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Originally published online as doi:10.1189/jlb.0507312 on April 18, 2008

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(Journal of Leukocyte Biology. 2008;84:182-190.)
© 2008 by Society for Leukocyte Biology

Mannosylated self-peptide inhibits the development of experimental autoimmune encephalomyelitis via expansion of nonencephalitogenic T cells

Junda M. Kel*,{dagger},1, Bram Slütter*, Jan Wouter Drijfhout{dagger}, Frits Koning{dagger} and Lex Nagelkerken*

* Business Unit Biosciences, TNO Quality of Life, Leiden, The Netherlands; and
{dagger} Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands

1Correspondence at current address: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: j.m.kel{at}amc.uva.nl


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ABSTRACT
 
Tolerance to experimental autoimmune encephalomyelitis (EAE) in SJL mice can be induced by immunization with a mannosylated form of the proteolipid protein (M-PLP139–151), despite the presence of CFA. The state of tolerance is characterized by poor delayed-type hypersensitivity responses and the absence of clinical EAE symptoms. In vivo monitoring of CFSE-labeled PLP139–151-specific TCR-transgenic (5B6) T cells revealed that immunization with M-PLP139–151 increases the clonal expansion of 5B6 T cells that do not develop full effector functions. Moreover, nonfunctional T cells obtained from M-PLP139–151-immunized mice showed poor blastogenesis and were unable to transfer EAE to naïve recipients. Nevertheless, the in vitro production of cytokines and chemokines associated with EAE was unaffected. Importantly, tolerance induced by M-PLP139–151 was abrogated by the administration of pertussis toxin, resulting in EAE development. Our results suggest that M-PLP139–151 inhibits EAE development by affecting the differentiation of T cells into encephalitogenic effector cells.

Key Words: delayed-type hypersensitivity (DTH) • tolerance • C-type lectins


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INTRODUCTION
 
Multiple sclerosis (MS) is an inflammatory disease that affects the CNS, and although little is known about its etiology, a role for autoimmune responses against myelin has been suggested [1 2 3 ]. Inflammation of the CNS, as observed in MS patients, can be mimicked in rodents by immunization with myelin components in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in development of experimental autoimmune encephalomyelitis (EAE) [4 5 ], which is mediated by autoreactive CD4+ T cells that are primed in lymphoid organs after immunization and subsequently infiltrate the CNS to mediate inflammation [6 7 ]. Frequently, pertussis toxin (PTX) is applied to facilitate EAE development, as it affects blood brain barrier (BBB) integrity [8 9 ]. Moreover, PTX affects immune regulatory mechanisms [10 ] and activates APC, resulting in enhanced Th1 immunity [11 12 13 ].

Re-establishment of self-tolerance can ameliorate symptoms in autoimmune disease, and it has recently been demonstrated that signaling via C-type lectin receptors (CLR) can result in the suppression of immunity in an antigen-specific manner. CLR family members were first discovered as pathogen recognition receptors, expressed on APC. Ligation of these receptors by sugar moieties present in pathogenic cell walls can contribute to the induction of immunity [14 15 ]. However, CLR targeting may also contribute to immune suppression. For example, various pathogens circumvent their elimination by the host via binding to dendritic cell (DC)-specific ICAM-grabbing nonintegrin, a CLR family member expressed on DC [16 17 18 ]. In addition, targeting immature DC by fusion proteins consisting of a T cell epitope and an anti-DEC-205 antibody mediated immunological unresponsiveness to class I- and class II-restricted T cell epitopes [19 20 ]. A similar targeting strategy resulted in tolerance induction to EAE, which was associated with increased CD5 expression of T cells [21 ]. In line with this, we have shown previously that immunization with a mannosylated self-peptide induces antigen-specific tolerance to EAE in SJL mice, despite the presence of complete adjuvant. This state of tolerance was characterized by poor DTH reactivity, absence of clinical EAE symptoms, and reduced inflammation in the CNS [22 ]. In addition, treatment with this mannosylated self-peptide during ongoing autoimmune responses can ameliorate EAE symptoms [23 ].

