination of IFN-{gamma} production from CD8+ cells that cannot be stimulated by MOG peptide (Fig. 6, B and C). CD8+ cells obtained from anti-CD122-treated EAE mice also produced more IFN-{gamma} than simple EAE mice (Fig. 6C). This increased production of IFN-{gamma} in cells obtained from anti-CD122-treated EAE mice was evident on day 14 after immunization (Fig. 6D). On day 11 after immunization, however, CD4+ cells obtained from simple EAE mice produced similar levels of IFN-{gamma} to those from anti-CD122-treated EAE mice (Fig. 6D). These results suggest that the level of IFN-{gamma} production may predict EAE symptoms because increased production of IFN-{gamma} continued beyond day 14 after immunization in anti-CD122-treated EAE mice, in which severe EAE symptoms continued (Figs. 2A and 4A).

We also examined IL-17 production in CD4+ cells obtained from simple EAE mice and stimulated them in vitro with plate-bound anti-CD3 mAb or MOG35–65 peptide presented on APCs (Fig. 6E). Production of IL-17 was clearly observed in CD4+ cells suffering from EAE, but was not different between anti-CD122-treated EAE and simple EAE mice, suggesting that the action of IL-17 (Th17) was independent of the regulatory action of CD8+CD122+ T cells.

IL-10 production is decreased in CD8+CD122+-depleted EAE mice and increased in CD8+CD122+-transferred EAE mice

We investigated the production of IL-10 in T cells derived from EAE-induced mice. IL-10 production from splenic cells of anti-CD122 mAb-treated EAE mice was significantly less than that from simple EAE mice (Fig. 7A). In contrast, IL-10 production from splenic cells of CD8+CD122+- transferred EAE mice dramatically increased compared with anti-CD122-treated EAE mice (Fig. 7A). When we examined the IL-10 production from CD4+ cells and CD8+ cells separately, the IL-10 production level from CD4+ cells peaked on day 11 after MOG immunization in simple EAE mice, while there was no such peak of IL-10 production in anti-CD122-treated EAE mice (Fig. 7B). In contrast, IL-10 production from CD8+ cells dramatically increased on day 14 after immunization in simple EAE mice, whereas there was no increase in IL-10 production in anti-CD122-treated EAE mice (Fig. 7C). This time course examination suggests that IL-10 production from T cells works to improve EAE symptoms and increased production of IL-10 from CD8+ cells is better correlated with EAE symptoms than that of CD4+ cells. IL-10 production of CD4+ cells may work in an earlier phase of EAE than that of CD8+ cells.


Figure 7
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  		FIGURE 7. IL-10 production is decreased in CD8+CD122+ cell-depleted EAE mice but is recovered in CD8+CD122+ cell-transferred EAE mice. A, C57BL/6 mice were treated or not treated with 25 µg/mouse of anti-CD122 mAb and immunized with MOG33–55 peptide to induce EAE. CD8+CD122+ cells were transferred into anti-CD122-treated EAE mice on day 18 after MOG immunization. On day 23 after immunization, mice were sacrificed, and total splenic cells were cultured with APC and MOG peptide for 3 days. After the culture, the supernatant was analyzed for IL-10. Data are shown as mean value and SD obtained from three mice. B, At several time points after immunization, mice were sacrificed and CD4+ cells and CD8+ cells were obtained separately. CD4+ cells were cultured under stimulation with plate-bound anti-CD3 mAb for 24 h, and IL-10 production in culture supernatant was measured by ELISA. Data are mean ± SD obtained from three experiments. The p value obtained by Student’s t test is shown. C, Cells were prepared as in B, and CD8+ cells were cultured under stimulation with plate-bound anti-CD3 mAb for 24 h. The level of IL-10 in the culture supernatant was measured by ELISA. The p value obtained by Student’s t test is shown.

 
CD8+CD122+ cells work independently of CD4+CD25+ natural regulatory T cells (nTreg)

We examined whether the suppressive action of CD8+CD122+ cells was mediated by the action of naturally occurring CD4+CD25+ regulatory T cells (nTreg). As is shown in Fig. 8A, IFN-{gamma} production from CD4+CD25– effector cells was directly suppressed by CD8+CD122+ cells and also by CD4+CD25+ cells, although to a lesser extent than by CD8+CD122+ cells. To measure IFN-{gamma} production, we chose the method of intracellular cytokine staining in CFSE-labeled CD4+CD25– cells because CD8+CD122+ cells also expressed high levels of IFN-{gamma} (data not shown), which made it difficult to evaluate the IFN-{gamma} production from CD4+CD25– cells by ELISA of the culture supernatant. Moreover, IL-10 levels in the culture supernatant were not significantly increased in cocultures of CD4+CD25– cells, CD4+CD25+ cells, and CD8+CD122+ cells compared with cocultures of CD4+CD25– cells and CD8+CD122+ cells (Fig. 8B). Thus, these results suggest that CD8+CD122+ cells work independently of CD4+CD25+ nTreg in the control of EAE pathogenesis.


