Regulation of lymphocyte trafficking in central nervous system autoimmunity

Abstract

CD4⁺ T helper (Th) cells play a central role in orchestrating protective immunity but also in autoimmunity. Multiple Sclerosis (MS) is a human autoimmune disease of the central nervous system (CNS) characterized by the infiltration of inflammatory lymphocytes and myeloid cells into the brain and spinal cord, leading to demyelination, axonal damage, and progressive loss of motor functions. The release of T cells in the circulation and their migration in the central nervous system are key and tightly regulated processes which have been targeted to decrease CD4+ T cell presence in the CNS and limit disease progression. Here, we review two of these pathways and discuss how their blockade modulate different subsets of CD4+ T cells.

Introduction

CD4⁺ T helper (Th) cells play a central role in protective immunity but also in autoimmunity. Multiple sclerosis is an autoimmune disease, which results from the autoimmune attack by myelin-reactive T cells and recruitment of other immune cells in the central nervous system (CNS) and progressive demyelination [1]. The long-standing hypothesis is that MS is initiated by myelin-specific CD4⁺ T cells activated by some environmental factors [2]. Once activated, CD4+ T cells differentiate into different subsets of T helper cells including but not limited to Th1 and Th17 cells with specific cytokine production and specialized effector functions [3,4]. CD4+ T cells which are believed to be pathogenic in MS and its animal model, experimental autoimmune encephalomyelitis (EAE) produce IFN-g, IL-17 and GM-CSF [5–10]. The activation and proliferation of CD4+ T cells can be limited effectively by regulatory T cells (Treg) [11]. After activation and differentiation, CD4+ T cells egress from lymphoid organs and migrate through the blood brain barrier (BBB). These, steps are critical for CD4+ T cells to access the CNS. While, some of the pathways that regulate these processes have previously been described, recent data show that the paths that control egress and migration might be more selective for specific Th cells types and that their blockade might have additional effect beyond migration.

Regulation of T cells by S1P

Sphingosine-1-phosphate (S1P) is a sphingosine-containing lipid generated from ceramide, which binds G protein-coupled receptors Sphingosine 1-phospate receptors (S1PRs) 1–5 and promotes T cell egress from secondary lymphoid organs (SLO) into the lymph and the blood [12]. T cell egress is dependent on the existence of an S1P gradient between lymphoid organs (where S1P concentrations are low) and the lymphatic and blood vessels (where S1P levels are intermediate and high, respectively). In the blood, high concentrations of S1P induce the internalization of S1P1 allowing for potential relocation of T cells into SLO or tissues [13]. Naive T cells constantly patrol and enter the lymph nodes until they encounter their specific antigen presented by an antigen-presenting cell (APC). Upon activation, TCR stimulation downregulates the expression of S1P1 and the very early activation antigen marker, CD69 transiently traps S1P1 inside the cells, inducing T-cell arrest into the lymph node [14]. This allow for the proliferation and differentiation of antigen specific T cells. S1P1 is then re-expressed by both effector and central memory T cells, allowing them to exit the lymph nodes [14,15]. S1P1/S1P mediated T cell egress is counteracted by retention signals such as the chemokine receptor CCR7 [15].

Naive T cells that do not encounter their cognate antigen in the lymph nodes do not down-regulate S1P1 on their cell surface and re-enter into the circulation following the S1P gradient. S1P induced S1P1 downregulation on T cells in the blood, allowing T cells to recirculation in the SLO. Their entry in non-lymphoid tissues is further controlled by local S1P concentration and receptor desensitization is mediated by GPCR kinase 2 (GRK2), which phosphorylates serine residues in the cytoplasmic tail of S1PR1 [16] [17]. S1P and S1P receptors have been specifically targeted as treatments for immune-mediated diseases. For example, Fingolimod, an S1P analogue has been the first approved oral therapy for relapsing Multiple Sclerosis [18–21]. It is a sphingolipid mimetic which binds in its phosphorylated form to S1P receptors and activates them. Fingolimod also downregulates S1P1 and as such, is believed to act as a functional antagonist of the S1P/S1Ps axis [13]. S1P is an important regulator of T cell trafficking [13,22] and genetic approaches highlight an intrinsic requirement for S1P1 in T cells [13,22]. Fingolimod has profound inhibitory effects on T cell egress and traficking [23]. Therefore, the immunosuppressive and disease-ameliorating effects of Fingolimod in RRMS have mostly been attributed to the blockade of inflammatory cell migration into the CNS [24,25]. In this context, it is interesting to note that Fingolimod differentially affects the recirculation of lymphocyte subsets.

