Mouse microglia express mRNA for TLR1–9 [58]. In addition, TLR stimulation activates the expression of MHC class II and costimulatory molecules, enabling the microglia to efficiently activate CD4⁺ T cells [58]. Infection of many different glial cells (neurons, microglia, oligodendrocytes, and astrocytes) and professional antigen-presenting cells (macrophages, dendritic cells, and B cells) with TMEV activates the production of various cytokines via TLR2, 3, 4, and 7 [30,38,70,71,72]. TMEV contains a single RNA genome recognized by TLR7 and the dsRNA intermediate by TLR3, and consequently TMEV infection activates NF-κB, AP-1, and IRFs, resulting in the production of various cytokines. These signals lead to further activation of NF-κB and increased production of various inflammatory cytokines such as IL-1β, IL-6, and IFNα/β, which augment the development of the pathogenic Th17 cell type [30,38,63,65,73]. In addition, the melanoma differentiation-associated gene 5 (MDA5) also recognizes viral messages leading to the activation of NF-κB and preferentially promotes the production of IFNα/β [74]. Interestingly, NF-κB signaling is necessary for viral replication and the expression of Bcl-2 and Bcl-xL, which prevent TMEV-induced cellular apoptosis extending viral replication and cytokine production [61,75]. Activation of chemokine and cytokine genes by TMEV is largely independent of the IFNαβ pathway and partly dependent on the dsRNA-dependent protein kinase (PKR) and MAP kinase pathways [73,76]. Interestingly, the activation of NF-κB is necessary for TMEV infection/replication [61] and, thus, the pattern recognition receptor-mediated cellular activation also serves as a mediator of viral persistence (Figure 2).

TLR-mediated signaling leads to the polymerization of the node-like receptor protein 3 (NLRP3) inflammasome, resulting in the activation of caspase-1 and the subsequent production of IL-1β and IL-18 [77,78,79]. The MDA-5 signaling also promotes the activation of NLRP3 [80,81]. Consequently, TMEV infection induces strong NLRP3 inflammasome and downstream prostaglandin E2 (PGE₂) signaling in dendritic cells (DCs) and macrophages/microglia from susceptible SJL mice compared to the cells from resistant B6 mice [82]. Inhibition of PGE₂ signaling using AH23848, an inhibitor of PGE₂ receptor, decreases pathogenesis of TMEV-IDD and viral loads in the CNS, indicating the pathogenic role of PGE₂ [82]. Furthermore, administration of IL-1β renders resistant B6 mice permissive to the development of TMEV-IDD, consistent with that the NLRP3 activation plays an important role in the pathogenesis [48,82]. The presence of a high IL-1β level elevates the pathogenesis by preferentially promoting the development of pathogenic Th17 responses [83]. These results suggest that the NLRP3 inflammasome signaling contributes to the pathogenesis of TMEV-induced demyelinating disease (Figure 2).

Viral and bacterial infections result in the production of chemokines and cytokines, which further activate additional cytokine and chemokine gene expression, promoting cellular infiltration and subsequent induction of adaptive immune responses [84,85,86]. Various chemokines and cytokines are produced in the CNS of mice after viral infections, including TMEV [87,88,89,90,91,92]. TMEV infection upregulates various chemokine gene expressions in glial cells and other antigen-presenting cells as early as 1–3 h after infection [51,93]. The infection of primary glial cells, including astrocytes and microglia, results in the activation of chemokine genes (CXCL1, CXCL2, CXCL10, CCL2, CCL3, CCL4, CCL5, CCL7, and CCL12) that are important in the initiation of an inflammatory response [93,94,95]. These results suggest that glial cells play critical roles in the development of, or protection from, TMEV-induced, immune-mediated inflammatory disease. Treatment with anti-CCL2 antibodies results in a significant decrease in the clinical disease progression, decreased CNS inflammation, and reduced viral load in the CNS [96]. This study suggests a protective role of CCL2 in the development of TMEV-IDD. In contrast to CCL2, both the lack of CXCL1 during TMEV infection and the excessive presence of this chemokine promote the pathogenesis of demyelinating disease. Therefore, a balance in the level of CXCL1 during TMEV infection is critically important in controlling the pathogenesis of demyelinating disease, although the level of CXCL1 produced is significantly higher in cells from susceptible SJL mice compared to that in cells from resistant BALB/c or B6 mice [95,97].

