The Role of Gamma-Delta T Cells in Diseases of the Central Nervous System

Gamma-delta (γδ) T cells are a subset of T cells that promote the inflammatory responses of lymphoid and myeloid lineages, and are especially vital to the initial inflammatory and immune responses. Given the capability to connect crux inflammations of adaptive and innate immunity, γδ T cells are responsive to multiple molecular cues and can acquire the capacity to induce various cytokines, such as GM-CSF, IL-4, IL-17, IL-21, IL-22, and IFN-γ. Nevertheless, the exact mechanisms responsible for γδ T cell proinflammatory functions remain poorly understood, particularly in the context of the central nervous system (CNS) diseases. CNS disease, usually leading to irreversible cognitive and physical disability, is becoming a worldwide public health problem. Here, we offer a review of the neuro-inflammatory and immune functions of γδ T cells, intending to understand their roles in CNS diseases, which may be crucial for the development of novel clinical applications.


INTRODUCTION
Together, gamma-delta (gd) and alpha-beta (ab) T cells represent two different T cell lineages that have been defined by their expression of ab or gd T cell receptors (TCRs) (1). Although gd T cells share many effector capabilities with ab T cells (for example, cytotoxicity and cytokine production), the lineages exhibit different biological properties, such as thymic-dependent or -independent development, major histocompatibility complex (MHC) restriction, and recognition of soluble protein and non-protein antigens of endogenous origin (2)(3)(4)(5).
Unlike ab T cells, gd T cells are a relatively minor subset of T lymphocytes in the peripheral blood (PB), comprising only 1-5% of lymphocytes circulating (6). However, gd T cells are abundant at barrier sites such as the skin, gut, lung, and reproductive tract; up to 20% of intraepithelial lymphocytes in the human colon express the gd TCRs (7).
gd T cells are divided according to the type of Vg and Vd chain they express at the TCRs. Concerning the Vg chains, a unique feature of murine gd T cells is the preferential expression of different Vg segments in different tissues. For example, Vg5 + gd T cells are present in the skin, Vg7 + gd T cells lie in the intestinal, Vg6 + gd T cells localize to the reproductive mucosa, and Vg1 + or Vg4 + gd T cells are found in secondary lymphoid organs (8,9). The previous studies related to human gd T cells have identified Vg9 as the most frequently used Vg chain in the PB (10). Vg9 chain associates with Vd2 in most cases, defining a Vg9Vd2 T cell population (account for 50-95% of gd T cells in the PB) that is unique to humans and other primates (11,12).
Recently, some discrete population of T cells that coexpressed ab-gd TCRs and Vg-Cb TCRs have been identified (30,31). Among them, the ab-gd T cells protected against infection by licensing encephalitogenic Th17 cells, triggered inflammatory and immune in the central nervous system (CNS). Moreover, our research group found that, in addition to diseases of the CNS, such as multiple sclerosis (MS) and stroke, immune responses induced by gd T cells are also critically implicated in neuroinflammation associated with spinal cord injury (SCI) (32)(33)(34). These findings raise significant questions concerning the inflammatory and immune functions of gd T cells in CNS disease that have yet to be addressed (35)(36)(37). CNS disease, which can result in irreversible sensory, motor, and autonomic impairments, is a severe health problem worldwide. As a central pathological process in CNS diseases, the inflammatory response is vital to clinical prognosis. Here, we provide a review of recent advances in the understanding of gd T cells with relevance to their inflammatory and immune roles in CNS disease, which suggest potential approaches for future treatment of CNS diseases ( Table 1).

