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The Journal of Immunology, 2006, 176: 3480-3489.
Copyright © 2006 by The American Association of Immunologists

Differential Role of Programmed Death-Ligand 1 and Programmed Death-Ligand 2 in Regulating the Susceptibility and Chronic Progression of Experimental Autoimmune Encephalomyelitis1

Bing Zhu*, Indira Guleria{dagger}, Arezou Khosroshahi{dagger}, Tanuja Chitnis*, Jaime Imitola*, Miyuki Azuma{ddagger}, Hideo Yagita§, Mohamed H. Sayegh{dagger} and Samia J. Khoury2,*

* Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; {dagger} Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115; {ddagger} Department of Molecular Immunology, Tokyo Medical and Dental University, Tokyo, Japan; and § Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Programmed death-1 (PD-1) is a negative costimulatory molecule, and blocking the interaction of PD-1 with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), enhances autoimmune disease in several animal models. We have studied the role of PD-1 ligands in disease susceptibility and chronic progression in experimental autoimmune encephalomyelitis (EAE). In BALB/c mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55, PD-L1 but not PD-L2 blockade significantly increased EAE incidence. In B10.S mice immunized with myelin proteolipid protein (PLP) peptide 139–151, both PD-L1 and PD-L2 blockade markedly enhanced EAE severity. In prediabetic NOD mice immunized with PLP48-70, PD-L2 blockade worsened EAE but did not induce diabetes, whereas PD-L1 blockade precipitated diabetes but did not worsen EAE, suggesting different regulatory roles of these two ligands in EAE and diabetes. B6 mice immunized with MOG35-55 developed chronic persistent EAE, and PD-L2 blockade in the chronic phase exacerbated EAE, whereas PD-L1 blockade did not. In contrast, SJL/J mice immunized with PLP139-151 developed chronic relapsing-remitting EAE, and only PD-L1 blockade during remission precipitated EAE relapse. The strain-specific effects of PD-1 ligand blockade did not correlate with the expression of PD-L1 and PD-L2 on dendritic cells and macrophages in lymphoid tissue, or on inflammatory cells in the CNS. However, EAE enhancement is correlated with less prominent Th2 cytokine induction after specific PD-1 ligand blockade. In conclusion, PD-L1 and PD-L2 differentially regulate the susceptibility and chronic progression of EAE in a strain-specific manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Increasing evidence indicate that the dysfunction of immune regulation contributes to the development of autoimmune diseases (1, 2, 3, 4). Immune regulation is mainly mediated by regulatory T cells (5, 6), negative costimulatory molecules (7, 8), cytokines with regulatory functions (9, 10), and molecules signaling apoptosis in activated immune cells (11, 12). Programmed death-1 (PD-1)3 (CD279) is an immunoreceptor that transduces inhibitory signals in activated T cells and B cells (13, 14, 15, 16). Little constitutive expression with strong and rapid up-regulation after activation suggests that the PD-1 pathway may function as a general mechanism in setting the threshold and controlling the degree of immune reactions (13). Interestingly, PD-1-deficient mice on C57BL/6, BALB/c, and NOD background spontaneously developed lupus-like arthritis and glomerulonephritis, autoimmune cardiomyopathy, and accelerated diabetes, respectively (17, 18, 19). In human, PD-1 gene polymorphism has been found to be associated with the susceptibility to lupus (20) and with the progressive course in multiple sclerosis (21).

PD-1 ligands (PD-L1) (B7-H1, CD274) and PD-L2 (B7-DC, CD273) are the ligands that signal through PD-1 (22, 23). PD-L1 is widely expressed on lymphocytes, APCs, endothelial cells, and some organ parenchymal cells (22, 24), whereas PD-L2 is largely restricted to activated dendritic cells and macrophages (22, 23, 24). Our group reported that in B6 mice, blockade of PD-1 as well as PD-L2 during the priming phase after myelin oligodendrocyte glycoprotein (MOG)35-55 immunization significantly enhanced experimental autoimmune encephalomyelitis (EAE) severity, whereas blockade of PD-L1 had no effect on disease (25). In contrast, PD-L1–/– 129 mice developed severe EAE after both active and passive induction (25, 26). Studies in other autoimmune disease models suggest that PD-L1 signaling suppresses spontaneous diabetes (27) and hapten-induced contact hypersensitivity (28), whereas PD-L2 signaling suppresses asthmatic response upon Ag challenge (29). In alloimmune models of transplantation and pregnancy, PD-L1 blockade or gene deficiency enhanced rejection (30, 31, 32). Some in vitro studies found PD-L can also stimulate T cell proliferation, possibly through a second receptor with function similar to CD28 (33, 34, 35, 36).