Figure 8
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  		FIGURE 8. CD8+CD122+ cells suppress CD4+CD25– effector cells independently of CD4+CD25+ regulatory cells. A, CD4+CD25– cells taken from EAE-induced C57BL/6 mice on day 14 after MOG immunization were labeled with CFSE and either cultured alone (5 x 105/well) or cocultured with CD4+CD25+ cells (1 x 105/well), CD8+CD122+ cells (1 x 105/well), or both CD4+CD25+ and CD8+CD122+ cells (1 x 105/well each) under stimulation with plate-bound anti-CD3 Ab for 48 h. CD4+CD25+ cells and CD8+CD122+ cells were obtained from naive C57BL/6 mice. Cultured cells were subjected to staining for intracellular IFN-{gamma}. Panels are dot plot analyses of IFN-{gamma} expression in CD4+CD25– (CFSE+) cells. Percentages of cells positively stained with IFN-{gamma} in total CD4+CD25– cells are shown in each panel. B, Cells were prepared as in A, and their culture supernatant was analyzed by ELISA. Data are mean ± SD obtained from three experiments. The p value obtained by Student’s t test is shown.

 
In vitro-activated CD8+CD122+ cells have the potential to improve EAE symptoms

We tried to diminish the course of EAE, especially the peak symptoms, by using CD8+CD122+ cells. CD8+CD122+ cells need to be activated to perform their regulatory activity (manuscript submitted for publication, M. Rifa’i, Z. Shi, Y. H. Lee, H. Shiku, K.-I. Isobe, and H. Suzuki); thus, we prepared CD8+CD122+ cells taken from the syngeneic strain of C57BL/6 mice and stimulated them with anti-CD3/CD28 Ab-coated microbeads. Thus, 5 x 105 in vitro-activated CD8+CD122+ cells were transferred into EAE-induced mice 4 days after immunization with MOG peptide. These CD8+CD122+ cell-transferred EAE mice showed diminished symptoms at the peak of the EAE course compared with CD8+CD122– cell-transferred EAE mice or simple EAE mice (Fig. 9). These CD8+CD122+ cell-transferred EAE mice also had decreased EAE symptoms in the recovery phase compared with simple EAE mice. This result provided evidence that preactivated CD8+CD122+ cells may work to prevent EAE symptoms.


Figure 9
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  		FIGURE 9. Transfer of in vitro-activated CD8+CD122+ cells into EAE-induced mice potentially diminishes EAE symptoms. CD8+CD122+ and CD8+CD122– cells were obtained from naive C57BL/6 mice and cultured under stimulation with anti-CD3/CD28 Ab-coated microbeads (Dynal) for 48 h. These prepared cells (5 x 105) were transferred into EAE-induced mice on day 4 after immunization with MOG peptide. The clinical course of EAE was evaluated and EAE symptoms were scored. Data were obtained from three mice in each group and their mean values with error bars of SDs are shown. Statistically significant (p < 0.05) values from CD8+CD122+ cell-transferred EAE mice compared with simple EAE mice are marked with asterisks.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Based on similarities in disease susceptibility, course, and histology, EAE is currently regarded as an experimental animal model for human multiple sclerosis (MS) (13). In the case of MS, autoreactive myelin-specific T lymphocytes invade the CNS, recruit peripheral mononuclear phagocytes, and cause demyelination in brain and spinal cord tissue, ultimately leading to impaired neuronal transmission (14). EAE in mice also results in inflammation and demyelination in the CNS that leads to limb weakness and eventual paralysis (15). EAE has been induced in rodents by many kinds of myelin proteins, including myelin basic protein (MBP) (16, 17), proteolipid protein (PLP) (18, 19), MOG (20, 21, 22), myelin-associated glycoprotein (23), and myelin oligodendrocyte basic protein (24). In particular, MOG35–55 peptide is a well-characterized target Ag of encephalitogenic T cells and is used to initiate EAE in the H-2b C57BL/6 mouse model (25). Recently, MOG-induced EAE attracted interest because MOG-reactive T cells were readily found circulating in MS patients (26, 27). MOG35–55 peptide induces both T cell and Ab responses, and the severity of EAE correlates with the presence of MOG-specific autoantibodies and the secretion of Th1 cytokines such as IFN-{gamma} (28, 29).