Proinflammatory cytokines, such as IL-1β, IL-6, IFNα/β, and TNFα, produced upon TMEV infection via TLR-mediated signals, further upregulate the production of chemokines [67,70,73,98]. In addition, TMEV infection upregulates the production of fibrin deposition, different adhesion molecules such as ICAM and VCAM, and endothelin-1 associated with blood–brain barrier permeability and cellular infiltration, which appear to participate in the pathogenesis of demyelinating disease [56,99,100,101,102]. TNF-α also appears to play an important pathogenic role in the development of TMEV-IDD because higher numbers of TNF-α producing cells are found in TMEV-infected susceptible mice [103]. However, TNF-α may also play a protective role because the absence of TNF-α worsens the pathology in TMEV-infected mice [104]. The signaling by NLRP3 inflammasome leads to the production of IL-1β and PEG₂ [105]. TMEV infection activates NLRP3 via TLR signaling [82]. IL-1 and other inflammatory cytokines produced after TMEV infection are greater in susceptible mice compared to resistant mice [67]. A high IL-1 level plays a pathogenic role by elevating pathogenic Th17 responses, whereas a lack of IL-1 signals promotes viral persistence in the spinal cord due to insufficient T cell activation by elevating the production of inhibitory cytokines and regulatory molecules [48,83]. The inhibition of virus-induced PGE₂ signaling results in decreased pathogenesis for TMEV-IDD, suggesting that the excessive activation of the NLRP3 inflammasome leading to the PGE₂ signaling contributes to the pathogenesis [82]. The excessive PEG₂ levels may prevent the T cell killing of target cells by inhibiting CD95L expression [106] and type I INF production [82]. TMEV infection upregulates the expression of PD-1 and PDL-1 in the CNS via type I IFN signaling in conjunction with the presence of IL-6 signaling [107,108].

TMEV infection results in the production of relatively high levels of Type I IFNs in various cell types from susceptible mice [73,109,110]. Type I IFNs stimulate T cell responses after TMEV infection by activating the expression of costimulatory molecules on APCs [30,63,111,112,113]. IFN-IR KO mice develop rapid fatal encephalitis accompanied with greater viral load and infiltration of immune cells of the CNS compared to the wild type mice [113]. The less efficient stimulation of virus-specific T cells in IFN-IR KO mice is attributable to the poor expression of costimulatory molecules on APCs. However, IFN-I also controls cellular infiltration to the CNS and shapes local T cell immune responses and B cell activation [30,63,113]. SJL DCs infected with TMEV induce rapid production of several different Type I IFNs (IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α9, IFN-α11, IFN-αβ, and IFN-β), and a type II IFN (IFN-γ) [63]. TMEV-infected DCs from susceptible mice produce higher levels of type I IFNs and IFN-γ compared to virus-infected DCs from resistant mice. The difference in the production of IFNs contributes to the significantly different virus-induced apoptosis, inhibition of development, and function of DCs [63]. The antiviral effect of Type I IFNs on TMEV replication is rather narrowly limited to just before viral infection. Therefore, the presence of high levels of Type IFNs in susceptible SJL mice may not necessarily be beneficial in controlling viral loads in the TMEV-induced demyelinating disease.

IL-6 and IL-1β play particularly important roles in the pathogenic T cell responses in the development of TMEV-induced demyelinating disease [65,83,113]. In addition, the IL-1β signal amplifies IL-6 production and activates CD4⁺ T cell expansion [48,114]. IL-6 participates in initiating and amplifying the pathogenesis of TMEV-induced demyelinating disease. In fact, mice lacking IL-6 signaling fail to develop demyelinating disease following TMEV infection [65,75]. However, the effect of IL-6 could be different depending on the TMEV strains because SJL mice receiving IL-6 prior to TMEV DA infection are free from the disease [115]. In addition, the presence of IL-6 is essential for the survival of mice infected with other viruses, such as influenza virus or lymphocyte choriomeningitis virus [116,117].

