The function and regulation of IL-17A-producing T cell subsets in central nervous system autoimmunity

Abstract

Multiple sclerosis (MS) is a chronic progressive autoimmune disease of the central nervous system (CNS). MS is characterised by the presence of demyelinating lesions in the CNS and is the leading cause of disability among young adults between the ages of 20-30. Experimental autoimmune encephalomyelitis (EAE) is the murine model for MS and is a crucial tool for dissecting the immune responses that mediate pathology in this disease. EAE is a T cell- mediated autoimmune disease that results in ascending paralysis of the mouse. Autoantigen- specific IL-17A-producing CD4 T (Th17) cells and γδ T cells have emerged as central players in the pathogenesis of EAE. Activation of naïve CD4 T cells and their differentiation into IL-17A- producing antigen-specific Th17 cells requires several signals, provided by antigen presenting cells (APCs). APCs present the myelin antigen, myelin oligodendrocyte glycoprotein (MOG) to CD4 T cells, hence regulating the differentiation of Th17 cells. Conversely, IL-17A-producing γδ T cells can be activated with the cytokines IL-1β and IL-23, in the absence of T cell receptor (TCR) engagement, thereby becoming activated under less stringent conditions than CD4 T cells. γδ T cells are a crucial early source of IL-17A that precipitate CNS autoimmunity. As CD4 T cells require antigen to be presented to them in the context of MHC molecules expressed by APCs, they are subject to regulatory T (Treg) cell-mediated suppression. Furthermore, the expression of checkpoints, such as programmed cell death protein-1 (PD-1) by CD4 T cells contributes to the regulation of the antigen-specific immune response. Engagement of PD-1 expressed by CD4 T cells negatively regulates T cell function. Immune checkpoint inhibitors (ICIs) are used in the treatment of cancer, but patients that respond to this immunotherapy develop autoimmune diseases. Since γδ T cells can be activated without TCR engagement with MHC and co-stimulatory molecules expressed by APCs we hypothesised that IL-17A-producing γδ T cells are not subject to the same regulatory mechanisms that control CD4 T cell function. This lack of conventional regulation may allow γδ T cells to become activated in an unrestrained manner enabling them to precipitate autoimmunity. The present study revealed that CD27- Vγ4-expressing γδ T cells express high levels of the immune checkpoint, PD-1 in the lymph node (LN) of naïve mice and this expression is further augmented during EAE. Anti-PD-1 treatment during EAE significantly enhanced the severity of disease in wildtype mice, but not in TCRδ-/- mice. Furthermore, anti-PD-1 enhanced IL-17A production by Vγ4 γδ T cells in the LN at the induction phase of EAE and also promoted the development of Th17 cells. However, anti-PD-1 treatment did not enhance the severity of EAE or the development of Th17 cells in TCRδ-/- mice. Studies using recombinant PD-L1 demonstrated that Vγ4 γδ T cells activated via their TCR were regulated by PD-1 engagement in vitro whereas PD-1 engagement of IL-1β+IL-23-activated Vγ4 γδ T cells did not impact their function. Additionally, the anti-PD-1-mediated increase in IL-17A production by Vγ4 γδ T cells was dependent on MOG, which suggests that during EAE, IL-17A-producing Vγ4 γδ T cells are MOG-specific and are regulated via PD-1 in a TCR-dependent manner. An examination of the role of IL-10 in regulating T cell responses during EAE revealed that neutralisation of the IL-10R enhanced the severity of disease and was associated with enhanced expression of the integrins LFA-1 and VLA-4 by CD4, CD8, and γδ T cells in the LN early in disease. Furthermore, neutralisation of the IL-10R enhanced the infiltration of VLA-4- expressing CD4, CD8, and CD27- γδ T cells into the brain at the peak of EAE. MOG-specific type 1 regulatory T (Tr1) cells regulated IL-17A-and IFN-γ-producing MOG-specific CD4 T cells in an IL-10-dependent manner. In contrast, IL-17A production by IL-1β+IL-23 activated CD27- γδ T cells was not regulated by MOG-specific Tr1 cells demonstrating that innately activated γδ T cells escape the regulatory mechanisms of TCR-activated T cells. An examination of the role of TGF-β in regulating the development of IL-17A-producing CD4 and γδ T cells during EAE has demonstrated that IL-17A production by IL-1β+IL-23-activated CD27- γδ T cells was not suppressed, but rather enhanced by TGF-β, whereas IFN-γ production by IL-12+IL-18-stimulated CD27+ γδ T cells was suppressed by TGF-β. Neutralisation of TGF-β- mediated signalling during EAE reduced the severity of disease and this was associated with decreased infiltration of IL-17A and TNF-producing γδ and CD4 T cells into the CNS at the peak of EAE and increased IFN-γ and GM-CSF production by CNS-infiltrated γδ and CD4 T cells. Neutralisation of IFN-γ reversed the suppressive effect of TGF-β neutralisation on IL-1β+IL-23- activated CD27- γδ T cells, suggesting that TGF-β supports IL-17A production by γδ and CD4 T cells in an IFN-γ-dependent manner. The findings of this study have demonstrated that IL-17A-producing γδ T cells are resistant to suppression mediated by TGF-β and IL-10. However, PD-1 engagement significantly suppressed IL-17A production by Vγ4 γδ T cells during EAE, highlighting the therapeutic potential of PD-1 agonistic monoclonal antibodies for the treatment of autoimmunity.