Regulation of autoreactive CD4 T cells by FoxO1 signaling in CNS autoimmunity

Background Myelin-specific CD4 T effector cells (Teffs), Th1 and Th17 cells, are encephalitogenic in experimental autoimmune encephalomyelitis (EAE), a well-defined murine model of multiple sclerosis (MS) and implicated in MS pathogenesis. Forkhead box O 1 (FoxO1) is a conserved effector molecule in PI3K/Akt signaling and critical in the differentiation of CD4 T cells into T helper subsets. However, it is still unclear whether FoxO1 may be a target for redirecting CD4 T cell differentiation and benefit CNS autoimmunity. Methods Using a selective FoxO1 inhibitor AS1842856, we determined the effects of FoxO1 inhibition in regulating myelin-specific Th1 and Th17 cells, and the transcriptional balance of T-bet and Foxp3 in myelin-specific CD4 T cells from EAE mice. The effects of AS1842856 in regulating the encephalitogenicity of myelin-specific T cells and the expansion of human Th1 cells from MS patients were also characterized. Furthermore, we characterized the potential role of FoxO1 in mediating PD-1 signaling in CD4 T cells, critical for regulating Teff and Treg cells. suppressed the expansion of Th1 cells. Furthermore, the immune checkpoint programmed cell death protein-1 (PD-1)-induced Foxp3 expression in CD4 T cells was impaired by FoxO1 inhibition, consistent with a bias toward Treg induction. Conclusions: These data illustrate an important role of FoxO1 signaling in CNS autoimmunity via regulating autoreactive Teff and Treg balance.


Introduction
Multiple Sclerosis (MS) is an immune-mediated central nervous system (CNS) disease characterized by neuroinflammation, demyelination, and neuronal degeneration. MS is the leading cause of non-traumatic neurologic disability in young adults and affects over 1 million people in US (1). Myelin-specific CD4 T effector cells (Teff), Th1 and Th17 cells, drive the formation of acute inflammatory demyelinating lesions and clinical relapses in experimental autoimmune encephalomyelitis (EAE) model of MS and have been implicated in MS pathogenesis (2,3). Early studies demonstrated Th1 cells that express key transcription factor T-bet and signature cytokine IFN to be highly encephalitogenic in EAE (4). Subsequent studies have identified Th17 cells as another encephalitogenic CD4 Teff population in EAE. Th17 cells display a high degree of plasticity upon antigen reencounter, depending on the inflammatory milieu in the microenvironment (5). TGFβ/IL-6 induces the development of Th17 cells that express the transcription factor RORt and cytokine IL-17. However, they are not encephalitogenic in EAE adoptive transfer studies (6)(7)(8). In a microenvironment that is rich in IL-12 and/or IL-23, nonencephalitogenic RORt + Th17 cells may transdifferentiate into RORt + T-bet + Th1-like cells and further convert into RORt -T-bet + Th1 cells, both of which are highly pathogenic in MS/EAE and other autoimmune diseases (9)(10)(11)(12).
Forkhead box O 1 (FoxO1) is a conserved effector molecule in PI3k/Akt signaling that regulates CD4 T cell development and function. The role of FoxO1 in regulating myelin-specific Th1 in CNS autoimmunity has not been well-characterized. Although FoxO1 has been shown to suppress TGFβ/IL-6-induced differentiation of naïve CD4 T cells into RORt + Th17 cells (13), its role in regulating Th17 transdifferentiation is unclear. FoxO1 plays a critical role in maintaining naive T cell quiescence and survival. As several FoxO-targeted genes are important regulators of 5 naïve T cell trafficking and functions (14)(15)(16), FoxO1 deficiency may alter the quiescence of naïve CD4 T cells. Therefore, we took a pharmacological approach to characterize the potential role of FoxO1 signaling in regulating autoreactive CD4 Teff and Treg cells. AS1842856 is a small molecule compound that binds FoxO1 and inhibits FoxO1 transactivation (17). AS1842856 has been used extensively to determine the role of FoxO1 in regulating glucose metabolism, pulmonary hypertension, adipocyte differentiation and cancer development (17)(18)(19)(20)(21). To understand the potential role of FoxO1 signaling in CNS autoimmunity, we determined the effects of AS1842856 in regulating myelin-specific Th1 and Th17 cells, and the transcriptional balance of T-bet and Foxp3 in myelin-specific CD4 T cells from EAE mice. The effects of AS1842856 in regulating the encephalitogenicity of myelin-specific T cells and the expansion of human Th1 cells from MS patients were also characterized. Furthermore, we characterized the potential role of FoxO1 in mediating PD-1 signaling in CD4 T cells, critical for regulating Teff and Treg cells.