The demyelination induced after TMEV-inoculation is immune-mediated based on early study results indicating that the treatment of mice with anti-thymocyte, anti-Ia (MHC class II), or anti-CD4 antibodies delays the onset of disease [118,119,120], and that the level of the delayed-type hypersensitivity response specific for viral antigens correlates with the course of clinical signs of disease [27]. During chronic TMEV infection, susceptible mice display prolonged T cell immune responses toward viral antigens [13,121,122]. The association between susceptibility to TMEV-induced demyelinating disease (TMEV-IDD) and the MHC [34,121], in addition to the T cell receptor (TCR) β-chain genes [123,124], further supports involvement of T cell responses in the pathogenesis. Moreover, many studies have suggested that the CD4⁺ Th response is pathogenic in susceptible mice [125,126,127]. Alternatively, some studies indicate that CD4⁺ T cells confer protection from disease [128,129,130]. However, these early studies potentially include different CD4⁺ T cell subpopulations, including Th1, Th17, and Treg cells, which are functionally different. I-A^s restricted CD4⁺ T cell epitopes of TMEV in susceptible SJL mice have been identified: one major (VP2_72–86) and two minor (VP1_233–244, VP3_24–37) epitopes on capsid proteins and one predominant epitope (3D_21–36) on RNA polymerase [126,131,132,133]. Similarly, two predominant I-A^b-restricted epitopes in resistant B6 mice have been found in the capsid region [134]. A diagram displaying the CD4⁺ T cell (I-A^s and I-A^b) and CD8⁺ T cell epitopes (H-2K^s and H-2D^b) of TMEV BeAn is shown in Figure 3.

As CD4⁺ T cells include several subpopulations (Th1, Th17, and Treg), which have distinct functions in the development of antiviral immunity and inflammatory diseases, the availability of naïve undifferentiated virus-specific CD4⁺ T cells is critically important for investigating CD4⁺ T cell differentiation during TMEV infection. TCR transgenic mice specific for a major CD4⁺ T cell epitope (VP2_72–86) of TMEV restricted with I-A^s (018030, SJL.Cg-Tg (TcraTcrbVP2) 1 Bkim/J from the Jackson Laboratory) have been used to investigate the differentiation of CD4⁺ T cell types during viral infection [65,136]. CD4⁺ T cells from VP2_72–86-specific TCR-Tg mice in the absence of TMEV infection can be the source of naïve CD4⁺ T cells and the T cell differentiation could be investigated in the presence of virus-infected APCs (Figure 4). The differentiation of CD4⁺ T cell types were investigated by comparing T cell development in TMEV-infected TCR-Tg mice with those infected with UV-inactivated TMEV. The differentiation was also investigated following the adoptive transfer of CFSE-labeled naïve VP2 TCR-Tg CD4⁺ T cells into SJL mice followed by TMEV infection [136]. Alternatively, the differentiation of naïve TCR-Tg CD4⁺ T cells has been investigated in vitro in the presence of TMEV-infected DCs or UV-inactivated TMEV-infected DCs [65].

TMEV-specific Th1 cells producing IFN-γ mediate lysis of the virus-infected glial cells in a Fas-dependent mechanism [137]. Preimmunization of SJL mice with capsid-epitope peptides significantly increased capsid-specific CD4⁺ T cell numbers in the CNS during the early stages of viral infection [138]. These preimmunized mice delay the development of demyelinating disease in SJL mice, suggesting a protective role of capsid-reactive Th1 cells. Genetically resistant mice with deficiencies in the IFN-γ or its receptor genes fail to clear TMEV and develop extensive demyelinating disease [139,140]. Similarly, the intraperitoneal (i.p.) injection of susceptible mice with an IFN-γ-neutralizing monoclonal antibody (mAb) or of mice deficient in the Type I IFN receptor significantly accelerates the onset of disease [112,141,142]. The treatment of mice with anti-IFN-γ mAb does not reduce the delayed-type hypersensitivity (DTH) response to the virus and does not produce significant effects in the clinical course of disease, suggesting that DTH response may not reflect their Th1 response [141]. However, intracerebral administration of recombinant IFN-γ significantly accelerates the onset of TMEV-induced disease and the enhancing effect of IFN-γ is abrogated with treatment with anti-IFN-γ mAb [141]. Therefore, the level of IFN-γ appears to play a key role in the TMEV-induced inflammatory response and the perturbation of this cytokine may alter the course of demyelinating disease. IFN-γ is produced by natural killer (NK) cells, and CD4⁺ and CD8⁺ T cells, and thus, the effect of IFN-γ may not entirely reflect the function of the CD4⁺ T cell population. In addition, the presence of IFN-γ receptors on the CNS glia suggests the importance of the target cells in the function of IFN-γ during TMEV-induced demyelination [143]. The effects of preimmunization and tolerization with individual epitopes indicate that capsid-specific CD4⁺ T cells are protective, whereas viral RNA polymerase (3D_21–36)-specific CD4⁺ T cells exacerbate the development of TMEV-induced demyelinating disease [133]. These results suggest the location and abundance of Th1 responses also play a role in the protection from the pathogenesis of TMEV-induced demyelinating disease.

