Priscilla W. Lee

Treatment of experimental autoimmune encephalomyelitis by codelivery of disease associated Peptide and dexamethasone in acetalated dextran microparticles.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system that can cause loss of motor function and is thought to result, in part, from chronic inflammation due to an antigen-specific T cell immune response. Current treatments suppress the immune system without antigen specificity, increasing the risks of cancer, chronic infection, and other long-term side effects. In this study, we show treatment of experimental autoimmune encephalomyelitis (EAE), a model of MS, by coencapsulating the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) with dexamethasone (DXM) into acetalated dextran (Ac-DEX) microparticles (DXM/MOG/MPs) and administering the microparticles subcutaneously. The clinical score of the mice was reduced from 3.4 to 1.6 after 3 injections 3 days apart with the coencapsulated microparticulate formulation (MOG 17.6 μg and DXM 8 μg). This change in clinical score was significantly greater than observed with phosphate-buffered saline (PBS), empty MPs, free DXM and MOG, DXM/MPs, and MOG/MPs. Additionally, treatment with DXM/MOG/MPs significantly inhibited disease-associated cytokine (e.g., IL-17, GM-CSF) expression in splenocytes isolated in treated mice. Here we show a promising approach for the therapeutic treatment of MS using a polymer-based microparticle delivery platform.

Analysis of TGF-β1 and TGF-β3 as regulators of encephalitogenic Th17 cells: Implications for multiple sclerosis

The phenotype of the CD4(+) T cells that mediate the CNS pathology in multiple sclerosis is still unclear, and yet a vital question for developing therapies. One of the conundrums is the role of TGF-β in the development of encephalitogenic Th17 cells. In the present study, TGF-β1 and TGF-β3 were directly compared in their capacity to promote the differentiation of myelin-specific Th17 cells that could induce experimental autoimmune encephalomyelitis (EAE). Myelin-specific CD4(+) T cell receptor transgenic cells differentiated with antigen in the presence of IL-6+TGF-β1 or IL-6+TGF-β3 generated T cells that produced robust amounts of IL-17, but were incapable of inducing EAE when transferred into mice. Further analysis of these non-encephalitogenic Th17 cells found that they expressed lower amounts of GM-CSF or IL-23R, both molecules necessary for encephalitogenicity. Thus, TGF-β, irrespective of isoform, negatively regulates the differentiation of encephalitogenic Th17 cells.

MicroRNAs targeting TGFβ signalling underlie the regulatory T cell defect in multiple sclerosis

Transforming growth factor beta (TGFβ) signalling is critical for regulatory T cell development and function, and regulatory T cell dysregulation is a common observation in autoimmune diseases, including multiple sclerosis. In a comprehensive miRNA profiling study of patients with multiple sclerosis naïve CD4 T cells, 19 differentially expressed miRNAs predicted to target the TGFβ signalling pathway were identified, leading to the hypothesis that miRNAs may be responsible for the regulatory T cell defect observed in patients with multiple sclerosis. Patients with multiple sclerosis had reduced levels of TGFβ signalling components in their naïve CD4 T cells. The differentially expressed miRNAs negatively regulated the TGFβ pathway, resulting in a reduced capacity of naïve CD4 T cells to differentiate into regulatory T cells. Interestingly, the limited number of regulatory T cells, that did develop when these TGFβ-targeting miRNAs were overexpressed, were capable of suppressing effector T cells. As it has previously been demonstrated that compromising TGFβ signalling results in a reduced regulatory T cell repertoire insufficient to control autoimmunity, and patients with multiple sclerosis have a reduced regulatory T cell repertoire, these data indicate that the elevated expression of multiple TGFβ-targeting miRNAs in naïve CD4 T cells of patients with multiple sclerosis impairs TGFβ signalling, and dampens regulatory T cell development, thereby enhancing susceptibility to developing multiple sclerosis.

Impact of suppressing retinoic acid‐related orphan receptor gamma t (ROR)γt in ameliorating central nervous system autoimmunity