Materials and Methods
Animals C57BL/6, SJL/J mice and TCR transgenic 2D2 mice that are specific for myelin oligodendrocyte glycoprotein (MOG) 35-55 were purchased from the Jackson Laboratory. B10.PL mice transgenic for the myelin basic protein (MBP) Ac1-11-specific TCR Vα2.3 or Vβ8.2 (22) were bred in a pathogen-free animal facility at Ohio State University. Age 8-12 wks male and female C57BL/6 and B10.PL mice and female SJL/J mice were used for EAE studies as male SJL/J mice are resistant to EAE induction. Animal protocols were approved by the OSU Institutional Animal Care and Use Committee. 6

Human subjects
All MS patients were treatment-naive for immunomodulatory drugs. Blood was obtained by leukapheresis from MS patients after informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated over a Ficoll gradient and stored in liquid nitrogen until further use. The study was performed under OSU Internal Review Board protocol # 2015H0076 with written informed consent received from participants prior to inclusion in the study.

EAE induction
Immunization: EAE was induced in 8-10 week old female SJL/J mice by subcutaneously injection (s.c.) over four sites in the flank with 200 µg proteolipid protein (PLP) 139-151 respectively (C S bio) in an emulsion with CFA (Difco). 200 ng pertussis toxin (List) per mouse in PBS was injected intraperitoneally (i.p.) at the time of immunization.

In vitro induction and expansion of Th1 and iTreg cells
Naïve CD4 T cells were purified from splenocytes of WT mice using Miltenyi naïve CD4 isolation kit. For Th1 induction in Figure 1A, naïve CD4 T cells were cultured on 24-well plates coated with 1 g/ml of αCD3/CD28 (Biolegend) plus IL-12 (0.5 ng/ml) and AS1842856 (or DMSO) for 72h.

Statistics
GraphPad software (GraphPad Prism Software, Inc., San Diego, CA, USA) was utilized for statistical analysis. Quantitated flow data comparisons were performed using two-tailed unpaired Student's t-test with two groups, one-way ANOVA with three or more groups or twoway ANOVA in Figure 2 G-H and Figure 3D. Mann-Whitney U-test was used to compare EAE data in Figure 4 A-B. Wilcoxon matched-pairs signed rank test was used to compare flow data in

FoxO1 inhibition with AS1842856 suppresses Th1 differentiation and expansion
Th1 cells are highly encephalitogenic in EAE and implicated in MS pathogenesis. To understand the potential role of FoxO1 signaling in CNS autoimmunity, we first determined whether FoxO1 inhibition with AS1842856 affects the differentiation of naïve CD4 T cells into Th1 cells. AS1842856 potently blocks FoxO1 activity at 0.05-1 µM without showing significant cellular cytotoxicity (17). Thus, AS1842856 was used at 0.05 and 0.1 µM during Th1 differentiation. Naive CD4 T cells were activated with αCD3/CD28 plus IL-12, in the presence of AS1842856 or vehicle control DMSO ( Figure 1A-B). Th1 key transcription factor T-bet and 9 signature cytokine IFN were analyzed. T-bet + ( Figure 1A) and IFN + CD4 T cells ( Figure 1B) were significantly lower in AS1842856-treated groups compared to the control group, suggesting FoxO1 inhibition with AS1842856 suppresses the differentiation of naïve CD4 T cells into Th1 cells. To determine whether FoxO1 inhibition alters the expansion of myelin-specific Th1 cells from EAE mice, dLNs from immunized SJL/J mice were activated in vitro with PLP 139-151 alone or plus IL-12 for 3-6 days, in the presence of AS1842856 or vehicle control DMSO ( Figure   1C-D). There was no significant difference of IFN + CD4 T cells between AS1842856 and the control group when activated with myelin antigen alone ( Figure 1C-D upper panels). However, in the presence of IL-12, IFN + Th1 cells were significantly lower in AS1842856 groups compared to control groups at both day 3 and day 6 post reactivation ( Figure 1C-D lower panels), suggesting FoxO1 inhibition significantly suppresses IL-12-induced IFN expression of effector/memory myelin-specific Th1 cells from EAE mice. These data illustrate the role of FoxO1 inhibition in suppressing the differentiation and expansion of myelin-specific Th1 cells.

FoxO1 inhibition suppresses the transdifferentiation of myelin-specific Th17 cells
Th17 cells are also a subset of CD4 Teff cells that can be encephalitogenic in EAE and implicated in MS pathogenesis (4). Although TGFβ/IL-6 induces the differentiation of naïve CD4 T cells into Th17 cells that express transcription factor RORγt and cytokine IL-17, those Th17 cells are not encephalitogenic in adoptively transferred EAE studies (6)(7)(8). Upon antigen reencounter, non-encephalitogenic RORγt + Th17 cells may transdifferentiate into Th1-like Th17 cells that co-express RORγt and the Th1 key transcription factor T-bet (RORγt + T-bet + ), which are highly encephalitogenic in adoptive transfer, and may further convert into Th1 cells that do not express RORγt (RORγt -T-bet + ) (5,(9)(10)(11)(12). While Th17 transdifferentiation may occur naturally, IL-12 and IL-23 promote Th17 transdifferentiation. mTORC1 has been shown to be important for Th17 transdifferentiation (23). As mTORC1 and FoxO1 are two conserved effector molecules of PI3K/Akt signaling, we determined whether FoxO1 plays a role in regulating the transdifferentiation of myelin-specific Th17 cells. dLN cells from EAE mice were reactivated in vitro with myelin antigen, or plus IL-12 or IL-23 for 3-6 days, in the presence of AS1842856 or vehicle control DMSO ( Figure 2). Subpopulations of CD4 T cells differentially expressing RORγt and T-bet were analyzed to determine whether FoxO1 regulates specific subsets of Th1 or Th17