Th17 cells producing IL-17, which are a distinct subset of CD4₊ T cells, are involved in the pathogenesis of various autoimmune diseases [144,145,146,147]. Th17 cells preferentially develop in an IL-6-dependent manner after TMEV infection [65,75]. The presence of IL-6 is necessary for the development of Th17 responses and the pathogenesis of TMEV-IDD as shown with IL-6 KO mice [75]. Th17 cells promote the pathogenesis of chronic demyelinating disease by increasing viral persistence via upregulating antiapoptotic molecules and blocking target cell killing by cytotoxic T cells. Administration of the neutralizing antibody against IL-17 augments viral clearance and prevents the pathogenesis of TMEV-induced demyelinating disease [65]. The presence of IL-6 further synergistically enhances the antiapoptotic function of IL-17 function, which further facilitates the TMEV persistence in the CNS [75]. Conversely, IL-17 may also further induce IL-6, which amplifies the IL-17/IL-6 cytokine circuit [148]. The association of Th17 responses to the pathogenesis of TMEV-induced demyelinating disease has been confirmed using Th17-biased RORγt Tg mice with the B6 background, which render the development of TMEV-IDD with elevated Th17 responses, in contrast to the control B6 mice [149]. Using isolated DCs infected with live TMEV and UV-inactivated TMEV, it has been demonstrated that the development of Th17 cells is dependent on DCs infected with live TMEV producing various innate immune responses [65]. Thus, these results indicate a pathogenic role of Th17 cells in persistent TMEV infection and the pathogenesis of associated chronic inflammatory demyelinating diseases (Figure 4).

High levels of FoxP3+CD4+ T cells are present in the CNS of virus-infected mice as early as 3 d after viral infection [150,151]. The early induction of FoxP3⁺CD4⁺ T cells may depend on the function of the TCR2-mediated signal [152,153] upregulated after TMEV infection [72]. When FoxP3⁺CD4⁺ T cells were removed, viral loads in the CNS and the development of clinical signs were significantly reduced in susceptible SJL mice [150], but not in resistant C57BL/6 mice [154]. These results suggest that the presence of a high level of FoxP3⁺CD4⁺ T cells promotes the pathogenesis of demyelinating disease. However, as much as a two-fold higher proportion of FoxP3⁺CD4⁺ T cells in the CNS of virus-infected SJL mice displaced CD25^lo [136]. Thus, FoxP3⁺CD4⁺ T cells in the CNS may require further activation to be functional regulatory T cells [155,156,157]. High levels of CD25^-FoxP3⁺CD4⁺ T cells were also observed in chronic hepatitis B virus-infected patients [158] and patients with systemic lupus [159]. CD25^loFoxP3⁺CD4⁺ T cells may lose FoxP3 expression and undergo trans-differentiation into pathogenic Th17 cells [160,161]. Therefore, it is conceivable that some or most of the CD25^loFoxP3⁺CD4⁺ T cells may be converted into pathogenic Th17 cells under the environment of abundant cytokines such as IL-6 and IL-1β in the CNS of TMEV-infected mice [65,83,136].