Memory T cells have classically been subdivided into central memory T (TCM) cell and effector memory T (TEM) cell subtypes. TCM cells retain expression of the lymph node homing receptors CCR7 and CD62L, and migrate to secondary lymphoid organs, whereas TEM cells do not express CCR7 and can respond to chemokines and migrate into inflamed tissues. Tissue-resident memory T cells (TRM) are memory T cells that express CD69 and CD103 (aEb7 integrin) and reside in peripheral tissues and do not recirculate. These cells respond rapidly without a need for recruitment of T cells from the circulation. CCR7⁺ TCM [26] are enriched in the CSF MS patients and effector memory T cells are enriched in normal-appearing white matter and white matter lesions [27]. In MS patients, Fingolimod has primarily been shown to reduce numbers of CCR7⁺CD45RA⁺ naïve T cells and of CCR7⁺CD45RA^– TCM cells in blood, whereas CCR7^– CD45RA^– and CCR7^–CD45RA⁺ TEM subsets remained largely unaffected [28]. Despite a decrease in numbers of blood circulating lymphocytes and Th17 TCM in MS patients treated with Fingolimod, [29–31], retention of CCR7+TCM in the cerebrospinal fluid (CSF) of some fingolimod treated patients has been observed [32]. This raises the question whether the efficacy of fingolimod as a DMT in MS is due to its effects on pathogenic T cells [31,33]. Indeed, it is conceivable that TCM from the CSF can differentiate into effector memory T cells upon restimulation and enter the brain parenchyma to induce relapses [34]. Furthermore, TRM cells could also participate in CNS damage and not be affected by Fingolimod therapy since they do not express S1P1. In other systems, Fingolimod treatment has been associated with tissue local expansion of TRM cells [35]. This can be beneficial during infection but may be problematic for autoimmune diseases. Together these data highlight the complexity and pleiotropic effect of the S1P1/S1P axis and question whether the effect on T cells is sufficient to provide protection against relapses. Recent data in mice for example show that Fingolimod beneficial effect in EAE extends beyond its effect on T cells and had a direct protective effect on CNS resident cells [36–38].

In addition to its effect on T cell migration, recent data demonstrate that S1P can regulate T cells by other mechanisms. The S1P/S1P1 axis has been shown to maintain the mitochondrial content of T cells [39] and to regulate their activation and differentiation, which could account for the beneficial effect of its blockade. Mice expressing an internalization-defective S1P1 (S5A), developed more Th17 cells and had more severe EAE [40]. In support for a role of the S1P/S1P1 axis in the development of Th17 cells, we have demonstrated that mice which lacked S1P1 in IL-17A+ cells had limited numbers of Th17 cells in the lymph nodes after immunization with antigen and were completely protected from the development of EAE [41]. Regulatory T cells play an important role in maintaining peripheral tolerance, but have been shown to be less efficient in patients with autoimmune diseases such as MS compared to healthy controls. An important role of S1P1/S1P axis for Treg egress from the thymus has now been well described [15,42–44]. However, such function and the possible interference of Fingolimod with T cell egress from the thymus becomes less relevant in adult patients in whom thymic output is limited. In human patients, Fingolimod significantly increased the number of T regulatory cells while decreasing central memory T cells [43], however, it was unclear whether this was due to a direct effect on Treg and whether these Treg were functional. Using conditional and temporal deletion of S1P1 in Treg, we have demonstrated that S1P1 not only regulated the egress of T_reg cells out of lymphoid organs and subsequent non-lymphoid tissue distribution but also their phenotypic diversity. Most of the T_reg cells found in S1P1-deficient mice as well as MS patients on fingolimod therapy had an activated phenotype, converted to effector T_reg and were more prone to apoptosis [41]. Treg from mice with conditional deletion of S1P1 in established Treg, were not capable of controlling autoimmunity development raising the possibility that human Treg function from MS patients treated with Fingolimod might be less suppressive [41] (Figure 1).

Figure 1. Control of T cell migration by fingolimod and natalizumab.

(A) Activated Th cells exit the secondary lymphoid organs (SLO) to the circulation following the S1P gradient. The transmigration of CD4+ T cells across the blood brain barrier (BBB) is in part mediated by VLA- 4-VCAM-1 interactions. (B) Fingolimod limit the egress of T cells from the SLO but also has other effects such as changing the phenotype of Treg. (C) Natalizumab blocks the transmigration of CD4+ T cells in the CNS. This blockade affects preferentially IFN-g+ Th1 cells.