In this study, we investigated the role of PD-L in regulating the natural resistance and chronic progression of EAE. We show that blockade of PD-L1 or PD-L2 in the priming phase increases EAE susceptibility, and blockade in the chronic phase enhances EAE severity and accelerates relapses. Which PD-L plays the dominant regulatory role is strain-specific, and may be related to their distinct function in regulating Th1/Th2 differentiation on specific strain background. These data demonstrate the role of genetic background in regulating immune responses and suggest that multiple regulatory mechanisms may interact with each other in controlling autoimmunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and reagents

Female mice including BALB/c, B10.S-H2s/SgMcd (B10.S), NOD/Lt, C57BL/6 (B6), B6.129S2-Igh-6tm1Cgn (B cell-deficient), and SJL/J strains were obtained from The Jackson Laboratory and housed according to local and National Institutes of Health guidelines. All animals were used at 6–8 wk of age. Purified anti-mouse PD-L1 (MIH6, rat IgG2a) and anti-mouse PD-L2 (TY25, rat IgG2a) mAbs were produced by Bioexpress Cell Culture. Specific binding of anti-PD-L1 and anti-PD-L2 Abs to their ligands, as well as their blocking effects have been demonstrated in the previous studies (25, 27, 37). Control rat IgG was obtained from Sigma-Aldrich.

EAE induction

Myelin protein peptides including MOG35-55 (MEVGWYRSPFSRVVHLYRNGK), myelin proteolipid protein (PLP)48-70 (TYFSKNYQDYEYLINIHAFQYV), and PLP139-151 (HSLGKWLGHPDKF) were synthesized by Quality Controlled Biochemicals (Division of BioSource International) and were purified to >99% by HPLC. Mice were immunized s.c. in the flank with the emulsion made of 75 µl of Ag peptide (150 µg of MOG35-55, 150 µg of PLP48-70, or 75 µg of PLP139-151 as specified) and 75 µl of complete Freund’s adjuvant containing 0.3 mg of heat-inactivated Mycobacterium tuberculosis (H37Ra; Difco Laboratories). Each animal also received 200 ng of pertussis toxin (List Biological Laboratories) through i.v. injection on days 0 and 2 postimmunization. EAE clinical score was determined in a blinded fashion as described previously (38): 0, no disease; 1, limp tail or isolated weakness of gait without limp tail; 2, partial hind limb paralysis; 3, total hind limb or partial hind and front limb paralysis; 4, total hind leg and partial front leg paralysis; and 5, moribund or dead animal. EAE disease curves in the figures represent data from animals both with and without EAE.

Ab treatment

For treatment during the priming phase, PD-L1 and PD-L2 blocking Abs as well as control rat IgG were given i.p. at 0.5 mg/mouse on the same day of immunization (day 0), followed by 0.25 mg/mouse on days 2, 4, 6, 8, and 10. For treatment in the chronic stage of EAE, B6 mice received 0.5 mg Ab/mouse on day 30 and 0.25 mg/mouse on alternative days from days 32 to 40. SJL mice received same doses of Abs on alternate days during EAE remission from days 23 to 33 postimmunization.

Histology

Animals were sacrificed and perfused with 10 ml of cold PBS. The entire spinal cord was dissected out, cut into nine segments, and then snap frozen in OCT medium. Ten-micron spinal cord cross-sections were prepared on a cryostat, and then stored at –20°C until use. Serial sections were stained with H&E, and immunostained with anti-mouse CD4 Ab (BD Biosciences) at 1/50, F4/80 Ab (Serotec) at 1/100, anti-PD-L1 (eBioscience) at 1/100, and anti-PD-L2 (eBioscience) at 1/100. Immunostaining was performed following a standard avidin-biotin protocol. Briefly, tissue sections were fixed in 4% paraformaldehyde, blocked with 10% normal goat serum and 1% BSA, and then incubated with primary Abs at 4°C overnight. After blocking endogenous peroxidase activity, the sections were incubated with biotinylated secondary Ab (Jackson ImmunoResearch Laboratories), avidin-biotin-peroxidase complex (Vector Laboratories), and then visualized with 0.5 mg/ml 3,3'-diaminobenzidine solution and 0.03% H2O2. The sections were counterstained in Gill’s hematoxylin (Sigma-Aldrich). For quantitation, inflammatory foci were identified as the aggregation of 20 or more cells in small focal areas, and positively stained cells were identified as positive staining surrounding counterstained nuclei. The numbers of inflammatory foci, CD4+, and F4/80+ cells were counted from nine levels of spinal cord sections, and the average for a single-level tissue section was calculated. PD-L1- or PD-L2-positive cells and total hematoxylin-counterstained cells were also counted from individual inflammatory foci, and the percentage of inflammatory cells expressing PD-L1 or PD-L2 was calculated. Each pathology group included tissue sections from three to four animals.

