CD137L, a driver of harmful inflammation in the nervous system

CD137 (TNFRSF9, 4-1BB) is a member of the tumor necrosis factor (TNF) receptor family and a potent costimulatory molecule. High levels of CD137 are expressed on T cells upon activation. CD137 signaling in T cells, either by cognate interaction with antigen-presenting cells (APC) or by agonistic anti-CD137 antibodies, strongly enhances proliferation, interferon-γ secretion, and cytolytic activity of T cells. Thus, CD137 signaling is a main driver of cellular, type 1 helper T cells (Th1) and type 1 cytolytic T cells (Tc1) polarised immune responses. CD137 costimulation enables the immune system to eliminate tumors as shown in a wide plethora of murine tumor models. Agonistic antibodies against human CD137 currently prove to be effective in clinical cancer therapy trials (Dharmadhikari et al., 2016).

When CD137 ligand (CD137L) binds to CD137, the adaptor proteins tumor necrosis factor receptor associated factor (TRAF) 1, 2, and 3 are recruited to the cytoplasmic domain of CD137, possibly as homo- and/or heterotrimers. The RING domains of TRAF2 from adjacent CD137 trimers may dimerize and bridge individual CD137 trimers, leading to the formation of a multimeric structure. The ubiquitin ligases cIAP-1 and cIAP-2 (cellular inhibitor of apoptosis protein) physically associate with TRAF-2. Elongating K63 polyubiquitin chains then recruit TAK-1-TAB1/2 (transforming growth factor beta-activated kinase 1-TAK-1 binding proteins 1 and 2) to the complex which phosphorylates other substrates, that activate nuclear factor-κB via IKKβ and NEMO (nuclear factor-κB essential modulator) as well as MAP kinases via MEKK1, thus forming a CD137 signalosome (Zapata et al., 2018).

The ligand for CD137, CD137L, is expressed by APC, and APC use the CD137-CD137L system to costimulate T cells. Although low levels of CD137L may be present on non-activated APC, its expression is much enhanced upon APC activation, e.g. by signaling of pattern recognition receptors such as TLR4 (Kang et al., 2007).

Microglial cells that form the major population of resident APC in the central nervous system (CNS), also express CD137L. Engagement of CD137L on microglial cell lines and primary microglia activates CD137L signaling, enhancing pro-inflammatory cytokine secretion (Yeo et al., 2012).

Here we summarize the current data on the regulation of CNS inflammation by CD137L, in order to stimulate research in the underlying mechanisms that may lay the foundations for gaining new insights into the CD137/CD137L biology, and a later translation to novel therapeutic approaches.

CD137L in experimental autoimmune encephalitis: Multiple sclerosis (MS) is a neuroinflammatory disease with autoreactive immune cells activated against myelin, leading to inflammation and demyelination in the CNS. The role of CD137L has been explored in experimental autoimmune encephalitis (EAE), a murine model of MS, in which neuroinflammation is induced by injecting mice with a peptide (MOG_35–55) of myelin oligodendrocyte glycoprotein. The absence of CD137L led to an amelioration of EAE-related symptoms and a significant drop in mean clinical score. Additionally, histopathological changes in demyelination and immune filtration were found in wild-type (WT) but not in CD137L^–/– C57BL/6 mice. These observations suggest that CD137L expression is critical for the initiation and progression of EAE (Martinez Gomez et al., 2012).

In order to elucidate the potential pathogenic mechanism of CD137L in neuroinflammation, the brain, draining lymph nodes, spinal cord, and vascular endothelium were analyzed. Impaired secretion of Th1 (interferon-γ and TNF) and Th17 [interleukin (IL)-17] cytokines by T cells from draining lymph nodes and CNS-infiltrating T cells was observed in CD137L^–/– mice. Moreover, infiltration of monocytes and neutrophils was significantly reduced in the spinal cord of CD137L^–/– mice. A possible reason for the reduction of immune cell infiltration into the CNS could be the downregulation of vascular cell adhesion molecule-1 (VCAM-1), expressed on vascular endothelial cells. VCAM-1 mediates the trafficking of leukocytes across the blood-brain barrier, and its expression is upregulated when the vascular endothelium is inflamed. In CD137L^–/–mice, VCAM-1 expression on endothelial cells was decreased compared to WT mice (Martinez Gomez et al., 2012).

