Background
Multiple sclerosis (MS) is the leading cause of neurologic disability in the US in young adults after trauma; thus, most patients suffer from the effects of MS for most of their adult life. Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated autoimmune disease of the central nervous system (CNS), which has served as an animal model for MS for several decades. The formation of acute inflammatory MS lesions is mediated by myelin-specific, autoreactive T cells [
1]. Previous EAE studies have shown that both IFNγ producing Th1 cells and IL-17 producing Th17 cells can be highly encephalitogenic effector T cells, although they have distinct cytokine profiles [
2‐
6]. However, both IFNγ and IL-17 deficient mice are still susceptible to EAE induction [
7,
8], suggesting that molecules other than the signature cytokines may contribute to the regulation of the effector function and encephalitogenicity of myelin-specific Th1 and Th17 cells.
The inhibitory receptors are important immune checkpoints that negatively regulate immune responses to prevent tissue damage and autoimmunity. The roles of inhibitory receptors in the regulation of T cell effector function have been well-established in T cell exhaustion, which was identified during chronic viral infection and observed in tumor microenvironment. The axis of PD-1 and its ligand is a central regulator of T cell exhaustion, although multiple inhibitory receptors, including Lag-3, CTLA-4, Tim3, CD244/2B4, CD160, TIGIT, are involved [
9,
10]. Blockade of the PD-1 pathway partially reversed T cell exhaustion and reduced viral or tumor load [
11‐
13], which indicated that dysfunctional T cells could be modulated by manipulating the PD-1 pathway, with implications for the treatment of diseases including chronic infections and cancer. As a result, anti-PD-1 therapy has been developed and shown remarkable success for treating human cancer. Meanwhile, in the context of autoimmunity, recent studies have identified the antagonistic effects of IL-7Rα and the inhibitory receptor PD-1 on effector T cells as essential parts of the cell-intrinsic immunoregulatory program of T cell effector function. The IL-7Rα expression on T effector/memory cells serves as an on-switch of T effector cell function, while the expression of the inhibitory receptor PD-1 serves as an off-switch to suppress the effector function of T cells, which plays an important role in the pathogenesis of autoimmune diabetes [
14,
15]. Although both IL-7Rα [
16‐
21] and the inhibitory receptor PD-1 [
22‐
24] have been implicated in MS/EAE pathogenesis, it is not clear whether the key cytokines and/or transcription factors that are critical for T cell encephalitogenicity regulate IL-7Rα/PD-1 balance of myelin-specific CD4 T effector/memory cells during EAE development. Therefore, in this study, we first analyzed the roles of the transcription factor T-bet in the regulation of the expression of IL-7Rα and inhibitory receptors in myelin-specific CD4 T cells in vitro and in vivo. Furthermore, we compared the effects of different inflammatory cytokines that are crucial for Th1 and Th17 development in regulating the IL-7Rα/PD-1 balance in vitro and in vivo.
Methods
Animals
B6/WT and B6/T-bet
−/− mice were purchased from the Jackson Laboratory and bred in a specific pathogen-free animal facility at the Ohio State University (OSU) Wexner Medical Center. B10.PL mice transgenic for the MBP Ac1-11-specific TCR chains Vα2.3 or Vβ8.2 [
25] were also bred in a specific pathogen-free animal facility at the OSU Wexner Medical Center. All animal protocols were approved by the OSU Institutional Animal Care and Use Committee.
In vitro culture of splenocytes from TCR transgenic mice
Splenocytes were prepared from naive 5–10-week-old Vα2.3/Vβ8.2 TCR transgenic mice and cultured in 24-well plates at 2 × 10
6 cells/well with irradiated B10.PL splenocytes (6 × 10
6 cells/well). Cells were activated with MBP Ac1-11 (2 μg/ml) and different combination of cytokines or neutralizing antibodies for cytokines to differentiate effector T helper cells. Cytokines and antibody concentrations were as follows: 0.5 ng/ml IL-12, 25 ng/ml IL-6, 1 ng/ml TGFβ1, 2 μg/ml anti-IFNγ, 1 μg/ml anti-IL-12, 2 μg/ml anti-IL-4, and 0.35 μg/ml anti-TGFβ [
6].
EAE induction
Immunization
The 8–10-week-old B6/WT, B6/T-bet+/−, or B6/T-bet−/− mice were s.c. injected over four sites in the flank with 200 μg MOG 35-55 (CSBio Company Inc.) in an emulsion with CFA (Difco); 200 ng pertussis toxin (List) per mouse in PBS was injected i.p. at the time of immunization and 48 h later.
Adoptive transfer
Splenocytes were isolated from naïve 5–10-week-old Vα2.3/Vβ8.2 TCR transgenic mice and activated with 2 μg/ml of MBP Ac1-11 with or without rmIL-7 (10 ng/ml) or αIL-7Rα (0.5 μg/ml) in 24-well plates at 2 × 106 cells/well with irradiated B10.PL splenocytes (6 × 106 cells/well). After 72 h, the cells were washed with PBS and 8 × 106 cells/mouse were injected i.p. into naive B10.PL mice.