Control of T cell entry in the CNS by VLA4

The migratory properties of effector Th cells are in part imprinted during differentiation with induction of chemokine receptors that enable their differential trafficking to inflammatory lesions [45]. In EAE, Th1 cells preferentially migrate into the spinal cord, while Th17 cells mainly infiltrate the brain [46,47]. It is not known however whether Treg can differentially enter selective areas of the CNS during Th1 or Th17-mediated disease. Migration of immune cells into the CNS parenchyma is a highly regulated process which occurs at various anatomical sites of the CNS, such as at the choroid plexus and the BBB at post-capillary venules. In addition to chemokine receptors, activated T cells upregulate many integrins and adhesion molecules, enabling their rolling and adhesion to vessel walls. The interactions of LFA-1–ICAM-1 and VLA-4–VCAM-1 are critically involved in the firm arrest of CD4⁺ T cells onto the inflamed cerebral vessels or primary brain EC monolayers. The anti-α4-integrin antibody, natalizumab is used as a disease modifying therapy for relapsing-remitting MS [48]. While clinical trials demonstrate a drastic reduction in the relapse rate [49–51], a number of Natalizumab treated patients have developed lethal progressive multifocal leukoencephalopathy (PML), a serious opportunistic brain infection caused by a neurotropic strain of the John Cunningham virus (JCV) [48,52]. Patients taking natalizumab are closely monitored for JCV antibodies since seropositivity significantly increase the risk of developing PML. Furthermore, because the risk of PML increases significantly in immune compromised patients, it has been proposed that PML occurs when CNS immunosurveillance is weakened [53][48]. Although blockade of VLA-4 was thought to completely abrogate CNS entry of T lymphocytes, patients under natalizumab treatment have lower but still detectable numbers of immune cells in the cerebrospinal fluid (CSF) [54,55], suggesting that there are alternative or compensatory molecular mechanisms for some immune cell populations to enter the CNS. We and others have shown that interfering in the interaction of Itga4b1 and V- CAM1 prevent the entry of Th1 cells but not Th17 cells in the CNS. In contrast, LFA-1–ICAM-1/2 interactions regulated Th17 adhesion to the endothelial barrier in the brain [*56,*57]. In MS patients, a population of CCR6⁺CXCR3⁺CCR4⁻ cells was shown to be selectively targeted by natalizumab treatment [58]. Alternative adhesion molecules might also be involved since PSGL-1 was shown to support extravasation of CD4⁺ T cells into the CNS in conditions where VLA-4-mediated processes were blocked and Th17 cells were shown to use MCAM in conjunction with VLA-4 to mediate adhesion to brain endothelium [59]. In addition, we demonstrated that Treg could enter the CNS and control EAE progression independently of Itga4 [60] (Figure 1). In accordance with these data, Natalizumab treatment did not change the frequency of peripheral blood Tregs or their suppressive capacity in MS patients [55]. Together, these observations suggest that efficacy of Itga4 neutralization on MS progression may be associated with the maintenance of Tregs migration into the CNS.

Conclusions

In conclusion, the pathways that regulate the trafficking of CD4+ T cells, i.e. Itga4b1 and S1P, which were discussed here prove more complex and selective than originally anticipated. In autoimmune diseases, the balance between effector/memory T cells and Treg cells is critical in determining disease progression and relapse occurrence. Our data, as well as those from others suggest that S1P and Itga4 can have contrasting effects on effector/memory T cells and Treg. Itga4 neutralization, while limiting the access of Th1 cells in the CNS, has limited effects on Treg cells entry. An increased proportion of Treg in the CNS could confer better control of effector/memory T cells and provide a significant therapeutic efficacy but might also create an immunosuppressive environment where infection might occur more easily. Regarding S1P, while Fingolimod effectively decreases relapse rates better than interferon beta therapy; a subset of MS patients treated with Fingolimod develop very severe relapses and even tumefactive lesions [61–63]. A very recent study showed that, while Fingolimod treatment prevents overall immune cell infiltration into the CNS; a subset of CCR6⁺ immune cells appears to escape the action of Fingolimod [64]. These observations and others suggest that S1P1-mediated signaling regulate specific immune cells differently and/or some immune cells may become over time insensitive to Fingolimod. Based on our findings, we postulate that Fingolimod may mediate its therapeutic effect by blocking the function of Th17 cells [41]. However, this drug may also, over time, disable the function of Tregs and increase the generation of autoimmune T cells such as GMCSF-producing T cells, which may be responsible for disease worsening, especially when the drug is withdrawn. Since much effort has been expended in the generation of therapeutics that interfere with the S1P receptor/S1P axis and that fingolimod continues to be used as a DMT in MS, it is critical to better understand the mechanism of S1P1 action through S1P1 and other relevant S1P receptors.

Highlights.

• Sphingosine-1-phosphate (S1P) blocks the egress of naïve T cells, Th17, and TCM cells from the lymph nodes.

• Inhibition of the S1P/S1P1 signaling alters the phenotype and function of Treg.

• Treg can enter the CNS independently of VLA4.

• In the absence of VLA4/VCAM-1 interaction, other adhesion molecules promote the entry of Treg and Th17 cells in the CNS.