Examination of PD-L expression on APCs using flow cytometry

Splenocytes from various strains of mice were prepared after lysing RBC, and CD90– cells were negatively selected with MACS CD90 microbeads (Miltenyi Biotec). Cells were blocked with 10 µg/ml Mouse BD Fc Block at 4°C for 5 min, and triple-labeled with FITC-conjugated anti-mouse CD11c or F4/80 Abs (BD Biosciences), PE-conjugated anti-mouse PD-L1 or PD-L2 Abs (eBioscience), and 7-aminoactinomycin D (7-AAD) (BD Biosciences) for 30 min at 4°C including proper isotype controls. After two steps of wash, cells were analyzed on a FACSort (BD Biosciences) using CellQuest software (BD Biosciences). Only 7-AAD– cells were included in data analysis.

T cell proliferation, ELISPOT, and ELISA

For T cell proliferation assay, splenocytes from three mice in each group were harvested on day 20 postimmunization and were cultured at 5 x 105 cells/well with 0, 1, 5, 25 µg/ml Ag peptide in a 96-well plate. RPMI 1640 culture medium was supplemented with 10% FBS, glutamine, 2-ME, sodium pyruvate, nonessential amino acid, HEPES, and antibiotics (BioWhittaker). After 48 h, 1 µCi [3H] was added into each well, and cells were harvested 16 h later. For ELISPOT assay, nitrocellulose plates (Millipore) were coated overnight with 4 µg/ml anti-IFN-{gamma} (R4-6A2), anti-IL-2 (JES6-1A12), anti-IL-4 (11B11), and anti-IL-5 (TRFK5) (all obtained from BD Biosciences) at 4°C. After blocking with 1% BSA, 5 x 105 cells were loaded in each well and incubated with 0–25 µg/ml Ag peptide for 36 h at 37°C. After wash, corresponding plates were incubated with 2 µg/ml biotinylated anti-IFN-{gamma} (XMG1.2), anti-IL-2 (JES6-5H4), anti-IL-4 (BVD6–24G2), and anti-IL-5 (TRFK4) (all obtained from BD Biosciences) overnight at 4°C. After wash, plates were incubated with alkaline phosphatase (Sigma-Aldrich) at 1/1000 for 2 h at room temperature, and developed in BCIP/NBT (Sigma-Aldrich) solution. Spots were counted on a computer-assisted ELISASpot Image Analyzer (Cellular Technology). For SearchLight multiplex cytokine ELISA, cell culture supernatant was collected after 48-h stimulation with 5 µg/ml Ag peptide and sent to Pierce Biotechnology for analysis. Each sample was analyzed in triplicates.

Statistical analysis

A {chi}2 test was used to analyze the difference in EAE incidence, and one-way ANOVA was used to analyze the ELISA data. Unpaired t test was used for other statistical analysis. p < 0.05 was considered as statistically significant. The error bars in figures represent SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PD-L blockade during the induction phase of EAE

BALB/c mice are resistant to EAE induction after immunization with syngeneic spinal cord homogenate (39), myelin basic protein (MBP) (40), MBP59-76 (41), or PLP40-59 peptide (39). We tested PD-1 ligand blockade in BALB/c mice during days 0–10 after MOG35-55 immunization. In the control IgG-treated group, 36% (9 of 25) of BALB/c mice developed severe clinical EAE with disease scores of 2.5 and 3 (Table I), but the remaining animals did not show any EAE signs. Sick mice rapidly recovered and the entire duration of disease was only 3.8 days on average. In mice treated with anti-PD-L1, EAE incidence was increased to 65.2% (15 of 23; p < 0.05). The peak of disease occurred earlier, around day 12 after immunization compared with day 14 in the control group, although disease onset and severity were not significantly different from the control group (Table I and Fig. 1A). PD-L2 blockade had no significant effect on EAE incidence (23.8% vs control 37.5%; p > 0.05) or other parameters (Table I and Fig. 1B). Pathological study shows that in control IgG-, anti-PD-L1-, and anti-PD-L2-treated mice, animals with no clinical EAE had few inflammatory foci in the spinal cord, whereas in animals with clinical EAE similar numbers of inflammatory foci were present in the meningeal and perivascular areas (Fig. 1C). Quantitation of CD4+ cells and F4/80+ macrophages in the CNS shows similar results (data not shown). These data suggest that EAE resistance in MOG35-55-immunized BALB/c mice is mainly reflected on EAE incidence, and PD-L1 but not PD-L2 plays a role in regulating the EAE resistance.