Multiple sclerosis (MS) is an immune‐mediated chronic central nervous system (CNS) disease affecting more than 400 000 people in the United States. Myelin‐reactive CD4 T cells play critical roles in the formation of acute inflammatory lesions and disease progression in MS and experimental autoimmune encephalomyelitis (EAE), a well‐defined mouse model for MS. Current MS therapies are only partially effective, making it necessary to develop more effective therapies that specifically target pathogenic myelin‐specific CD4 T cells for MS treatment. While suppressing T‐bet, the key transcription factor in T helper type 1 (Th1) cells, has been demonstrated to be beneficial in prevention and treatment of EAE, the therapeutic potential of retinoic acid‐related orphan receptor gamma t (ROR)γt, the key transcription factor for Th17 cells, has not been well‐characterized. In this study, we characterized the correlation between RORγt expression and other factors affecting T cell encephalitogenicity and evaluated the therapeutic potential of targeting RORγt by siRNA inhibition of RORγt. Our data showed that RORγt expression correlates with interleukin (IL)‐17 production, but not with the encephalitogenicity of myelin‐specific CD4 T cells. IL‐23, a cytokine that enhances encephalitogenicity, does not enhance RORγt expression significantly. Additionally, granulocyte–macrophage colony‐stimulating factor (GM‐CSF) levels, which correlate with the encephalitogenicity of different myelin‐specific CD4 T cell populations, do not correlate with RORγt. More importantly, inhibiting RORγt expression in myelin‐specific CD4 T cells with an siRNA does not reduce disease severity significantly in adoptively transferred EAE. Thus, RORγt is unlikely to be a more effective therapeutic target for ameliorating pathogenicity of encephalitogenic CD4 T cells.

IL-23R–activated STAT3/STAT4 is essential for Th1/Th17-mediated CNS autoimmunity

The factors that promote the differentiation of pathogenic T cells in autoimmune diseases are poorly defined. Use of genetically modified mice has provided insight into molecules necessary for the development of autoimmunity, but the sum of the data has led to contradictory observations based on what is currently known about specific molecules in specific signaling pathways. To define the minimum signals required for development of encephalitogenic T cells that cause CNS autoimmunity, myelin-specific T cells were differentiated with various cytokine cocktails, and pathogenicity was determined by transfer into mice. IL-6+IL-23 or IL-12+IL-23 generated encephalitogenic T cells and recapitulated the essential cytokine signals provided by antigen-presenting cells, and both IL-6 and IL-12 induced IL-23 receptor expression on both mouse and human naive T cells. IL-23 signaled through both STAT3 and STAT4, and disruption in STAT4 signaling impaired CNS autoimmunity independent of IL-12. These data explain why IL-12-deficient mice develop CNS autoimmunity, while STAT4-deficient mice are resistant. CD4+ memory T cells from multiple sclerosis patients had significantly higher levels of p-STAT3/p-STAT4, and p-STAT3/p-STAT4 heterodimers were observed upon IL-23 signaling, suggesting that p-STAT3/p-STAT4 induced by IL-23 signaling orchestrate the generation of pathogenic T cells in CNS autoimmunity, regardless of Th1 or Th17 phenotype.

TGF-β regulation of encephalitogenic and regulatory T cells in multiple sclerosis

Transforming growth factor beta (TGF‐β) is a pleiotropic cytokine that has been shown to influence the differentiation and function of T cells. The role that TGF‐β plays in immune‐mediated disease, such as multiple sclerosis (MS), has become a major area of investigation since CD4+ T cells appear to be a major mediator of autoimmunity. This review provides an analysis of the literature on the role that TGF‐β plays in the generation and regulation of encephalitogenic and regulatory T cells (Treg) in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as well as in T cells of MS patients. Since TGF‐β plays a major role in the development and function of both CD4+ effector and Treg, which are defective in MS patients, recent studies have found potential mechanisms to explain the basis for these T‐cell defects to establish a foundation for potentially modulating TGF‐β signaling to restore normal T‐cell function in MS patients.

IL-3 Is a Marker of Encephalitogenic T Cells, but Not Essential for CNS Autoimmunity

Identifying molecules that are differentially expressed in encephalitogenic T cells is critical to the development of novel and specific therapies for multiple sclerosis (MS). In this study, IL-3 was identified as a molecule highly expressed in encephalitogenic Th1 and Th17 cells, but not in myelin-specific non-encephalitogenic Th1 and Th17 cells. However, B10.PL IL-3-deficient mice remained susceptible to experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Furthermore, B10.PL myelin-specific T cell receptor transgenic IL-3−/− Th1 and Th17 cells were capable of transferring EAE to wild-type mice. Antibody neutralization of IL-3 produced by encephalitogenic Th1 and Th17 cells failed to alter their ability to transfer EAE. Thus, IL-3 is highly expressed in myelin-specific T cells capable of inducing EAE compared to activated, non-encephalitogenic myelin-specific T cells. However, loss of IL-3 in encephalitogenic T cells does not reduce their pathogenicity, indicating that IL-3 is a marker of encephalitogenic T cells, but not a critical element in their pathogenic capacity.