CD137L-activated microglia was found to induce cell death in oligodendrocytes in vitro through reactive oxygen species production, and microglia of CD137L^–/– mice showed profoundly less activation during EAE, accompanied by lower degrees of oligodendrocyte apoptosis and demyelination (Yeo et al., 2012).

Thus, data from the EAE model suggest that CD137L plays an important role in the initiation of neuroinflammation by regulating VCAM-1 expression on vascular endothelial cells, followed by leukocyte infiltration into the CNS. CD137L initiates CD137 signaling in encephalitogenic T cells, thereby inducing the secretion of Th1 and Th17 cytokines, which leads to a progression of EAE. In addition, reverse CD137L signaling further amplifies the pro-inflammatory activity of microglia.

CD137L in multiple sclerosis: The EAE model in CD137L^–/– mice provided evidence of how CD137L regulates neuroinflammation by providing costimulation to T cells. Interestingly, data from post-mortem human brain sections of patients with MS suggests that CD137 - CD137L interaction also activates B cells in MS. CD137-expressing B cells were identified in MS brain lesions and meningeal infiltrates (Wong et al., 2020). Meningeal infiltrates with clusters of B cells are key features of compartmentalized inflammation found in MS. Increased proinflammatory cytokine release by B cells has been observed in cerebrospinal fluids of MS patients with high-grade meningeal inflammation. The identification of CD137-expressing B cells prompted a further investigation on how these cells regulate inflammation. The most interesting observation was that the induction of CD137 expression on B cells requires crosslinking of CD40 as well as of cell surface IgM (Wong et al., 2020). In MS, infiltration of autoreactive T cells, macrophages, and B cells provide a potential environment, which allows the induction of CD137 expression on B cells, and its engagement with CD137L on APC.

In vitro studies with transfected B cell lines and activated primary B cells show that CD137 expressed on B cells, interacts with CD137L which increases the secretion of inflammatory cytokines (TNF) immediately upon activation. Furthermore, increased proliferation of B cells at a later phase (7 days after activation) with the prolonged secretion of IL-6 was observed. Using an organoid model, it was found that CD137 expression on B cells increases their rate of infiltration. In summary, the engagement of CD137 with subsequent activation of B cells, promotes the secretion of inflammatory cytokines, and increases their proliferative capacity, which may contribute to meningeal inflammation.

CD137L in CNS manifestations of systemic lupus erythematosus: Systemic lupus erythematosus (SLE), an immune complex-mediated autoimmune condition, may affect any organ, including the CNS. To address the impact of CD137L on the CNS in an SLE setting, C57BL/6^lpr–/– (B6.lpr) mice were crossed to C57BL/6.CD137L^–/– mice. The absence of CD137L in these double knock out mice is associated with an immune deviation towards the Th17 subset and reduced numbers of IL-10-producing CD11b⁺ cells, resulting in more severe glomerulonephritis compared to the B6.lpr mice or WT mice. As demyelination is evident in SLE, and since microglia activation was enhanced by CD137L in the EAE model, it was hypothesized that microglial activation might also be influenced by the presence of CD137L in lupus brains. Indeed, there was a significant reduction of activated microglia and of the severity of demyelination in the double knock out mice. This was accompanied by reduced inflammation, cell infiltration and cell death in the cerebral cortex, hippocampus, thalamus and hypothalamus in the double knock out mice (Mak et al., 2019). Similar to EAE and MS, CD137L was identified as a driver of inflammation and CNS pathology in lupus.

CD137L in stroke: Ischaemic stroke occurs when there is an occlusion of blood flow to the brain by either an embolus or a thrombus. Cerebral ischemia induces cell death and the release of necrotic materials, which subsequently leads to inflammation that can be harmful to neurons and glial cells. In particular, inflammasomes are key mediators in detecting cell damage and promoting inflammatory processes. In ischemic stroke, CD137 and CD137L play a role in inflammasome-mediated brain injury.

In the middle cerebral artery occlusion and reperfusion stroke model, CD137L^–/– mice displayed a decreased infarct volume and fewer neurological deficits compared to WT mice (Fann et al., 2020). In CD137L^–/– mice, the inflammasome receptors NLRP1 and AIM2 were expressed at lower levels 24 hours after reperfusion. In addition, levels of cleaved caspase-1 and -8, and mature IL-1β were much reduced, reflecting a reduced inflammasome activation. The suppressed inflammasome activity was accompanied by the dampening of cell death-related pathways (Fann et al., 2020). These results suggest that CD137 - CD137L interaction contributes to inflammasome activation and inflammasome-associated cell death during ischemic stroke.