In many chronic viral infections, including TMEV, FoxP3⁺CD4⁺ T cells appear to contribute to the pathogenesis by inhibiting protective T cell function and consequently promoting viral persistence [150,162]. In contrast, FoxP3⁺CD4⁺ T cells may be beneficial in controlling acute viral infections [163,164]. CD25^loFoxP3⁺CD4⁺ T cells do not appear to display regulatory function [157]. After activation of CD25^loFoxP3⁺CD4⁺ T cells in vitro under experimental conditions with various cytokines and peptides [156,165,166], up to 60% of CD4⁺ T cells bear FoxP3 and CD25^hi [136]. The TMEV VP2-specific FoxP3⁺CD4⁺ T cells activated in vitro inhibit the production of IFN-γ but not IL-17 by VP2-specific CD4⁺ T cells [136]. In contrast, activated epitope-specific FoxP3⁺CD4⁺ T cells in MHV-infected mice efficiently inhibit the proliferation of the same epitope-specific CD4⁺ T cells [151]. The previous studies indicating the inhibition of proliferation of Th cells utilized FoxP3⁺CD4⁺ T cells generated in vivo from TMEV-infected mice [150]. In addition, MHV-specific FoxP3⁺ CD4⁺ T cells were stimulated in vitro with anti-CD3 and anti-CD28 antibodies instead of using the epitope peptide [151]. Therefore, activating antigens for FoxP3⁺ CD4⁺ T cells and Th cells, in addition to the heterogeneity of target Th cells during the inhibition assays, may be important. TMEV-infected mice treated with ex vivo-generated FoxP3⁺ Tregs at an early stage of viral infection worsened the clinical signs, whereas the mice treated with the Tregs at a later stage decreased immune cell recruitment in the CNS [167]. Nevertheless, VP2 epitope-specific FoxP3⁺CD4⁺ T cells preferentially inhibited the production of IFN-γ, but not IL-17, by the same epitope-reactive Th cells [136]. As virus-reactive IFN-γ-producing Th1 cells are protective but IL-17-producing Th17 cells inhibit Th1 development and cytotoxic T cell function [65,168], FoxP3⁺CD4⁺ T cells together with Th17 cells may promote the pathogenesis of TMEV-induced demyelinating disease in an epitope-dependent manner (Figure 5).

As demyelination is closely linked to viral persistence [42,43,44], TMEV-specific cytotoxic CD8⁺ T cells producing IFN-γ and perforin (Tc1) are likely to play an important role in protection and/or resistance [39,169,170]. TMEV-specific cytotoxic CD8⁺ T lymphocytes (CTL) appear to damage virus-infected, myelin-producing oligodendrocytes and other cell types [171,172,173,174]. Many investigations further confirmed the role of Tc1 cells by antibody-mediated CD8⁺ T cell depletion [175], and using Class I deficient mice [176,177,178]. Rodriguez and his colleagues proposed that CD8⁺ T cells are necessary for the manifestation of clinical symptoms using the DA strain of TMEV [169,173]. However, β₂M-deficient or perforin-deficient mice on a resistant background are susceptible to both demyelination and clinical disease [170,176,177,178]. Furthermore, β₂M-deficient mice with the susceptible SJL background displayed similar exacerbation of TMEV-IDD [179]. These results indicated a protective role of CD8⁺ T cells in the development of TMEV-induced demyelinating disease in both resistant and susceptible mice. Moreover, the presence of a high level of CTL in resistant mice and a low level in susceptible mice [180], and the resistance to TMEV-IDD in susceptible mice adoptively received CD8⁺ T cells [181], further support the protective function of CTL. In resistant B6 mice, the majority (50% to 70%) of CNS-infiltrating CD8⁺ T cells recognize VP2_121–130 [182,183], and two minor populations (<10%) react with VP2_165–173 and VP3_110–120 capsid epitopes [184] based on the production of IFN-γ (Figure 3). Similarly, CNS-infiltrating CD8⁺ T cells of virus-infected SJL mice react with one predominant (VP3_159–166,) and two subdominant capsid epitopes (VP3_173–181, and VP1_11–20) [35]. During the early stages of viral infection, a lower level of virus-specific CD8⁺ T cells in SJL mice was observed [184]. In addition, the resistance of (B6xSJL)F1 mice is associated with a higher level of the initial virus-specific H-2b-restricted CD8⁺ T cell responses compared to the H-2s-restricted CD8⁺ T cell responses [37]. These results further suggest that Tc1 cells play an important protective role in preventing TMEV-IDD by clearing viral loads from the CNS. There is, however, a possibility that certain CD8⁺ T cell populations play a pathogenic role, perhaps depending on epitope-reactivity or cytokine production, in TMEV-induced demyelination [171,172,173,185]. Similar CTL-mediated immunopathology was reported with the lymphochoriomeningitis virus (LCMV) and Coxsackie B virus in mice [186,187,188].