Regulation of effector function of CNS autoreactive CD4 T cells through inhibitory receptors and IL-7Rα

BackgroundMultiple sclerosis (MS) is a chronic CNS autoimmune disease characterized by inflammation, demyelination, and neuronal degeneration, where myelin-specific CD4 T cells play critical roles in the formation of acute MS lesions and disease progression. The suppression of IL-7Rα expression and the upregulation of inhibitory receptors (PD-1, etc.) are essential parts of the cell-intrinsic immunosuppressive program regulating T effector functions to prevent autoimmunity. However, little is known on the factors regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T effector/memory cells during the development of CNS autoimmunity.MethodsWe analyzed the roles of the transcription factor T-bet in regulating the expression of IL-7Rα and inhibitory receptors in myelin-specific CD4 T cells. 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.ResultsWe discovered that T-bet suppresses the expression of inhibitory receptors (PD-1 and LAG-3) and promotes IL-7Rα expression in myelin-specific CD4 T cells in vitro and in vivo. As a result, T-bet skews IL-7Rα/PD-1 balance towards IL-7Rα and promotes enhanced effector function. Furthermore, IL-12 enhances IL-7Rα expression in a T-bet independent manner in myelin-specific Th1 cells. Meanwhile, IL-6, the cytokine inducing highly encephalitogenic Th17 differentiation, suppresses PD-1 while upregulating IL-7Rα, skewing IL-7Rα/PD-1 balance towards IL-7Rα, and promoting enhanced effector function. Moreover, blocking IL-7 signaling in myelin-specific CD4 T cells by αIL-7Rα significantly delays experimental autoimmune encephalomyelitis (EAE) onset and reduces disease severity.ConclusionsT-bet is a major transcription factor regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells during EAE development, 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. Furthermore, 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.

Critical contribution from antigen presenting cells for encephalitogenic T cells

umbelliferone (4-MU), that prevents development of EAE. We found that treatment with 4-MU significantly decreases the incidence (13/21 vs. 28/30 in untreated animals), delays the onset (24.9 ± 1.5 days post immunization vs. 13.3 ± 0.6 days) and reduces the severity (average score of 1.5 ± 0.1 vs. 2.8 ± 0.2, peak score of 1.9 ± 0.1 vs. 3.6 ± 0.2) of EAE. In addition, 4-MU treatment substantially reduces clinical deficits in already established EAE (average score of 1.9 ± 0.2 vs. 2.8 ± 0.1, peak score of 2.6 ± 0.2 vs. 3.7 ± 0.2). Using multi-parameter flow cytometry, we further analyzed the immunological mechanism of this therapeutic effect. We found that 4-MU treatment skews the immune response towards an antiinflammatory profile, characterized by an increase in the number of FoxP3 regulatory T-cells. Together, our data provides the therapeutic profile and immunological mechanism of 4-MU treatment in EAE. Considering that 4-MU is already an approved drug currently used in people throughout Europe and Asia, we therefore propose that 4-MU has great promise for the treatment of MS.

Differentially expressed microRNAs in multiple sclerosis patients alter regulatory T cells

expression of Ly6C and Ly6G, very important in infectious, autoimmune and tumor models. The present work will further characterize the potential role of miR-223 in the EAE model and MS. First we found an upregulation of miR-233 in the Peripheral Blood Mononuclear Cell (PBMC) of 20 MS samples vs. 20 controls (fold change over controls 1.64 ± 1.25 vs. 1.20 ± 0.95, P = 0.018). This result was confirmed in a different cohort of subjects, including 15 untreated MS subjects (population from Italy: 11 RRMS, 4 PPMS) and 12 healthy controls. In this cohort, miR-233 was upregulated in MS vs. control subjects (fold change over controls 0.81 ± 0.65 vs. 0.40 ± 0.26, P = 0.010). We also performed several active EAE experiments in miR-223 knockout (miR-223 KO) mice and littermate control mice. MiR-223 KO mice developed a significantly less severe disease (P b 0.0001 by two-way ANOVA) with a significantly higher percentage of PMN-MDSC (CD11b/Ly6G positive cells) and MO-MDSC (CD11b/Ly6C positive cells) in the spleens and spinal cords compared to control mice. We found also that MO-MDSC from miR-223 KO mice had greater immune-suppressive effects on CD4 T cell proliferation than controls in antigen T cell stimulatory conditions. It is established that MO-MDSCs inhibit CD4 and CD8 T cell proliferation mostly via ARG1 action. ARG1 was promptly upregulated in MO-MDSC from miR-223 KO cells corresponding to their high immunosuppressive function. These results demonstrate altered levels of miR 223 in the PBMC of MS patients and suggest that miR-223 plays a role in EAE. This may lead to the identification of new disease biomarkers of therapeutic targets.