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  		Table I. PD-L1 and PD-L2 blockade in EAE-resistant modelsa

 

Figure 1
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  		FIGURE 1. PD-1 ligand blockade during days 0–10 postimmunization enhanced EAE in resistant strains. BALB/c mice were immunized with MOG35-55 peptide and treated with anti-PD-L1 (A) and anti-PD-L2 (B). EAE curves represent data from animals both with and without EAE. PD-L1 blockade results in an enhanced and accelerated peak on day 12, but PD-L2 blockade did not change the disease pattern. C, Spinal cord tissue was obtained on day 14 from three BALB/c mice without clinical EAE in each of rat IgG-treated, anti-PD-L1-treated, and anti-PD-L2-treated groups, and from three mice with 2.5–3 EAE grade in each group. Cross-sections of spinal cord were stained with H&E, and the numbers of inflammatory foci were counted. D, All B10.S mice immunized with PLP139-151 developed mild EAE in the control group with a mean maximal grade of 1.3 ± 0.1, compared with 3.4 ± 0.5 in anti-PD-L1 and 3.1 ± 0.4 in anti-PD-L2 treated mice (p < 0.01). E, All NOD mice immunized with PLP48-70 also developed mild EAE in the control group (mean maximal grade, 1.2 ± 0.2). PD-L1 blockade did not worsen EAE. Because some animals died from diabetes, EAE observation was terminated at day 60. F, PD-L2 blockade in NOD mice significantly exacerbated EAE especially in the chronic phase (mean maximal grade, 1.6 ± 0.2 vs 1.1 ± 0.1 in control group; p < 0.05).

 
B10.S mice are congenic with SJL/J mice (both H-2s) but are resistant to EAE induction with PLP139-151 immunization (42). We found that all B10.S mice in the control group developed mild EAE (mean maximal grade, 1.3 ± 0.1) with an average peak of disease on day 20 (Table I and Fig. 1D). Both PD-L1 and PD-L2 blockade resulted in full-blown EAE with mean maximal grade of 3.4 ± 0.5 and 3.1 ± 0.4, respectively (p < 0.01 compared with control in both groups). The chronic EAE signs were also more severe in anti-PD-L1- and anti-PD-L2-treated groups (mean disease scores at day 50 were 1.3 ± 0.3 and 1.6 ± 0.4 in anti-PD-L1 and anti-PD-L2 groups and 0.5 in all rat IgG-treated animals). These data suggest that in B10.S mice, both PD-L1 and PD-L2 contribute to EAE resistance.

NOD mice are known to develop mild EAE after PLP56-70 or PLP48-70 immunization (43, 44). We have previously reported that PD-L1 but not PD-L2 blockade rapidly precipitated diabetes in prediabetic NOD mice (27). In this study, we investigated how PD-1 ligand blockade would affect PLP48-70-induced EAE in NOD mice (Table I). We found that all rat IgG-treated animals developed mild EAE (mean maximal grade of 1.2 and 1.1 in two control groups; Fig. 1, E and F), but none developed diabetes up to 50 days after immunization, when animals were ~14 wk of age. Consistent with our previous report, PD-L1 blockade rapidly precipitated diabetes in 71% of the mice within 10 days of treatment, but the course and severity of EAE were similar to the control group (Fig. 1E). In contrast, PD-L2 blockade did not induce diabetes in any treated animals, but significantly worsened EAE severity, especially in the chronic phase (Fig. 1F). Therefore, in NOD mice, PD-L2 regulates EAE susceptibility, whereas PD-L1 regulates the development of diabetes, suggesting that PD-L differentially regulate two autoimmune diseases in the same strain.

PD-L blockade in chronic and relapsing EAE

We further examined the role of PD-L1 and PD-L2 in chronic persistent disease (B6 mice immunized with MOG35-55) and in relapsing disease (SJL mice immunized with PLP139-151) (Table II). We have previously shown that in B6 mice, PD-L2 but not PDL1 blockade during the priming phase of EAE worsened disease (25). In this study, we found consistently that PD-L2 but not PD-L1 blockade on days 30–40 rapidly worsened chronic EAE and half of the animals died from severe disease during the treatment period (Fig. 2A). In contrast, in PLP139-151-immunized SJL mice, PD-L1 blockade during remission (day 23–33) immediately brought out EAE relapses, whereas PD-L2 blockade had no significant effect (Fig. 2, B and C). Pathological analysis of spinal cord tissue obtained on day 30 from SJL mice showed that the anti-PD-L1-treated group but not the anti-PD-L2-treated group had significantly more inflammatory foci than the rat IgG-treated group (numbers of inflammatory foci were 7.1 ± 0.9, 2.5 ± 0.3, and 3.3 ± 0.5, respectively; p < 0.05). These data indicate that PD-1 ligands play an important role in regulating the chronic phase of EAE, but PD-L1 and PD-L2 have differential roles in different strains.