Here, we investigate the fate of autoreactive T cells after immunization with a mannosylated peptide derived from proteolipid protein (M-PLP139–151). Using TCR-transgenic PLP139–151-specific CD4+ T cells (5B6 cells [24 ]), we show that immunization with M-PLP139–151 enhances the clonal expansion of T cells that do not develop effector functions. As PTX abrogated tolerance, resulting in mild EAE, our study favors the idea that a delicate balance between pro- and anti-inflammmatory signals determines the development of autoimmunity.


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MATERIALS AND METHODS
 
Animals
5B6 mice (kindly provided by Dr. Vijay K. Kuchroo, Harvard University, Boston, MA, USA; and Dr. David C. Wraith, University of Bristol, Bristol, UK) were bred and housed in our own animal facilities in individually ventilated cages with unlimited access to food and water. Female SJL mice were obtained from Janvier (France). All experimental procedures were approved by the Animal Welfare Committee of TNO Quality of Life (The Netherlands).

Peptide synthesis
PLP139–151 (HSLGKWLGHPDKF) and its counterpart M-PLP139–151 were produced as described elsewhere [25 26 ]. In short, peptides were synthesized using solid-phase synthesis. Mannosylation was accomplished by N-terminal elongation of the peptide with a building block containing lysine coupled to two tetra-acetyl-protected mannose groups. PLP139–151 was elongated with bis-acetyl lysine only, and the acetyl-protecting groups on the mannose moieties were removed using Tesser’s base. All peptides were analyzed with reversed-phase HPLC and MALDI-TOF mass spectrometry and showed the expected masses.

Cell separation and in vitro cultures
Spleens were isolated from 5B6 mice, and single-cell suspensions were prepared using a 40-µm filter (BD Falcon, Bedford, MA, USA). For CFSE labeling, splenocytes were incubated with 5 µM CFSE in RPMI 1640 (Cambrex, East Rutherford, NJ, USA) for 10 min and subsequently washed twice with ice-cold PBS, as described by Lyons and Parish [27 ]. To study in vitro proliferation, these cells were resuspended in RPMI 1640 containing 5% FCS (Cambrex), 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM glutamine, and 50 µM β-ME and seeded in 96-well flat-bottom microtiter plates (Costar, Cambridge, MA, USA) at a density of 2 x 105 cells per well. The 5B6 cells were stimulated with 0.1–100 µg/ml PLP139–151 or M-PLP139–151 for a period of 4 days.

For coculture experiments, naive CD4+ 5B6 cells were isolated from spleens via negative isolation using Dynal beads (Dynal Biotech, Oslo, Norway). Naive SJL spleens were used for the isolation of CD11c+ DC, CD11b+ macrophages, and CD19+ B cells using MACS beads and columns according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA, USA). The APC populations were irradiated (30 Gy) and cocultured with 5 x 104 CD4+ 5B6 cells (ratios, 2:1 and 1:4) in the presence of 30 µg/ml PLP139–151 or M-PLP139–151 for 3 days. Supernatants were collected from all cultures, and proliferation was assessed by addition of 0.5 µCi 3H-labeled thymidine for 6 h (2 Ci/mmol, Amersham Biosciences, Buckinghamshire, UK).

In vivo transfer of 5B6 cells
Female 5B6 mice were killed, and lymph nodes (LN) and spleens were isolated. A single-cell suspension was prepared in RPMI medium and incubated for 45 min on a nylon wool column. This procedure depleted 50% of B cells and macrophages and resulted in an enriched cell suspension containing 50% CD4+ T cells. The 5B6 cells were CFSE-labeled as described above and extensively washed with PBS before injection. i.v. injection of 10 x 106 5B6 cells into naïve, female SJL mice occurred 1 day before immunization.