The mice were evaluated daily for clinical signs of EAE. Mice were scored on scale of 0 to 6: 0, no clinical disease; 1, limp/flaccid tail; 2, moderate hind limb weakness; 3, severe hind limb weakness; 4, complete hind limb paralysis; 5, quadriplegia or premoribund state; and 6, death.
ELISA
ELISA was performed to detect the expression of IL-17 and IFNγ in supernatant. Purified anti-mouse IL-17 primary antibody (BD Biosciences) was diluted in 0.1 M NaHCO3 (pH 8.2) at 2 μg/ml while purified anti-mouse IFNγ primary antibody was diluted in 0.1 M NaHCO3 (pH 9.5) at 2 ug/ml. Immunolon II plates (Dynatech Laboratories) were coated with 50 μl of primary antibodies per well and incubated overnight at 4 °C. The plates were washed twice with PBS/0.05% Tween 20. The plates were blocked with 200 μl of 1% BSA in PBS per well for 2 h. The plates were washed twice with PBS/0.05% Tween 20, and 100 μl of supernatants were added in duplicate. The plates were incubated over-night at 4 °C and washed four times with PBS/0.05% Tween 20. Biotinylated rat anti-mouse secondary antibody (BD Biosciences) were diluted in PBS/1% BSA, 100 μl of 1 μg/ml biotinylated antibody was added to each well, and plates were incubated at room temperature for 1 h. The plates were washed six times with PBS/0.05%Tween 20, and 100 μl avidin-peroxidase was added at 2.5 μg/ml and incubated for 30 min. The plates were washed eight times with PBS/0.05% Tween 20, and 100 μl ABTS substrate containing 0.03% H2O2 (for IL-17) or TMB substrate (for IFNγ) was added to each well. The plate was monitored for 10–20 min for color development and read at A 405. A standard curve was generated from cytokine standard, and the cytokine concentration in the samples was calculated.
Intracellular staining and flow cytometric analysis
Flow cytometric analysis was performed to evaluate the expression of surface markers and T-bet in CD4 T cells, as previously described [
6]. Briefly, splenocytes were activated with antigen or αCD3/CD28 for 48 to 72 h. Cells were then collected, washed, and resuspended in staining buffer (1% BSA in PBS). The cells were incubated with mAbs to the cell-surface markers for 30 min at 4 °C. After washing twice with staining buffer, cells were fixed and permeabilized using Cytofix/Cytoperm solution for 20 min at 4 °C. Cells were stained for intracellular cytokines and T-bet for 30 min at 4 °C. The 80,000–100,000 live cell events were acquired on a FACSCanto (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.). PerCP-anti-CD4 and Pacific Blue-anti-CD44 were purchased from BD Biosciences. PE-anti-PD-1, PE-Cy7-anti-IL-7Rα, and Pacific Blue-anti-T-bet were purchased from Biolegend Biotechnology, Inc.
Statistical analysis
GraphPad software (GraphPad Prism Software, Inc., San Diego, CA, USA) was utilized for statistical analysis. A statistically significant difference in EAE clinical scores was considered to be P < 0.05, as determined by Mann–Whitney U test. The Mann–Whitney U test is non-parametric, and therefore accounts for the fact that EAE scores are ordinal and not interval-scaled. ELISA and quantitated flow data comparisons were performed using two-tailed unpaired student’s t tests. Differences with P < 0.05 were considered significant.
Discussion
IFNγ producing Th1 cells and IL-17 producing Th17 cells are highly encephalitogenic in the EAE model of MS, although they have distinct signature cytokine profiles, prompting us to hypothesize that molecules other than the signature cytokines regulate the effector function and contribute to the encephalitogenicity of both myelin-specific Th1 and Th17 cells. IL-7Rα and the inhibitory receptor PD-1 are essential parts of the cell-intrinsic immunoregulatory program regulating CD4 T effector function. Although both IL-7Rα and PD-1 have been implicated in the pathogenesis of MS/EAE, the factors regulating their expression in myelin-specific CD4 T cells during EAE development are not well-elucidated. This study aims to determine if the key factors regulating T cell encephalitogenicity of myelin-specific Th1 and Th17 cells, including transcription factor T-bet and cytokines (IL-12, IL-6, and IL-23), may exert their function through regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells during EAE development.
T-bet is the key transcription factor regulating the differentiation of Th1 cells. T-bet deficient mice were originally shown to be resistant to EAE induction by active immunization [
31], but later studies showed that T-bet deficient mice are still susceptible to EAE induction and T-bet is essential for Th1-mediated, but not Th17-mediated, CNS autoimmune disease [
27,
37]. Although these results from genetically engineered mice appear to contradict each other, other studies support an important role of T-bet in EAE [
28‐
30] and MS [
38,
39] as a potential therapeutic target. Our data showed that T-bet is a major regulator of IL-7Rα/PD-1 balance in myelin-specific CD4 T effector/memory cells differentiated in vitro and during EAE development in vivo. T-bet suppresses the expression of inhibitory receptors, which is similar to what was observed in CD8 T cells during chronic infection [
32]. Meanwhile, T-bet enhances IL-7Rα expression in myelin-specific CD4 T cells. IL-7Rα expression in myelin-specific CD4 T cells is dysregulated when T-bet is deficient. Upon antigen encounter, myelin-specific CD4 T cells from WT mice upregulate IL-7Rα, but myelin-specific CD4 T cells from T-bet deficient mice fail to upregulate IL-7Rα after primary stimulation. After CD4 T cells are rested for 4 days, IL-7Rα expression is downregulated in myelin-specific CD4 T cells from WT mice but is upregulated in T-bet deficient myelin-specific CD4 T cells. After antigen restimulation, IL-7Rα expression is similar between two groups while T-bet deficient myelin-specific CD4 T cells have notably higher PD-1 expression. Altogether, our data suggest that T-bet is a key transcription factor regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells.
