Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis

Abstract

Experimental autoimmune encephalomyelitis (EAE) is mediated by autoantigen-specific T cells dependent on critical costimulatory signals for their full activation and regulation. We report that the programmed death-1 (PD-1) costimulatory pathway plays a critical role in regulating peripheral tolerance in murine EAE and appears to be a major contributor to the resistance of disease induction in CD28-deficient mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) there was a progressive increase in expression of PD-1 and its ligand PD-L1 but not PD-L2 within the central nervous system (CNS) of mice with EAE, peaking after 3 wk. In both wild-type (WT) and CD28-deficient mice, PD-1 blockade resulted in accelerated and more severe disease with increased CNS lymphocyte infiltration. Worsening of disease after PD-1 blockade was associated with a heightened autoimmune response to MOG, manifested by increased frequency of interferon gamma-producing T cells, increased delayed-type hypersensitivity responses, and higher serum levels of anti-MOG antibody. In vivo blockade of PD-1 resulted in increased antigen-specific T cell expansion, activation, and cytokine production. Interestingly, PD-L2 but not PD-L1 blockade in WT animals also resulted in disease augmentation. Our data are the first demonstration that the PD-1 pathway plays a critical role in regulating EAE.

Figure 1.

Expression of PD-1, PD-L1, and PD-L2 in the CNS of mice with EAE. (a) Time course of expression of PD-1, PD-L1, and PD-L2. Immunohistochemistry for PD-1, PD-L1, and PD-L2 expression in spinal cord sections from animals with EAE at different time points after immunization for up to 1 mo. The numbers above each plate represent the time after immunization when the animals were killed and the sections were stained. There is a progressive increase in the PD-1 and PD-L1 expression, which appears to peak by day 21 and begin to decline thereafter, mirroring the clinical tempo of disease (×100), whereas there is no PD-L2 staining until day 30 when it appears minimally. (b) Expression of PD-L1 on resident brain cells during EAE. Confocal immunohistochemistry demonstrating expression of PD-L1 on astrocytes (costained with GFAP on day 20 of EAE) and microglia (costained with lectin IB4 on day 30 of EAE; all ×40). No expression of PD-L2 was found on resident brain cells.

Figure 2.

EAE outcome in animals treated with PD-1 pathway blockade. The disease scores for control and anti–PD-1–treated animals >40 d of follow up are shown, for WT mice with early therapy (a) and delayed therapy (b), and for CD28-deficient mice (d). There is a highly significant difference in disease severity between the early therapy and control groups (P = 0.0053 for WT and P = 0.0074 for CD28-deficient mice by two-tailed Mann-Whitney U test). No difference was found with delayed therapy. PD-L2 blockade also augmented disease (P = 0.03 compared with control by two-tailed Mann-Whitney U test) whereas PD-L1 blockade had no effect (c).

Figure 3.

T cell and antibody responses to MOG in animals treated with PD-1 blockade. ELISPOT analysis from one representative experiment, demonstrating the frequency of MOG-specific IFN-γ–producing T cells at different concentrations of antigen for WT (a) and CD28-deficient animals (b). In the WT group, anti–PD-1–treated animals (solid bars) had a higher frequency of MOG-specific IFN-γ–producing T cells at all antigen concentrations compared with controls (hatched bars, *, P = 0.017 by one-way ANOVA). In the CD28-deficient mice, a significant difference was also seen at higher antigen concentrations (*, P = 0.0254 by one-way ANOVA). (c) DTH measurement assessed by an increase in footpad skin thickness after intradermal injection with 50 μg antigen. DTH response was significantly greater in the anti–PD-1–treated animals than controls (*, P = 0.0245 by two-tailed Mann-Whitney U test). (d) Serum level of anti-MOG antibodies obtained on day 14 after immunization were greater in anti–PD-1–treated animals than controls in both WT and CD28-deficient animals (*, P = 0.0476; **, P = 0.0009 by two-tailed Mann-Whitney U test).

Figure 4.

Immunohistology of the CNS in animals treated with PD-1 blockade. Spinal cord sections from WT and CD28-deficient animals treated with anti–PD-1 or control antibody were obtained on day 14 after immunization and stained with anti-CD4, anti-CD8, and anti-F4/80. There is a visible increase in the number of CD4⁺ and CD8⁺ T cells as well as the macrophages (F4/80⁺ cells) in the anti–PD-1–treated mice, compared with controls in both the WT and CD28-deficient animals, more apparent in the former (×200).

Figure 5.

Effect of PD-1 blockade on antigen-specific cells in vivo. DO11.10 TCR Tg cells (3 × 10⁶ cells per mouse) were transferred into BALB/c mice. 4 d after priming with OVA peptide, the draining lymph nodes were collected and the number of OVA-specific (KJ1-26⁺) CD4⁺ T cells was measured. (a) Flow cytometry plot of CD4⁺ KJ1-26⁺ cells from control IgG–treated animals, demonstrating that 1.54% of lymph node cells were TCR transgenic CD4⁺ T cells. (b) By comparison, in the anti–PD1–treated animal 2.5% of lymph node cells were TCR transgenic. (c) Calculating the absolute numbers of TCR transgenic cells demonstrates a significant increase in KJ1-26⁺ CD4⁺ T cells in the anti–PD-1–treated animals compared with controls (P < 0.0001 by two-tailed Mann-Whitney U test).