EAE induction
Female SJL mice (8–12 weeks old) were immunized s.c. with 50 µg PLP139–151 or M-PLP139–151, dissolved in PBS, and emulsified in an equal volume of complete adjuvant supplemented with 1 mg/ml Mycobacterium tuberculosis (H37RA, Difco Laboratories, Detroit, MI, USA). Control mice were immunized with PBS in adjuvant. Animals were weighed daily and monitored for EAE development. Clinical EAE was graded as follows: 0, no symptoms; 0.5, partial loss of tail tonus; 1, complete loss of tail tonus or partial limb weakness; 1.5, limb weakness and partial tail paralysis; 2, limb weakness and complete tail paralysis; 2.5, partial paresis; 3, complete paralysis of hind limbs; 3.5, complete paralysis from diaphragm and hind limbs; 4, moribund; and 5, death from EAE. Where indicated, 1 x 109 heat-hilled Bordetella pertussis bacteria (BP) or 200 ng PTX (Toxin Technology, Sarasota, FL, USA) were injected i.v. on Day 3.

Adoptive transfer EAE
Donor mice were immunized with 75 µg PLP139–151 or M-PLP139–151 in adjuvant as described above. After 14 days, these mice were killed, and draining LN were isolated. Single-cell suspensions were prepared, and LN cells were cultured with 30 µg/ml PLP139–151 or M-PLP139–151 for 3 days in the presence of 50 U/ml IL-2. Subsequently, cells were harvested and washed with PBS before i.p. injection into naive recipient mice (15x106 cells/mouse).

Delayed type hypersensitivity (DTH)
The DTH response was evaluated by injecting 12 nmol PLP139–151, dissolved in 10 µl saline into the dorsal side of the right ear using a Hamilton syringe fitted with a 30-gauge needle. As a control for nonspecific ear swelling, 10 µl saline was injected into the left ear. Ear thickness was measured before and 24 h after intradermal injection using of a Mitutoyo micrometer. Results are expressed as the percentage-specific ear swelling obtained by subtracting the percentage nonspecific ear swelling.

Flow cytometry
For FACS analysis, cells were collected from in vitro cultures, or single-cell suspensions were prepared from isolated organs. Blood samples were treated with lysing solution (PharMingen, San Diego, CA, USA) before staining. Cells were stained with different rat anti-mouse antibodies obtained from BD Biosciences [San Jose, CA, USA; CD4-PerCP, Vβ6-PE, CD19-FITC, CD8-allophycocyanin-Cy7, CD11c-Alexa647, CD11b-biotin, CD25-biotin, CD25-allophycocyanin, CD62 ligand (CD62L)-allophycocyanin, CD44-PE, forkhead box P3 (FoxP3)-PE, IL-4-PE, and IFN-{gamma}-allophycocyanin]. Where needed, isotype-matched controls were included (rat-anti-mouse-IgM-biotin, rat-anti-mouse-IgG1-allophycocyanin, rat-anti-mouse-IgG2a-PE, and rat-anti-mouse-IgG2a-allophycocyanin). Biotinylated antibodies were visualized with Streptavidin-allophycocyanin or Streptavidin-PE-Cy7. For intracellular FACS staining, cells were incubated for 4 h with Brefeldin A (Sigma Chemical Co., St. Louis, MO, USA), and fixation and permeabilization reagents (Caltag Laboratories, Burlingame, CA, USA) were used for staining.

Antibody arrays
The undiluted supernatants of the LN cultures used for transfer experiments were incubated on Mouse Inflammation Antibody Array I (Raybiotech, Norcross, GA, USA), according to the manufacturer’s instructions. The presence of cytokines was evaluated by chemiluminescent detection, using a chemiDoc-it imaging system (UVP Inc., Upland, CA, USA), and the signal intensities were quantified with Labworks software. On the array, duplicate spots for each cytokine were present, as well as positive and negative controls. For analysis, the OD of all spots was quantified, the background was subtracted, and the mean signal of duplicate cytokine spots was calculated (mean background, 500). All results were normalized toward the positive control to enable comparison of different arrays.

Statistics
Statistical analysis of data was performed with a Mann-Whitney test and a {chi}2 test.