After the identification of Th17 cells as another encephalitogenic CD4 T helper population in addition to Th1 cells in EAE, the search for the potential therapeutic targets that convey the encephalitogenicity to myelin-specific CD4 T cells becomes even more complicated. Although IFNγ producing Th1 and IL-17 producing Th17 cells are both encephalitogenic, they have distinct cytokine profile, which raises the question whether encephalitogenic CD4 T cells exert their function mainly through the production of signature cytokines. Both IFNγ and IL-17 deficient mice are still susceptible to EAE induction [
7,
8]. On a related note, we previously showed that myelin-specific Th17 cells induced with IL-6 in the absence of Th1 and Th2 signaling are highly encephalitogenic following adoptive transfer while myelin-specific Th17 cells induced with the combination of TGFβ and IL-6, although producing large amounts of IL-17, are not encephalitogenic [
6,
40]. These data clearly argue that there are molecules other than the signature cytokines responsible for the encephalitogenicity of myelin-specific CD4 T cells, although the detailed mechanisms are still unclear. IL-12 and IL-6 are two critical cytokines for the differentiation of encephalitogenic Th1 and Th17 differentiation, respectively. Our data showed that IL-12 and IL-6 have similar effects in regulating IL-7Rα/PD-1 balance by skewing the balance towards IL-7Rα in both Th1 and Th17 cells. On the other hand, TGFβ1/IL-6 induced non-encephalitogenic Th17 cells have an IL-7Rα/PD-1 balance skewed towards PD-1. These data suggest that IL-7Rα/PD-1 balance is a common mechanism shared by both Th1 and encephalitogenic Th17 cells to regulate effector function. Therefore, it may be possible to target both Th1 and Th17 cells by manipulating IL-7Rα/PD-1 balance.
Targeting PD-1/PD-L1 pathway for therapeutic purposes has been explored in cancer and chronic viral infection [
41]. Contrary to autoimmunity, tumor cells upregulate PD-L1 which binds its receptors (PD-1, etc.) on T effector cells, thus paralyzing T cells, suppressing tumor immunity, and allowing the tumor to evade immune attack. Therefore, anti-PD therapy has been developed and tested in clinical trials and these trials have shown remarkable success for treating human cancer, especially solid tumors. The FDA recently approved two PD-1 monoclonal antibodies to treat human cancers. Additionally, multiple monoclonal antibodies to either PD-1 or PD-L1 are under active development in clinical trials [
42,
43]. Similarly, in the scenario of chronic viral infection, after prolonged exposure to antigen and inflammation, exhausted T cells express high levels of PD-1 and other inhibitory receptors, resulting loss of robust effector function [
9]. Preclinical data have shown that targeting PD-1/PD-L1 pathway can improve T cell responses and viral clearance [
10,
42].
In the context of autoimmunity, our data demonstrate that several major determinants of T cell encephalitogenicity, including T-bet, IL-12, and IL-6, skew IL-7Rα/PD-1 balance towards IL-7Rα, favoring an encephalitogenic phenotype of myelin-specific CD4 T cells with enhanced effector function. Our data show that IL-7 signaling inhibits PD-1 expression in myelin-specific CD4 T cells and blockade of IL-7R signaling on myelin-specific CD4 T cells significantly decreased the encephalitogenic potential of those cells. Therefore, skewing IL-7Rα/PD-1 balance towards PD-1 by either stimulating PD-1/PD-L1 pathway or suppressing IL-7Rα signaling may have therapeutic potential for the treatment of autoimmune diseases, including MS.
Conclusions
In this study, we characterized the factors regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T effector/memory cells during EAE development. We have shown that T-bet is a major transcription factor regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells, and there is a positive correlation between several major determinants promoting T cell encephalitogenicity (T-bet, IL-6, IL-12) and an IL-7Rα/PD-1 balance skewed towards IL-7Rα, suggesting that those major determinants critical to T cell encephalitogenicity may exert their function through regulation of IL-7Rα/PD-1 balance. Additionally, IL-7 signaling inhibits PD-1 expression in myelin-specific CD4 T cells and blocking IL-7 signaling suppresses T cell encephalitogenicity. Therefore, interference with inhibitory pathways and IL-7Rα expression may suppress the encephalitogenic potential of myelin-specific CD4 T cells and have therapeutic benefits for MS patients.
Acknowledgements
Not applicable.