FoxO1 inhibition suppresses T cell encephalitogenicity and the expansion of Th1 cells of MS patients
To understand the impact of FoxO1 in CNS autoimmunity, we determined whether AS1842856 may affect T cell encephalitogenicity in EAE and the expansion of pathogenic Teff cells from MS patients. Although AS1842856 has been administered in vivo to diabetic mice for a short period of time, most studies were limited to in vitro studies as the plasma concentration of AS1842856 could not be detected 2 hrs after oral administration (17), making it unsuitable for EAE studies in vivo. Therefore, we determined whether AS1842856 treatment in vitro altered the encephalitogenic potential of myelin-specific CD4 T cells following adoptive transfer.
Splenocytes from MBP Ac1-11-specific TCR transgenic mice were activated with MBP Ac1-11 and IL-12/IL-23 for 3 days in the presence of 0.1 μM of AS1842856 or vehicle control DMSO, and injected into naive B10PL recipient mice ( Figure 4A). EAE severity in the mice receiving 13 AS1842856-treated myelin-specific CD4 T cells was significantly lower compared to the mice receiving control-treated cells ( Figure 4A), indicating that FoxO1 inhibition suppresses the encephalitogenic potential of myelin-specific CD4 T cells. To make certain that the suppression of T cell encephalitogenicity by AS1842856 is not specific to this EAE model MBP Ac1-11 or T cells were significantly lower in AS1842856-treated group compared to the control group ( Figure 4E). The percentage of CD45RAeffector/memory CD4 T cells that express IFN ( Figure   4F) and the percentage of T-bet + IFN + CD4 T cells ( Figure 4G) were also significantly lower in AS1842856-treated group compared to the control group, demonstrating that AS1842856 suppresses the expansion of Th1 cells from MS patients. These data suggest that FoxO1 inhibition may limit pathogenic Th1 cells and provide therapeutic benefits in MS. IC50 of AS1842856 to inhibit FoxO1 is 0.033 µM (17). Although FoxO1, FoxO3 and FoxO4 share a high degree of sequence homology, FoxO1-mediated promoter activity was decreased by more than 70% while FoxO3-and FoxO4-mediated promoter activity by 3 and 20% respectively when AS1842856 was used at 0.1 μM (17). FoxO3 deficiency in T cells does not alter Th17 development (13). FoxO4 deficient mice have no known phenotypic differences from WT mice (15). Therefore, the effects that we observed with AS1842856 appear due to inhibition of FoxO1. The plasma concentration of AS1842856 could not be detected 2 hours after orally administration (17), which is not suitable for in vivo EAE studies. Further investigation is needed to improve the pharmacokinetic properties of AS1842856 for evaluating the potential role of FoxO1 signaling in CNS autoimmunity in vivo.

FoxO1 inhibition impairs PD-1-induced Foxp3 expression
In addition to CD4 T cells, FoxO1 also plays a role in regulating antigen presenting cells (40,41). FoxO1 deficiency in DCs decreases IL-12 produced by DCs in mucosal surfaces. The role of FoxO1 in regulating Tregs is still controversial. FoxO1 deficiency in T cells impairs Treg development and leads to a mild autoimmune phenotype (14,43), suggesting FoxO1 promotes iTreg development. However, unprimed FoxO1 -/-CD4 T cells express relatively lower levels of TGFβRII compared to naïve FoxO1 +/+ CD4 T cells from WT mice (14), which could impair iTreg development since it is dependent on TGF signaling. Conversely, forced expression of constitutively active FoxO1 in T cells also impairs Treg development and leads to severe autoimmunity (44), suggesting FoxO1 suppresses Treg development. Our data show that FoxO1 inhibition with AS1842856 led to increased Foxp3 expression ( Figure 5), which appears to be consistent with the notion that FoxO1 suppresses Treg development. Further investigation is needed to further characterize the role of FoxO1 in regulating Tregs.
PD-1 ligation signals through PI3K/Akt signaling (38,39). But little is known about the major signaling nodes of PI3K/Akt pathway that mediate PD-1 signaling in T cells. Our data show that AS1842856 impairs PD-1-induced Foxp3 expression (

Ethics Statement
The protocols used for these experiments received prior approval by the OSU Institutional Animal Care and Use Committee and were conducted in accordance with the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals. The study on human cells was performed under OSU Internal Review Board protocol # 2015H0076 with written informed consent received from participants prior to inclusion in the study.

Availability of data and materials
The data from this manuscript are available from the corresponding author upon reasonable request.