CD137L in neuropathic pain: Myeloid cells are pivotal for the regulation of inflammation, and inflammation is a main mediator of pain. It is therefore not surprising that microglia is a critical factor in the manifestation of neuropathic pain. Wakley and colleagues investigated the role of CD137L in injury-induced neuropathic pain using a spinal nerve L5 transection (L5Tx) murine model. The absence of CD137L resulted in decreased mechanical and diminished heat hypersensitivity. Also, neutralization of CD137L by an antibody reduced L5Tx-induced mechanical hypersensitivity, if the antibody was administered early after injury, with later treatments having no effect. The lack of CD137L changed the infiltration of leukocytes and the expression of inflammatory markers, especially by the microglia. The authors concluded that CD137L promotes a pro-inflammatory environment immediately after injury in the lumbar spinal cord, thus facilitating the maintenance of neuropathic pain (Wakley et al., 2018).

Potential mechanism of action: What might be the reason for these pathogenic effects of CD137L in EAE, MS, lupus, stroke and neuropathic pain? The common denominator of these conditions is inflammation. The presence of CD137L exacerbates inflammation, and it seems to do so more in the nervous system than in the remaining tissues (Figure 1).

Figure 1.

CD137L is expressed on antigen-presenting cells, including monocytes and microglia in the nervous system.

CD137L associates with CD32a and TNFR1, and upon binding to CD137, which is expressed on activated T cells, CD137L and its associated molecules activate monocytes and microglia which release inflammatory mediators. Concurrently, T cells - and to a lesser extent - B cells, which express CD137 upon activation, are costimulated by CD137 signaling. These inflammatory activities can cause or exacerbate various pathologies in the central and peripheral nervous systems. IL-1b: Interleukin-1 beta; MHC: major histocompatibility complex; TCR: T cell receptor; Th1: helper T cell type 1.

The reason for that is currently not known but two possible theories may provide an explanation. Since the CD137 - CD137L system not only costimulates but also polarizes T cells to a Th1/Tc1 immune response, one could hypothesize that a CD137-costimulated, cellular response is particularly damaging for the nervous system.

However, a more likely explanation would be the receptor-independent signaling that has been shown for CD137L (Kang et al., 2007). CD137L can be part of larger signaling complexes even in the absence of CD137, and contribute to inflammatory signaling. Murine CD137L has been reported to associate with murine TLR-4, and to be involved in sustaining TLR-4-mediated signaling. Further, murine CD137L associates with TMEM126A, and is involved in reverse CD137L signaling. In the human system, CD137L associates with human TNFR1, and the two molecules mediate the induction of pro-inflammatory cytokines in monocytic cells (Moh et al., 2013). Finally, human CD137L also associates with CD32a, where it mediates the activation of monocytic cells (Zeng et al., 2020). It could be this participation of CD137L in the signaling of several inflammation-inducing supramolecular activation complexes (SMAC), which may be more abundant and/or active in the nervous system that forms the basis for the pathogenic role of CD137L in the nervous system.

Implications for CD137 agonistic therapy: CD137 agonists are novel and promising candidates for cancer immunotherapy that can potently induce or enhance anti-tumor immune responses, and that are being increasingly used in combination therapies (Sanmamed et al., 2019). This prompts the question of whether and how CD137 agonists may influence inflammation in the nervous system. In all of the above-mentioned cases, inflammation was enhanced by the presence of CD137L, and according to our hypothesis, it did so because CD137L participates in an inflammation-inducing SMAC, whose activity is not dependent on the presence of CD137. Going by this assumption, one could speculate that CD137 agonists may not have any or much effect on nervous system inflammation driven by CD137L. On the other hand, if CD137 binding to CD137L prevents CD137L from participating in the SMAC, then CD137 agonists may compete with CD137L for CD137, and free up CD137L which could then become part of the SMAC, thus enhancing its inflammatory activity in the nervous system. Future studies are required to elucidate the effect of CD137 agonists on nervous system inflammation.

We thank Emily Nickles for proofreading the manuscript.

Additional file: Open peer review report 1 ^{(76.1KB, pdf)} .