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RESULTS
 
Mannosylated self-peptide is well-recognized by antigen-specific T cells in vitro
To study the fate of myelin-specific T cells in response to immunization with M-PLP139–151, 5B6 cells were used. First, it was studied whether the mannosylated peptide influenced antigen recognition by 5B6 cells in vitro. Therefore, CFSE-labeled 5B6 splenocytes were cultured for 4 days in the presence of 0.1–100 µg/ml PLP139–151, M-PLP139–151, or medium. Cell division of CD4+ 5B6 cells was monitored daily by flow cytometric evaluation of CFSE dilution (Fig. 1 A ) and by 3H-thymidine incorporation (Fig. 1B) . Although comparable results for the two peptides were obtained with all tested concentrations, CD4+ T cells incidentally responded better to M-PLP139–151. Furthermore, flow cytometric analysis revealed similar activation of CD4+ 5B6 cells in response to either peptide, based on CD25 expression and IFN-{gamma} production. ELISA of culture supernatants confirmed that equal amounts of IFN-{gamma} were secreted in the absence of IL-4 and IL-10 production (data not shown). C-type lectins are widely expressed by innate immune cells [28 29 ], and to evaluate whether targeting of such receptors resulted in their proliferation, the CFSE dilution pattern of CD4-negative 5B6 splenocytes was studied, which revealed that it was primarily the CD4-positive T cell population that was responsible for the proliferative response to either peptide (data not shown).


Figure 1
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  		Figure 1. In vitro stimulation of 5B6 splenocytes with PLP139–151 and M-PLP139–151. Naïve, CFSE-labeled 5B6 splenocytes were cultured for 4 days with 10 µg/ml PLP139–151 (black bars), M-PLP139–151 (open bars), or medium (gray bars). Division of CD4+ 5B6 cells was evaluated via dilution of the CFSE signal (A; medium control in dotted histogram). Proliferation of 5B6 cells was also assessed via the incorporation of 3H-thymidine (B, *, P<0.05). Similar results were obtained with other peptide concentrations, and one out of three similar experiments is shown. CD4+-enriched 5B6 T cells were cocultured with irradiated splenocytes (SP), CD11c+-enriched cells (DC), CD11b-enriched cells [macrophages (M{Phi})], or CD19-enriched cells (B) in the presence of 30 µg/ml PLP139–151, M-PLP139–151, or medium. T cell proliferation was assessed by incorporation of 3H-thymidine (C). IFN-{gamma} levels in the supernatant of these cocultures were determined by ELISA (D).

To study whether APC populations discriminated between normal peptide and mannosylated peptide, CD4+-enriched 5B6 cells were stimulated for 3 days with irradiated spleen cells, CD11c-enriched DC, CD11b-enriched macrophages, or CD19-enriched B cells in the presence of PLP139–151, M-PLP139–151, or medium. Results from cocultures in a ratio of 2:1 are presented, but similar results were obtained with a 1:4 ratio (APC:T). In particular, DC induced proliferation (Fig. 1C) and IFN-{gamma}-production (Fig. 1D) by 5B6 T cells, and we obtained no indications that the APC populations under investigation differentially presented the two peptides. We conclude that both peptides were equally presented and recognized in vitro, resulting in the expansion of CD4+ T cells with a Th1 phenotype.

Increased clonal expansion of T cells after immunization with M-PLP139–151
To evaluate the clonal expansion of PLP139–151-specific T cells in response to immunization to PLP139–151 or M-PLP139–151, CFSE-labeled 5B6 cells were injected into naive recipient mice that were immunized 1 day later with peptide or PBS in complete adjuvant. Transfer of 5B6 cells itself or sham immunization did not induce EAE (Table 1 ). After immunization with PLP139–151, EAE developed around Day 11, and one animal died as a result of the disease (Table 1 and Fig. 2 A ). In contrast, only 50% of the mice developed EAE after immunization with M-PLP139–151 (Table 1 and Fig. 2B , P=0.08), and the disease onset was delayed significantly (P<0.01). In affected mice, we observed a mild disease pattern, resulting in a significant reduction of the maximal EAE score (P<0.01) and the cumulative EAE score (P<0.05).


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  		Table 1. EAE Development after Transfer of 5B6 Cells into SJL Mice


Figure 2
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  		Figure 2. Immunization with M-PLP139–151 induces poor effector T cells in vivo. CFSE-labeled 5B6 cells were transferred into naïve recipient mice, and these were immunized 1 day later with PBS (no EAE development), PLP139–151 (A), or M-PLP139–151 (B). Representative data from one experiment are shown; combined data and statistics are presented in Table 1 . The numbers of animals with clinical EAE are indicated in the graphs. On Day 3 (circles) or 6 (triangles) after immunization, PLP139–151 was injected into the right ear, and saline was injected in the left ear. Antigen-specific ear swelling was determined 48 h later (C). Combined data from three experiments are presented.