PROINFLAMMATORY CYTOKINES INDUCED BY gd T CELLS IN THE CNS
Activation and development of gd T cells promoting CNS inflammation are chiefly mediated by dendritic cells (DCs). The immunostimulatory component induces IL-1b, IL-6, IL-18, and IL-23 by DCs via caspase-1 and inflammasome complex. gd T cells secrete IL-17 in response to IL-1b, IL-18, and IL-23 in the absence of TCR (38)(39)(40). During this process, the retinoidrelated orphan receptor (ROR) -gt and IL-7 coordinate the B and T lymphocyte attenuator (BTLA) expression, thus regulating gd T cell inflammatory responses (41)(42)(43)(44). Moreover, Shibata et al. demonstrated that signal transducer and activator of transcription 3 (STAT3) is dispensable for the development of IL-17-producing gd T (gdT17) cells (45). Also, IL-23-activated gd T cells suppress the factor forkhead box P3 + (Foxp3) -expressing Treg cells conversion, as well as promoting effector T (Te) cells response (46,47). The capacity of gd T cells to produce a burst of IL-17 in the absence of activated ab T cells is crucial for the initiation of CNS inflammation (48).
During this process, IL-23 acts as a maturation factor for Th17 cells, and both IL-23 and IL-21 can induce IL-17 expression independently of IL-6 (55, 64-66). Therefore, mice lacking IL-23 are resistant to Th17-mediated CNS inflammation (46). To demonstrate the role of IL-23, Awasthi et al. substituted the green fluorescent protein for the intracellular domain of IL-23R, to generate a "knock-in" mouse, which demonstrated that IL-23 is crucial for Th17 cell function (67). IL-23 created a positive feedback loop, whereby GM-CSF secreted by Th17 cells induced the generation of IL-23 (68,69).
Alongside IL-17, GM-CSF is also essential for CNS inflammation. Further, the activation of the microglial cell, but not macrophage in the periphery, is a GM-CSF-dependent process (70). El-Behi et al. demonstrated that GM-CSF neutralization attenuated CNS inflammation (68). Although both IL-12 and IL-23 can induce Te cells to generate GM-CSF, IL-23 is crucially required for GM-SCF generation (69,71). In addition to DCs and Th17 cells, gd T cells generate large amounts of GM-CSF, resulting in neuroinflammation (72).

Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis
MS is a chronic inflammatory demyelinating CNS disease, resulting in progressive cognitive, sensory, and motor disorders. Experimental autoimmune encephalomyelitis (EAE), a murine MS model, is used to research the proinflammatory mechanism underlying CNS (73). Before the discovery of Th17 cells, IFN-g-producing Th1 cells were considered the primary pathogenic cell inducing MS and EAE, which puzzled immunologists for many years, since both IFN-g −/− and IFN-gR −/− mice enhanced EAE development (74)(75)(76)(77). Besides, deficiencies of IL-12 and IL-12R, which are critical to the development of Th1 cells, also exhibited exacerbated EAE (64). Together, findings to date indicate that Th1 cells are not the initial T cell involved in EAE. IL-12 and IFN-g (Th1-associated molecules) negatively regulate tissue inflammation in EAE (78). Nonetheless, Th1 cells are vital to EAE, as they are detected in active EAE.
Subsequently, the identification of IL-23 and Th17 cells partly worked out this issue (61, 65) ( Figure 2). IL-23p40 −/− and IL-23p19 −/− mouse strains are both resistant to EAE (64). The depletion of Th17 cells or IL-17 resulted in reduced EAE severity (78). Although Th17 cells are thought to be the major mediators of EAE, gdT cells can also be a potent producer of IL-17, and are dominant over Th17 cells in CNS inflammation (79). Several researchers demonstrated that gd T cells are frequently present in the peripheral blood (PB) and cerebrospinal fluid (CSF) of MS patients, as well as in the brains of mice with EAE (27,28,(80)(81)(82). During the chronic and acute phases of EAE, the absence of gd T cells notably reduces the CNS inflammation, suggesting that gd T cells are significant in EAE, and their inflammatory mobilization is related to the pathogenesis of CNS autoimmunity (83)(84)(85)(86)(87). Indeed, an enormous population of CD4 + T cells (IL-17 and IFN-g double-positive) is observed at the peak of EAE (88). Using a fate-tracking system, 5-10% of gdT17 cells were shown to express IFN-g in the CNS, indicating that IL-17-IFN-g-gd T cells might be consequential intermediates in EAE pathogenesis (89).
Moreover, Vg4 + gd T cells were identified as the major gdT17 cells in EAE, while Vg5 + and Vd6 + gd T cells were present (40). Vd1 + , Vd2 + , and Vg9 + gd T cells were also observed in acute demyelinating plaques of MS patients (27,90). Besides, the biological drugs designed to suppress the activity of gd T cells, such as fingolimod (FTY720) and Natalizumab, partly contribute to the clinical therapeutic effects of MS. (90)(91)(92). Further, HSP60 and HSP90 compared with normal CNS tissues are overexpressed in MS plaques, while Selmaj et al. showed the colocalization of HSP65 and gd T cells in immature oligodendrocytes in MS lesions (27,80,93). The multitude of gd T cells collected from MS patients proliferated in response to HSP70, but not to HSP65, revealing that HSPs may be the antigens responsible for promoting the gd T cells proliferation (28). CNS inflammation is associated with altered expression of HSPs, which may function as targets in the development of the chronic disease. Interestingly, IL-15-producing gd T (gdT15) cells, another subset of gd T cell, were recently discovered; however, whether these cells produce other proinflammatory cytokines in EAE is not well elucidated (94).
In contrast to the above observations, gd T cells are also reported have a protective function in EAE. Ponomarev et al. reported that gd T cells of wild-type (WT) reconstitute gd T cell −/− mice, but not FasL dysfunctional gd T cells, diminishing inflammation in EAE (95). These findings suggest that the gd T cell-mediated Fas/FasL-induced T cells apoptosis regulates CNS inflammation. Indeed, the mechanism by which gd T cells regulate proinflammatory chemokine and cytokine expression in CNS, as well as infiltrating cell heterogeneity, warrant detailed investigation.

