γδ T cell subsets play opposing roles in regulating experimental autoimmune encephalomyelitis

https://doi.org/10.1016/j.cellimm.2014.04.013Get rights and content

Highlights

  • γδ IL-17-producing T cells are pathogenic in SJL/J mice with EAE.

  • A γδ T cells are found in CNS inflammatory lesions and contact oligodendrocytes.

  • The Vγ4 subset exclusively produces IL-17 in the CNS.

  • The Vγ1 subset plays a protective role in regulating EAE priming in the periphery.

  • Transcriptional regulation of IL-17 in Vγ4 cells differs from CD4+ Th17 cells.

Abstract

γδ T cells are resident in cerebrospinal fluid and central nervous system (CNS) lesions of multiple sclerosis (MS) patients, but as multifaceted cells exhibiting innate and adaptive characteristics, their function remains unknown. Previous studies in experimental autoimmune encephalomyelitis (EAE) are contradictory and identified these cells as either promoting or suppressing disease pathogenesis. This study examines distinct γδ T cell subsets during EAE and indicates they mediate differential functions in CNS inflammation and demyelination resulting in pathogenesis or protection. We identified two γδ subsets in the CNS, Vγ1+ and Vγ4+, with distinct cytokine profiles and tissue specificity. Anti-γδ T cell receptor (TCR) monoclonal antibody (mAb) administration results in activation and downregulation of surface TCR, rendering the cells undetectable, but with opposing effects: anti-Vγ4 treatment exacerbates disease whereas anti-Vγ1 treatment is protective. The Vγ4+ subset produces multiple pro-inflammatory cytokines including high levels of IL-17, and accounts for 15–20% of the interleukin-17 (IL-17) producing cells in the CNS, but utilize a variant transcriptional program than CD4+ Th17 cells. In contrast, the Vγ1 subset produces CCR5 ligands, which may promote regulatory T cell differentiation. γδ T cell subsets thus play distinct and opposing roles during EAE, providing an explanation for previous reports and suggesting selective targeting to optimize regulation as a potential therapy for MS.

Introduction

MS and its murine model, EAE, are characterized by perivascular T cell and mononuclear cell infiltration in the central nervous system (CNS) with subsequent primary demyelination of axonal tracts leading to progressive paralysis. Autoreactive CD4+ T cells in MS patients and in EAE respond to a variety of myelin membrane constituents including myelin basic protein, myelin proteolipid protein (PLP), and/or myelin-oligodendrocyte glycoprotein, [1], [2] which induce CNS inflammation and demyelination. With the recent revelation that IL-17-mediated inflammation, rather than IFN-γ responses, are most critical during autoimmunity, the research focus has centered on understanding the differentiation and effector functions of CD4+ Th17 cells in EAE and MS [3], [4], [5]. However, conventional Th17 cells are not the sole producers of IL-17. γδ T cells secrete large amounts of IL-17, perhaps even without the clonal expansion or additional TCR stimulation required for the adaptive response [6], [7], [8]. Interestingly, IL-17 producing γδ T cells have been shown to be pathogenic in models of autoimmunity including collagen induced arthritis and protective for airway hyper-reactivity, indicating a pleiotropic role for γδ T cells in immune-mediated pathology [9], [10], [11].

Significant numbers of γδ T cells have been identified in the cerebral spinal fluid and the CNS demyelinating lesions of MS patients. In addition to clonally expanded αβ T cell populations, which use a restricted set of gene segments, γδ T cells also display a restricted repertoire that is over-expressed in MS plaques [12], [13]. Junctional sequence analysis of these expanded cells suggests they are oligoclonal in nature, perhaps indicating specific antigen stimulation. It has been proposed that γδ T cells respond to heat shock proteins, which could be released in response to inflammatory CNS tissue damage [14]. Although the antigen specificity and regulation of these cells is not well understood, it is clear γδ T cells are involved in the autoimmune CNS inflammation in MS.

Past attempts utilizing murine models of MS to study the role of γδ T cells in the pathogenesis of autoimmune demyelination have been contradictory [reviewed in [15]]. On the one hand, γδ T cells have been shown to play a protective role or no role at all during disease. It has been proposed that γδ T cells regulate autoimmune inflammation via Fas–FasL mediated killing of CNS antigen-specific T cells based on the observation that γδ T cell-deficient mice on the B10.PL background develop a chronic disease compared to the monophasic acute disease course seen in the control animals [16], [17]. However, an additional study concluded that regulation of autoreactive inflammation in EAE is specifically the role of T regulatory cells and not γδ T cells [18]. To further complicate the situation γδ T cells have been reported to enhance autoimmunity by restraining Treg responses [19]. Similarly, adoptive transfer of autoreactive CD4+ T cells into γδ-deficient recipient mice on the C57BL/6 (B6) background elicited similar disease as seen in the WT recipient controls suggesting γδ T cells do not play a significant role in the mediation or regulation of effector mechanisms in EAE [20].