DTH responses toward PLP139–151 were evaluated on Days 3 and 6 after immunization, and poor responses were measured in mice that had been immunized with M-PLP139–151 (Fig. 2C , P<0.001). Transfer of 5B6 cells itself and sham immunization was insufficient to elicit a DTH response (data not shown).

The presence of CD4+ 5B6 cells in the blood was monitored 1–4 days after immunization. Retrieval of the injected 5B6 cells was based on the CFSE signal within the CD4+ Vβ6+ population. Representative results from the analysis on Day 1 (Fig. 3 A ) and on Day 4 (Fig. 3B) are presented. Immunization with either peptide resulted in reduced numbers of 5B6 cells in the bloodstream up to 2 days after immunization, compared with sham-immunized mice. Subsequently, the numbers of 5B6 cells increased substantially, in particular, in response to M-PLP139–151 (Fig. 3C) . With respect to the division pattern of the 5B6 cells in the blood, we observed that the ratio between divided and undivided cells was significantly increased after immunization with M-PLP139–151 (Fig. 3D) . In all mice, 5B6 cells in the blood highly expressed CD62L (data not shown). After 4 days, multiple-divided 5B6 cells had lost their CFSE signal and could no longer be visualized as a result of the endogenous expression of Vβ6 in SJL mice.


Figure 3
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  		Figure 3. Expansion of 5B6 cells in vivo after immunization with M-PLP139–151. CFSE-labeled 5B6 cells were transferred to naïve recipient mice, and these were immunized 1 day later with PBS (gray bars), PLP139–151 (black bars), or M-PLP139–151 (open bars). The presence of 5B6 cells in the blood was monitored on Days 1–4 after immunization. After gating on the CD4+Vβ6+ population, the CFSE signal was used to identify the donor T cells from 5B6 origin. At least three individual mice per time-point were analyzed, and representative data obtained on Day 1 (A) and Day 4 (B) are presented. The percentages of 5B6 cells in the blood were monitored in time, and within the CD4+ population, the mean percentages are shown (C). To visualize the expansion of 5B6 cells, the ratio between CFSElow (divided) and CFSEhigh (undivided) cells was calculated (D). *, P < 0.01; **, P < 0.001; #, P< 0.05, compared with PLP139–151-immunized and sham-immunized mice.

Together, the data indicate that immunization with M-PLP139–151 increased the early in vivo expansion and circulation of PLP139–151-specific T cells that develop poor effector functions.

Reduced encephalitogenicity of T cells after immunization with M-PLP139–151
Adoptive transfer of EAE experiments was performed to study whether the intrinsic encephalitogenic capacities of autoreactive T cells were affected by immunization with M-PLP139–151. For this purpose, donor mice were immunized with 75 µg PLP139–151 or M-PLP139–151 and killed after 14 days when initial EAE symptoms in PLP139–151-immunized mice were present. Draining LN were isolated and cultured with the corresponding peptide for 3 days in the presence of IL-2. Although the viability in the pooled LN cell cultures was similar for both peptides, M-PLP139–151-stimulated cells showed less blastogenesis and reduced cell recovery after 3 days of culture (Fig. 4A , P<0.05). Equal numbers of expanded cells were injected into naive recipient mice. Transfer of PLP139–151-expanded T cells resulted in EAE development in five out of six recipient mice (mean day of onset, 15.4±7.0; maximal EAE score, 1.8±1.1), while M-PLP139–151-expanded T cells were unable to transfer EAE (Fig. 4B ). Moreover, these cells mounted significantly reduced DTH responses in the recipient mice after an ear challenge with PLP139–151 (Fig. 4C , P<0.01).