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  		Table II. PD-L1 and PD-L2 blockade in the chronic stage of EAEa

 

Figure 2
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  		FIGURE 2. PD-1 ligand blockade in chronic EAE worsened the disease. A, MOG35-55-immunized B6 mice developed chronic persistent EAE. PD-L2 but not PD-L1 blockade during days 30–40 markedly enhanced chronic disease (mean EAE scores at day 40 were 3.9 ± 0.3 and 2.3 ± 0.4, respectively, compared with control group 2.9 ± 0.1 (p < 0.05) when comparing between rat IgG and anti-PD-L2-treated groups), and 6 of 12 mice died from severe EAE. B and C, PLP139-151-immunized SJL/J mice developed relapsing-remitting EAE, and PD-L1 blockade started at EAE remission (days 23–33) immediately brought out EAE relapse, whereas PD-L2 blockade during days 23–33 did not precipitate EAE relapse. The treatment duration is shown by a black line in the graph.

 
Role of B cells in PD-L blockade

We have previously reported that MOG35-55-immunized B6 mice had significantly increased serum IgG against MOG35-55 after PD-1 blockade. B cells are known to express PD-1, and PD-1 signaling may inhibit their cell cycle progression (37, 45). To investigate the role of B cells in disease worsening in B6 mice, we immunized B cell-deficient mice on B6 background with MOG35-55, and found that PD-L2 but not PD-L1 blockade dramatically worsened EAE in these mice, and 4 of 5 mice died from severe disease around day 20 (Fig. 3) (mean maximal scores are as follows: rat IgG group, 3.1 ± 0.4; anti-PD-L1 group, 3.3 ± 0.1; and anti-PD-L2, 4.6 ± 0.4) (p < 0.05 compared with rat IgG group). Pathological examination of spinal cord tissue collected on day 20 showed that the anti-PD-L2-treated group, but not the anti-PD-L1-treated group, had significantly more inflammatory foci than the rat IgG-treated group (rat IgG group, 6.4 ± 0.6; anti-PD-L1 group, 6.8 ± 0.9; and anti-PD-L2 group, 11.5 ± 1.5) (p < 0.05). Thus, the effects of PD-L2 blockade in B6 mice is independent of enhanced humoral response to myelin Ag or B cell functions, and PD-L2 blockade consistently worsened EAE in mice on B6 background.


Figure 3
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  		FIGURE 3. PD-L2 but not PD-L1 blockade during days 0–10 after MOG35-55 immunization markedly enhanced EAE in B cell-deficient mice on B6 background. The mean maximal scores were 3.1 ± 0.4 in rat IgG-treated mice, 3.3 ± 0.1 in anti-PD-L1-treated mice, and 4.6 ± 0.4 in anti-PD-L2-treated mice (p < 0.05 when comparing rat IgG- and anti-PD-L2-treated group). Four of 5 anti-PD-L2-treated mice died from severe EAE on day 20 and day 21.

 
Expression of PD-L1 and PD-L2 in different strains

To study the mechanism of strain-specific responses to PD-1 ligand blockade, we examined whether lymphoid organ APCs from different strains express different levels of PD-L1 and PD-L2. We studied the expression of PD-L1 and PD-L2 by flow cytometry in spleen dendritic cells and macrophages from BALB/c, B10.S, and NOD mice on day 8 after immunization, as well as from B6 mice on day 30 and SJL mice on day 26. These time points were chosen based on when blocking Abs were administered in these strains. We found that PD-L1 expression varied little in dendritic cells among different strains, although more macrophages from NOD and SJL mice were PD-L1+ (Fig. 4A). PD-L2 was expressed on very small percentages of dendritic cells and macrophages, even in these immunized animals. NOD and SJL mice also had higher proportions of PD-L2+ cells (Fig. 4A). However, the expression of PD-L1 and PD-L2 did not correlate with specific PD-L blockade effects in the different strains.


Figure 4
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  		FIGURE 4. Comparison of PD-1 ligand expression in spleen APCs and in the CNS. A, Splenocytes were harvested from immunized BALB/c, B10.S, NOD mice on day 8, B6 mice with chronic EAE on day 30, and SJL/J mice at EAE remission on day 26. Splenocytes were depleted of CD90+ T cells, and then stained for PD-L1 or PD-L2 together with F4/80 (for macrophages) or anti-CD11c (for dendritic cells). The y-axis represents the percentage of cells expressing PD-1 ligands within the gated F4/80+ or CD11c+ population. B, PD-1 ligand expression in the CNS was quantified by analyzing the percentage of cells positive for PD-L1 or PD-L2 within the inflammatory foci in the spinal cord. Spinal cord tissue was obtained from BALB/c mice on day 14–15, B10.S and B cell-deficient mice on day 20 at EAE peak, B6 mice with chronic EAE on day 30, and SJL/J mice on day 26 during EAE remission. Spinal cord cross-sections were immunostained with anti-PD-L1 or anti-PD-L2, and counterstained with hematoxylin. PD-L1- or PD-L2-positive cells and total hematoxylin-counterstained cells were counted from individual inflammatory foci, and the percentage of inflammatory cells expressing PD-L1 or PD-L2 was calculated and averaged over nine levels of sectioning. There were three to four mice per group.