Unlike the aforementioned data, other reports support the hypothesis that γδ T cells play a pathogenic role during disease [21], [22], [23]. Targeting γδ T cells with monoclonal antibodies during various stages of disease resulted in the inability to detect the cells as well as decreased disease, suggesting γδ T cells play a critical role in the pathogenesis of EAE during both acute and chronic phases [24]. Similarly, in both actively induced and adoptively transferred EAE in B6 mice that genetically lack γδ T cells, EAE disease was significantly reduced [25]. These diverse and conflicting results obtained from animal model studies aimed to dissect the mechanisms of γδ T cell involvement in demyelinating disease could attributed to the use of a variety of mouse strains, inducing antigens and methods of γδ T cell manipulation, namely genetic depletions or monoclonal antibody targeting in vivo.

γδ T cells are a heterogeneous population and perhaps the pleiotropic nature of this cell subset and thus the variety of results from many different studies aimed at determining their role during EAE and MS may be explained by a dichotomy of γδ T cell subset function. We therefore sought to examine a possible dichotomy of these pleiotropic cells within the murine model of MS with the goal of clarifying the previous controversy surrounding the role of γδ T cells in EAE as well as to provide evidence for an alternative method of specifically targeting these cells as a possible treatment for MS. Our study indicates that γδ T cell subsets play opposing roles, such that targeted treatment could optimize the regulation of disease. We show that Vγ4-expressing γδ T cells constitute a significant proportion of IL-17-producing cells in the CNS during EAE pathogenesis and when activated in vivo, exacerbate disease symptoms due to their pathogenic nature. Conversely, the Vγ1 subset plays a protective role and perhaps eliciting function at the priming stage within the spleen rather than in the CNS. Using a γδ T cell reporter mouse we were able to show that in vivo antibody treatment resulted in activation of the γδ T cell subsets and not depletion. Collectively, these data provide some much needed explanation for the contradictory literature surrounding the role of γδ T cells during EAE. We propose that γδ T cell subsets show distinct and opposing functions, such that antibody targeting of these cells may allow a more carefully defined inhibition of the pathogenic response in MS, while maintaining the protective immune mechanisms of these critical immune cells.

Section snippets

Mice and peptides

Female SJL/J (Harlan Sprague Dawley), C57BL/6J and Tcrd/ (The Jackson Laboratory) and Tcrd-eGFP mice [26] were housed under specific pathogen-free conditions in the Northwestern University Animal Facility. All protocols were approved by Northwestern University Animal Care and Use Committee. PLP139–151 (HSLGKWLGHPDKF) and MOG35–55 (MEVGWYRSPFSRVVHLYRNGK), were purchased from Genemed Synthesis (San Francisco, CA).

Induction of EAE

Chronic EAE (C-EAE) was induced in Tcrd-eGFP, Tcrd/, and C57BL/6 mice with 200 μg

γδ T cell subsets accumulate in the CNS of SJL/J mice with PLP139–151-induced EAE correlates with relapsing-remitting disease severity and co-localize with CD4+ T cells

Interestingly, a limited repertoire of γδ T cells, defined by TCR chain usage, have been seen in the CSF and lesions of MS patients, however their function is not understood. The relapsing-remitting EAE model (R-EAE) closely represents the most common form of MS, characterized by repeating bouts of paralytic symptoms interrupted by remissions. Previous studies have examined the role of γδ T cells using multiple EAE models of MS, however the results are conflicting in that these cells have been

Discussion

γδ T cells have been identified in both lesions and the CSF of MS patients and display a limited repertoire, however their function is unknown. Past literature using the EAE model of MS is filled with contradictory data as to whether these cells play a pathogenic or protective role [15]. As a population, γδ T cells display a broad functional spectrum that may segregate into the multiple subsets defined by the TCR variable region. In this study we characterize the two main circulating subsets of

Acknowledgments

This work was supported by Grants from the Myelin Repair Foundation and NIH Grant NS-026543. We thank Dr. Liang Zhou for his assistance with real time PCR primers and for helpful discussions. We also thank Dr. William Karpus for the generous gift of PCR primers for chemokines. The authors declare no competing financial interests.

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