Figure 4
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  		Figure 4. Reduced encephalitogenicity after immunization with M-PLP139–151. Donor mice were immunized with PLP139–151 or M-PLP139–151, and after 14 days, the draining LN were isolated and cultured for 3 days with corresponding peptide and IL-2. M-PLP139–151-stimulated cells showed similar viability after culture but a significantly decreased yield and blastogenesis (A, *, P<0.05). Transfer of M-PLP139–151-stimulated cells into naïve recipient mice did not result in EAE development (B) and a poor DTH response after ear challenge with PLP139–151 (C). The numbers of mice with clinical EAE are indicated in the graph, and the DTH results were combined from three similar experiments. The supernatants of the transferred cells were analyzed with cytokine antibody arrays. The chemiluminescent signals (OD) of supernatants obtained from PLP139–151- and M-PLP139–151-stimulated LN cell cultures were plotted against each other to show that a similar cytokine production profile was observed (D). One out of two similar experiments is presented, and factors of interest are indicated by filled symbols. As IL-2 is added to the cultures, it was excluded from the analysis (indicated in gray). In a separate experiment, three individual mice per group were immunized with PLP139–151 or M-PLP139–151, and their LN cells were cultured in a similar way for flow cytometric analysis. TCA-3, T cell activation 3. The percentage (E) as well as the absolute number (F) of CD4+ T cells were decreased after 3 days of culture with M-PLP139–151 (*, P<0.05). No differences in FoxP3 expression were observed (G). Filled bars and symbols, PLP139–151; open bars and symbols, M-PLP139–151.

Supernatants from the LN cell cultures were incubated on cytokine antibody arrays. As depicted in Figure 4D , a similar spectrum of cytokines and chemokines was produced by PLP139–151- and M-PLP139–151-stimulated cells. Surprisingly, the T cells that were rendered nonencephalitogenic after immunization with M-PLP139–151 were not impaired with respect to the production of IFN-{gamma} and IL-17 in vitro, and also, the secretion levels of the chemokines TCA-3 (CCL1), MIP-1{alpha} (CCL3), and RANTES (CCL5) were similar. Furthermore, high levels of IL-3 and MIP-1{gamma} (CCL9) were detected, and IL-4 and IL-10 levels were low.

In a separate experiment, mice were immunized with PLP139–151 or M-PLP139–151 and killed on Day 10. For flow cytometric analysis, the draining LN cells from individual mice were cultured for 3 days in the presence of corresponding peptide and IL-2. Although the LN suspensions obtained from mice immunized with either peptide contained similar percentages of CD4+ T cells (i.e., results obtained on Day 0), the percentage of CD4+ T cells was significantly lower after 3 days of culture in the presence of M-PLP139–151 (Fig. 4E , P<0.05), and accordingly, the absolute numbers of CD4+ T cells recovered from the LN cultures tended to be lower (Fig. 4F) . However, the T cells seemed equally activated, based on the expression of CD44 and CD25 (data not shown). In addition, we observed similar FoxP3 expression by CD4+ T cells after 3 days of culture (Fig. 4G) .

These data suggest that the inability of LN cells of M-PLP139–151-immunized mice to transfer EAE may be partially a result of less expansion of CD4+ T cells, although these T cells appear well-activated and not impaired with respect to the in vitro production of cytokines and chemokines that are associated with EAE development.

PTX abrogates tolerance induction by M-PLP139–151
Immunization with M-PLP139–151 resulted in poor EAE development, despite the presence of complete adjuvant (Fig. 5A and B ). To increase the efficacy of EAE induction, heat-killed BP or PTX are frequently used as an additional adjuvant [8 ]. Importantly, treatment with BP and PTX abrogated the induction of tolerance associated with M-PLP139–151. Immunization with M-PLP139–151 combined with i.v. BP (Fig. 5C and 5D) or PTX injection (Fig. 5E and 5F) resulted in disease development, although EAE symptoms were mild compared with PLP139–151-immunized mice that received the same treatment (Table 2) .


Figure 5
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  		Figure 5. PTX abrogates tolerance induction by M-PLP139–151. Mice were immunized with 50 µg PLP139–151 or M-PLP139–151 and were injected i.v. with PBS (A and B), heat-killed BP (C and D), or PTX (E and F) on Day 3. Body weight and clinical scores were assessed daily. Mean data of four mice per group are shown, and statistics are described in Table 2 .