Ischemic Brain Injury
The main consequence of ischemic brain injury is manifested as the CNS tissue necrosis, due to the loss of nutrition. The tissue necrosis leads to a secondary inflammation, involving the accumulation of specific immune cells, especially neutrophils, macrophages, and T cells, which is a critical factor to the entire pathophysiology (96,97).
IL-17 has a specific role in the delayed phase of the ischemic brain injury inflammatory cascade (98). Shichita et al. demonstrated that gdT17 cells play a significant role during latestage ischemic brain injury, and that they, rather Th17 cells, are (surprisingly) the major origin of IL-17 (99). Moreover, increased IL-17 levels are present in the PB of patients who have suffered a stroke, relative to healthy individuals (100). IL-23, generated by macrophages or monocytes from stroke initiation, is an essential contributor for inducing IL-17 by gd T cells during the delayed phase of encephalic ischemia. Thereby, IL-23p19 −/− mice illustrated a diminishment in infarct extent only 1 day after the ischemic injury, whereas IL-17 deficiency led to reduced infarct size after 4 days. Long-term, deficiencies of IL-17 and IL23 demonstrated obviously diminished CNS injury, relative to WT, or even IFN-g −/− , mice (99). Gelderblom et al. demonstrated that injection of IL-17-neutralizing antibody to mice within the poststroke 3h could reduce infarct volume and improve disease prognosis after 3 days (101).
Overall, the available evidence demonstrates that gd T cells are the main source of IL-17. The mechanism of antigenindependent T cell activation post-stroke remains unclear; however, it has been owed chiefly to gd T cells. Nevertheless, Kleinschnitz et al. demonstrated that gd T cell-deficient mice remain susceptible to ischemic insult, indicating an extra function for other immune cells in ischemic brain injury. Furthermore, the fact that transgenic-TCR mice are susceptible to stroke implies that, besides gd T cells, Th17 cells may also have a prominent role in stroke, whereas the precise function of Th17 cells in inducing stroke is not exact (102).
In addition, astrocytes can respond to IL-17 and promote stroke induction and development (101). For instance, IL-17 produced by gd T cells and TNF-a secreted by macrophages act synergistically on astrocytes, by inducing the expression of CXCL1, a neutrophil chemoattractant (101,103). Recently, periventricular leukomalacia (PVL), a distinctive form of brain injury in premature infants, was demonstrated to be caused by developmental immaturity of the cerebral vasculature in mid to late gestational age, and large numbers of gd T cells were observed in postmortem brains from preterm infants (104). Although there were increased IL-17 and IL-22 in mouse brains after injury, neither cytokine contributes to preterm brain injury (104).
In summary, gd T cells and IL-17 have essential roles in ischemic brain injury. Hence, gd T cells and IL-17 should be considered potential therapeutic targets to decrease secondary inflammation after ischemic brain injury (105,106).

Central Nervous System Infection
The CNS infections commonly lead to the disruption of the blood-brain barrier (BBB) protectiveness and subsequent tissue inflammation; however, inflammation is also crucial to CNS immunity, as reduced gd T cell expansion leads to increased host vulnerability to viral infection (107,108). For example, MS patients treated with Natalizumab, a monoclonal antibody against a4-integrin, undergo fatal viral infections, due to the immune cells fail to infiltrate the CNS and eliminate the infection (109).
In contrast to viral infection, there are some (although limited) researches involving the function of gd T cells in models of CNS bacterial infection (110). For example, children with bacterial meningitis exhibit high gd T cell fractions in the CSF (111). Nichols et al. suggested that the gd T cell was an alternative pathway available to respond to Grampositive bacteria CNS infection. They found that TLR2 −/− brain abscess mice (TLR2 is a critical receptor for eliciting responses to Grampositive bacteria) were detected elevated IL-17, and gd T cells were the source of IL-17 (112)(113)(114). Similarly, IL-17R signaling regulates gd T cell infiltration, as well as bacterial clearance, during S. aureusinduced brain abscess formation (115). Also, IL-17 expression is augmented in the CNS of mice infected with Toxoplasma gondii (116). An increased percentage of gdT17 cells was observed in the PB and lesion in children with bacterial meningitis, and the condition was reversed after antibacterial therapy (111). High levels of IL-17 can also be detected in the abscess formation of humans; however, such researches are only associated, since no direct evidence can be demonstrated (117). Nevertheless, evidence for the involvement of gd T cells in any CNS infection is sparse, and more studies are needed to establish a relation between gd T cells and CNS infections.