Chronic inflammation promotes the induction of IL-17-producing CD8⁺ cells with reduced cytolytic function [189,190]. Therefore, it is conceivable that a subset of CD8⁺ T cells producing IL-17 may be associated with the pathogenesis of TMEV-IDD. To investigate the possible epitope-dependent function of CD8⁺ T cells in the protection and/or pathogenesis, a single amino acid substitution was introduced into the predominant viral epitope (VP3_159–166) and/or a subdominant viral epitope (VP3_173–181) of susceptible SJL/J mice by site-directed mutagenesis altering a single amino acid within the epitopes [191]. The resulting variant viruses (substituted at N160V, P179A, and N160V/P179A) failed to induce CD8⁺ T cell responses in the respective epitopes. TMEV (N160V and N160V/P179A) viruses, deficient in the predominant CD8⁺ T cell epitope, do not induce demyelinating disease [191]. The CD8⁺ T cells specific for the predominant VP3_159–166 epitope identified by reactivity with the tetramers containing VP3_159–166 showed strong cytolytic activity and produced high levels of IFN-γ. In contrast, VP3_173–181-specific CD8⁺ T cells produced higher levels of transforming growth factor beta, interleukin-22 (IL-22), and IL-17 mRNA, and exhibited minimal cytotoxicity. Therefore, it is conceivable that differences in the functional avidity toward their cognate epitopes and/or the type of cytokines produced may affect the function of CD8⁺ T cell populations in an epitope-dependent manner. IFN-γ producing VP3_159–166-specific CD8⁺ T cells with high cytolytic function, in contrast to low-cytolytic VP3_173–181-specific CD8⁺ T cells, may be necessary to initiate the pathogenic process by destroying infected neurons and/or releasing sequestered autoantigens followed by Tc-17- and Th17-mediated inhibition of cytolytic function promoting viral persistence. Thus, both Th17 and Tc1 populations reactive to TMEV epitopes may cooperatively participate in the pathogenesis of virally induced demyelinating disease in an epitope-dependent manner [191].

The major neutralizing antibody epitopes of TMEV reside on VP1, and these epitopes may also be involved in virus-binding to cellular receptors [192,193]. The antibodies found in the spinal fluids of TMEV-infected mice [194] and in MS patients [195] displayed relatively limited heterogeneity. Furthermore, B cells producing antibodies to VP1 and VP2 proteins are detectable in the demyelinating lesions [196]. The plasma cells producing anti-TMEV antibodies were found primarily in perivascular infiltrates and in the meninges of the CNS parenchyma [197].

Antibodies from TMEV-infected mice also recognize several linear epitopes of 13–15 amino acid peptides or recombinant proteins representing the capsid proteins, and some of these antibodies display varying degrees of virus neutralization accompanying the protection from demyelinating disease development [122,198,199,200]. Anti-TMEV antibody responses during the early stage of viral infection play a protective role [49,201,202]. In addition, treatment of mice with the monoclonal anti-CD20 antibody accelerate the pathogenesis of TMEV-induced demyelinating disease, suggesting a protective role of the B cells [203]. However, antibodies to viral determinants appear to play a relatively minor role in the protection of mice from the pathogenesis of demyelinating disease compared to CD4⁺ Th1 and CD8⁺ T cells [122,201,202].

An earlier study suggested that B cells are not permissive to TMEV infection [204]. However, a recent study showed as much as 40% of primary B cells in susceptible SJL mice are permissive to TMEV infection and replication [30]. TMEV-infected B cells expressed elevated levels of an activation marker, CD69. In addition, the expression of MHC class II and costimulatory molecules was upregulated in the infected B cells, accompanied by elevated levels of antigen-presenting function and antibody production [30]. The upregulation of these B cell activation markers induced with TMEV infection was replicated with the treatment of B cells with TLR ligands for TLR2, TLR3, TLR4, TLR7, and TLR9, suggesting that B cell activation after TMEV infection is associated with TLR signals [30]. These results are consistent with the previous findings that various TLR-mediated signals activate B cells to function as an important professional APC type involved in the activation of Th cell types [205,206,207]. The innate responses via TLRs following TMEV infection appear to trigger excessive production of IFN-α/β, IL-6, IL-1, and PGE₂ in B cells of susceptible mice [30,208,209,210]. These innate immune responses following TMEV infection also activate B cells to produce antibodies and facilitate elevated T cell responses [30]. In addition, these cytokines may, in turn, further enhance the viral persistence by inhibiting apoptosis of TMEV-infected B cells and protective Th1 cell responses, while enhancing pathogenic Th17 responses [65,82] (Figure 5). Furthermore, TMEV-infected B cells form a germinal center-like structure with close proximity of T cells in the CNS, which may promote the induction of pathogenic T cells, such as Th17 and FoxP3⁺ Treg, recognizing not only viral determinants but also autoantigens [30].