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  		Table 2. The Effect of PTX on EAE Development

To further investigate why PTX abrogates tolerance induction by M-PLP139–151, mice were immunized with PLP139–151 or M-PLP139–151 in CFA, with or without additional administration of PTX and killed on Day 10. At this time-point, clinical EAE was only observed in PLP139–151-immunized mice that had received PTX (data not shown). The draining LN of individual mice were collected, and a significantly increased number of LN cells was found in mice that were immunized with M-PLP139–151 compared with PLP139–151-immunized mice (Fig. 6A , P<0.001). Administration of PTX in M-PLP139–151-immunized mice decreased the number of LN cells (P<0.01), although the LN cellularity was still increased compared with mice that were immunized with the nonmannosylated peptide and PTX (P<0.05).


Figure 6
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  		Figure 6. Immunization with M-PLP139–151 increases cell numbers in the LN. Mice were immunized with PLP139–151 or M-PLP139–151, with or without i.v. PTX injection on Day 3. Ten days later, the draining LN were isolated, and the numbers of LN cells were counted (A). Flow cytometric analysis revealed that the LN of M-PLP139–151-immunized mice contained increased numbers of T cells and B cells (B) as well as increased numbers of CD11b+CD11clow and CD11b+CD11chigh cells (C). Injection of PTX diminished this effect in the M-PLP139–151-immunized mice. Three mice per group were studied (P, PLP139–151/CFA; P+, PLP139–151/CFA+PTX; M, M-PLP139–151/CFA; M+, M-PLP139–151/CFA+PTX).

Flow cytometric analysis revealed that the increased cellularity was a result of increased numbers of CD4+ and CD8+ T cells as well as B cells (Fig. 6B , P<0.05). Based on the expression of CD49b, only small numbers of NK cells were present (data not shown). As shown in Figure 6C , significantly increased numbers of CD11b+ cells were found in draining LN of M-PLP139–151-immunized mice, and these comprised the CD11chigh and the CD11clow population (P<.05).

Altogether, these data suggest that the inability M-PLP139–151 to induce EAE is associated with the accumulation of lymphocytes in draining LN. Tolerance was abrogated by PTX, possibly by driving DC maturation in conjunction with the development of effector T cells capable of migration to target tissues.


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DISCUSSION
 
In the search for an improved treatment of patients suffering from autoimmune diseases, such as MS, the antigen-specific inhibition of autoreactive T cells has received considerable attention. In this manuscript, we substantiated that the development of autoimmunity can be inhibited with high efficiency by immunization with a mannosylated self-peptide. The application of TCR transgenic 5B6 cells revealed that the absence of EAE is the result of increased expansion of T cells that develop poor effector functions.

In vitro experiments using 5B6 cells reveal that T cells respond equally to PLP139–151 and M-PLP139–151, regardless of the APC population used to present the peptides. This is consistent with our previous in vitro experiments using OTII cells that were stimulated with a mannosylated OVA peptide [30 ]. Immunization with M-PLP139–151 in complete adjuvant results in enhanced early in vivo expansion of autoreactive T cells that are impaired with regard to their encephalitogenic properties and the induction of DTH responses. The poor DTH responses suggest that T cells are already nonfunctional at early time-points and may therefore be ineffective in the subsequent induction of EAE. These data are in line with studies by Cua et al. [31], who showed a clear association between DTH responsiveness and EAE susceptibility as a result of the involvement of the same effector T cells population(s) as well as macrophages. Transfer of cells from M-PLP139–151-immunized mice into naive recipient mice does not result in EAE development, indicating that the absence of disease is not simply the consequence of a decoy effect mediated by a mannosylated self-peptide in donor mice.