 
We have also compared among different strains the expression of PD-L1 and PD-L2 in the CNS during EAE by immunohistochemistry (Fig. 4B). Spinal cord tissues were collected at EAE peak in acute EAE models (BALB/c mice on days 14–16 postimmunization, B10.S and B cell deficient mice on day 20) and in chronic EAE models (B6 mice on day 30, and SJL/J mice on day 26). We found both PD-L were largely restricted to the inflammatory foci in all strains. At the peak of acute disease as well as in chronic EAE stage, PD-L1 was expressed on ~50–70% of the inflammatory cells within the foci, whereas PD-L2 was expressed on about one-third of inflammatory cells with some strain variations. There was also no clear correlation of CNS PD-L expression with the effects of specific ligand blockade in different strains.

Differential up-regulation of Th2 cytokines by PD-L blockade in BALB/c and B cell-deficient mice

We next investigated the effects of PD-L blockade on peripheral autoimmune response and compared MOG35-55-induced T cell proliferation and cytokine production between BALB/c mice (in which PD-L1 blockade significantly increased EAE incidence) and B cell-deficient mice (in which PD-L2 blockade markedly worsened acute EAE) on day 20 after MOG35-55 immunization. In BALB/c mice, both PD-L1 and PD-L2 blockade increased MOG35-55-induced T cell proliferation, but PD-L2 blockade induced stronger proliferation even without in vitro Ag stimulation (Fig. 5A). ELISPOT assay showed that PD-L1 and PD-L2 blockade had a similar effect in increasing IFN-{gamma}-producing cells, although PD-L2 blockade showed a higher frequency of cells spontaneously producing IFN-{gamma} in vitro. In contrast, PD-L2 blockade resulted in larger increases in IL-2-, IL-4-, and especially IL-5-producing cells than PD-L1 blockade. In contrast to BALB/c data, PD-L1 blockade in B cell-deficient mice had a stronger effect than PD-L2 blockade in increasing the proliferation and induced frequency of IL-2-, IL-4-, and IL-5-producing cells, whereas IFN-{gamma}-producing cell numbers were not markedly changed by either treatment.


Figure 5
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  		FIGURE 5. PD-L1 and PD-L2 blockade had different effects on T cell proliferation and cytokine induction in BALB/c mice and B cell-deficient mice. BALB/c mice (A) and B cell-deficient mice (B) were immunized with MOG35-55, and treated with anti-PD-L1 and anti-PD-L2 during days 0–10 with rat IgG as control. Splenocytes from three mice per group were harvested on day 20. Five hundred thousand cells were seeded into each well, and cultured with 0, 1, 5, 25 µg/ml MOG35-55. T cell proliferation was tested after 48 h, and the frequencies of cells producing IFN-{gamma}, IL-2, IL-4, and IL-5 were analyzed by ELISPOT assay.

 
We also collected culture supernatant of BALB/c splenocytes that were stimulated with 5 µg/ml MOG35-55, and measured cytokine concentration using SearchLight multiplex ELISA (Fig. 6). Consistent with ELISPOT results, we found that PD-L1 and PD-L2 blockade significantly increased the production of both Th1 cytokines (IFN-{gamma} and TNF-{alpha}) and Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) compared with rat IgG group, but PD-L2 blockade induced higher up-regulation of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) than PD-L1 blockade (p < 0.05). In addition, PD-L1 and PD-L2 blockade also significantly increased the production of IL-1{alpha}, IL-6, IL-12, and CCL2 (p < 0.05), suggesting that PD-L blockade impacts cells of the innate immune system as well. Compared with PD-L1 blockade, PD-L2 blockade induced higher up-regulation of IL-1{alpha}, IL-12, and CCL2 but not IL-6 (p < 0.05). CCL5 and CXCL2 were increased <2-fold after PD-L1 and PD-L2 blockade, and there were no significant differences between these two treatment groups (data not shown).


Figure 6
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  		FIGURE 6. PD-L1 and PD-L2 blockade during days 0–10 after MOG35-55 immunization in BALB/c mice differentially increased the production of cytokines and chemokines upon in vitro restimulation. Splenocytes were collected on day 20 from three mice in each group, and were stimulated with 5 µg/ml MOG35-55 for 48 h. Supernatants were analyzed for cytokine and chemokine concentrations using SearchLight multiplex ELISA. The numbers on each bar represent the fold of increases in anti-PD-L1- or anti-PD-L2-treated groups over the rat IgG-treated group. Symbols (*) presented over anti-PD-L2 bars represent these cytokine/chemokine concentrations were significantly higher than those in the corresponding anti-PD-L1 group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Data from our group (25) and others (26) indicate that the PD-1 pathway regulates autoimmune response in EAE. This study provides evidence for two novel observations: first, PD-1-PD-L pathway participates in determining disease susceptibility and regulating disease progression including chronicity and relapses; second, there is a genetic influence for the differential role of PD-L1 and PD-L2 in different strains.