Central Nervous System Traumatic Diseases
Immune responses and neuroinflammation involving gd T cells are also critically involved in CNS traumatic diseases. Diseases resulting from CNS trauma usually involve irreversible sensory, motor, and autonomic impairments (118). Peripheral immune mechanisms establishment is related to the pathological processes of traumatic brain injury (TBI). Richard et al. found that CD4+ and CD8+ T cells, Tregs, and gd T cells, increased in number within 24 h after TBI (119). Further, recent results from our laboratory found that gd T cells, particularly Vg4 + gd T cells, exert a detrimental role in SCI, probably by providing an important origin of IFN-g, which induces macrophages to adopt the M1 phenotype, with increased secretion of inflammatory cytokines, such as TNF-a (34) (Figure 2). Moreover, one significant discovery from our studies was that bone marrow-derived macrophages (BMDMs) respond to IFN-g. This was supported by two sets of findings. First, IFN-gR −/− mice, chimeras with IFN-gR −/− bone marrow, and mice receiving adoptively transferred IFN-gR −/− peritoneal macrophages, all showed similar recovery following SCI. Second, numbers of M1 macrophages and proinflammatory cytokines are reduced in IFN-gR −/− compared with WT controls (34). Besides, the treatment of SCI with anti-Vg4 antibodies has a beneficial effect, similar to that obtained with anti-TNF-a (34). In conclusion, manipulation of gd T cell functions may be a potential treatment approach for future CNS traumatic diseases.

Other Central Nervous System Diseases
The pathological and clinical outcome of CNS diseases can also be affected by the intestinal microflora in the context of autoimmunity (120)(121)(122)(123)(124). This relationship has been particularly well established for the response to bacteria, including pathogens and commensals, within the intestinal compartment and its effects on the CNS, a connection that was recently termed the gut-brain axis. The gut environment has been found to significantly influence CNS diseases such as MS, EAE, and ischemic brain injury; however, immune cell mechanisms are unclear. In addition, the pathogenesis of intractable epilepsy is related to gd T cells, where proinflammatory gd T cells were concentrated in epileptogenic lesions, and their numbers positively associated with disease severity (125)(126)(127).

CONCLUSION
Since the identification of gd T cells, there has been a boom in related studies and discoveries. Equipped with functions of both innate and adaptive immune cells, gd T cells can provide consequential functions in the development of CNS diseases, such as recognizing a diverse array of antigens, rapid production of inflammatory mediators, and influencing the differentiation of their ab counterparts. Recently, understanding the inflammatory and immune roles of gd T cells has resulted in the development of many prospective therapies for CNS diseases. However, the exact mechanisms behind their contributions are yet to be fully elucidated.
The pandemic of 2019 coronavirus disease COVID-19, caused by the SARS-CoV-2 virus infection, has caused worldwide mortality (128). Past pandemics have demonstrated that COVID-19 is accompanied by diverse neuropsychiatric symptoms, such as encephalopathy, neuromuscular dysfunction, or demyelinating processes (129). Whether recovered SARS-CoV-2 patients will exhibit an increased incidence of MS symptomatology or other delayed neurologic sequelae, is an important, yet unanswered. Nevertheless, more substantial shreds of evidence are required on different subtypes of gd T cells for defining their opposing roles in CNS inflammation and explaining the confounding findings on their pathogenic or protective role in CNS diseases. In summary, this review discusses recent notable studies of the neuro-inflammatory and immune functions of gd T cells, intending to understand their roles in CNS disease, which may be crucial for the effective immunotherapies.

AUTHOR CONTRIBUTIONS
JW and FZ contributed to editing the manuscript. ZL and CS provided administrative support. GS and WZ helped the manuscript editing and discussions. All authors contributed to the article and approved the submitted version.