T Helper 17.1 Cells Associate With Multiple Sclerosis Disease Activity: Perspectives for Early Intervention

Jamie van Langelaar; Roos M. van der Vuurst de Vries; Malou Janssen; Annet F. Wierenga-Wolf; Isis M. Spilt; Theodora A. Siepman; Wendy Dankers; Georges M. G. M. Verjans; Helga E. de Vries; Erik Lubberts; Rogier Q. Hintzen; Marvin M. van Luijn

Disclosures

Brain. 2018;141(5):1334-1349. 

In This Article

Abstract and Introduction

Abstract

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.

Introduction

Multiple sclerosis is mediated by effector T cells trafficking from the periphery into the CNS to trigger local inflammation, demyelination and neurodegeneration (Dendrou et al., 2015). Although current T cell-directed treatment attenuates disease activity, it often causes serious complications and does not prevent disease progression in multiple sclerosis (Ransohoff et al., 2015). To improve treatment efficacy and risk management, more in-depth insight into human effector T cells during multiple sclerosis onset is warranted. In the earliest clinical presentation of multiple sclerosis, clinically isolated syndrome (CIS), increased peripheral CD4+ T cell activation is linked to the occurrence of a second attack (Corvol et al., 2008). However, substantial knowledge has been gained about specific human T helper (Th) functions, and the exact nature of the pro-inflammatory T helper subsets involved in multiple sclerosis is incompletely understood.

Both Th1 and Th17 cells are known to be encephalitogenic, but use distinct transmigration routes to enter the CNS. In experimental autoimmune encephalomyelitis, Th1 cells preferentially migrate into the spinal cord, while Th17 cells mainly infiltrate the brain (Stromnes et al., 2008). This is facilitated by their differential expression of pro-inflammatory cytokines, chemokine receptors and integrins (Bauer et al., 2009; Reboldi et al., 2009; Larochelle et al., 2011). Interleukin-17 (IL-17) and C-C chemokine receptor 6 (CCR6) are key determinants for Th17 transmigration across the blood–brain barrier (Kebir et al., 2007; Reboldi et al., 2009). IL-17 is generally considered as the signature cytokine produced by CCR6-positive Th17 cells. However, this greatly underestimates Th17 effector function, since subpopulations also (co-)produce interferon-gamma (IFN-γ) and granulocyte macrophage colony-stimulating factor (GM-CSF). Next to IL-17, also IFN-γ and GM-CSF are strongly produced by myelin-specific CCR6-positive T helper cells in multiple sclerosis (Cao et al., 2015). Th17 polyfunctionality is differently regulated between species, as reflected by the antagonistic regulation of IL-17 and GM-CSF expression in human compared to murine T helper cells (El-Behi et al., 2011; Noster et al., 2014; Paterka et al., 2016). Particularly GM-CSF produced by T helper cells is implicated as a critical mediator of multiple sclerosis onset (Codarri et al., 2011; Hartmann et al., 2014).

The surface expression of another chemokine receptor, CXCR3, defines Th17 cells with Th1-like features (Acosta-Rodriguez et al., 2007). CCR6 and CXCR3 expression on CD4+ T cells is controlled by transcription factors RORγt and T-bet, respectively, which were originally associated with IL-17/IFN-γ double-production (Acosta-Rodriguez et al., 2007). However, recent findings demonstrate more heterogeneous IL-17, IFN-γ and GM-CSF expression profiles in Th17 cells, depending on the inflammatory milieu (Duhen and Campbell, 2014). Besides CCR6 and CXCR3, also the presence of the α4β1 integrin very late antigen-4 (VLA-4), which is abundant on Th17 cells in multiple sclerosis CSF (Brucklacher-Waldert et al., 2009), determines T cell transmigration capacities. Anti-VLA-4 monoclonal antibody natalizumab is currently one of the most effective therapies in multiple sclerosis, but relapses are still encountered after 1 year in about one-third of treated patients (Polman et al., 2006). Understanding which distinct pro-inflammatory T helper subsets are targeted by natalizumab will help to better predict treatment response in multiple sclerosis (Prosperini et al., 2012).

Here, blood and CSF samples from CIS and both untreated and natalizumab-treated relapsing-remitting multiple sclerosis (RRMS) patients were assessed for distribution, memory phenotype, activation and pro-inflammatory capacity of Th17 subsets. We reveal that IFN-γ-/GM-CSF-producing (CCR6+CXCR3+), but not IL-17-producing (CCR6+CXCR3) Th17 effector cells are key regulators of multiple sclerosis onset. A Th1-like Th17 subpopulation termed Th17.1 (CCR6+CXCR3+CCR4) is selectively targeted by natalizumab in patients with multiple sclerosis who remained free of clinical relapses. This work supports the design and early use of therapeutic strategies against Th17.1 cells to prevent relapses in multiple sclerosis.

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