EAE resistance offers a unique opportunity to study the regulatory mechanisms that control the autoimmune response. In BALB/c mice, immunization with syngeneic spinal cord homogenate, MBP, MBP59-76, or PLP40-59 resulted in little if any clinical EAE (39, 40, 41). Although MHC restriction may contribute to EAE resistance in BALB/c mice, MBP-specific T cell clones generated after priming are able to adoptively transfer EAE (41, 46). The disease resistance in BALB/c mice has been attributed to a Th2 bias in their immune response (47, 48, 49) and to the regulation by CTLA-4 (39). We found that MOG35-55 immunization induced severe clinical and pathological EAE in approximately one-third of BALB/c mice, whereas two-thirds of the animals had no clinical or pathological evidence of disease. There were no significant differences in MOG35-55-induced T cell proliferation and cytokine production between animals developing EAE and those without disease (data not shown). These data suggest that the immune response in BALB/c mice against MOG35-55 immunization is very close to the threshold, and once it passes the threshold, effector mechanisms can efficiently lead to severe CNS inflammation and clinical EAE. Our data show that PD-L1 but not PD-L2 blockade increased EAE incidence in BALB/c mice, although both treatments markedly enhanced MOG35-55-induced T cell proliferation, suggesting that the expansion of Ag-specific T cells is not by itself sufficient to enhance EAE development. An interesting difference was observed in the cytokine profile, with PD-L2 blockade inducing larger increases in Th2 cytokine production than PD-L1 blockade, even in unstimulated cultures, suggesting that Th2 cells were strongly activated and cycling in vivo. Consistent with this relative preponderance of Th2 cytokine production, it has been reported that PD-L2 but not PD-L1 blockade worsened asthma in BALB/c mice (29, 50). Furthermore, adoptively transferred CD4+ T cells from DO11.10 mice were found to express increased IL-4 mRNA in vivo when recipient BALB/c mice were immunized with OVA peptide-pulsed dendritic cells from B7-DC–/– mice compared with dendritic cells from B7-DC+/+ mice (36). Because STAT4/T-bet and STAT6/GATA-3 are the major transcription factors of Th1 and Th2 differentiation, examination of EAE outcome upon PD-L blockade in mice knockout of these genes may further reveal the mechanisms by which PD-L regulate EAE pathogenesis.

APCs play an important role in determining the type and degree of T cell responses through providing costimulatory signals and establishing local cytokine milieu (51). Our data show that PD-L blockade markedly increased the production of IL-1, IL-6, IL-12, and CCL2 in BALB/c splenocyte culture, indicating enhanced activation of APCs after PD-L1 and PD-L2 blockade. We have found that spleen dendritic cells, macrophages, and B cells are able to up-regulate PD-1 expression after LPS and IFN-{gamma} stimulation (B. Zhu and S. J. Khoury, unpublished observations). It is possible that PD-1 signaling in APCs directly controls the level of their activation. In contrast, enhanced T cell activation upon PD-L blockade may provide stronger positive feedback to APCs through increased CD40 and cytokine signaling. In addition, we found that CCL2, which is an important Th2 differentiation factor, was preferentially induced after PD-L2 blockade. Intriguingly, it has been reported that CCL2 could enhance the expansion of alloreactive CD4+ T cells in vivo, and neutralization of CCL2 increased PD-L1 expression on these T cells (52). Collectively, these results may suggest an antagonistic relationship between PD-L and CCL2.

In contrast, EAE in B cell-deficient mice, which was induced with same Ag MOG35-55, was enhanced by PD-L2 rather than PD-L1 blockade. This is consistent with the effects of PD-L blockade in B6 mice at both priming and chronic stages. In this strain, the cytokine balance in favor of Th2 profile was induced by PD-L1 blockade rather than PD-L2 blockade. It has been reported that Th1 and Th2 cytokines may increase PD-L1 and PD-L2 expression, respectively, on macrophages (53). In general, BALB/c mice are Th2-prone, whereas B6 mice are Th1-prone in immune responses (54). Although our data did not reveal any correlation between PD-L expression on APCs and PD-L blocking effects in EAE, it remains possible that PD-L1 and PD-L2 are differentially up-regulated in specific APC subpopulations (e.g., CD8{alpha}+ and CD8{alpha}– dendritic cells) across strains due to differential cytokine milieu. In contrast, there may be strain differences in the interaction between other immune regulatory mechanisms and PD-L/PD-1 pathway. It has been shown in a transplantation model that PD-L1 blockade enhanced Th1 differentiation, but CD4+CD25+ T cell depletion abrogated this effect (55). It will be important to investigate the relationship between CD4+CD25+ T cells and PD-L/PD-1 pathway in different strains.