The absence of T cell effector functions in recipient mice may be partially explained by less vigorous expansion of CD4+ T cells in vitro and the decreased formation of lymphoblasts, suggesting that the T cells do not become fully activated or differentiated. However, the unaffected production of cytokines associated with encephalitogenicity, such as IFN-{gamma}, IL-17, and IL-3 [32 33 ], is supportive for an activated state of the T cells. Moreover, we observed considerable production of TCA-3 (CCL1), MIP-1{alpha} (CCL3), and RANTES (CCL5), chemokines involved in EAE [34 ]. Although MIP-1{gamma} (CCL9) is known as a proinflammatory chemokine, the high levels in culture supernatants of myelin-specific cells were less expected, as a role for MIP-1{gamma} (CCL9) in autoimmunity has not been described so far. The limited production of IL-4 and IL-10 suggests that a mannosylated self-peptide does not promote the development of Th2 cells or IL-10-secreting regulatory T cells (Tregs). Furthermore, the evaluation of the numbers of FoxP3-expressing CD4+ T cells did not reveal that increased numbers of Tregs developed in response to a mannosylated peptide. In line with these observations, we found that tolerance induction to EAE by immunization with mannosylated myelin peptide is successful in IL-10-deficient mice, indicating that IL-10 does not play a pivotal role (data not shown).

In earlier studies, we have shown that proliferative responses of T cells isolated from mice that were rendered tolerant by immunization with M-PLP139–151 were decreased after Day 50, although normal in vitro proliferation was observed at earlier time-points [22 ]. Unfortunately, the long-term monitoring of 5B6 cells after immunization with a mannosylated self-peptide in current studies was hampered by the lack of a congenic marker. The possible deletion of nonfunctional T cells at later time-points can therefore not be excluded.

Interestingly, tolerance induction by a mannosylated self-peptide in SJL mice can be established despite strong proinflammatory signals provided by a complete adjuvant containing M. tuberculosis that results in triggering of TLR2 and TLR4 [35 ]. We show here that the absence of EAE in mice that were immunized with a mannosylated peptide is associated with accumulation of lymphocytes in the draining LN, which is suggestive for disturbed migration of lymphocytes toward the periphery. Importantly, disease inhibition was abrogated by PTX injection, and in line with this, we observed that immunization with a mannosylated myelin oligodendrocyte glycoprotein peptide in the presence of PTX did not induce tolerance but severe disease in C57Bl/6 mice (unpublished observations). PTX affects the autoimmune responses in EAE in multiple ways. Administration of PTX prevents T cell anergy [36 ] and can enhance Th1-mediated autoimmunity in a TLR-independent way [37 ]. On the other hand, PTX affects G-protein-coupled receptor signaling, which promotes BBB permeability [9 ], but also disrupts chemokine receptor signaling in lymphocytes in a dose-dependent way [13 ]. At present, the mechanism for the opposing effects of mannosylated myelin peptide and PTX remains unknown.

The balance between inflammatory and tolerizing signals delivered to APC determines the further instruction of T cells [38 ]. Possibly, a mannosylated peptide elicits an immune-suppressive signal in APC, for example, via the inhibition of proinflammatory signaling mediated by TLR. Mannosylated antigens may be targeted toward CLR expressed by APC, and this can result in the suppression of NF-{kappa}B signaling and IL-12 production [39 40 41 ]. Recent studies have demonstrated that in the mouse, DC and macrophages can express the mannose receptor [42 ]. Although the in vitro experiments suggest that, in particular, DC are involved in the presentation of a mannosylated peptide, increased numbers of CD11b+CD11chigh as well as CD11b+CD11clow APC were found in the draining LN of mice that were immunized with a mannosylated self-peptide. Therefore, additional studies are required to reveal the identity and the activation status of APC that are targeted by a mannosylated self-peptide.

We show here that a mannosylated self-peptide inhibits EAE development by affecting the encephalitogenicity of T cells. More research is needed to elucidate whether tolerance induction by M-PLP139–151 is the result of early T cell exhaustion, impaired T cell migration, or incomplete differentiation. This may further support the development of antigen-specific therapy in autoimmunity based on mannosylated self-antigens.


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ACKNOWLEDGEMENTS
 
This research was supported by Grant 00-432 from the Dutch Society for MS research. We thank Wyeth Pharmaceuticals, Dr. V. K. Kuchroo, and Dr. D. C. Wraith for kindly providing us the 5B6 mice and Inge Haspels and Willemien Benckhuijsen for their technical assistance.

Received May 21, 2007; revised February 27, 2008; accepted March 11, 2008.


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