Numerous studies have reported the role of CD4+CD25+ cells in EAE (30, 31). PLP139–151-induced EAE is enhanced significantly by treatment with anti-CD25 mAb to deplete CD4+CD25+ regulatory T cells. Production of IL-10 decreases and that of IFN-{gamma} increases in lymph node cells stimulated with PLP130–151 in vitro in these mice (32). Thus, B10.S mice are usually resistant to PLP139–151-induced EAE, but when CD4+CD25+ cells were depleted by anti-CD25 mAb, the incidence of EAE increases (33). CD4+CD25+ regulatory T cells could also inhibit both the proliferation and IFN-{gamma} production of a MOG35–55-specific T cell line in vitro. Furthermore, supplementation of CD4+CD25+ regulatory T cells by adoptive transfer before active and adoptive EAE induction significantly reduced the severity of the clinical score (34). In contrast, one recent report demonstrates that the depletion of CD4+CD25+ regulatory T cells could accelerate disease onset and severity. In these CD4+CD25+ regulatory T cell-depleted mice, initial remission of EAE still occurred but the secondary remission was lost (35).

Compared with the conclusion that CD4+ regulatory T cells are involved in the improvement or deterioration of EAE, the role of CD8+ regulatory T cells in EAE is still poorly understood. CD4+ T cells were required for the initial induction of EAE and CD8+ or CD25+ T cells could decrease EAE symptoms (36). Studies using MBP and MOG systems demonstrated that CD8+ T cells could adoptively transfer severe EAE into naive recipients (37). Additionally, a recent report described a potentially pathogenic role of CD8+ T cells in MS patients, as MBP-restricted CD8+ T cells isolated from MS patients had a higher frequency and increased cytotoxicity relative to cells isolated from normal healthy controls (38). It was also reported that MOG35–55-primed CD8+ T cells could adoptively transfer EAE into the recipient mice and that MOG37–50-H-2Db MHC tetramers allowed direct visualization of MOG-specific CD8+ T cells in the peripheral lymphoid organs and CNS (39).

In the present study, we first demonstrated the strict effect of CD8+ regulatory T cells on EAE. The success of this study is achieved by concentrating on the CD8+CD122+ regulatory T cells, which are naturally occurring regulatory T cells like CD4+CD25+ cells. The importance of CD8+ regulatory T cells in immune homeostasis is clearly proved by in vivo experiments (10). EAE symptoms did not recover when CD8+CD122+ cells were depleted by treating mice with anti-CD122 mAb and this depletion did not affect the peak clinical score and the day of onset of EAE symptoms (Figs. 2A and 4A). This result suggests that CD8+CD122+ cells have a regulatory activity in the late phase of disease. Significantly higher levels of IFN-{gamma} were observed in anti-CD122-treated EAE mice than in simple EAE mice on day 14 after immunization, but not on day 11 (Fig. 6D), further supporting this idea.

Because CD8+CD122+ cells are known to produce IL-10 to regulate other T cells (11) and the increased IL-10 production from CD8+ cells correlates with EAE symptoms (Figs. 2A, 4A, and 7C), it may be reasonable that CD8+CD122+ cells reduce EAE symptoms via the action of IL-10. When CD8+CD122+ regulatory T cells were transferred into CD122-depleted mice on the day of peak clinical score, the symptoms of EAE clearly improved (Fig. 4A) but expression levels of IFN-{gamma} were still high and were not different from those of anti-CD122- treated EAE mice (Fig. 6A). In contrast, IL-10 was dramatically increased in CD8+CD122+ cell-transferred EAE mice (Fig. 7A). Taken together, we conclude that CD8+CD122+ regulatory T cells can improve EAE symptoms via the effects of IL-10 and that the depletion of CD8+CD122+ cells induces an increase in IFN-{gamma} expression that causes severe EAE symptoms. When we depleted the CD122+ cells with anti-CD122 mAb, this depletion also affected CD4+ cells as shown by ELISA and histological analyses. Interestingly, IL-10 production from CD4+ cells was evident in EAE mice and such IL-10 production from CD4+ cells is reduced in CD8+CD122+ cell-depleted EAE mice (Fig. 7B). This result may suggest that CD8+CD122+ cells not only produce IL-10 but also induce IL-10 production from CD4+ cells (possibly inducing IL-10-producing CD4+ cells such as Tr1). We suspect that CD8+CD122+ regulatory T cells perform their regulatory activity in cooperation with CD4+ cells and form a network to maintain immune homeostasis.

In this study, we demonstrated the action of CD8+CD122+ regulatory T cells in reducing the symptoms of EAE, especially in the recovery phase. CD8+CD122+ regulatory T cell may be a potential clinical tool for controlling autoimmune-based encephalomyelitis such as MS.


   Acknowledgments
 
We thank Dr. H. Shiku for providing the anti-CD122 Ab and Dr. T. Yamamura for useful discussion.


   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 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Japan Society for the Promotion of Science. Back

2 Address correspondence and reprint requests to Dr. Haruhiko Suzuki, Department of Immunology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail address: k46200a{at}nucc.cc.nagoya-u.ac.jp Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; MBP, myelin basic protein; nTreg, naturally occurring regulatory T cell; EGFP, enhanced GFP; PLP, proteolipid protein. Back

Received for publication July 5, 2007. Accepted for publication October 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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