Although PD-L2 blockade in BALB/c mice and PD-L1 blockade in B6 mice greatly increased Th2 responses, we did not observe significant EAE suppression with these treatments. Markedly enhanced T cell proliferation suggests that the pool of Ag-specific T cells may be greatly increased after PD-L blockade. It has also been reported that PD-L may regulate the apoptosis of activated T cells (30). Therefore, PD-L may control T cell expansion, differentiation, and cell death in EAE pathogenesis. Our data suggest that Ag-specific T cell expansion induced by PD-L blockade may enhance EAE when Th1/Th2 balance is little altered, but this effect could be offset when a strong Th2 shift occurs.

We have also studied PD-L function in B10.S and NOD EAE models. B10.S mice have multiple genetic loci associated with their resistance to EAE induction by PLP139-151 (42, 56). It has recently been reported that depletion of regulatory T cells significantly worsened EAE in B10.S mice (57). Interestingly, we found that both PD-L1 and PD-L2 blockade markedly enhanced EAE, suggesting that the suppression by PD-L is another mechanism controlling EAE susceptibility in B10.S mice. In NOD mice, we have previously reported that PD-L1 but not PD-L2 blockade precipitates diabetes in NOD mice (27). It is known that immunization with Freund’s adjuvant can prevent NOD mice from developing spontaneous diabetes (58). We found PD-L1 blockade still rapidly induced diabetes in the majority of NOD mice even after MOG35-55 immunization. In addition, we show in this study that PD-L2 but not PD-L1 blockade worsens PLP48-70-induced EAE. Further investigations are needed to determine whether Th1/Th2 responses for EAE and diabetes are differentially regulated by the same PD-L blockade, or whether other regulatory pathways play a more critical role in this model. Different pathogenic mechanisms in these two autoimmune diseases have been suggested by differential genetic regulation. Idd5.1 on chromosome 1 contains the ctla4 and icos genes, and is a susceptibility locus for type 1 diabetes in NOD mice (59). Activated T cells from NOD mice express higher ICOS and lower full-length CTLA-4 compared with cells from B6 mice and C57BL/10 mice (59). NOD.B10 Idd5 congenic mice were found to be resistant to diabetes, but were more susceptible to MOG35-55-induced EAE than NOD mice (59). The functional interaction of PD-L with ICOS and CTLA-4 remains to be investigated.

Furthermore, we found that PD-L have important regulatory function in chronic and relapsing EAE. PD-L2 blockade rapidly worsened chronic persistent EAE in B6 mice, whereas PD-L1 blockade in EAE remission precipitated relapses in SJL/J mice. It has recently been reported that PD-1 ligand blockade in vitro enhanced proliferation and cytokine production in memory T cells (60), and our chronic EAE models are useful in studying how PD-L regulate memory T cell activation in vivo. Recently, it was reported that transfer of genetically modified dendritic cells overexpressing MOG peptide/MHC II and PD-L1 suppressed EAE (61), opening the door for potential therapeutic strategies using PD-1 signaling molecules, such as PD-L1-Ig (22) and PD-L2-Ig (23).

In summary, our data indicate that PD-1 ligands play important roles in determining EAE susceptibility and in controlling chronic disease progression. The strain-specific effects in response to PD-L blockade may relate to genetically determined mechanisms in regulating Th1/Th2 differentiation. Defining the exact nature of these interactions will further our understanding of the susceptibility and pathogenic mechanisms of autoimmune responses, and may lead to the development of novel therapies for autoimmune diseases.


   Acknowledgments
 
We thank Dr. Byron Waksman for helpful discussions.


   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 AI058680 from National Institutes of Health (NIH) and RG3504 from National Multiple Sclerosis Society (NMSS) (to S.J.K.), Grant PO1 AI50157 from NIH and Juvenile Diabetes Research Foundation Center Grant on Immunological Tolerance in Type 1 Diabetes (to M.H.S.), American Society of Transplantation Basic Scientist Faculty Grant (to I.G.), and Grant FG1569-A-1 from NMSS (to B.Z.). Back

2 Address correspondence and reprint requests to Dr. Samia J. Khoury, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, HIM 712, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: skhoury{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: PD-1, programmed death-1; PD-L, PD-1 ligands; MOG, myelin oligodendrocyte glycoprotein; EAE, experimental autoimmune encephalomyelitis; PLP, myelin proteolipid protein; 7-AAD, 7-aminoactinomycin D; MBP, myelin basic protein. Back

Received for publication October 24, 2005. Accepted for publication December 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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