Lymphocytes and autoimmunity after spinal cord injury.

Experimental Neurology 258 (2014) 78–90

Contents lists available at ScienceDirect

Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

Review

Lymphocytes and autoimmunity after spinal cord injury T. Bucky Jones ⁎ Department of Anatomy, Arizona College of Medicine, Midwestern University, Glendale, AZ, USA

a r t i c l e

i n f o

Article history: Received 2 October 2013 Revised 5 March 2014 Accepted 6 March 2014 Keywords: Autoimmunity Spinal cord injury Effector T-lymphocytes B-lymphocytes Autoantibodies Adaptive immunity Regulatory T-cells

a b s t r a c t Over the past 15 years an immense amount of data has accumulated regarding the infiltration and activation of lymphocytes in the traumatized spinal cord. Although the impact of the intraspinal accumulation of lymphocytes is still unclear, modulation of the adaptive immune response via active and passive vaccination is being evaluated for its preclinical efficacy in improving the outcome for spinal-injured individuals. The complexity of the interaction between the nervous and the immune systems is highlighted in the contradictions that appear in response to these modulations. Current evidence regarding augmentation and inhibition of the adaptive immune response to spinal cord injury is reviewed with an aim toward reconciling conflicting data and providing consensus issues that may be exploited in future therapies. Opportunities such an approach may provide are highlighted as well as the obstacles that must be overcome before such approaches can be translated into clinical trials. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adaptive immunity in the injured spinal cord . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic manipulation of lymphocytes . . . . . . . . . . . . . . . . Pharmacologic suppression of adaptive immunity . . . . . . . . . . . Pharmacologic modulation of lymphocyte trafficking into the injured CNS Trauma-induced autoimmunity (TIA) . . . . . . . . . . . . . . . . . . . SCI activates CNS-specific lymphocytes . . . . . . . . . . . . . . . . Functional impact of TIA . . . . . . . . . . . . . . . . . . . . . . Experimental manipulation of CNS autoimmune lymphocytes in SCI . . . . . Passive immunization with myelin-reactive T-cells . . . . . . . . . . Vaccination with antigen-pulsed DCs . . . . . . . . . . . . . . . . . Active immunization with myelin antigens . . . . . . . . . . . . . . Epitope vaccines using APLs . . . . . . . . . . . . . . . . . . . . . Treg-mediated neuroprotection . . . . . . . . . . . . . . . . . . . . . Translational potential of neuroprotective immunity . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

79 79 79 79 79 80 80 80 80 82 82 83 84 84 85 85 86 86 87

Abbreviations: DC, dendritic cell; NK, natural killer; RAG, recombination-activating gene; BCKO, B-cell knockout; BMS, Basso Mouse Scale; BBB, Basso–Beattie–Bresnahan; Ig, immunoglobulin; CR3, complement receptor 3; FcγR, Fc gamma receptor; CsA, cyclosporin A; Treg, regulatory T-cell; nTreg, naturally occurring Treg; iTreg, induced Treg; MS, multiple sclerosis; PD, Parkinson's disease; AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MOG, myelin oligodendrocyte protein; MAG, myelin-associated glycoprotein; OMgp, oligodendrocyte myelin glycoprotein; GluR, glutamate receptor; NgR, Nogo receptor; TBI, traumatic brain injury; APL, altered peptide ligand; SCH, spinal cord homogenate; APC, antigen presenting cell; TNF, tumor necrosis factor; TGF, transforming growth factor; IFN, interferon; IL, interleukin; TCR, T-cell receptor; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; T-MBP, MBP-specific T-cells; CFA, complete Freund's adjuvant; IFA, incomplete Freund's adjuvant; VIP, vasoactive intestinal peptide; BCG, Bacillus Calmette–Guerin; FoxP3, forkhead box P3; Aβ, amyloid beta; SOD1, superoxide dismutase-1; Cop-1, Copolymer1; ROS, reactive oxygen species. ⁎ Midwestern University, 19555 N. 59th Ave., Glendale, AZ 85308, USA. E-mail address: [email protected].

http://dx.doi.org/10.1016/j.expneurol.2014.03.003 0014-4886/© 2014 Elsevier Inc. All rights reserved.

T.B. Jones / Experimental Neurology 258 (2014) 78–90

Introduction Over the past 15 years an immense amount of data has accumulated regarding the role of lymphocytes in the traumatized spinal cord. Despite this focus, it remains unclear whether the net impact of this response is beneficial or detrimental to the host. Indeed, it may be both. The purpose of this review is to discuss and critically evaluate the current status of the field regarding lymphocytes that accumulate within the injured spinal cord. Before modulation of the lymphocytic response can be considered clinically for its neuroprotective and regenerative potential in the context of spinal cord injury (SCI), it is imperative that we understand the complex effects lymphocytes exert on preserved or damaged tissues within the injury site. Adaptive immunity in the injured spinal cord Overview The primary cellular effectors of adaptive immunity are the T- and B-lymphocytes. Lymphocyte activation requires the selective recognition of antigens via highly specific cell surface receptors (Chen and Flies, 2013; Yuseff et al., 2013), in contrast to the comparatively nonselective activation of innate immune components, which include macrophages, dendritic cells (DCs), natural killer (NK) cells and complement. T- and B-lymphocytes responsive to the same antigen interact within secondary lymphoid organs then migrate to the injury site to mount a multifaceted adaptive immune response. Lymphocytic infiltration of the injury site occurs during the first week post-injury and is maintained chronically (Ankeny et al., 2006; Beck et al., 2010; Sroga et al., 2003; Vaughn et al., 2013). Whether lymphocytes contribute to the progression or the resolution of pathophysiological events within the injury site is not well defined. Several lines of evidence implicate intraspinal lymphocytes as effectors of pathology. The use of animal models with genetic mutations in genes associated with lymphocyte development allows insight into the role of certain lymphocyte populations in SCI. Additional evidence comes from pharmacological manipulation of lymphocyte activation, function, or migration to the injury site. These data are reviewed in the next section. Genetic manipulation of lymphocytes In genetic models of mice and rats that lack T-cells, the absence of T-cells is generally associated with improvements in function and/or tissue preservation following SCI. In athymic (nude) rats, improvements in hind limb movements were observed after complete spinal cord transection (Potas et al., 2006). This was attributed to improved spinal reflexes rather than regeneration of descending motor systems as there were no axons present caudal to the transection site in either the nude rats or the controls. Rostral to the transection site there was improved tissue architecture associated with a reduction in activated macrophages (Potas et al., 2006). Improvements in locomotor recovery have also been reported following contusion SCI in non-obese diabetic severe combined immunodeficient mice and following compression SCI in recombinationactivating gene (RAG) 2-deficient mice (Luchetti et al., 2010; Wu et al., 2012). These mice each have a genetic mutation that affects the generation of mature lymphocytes, thus lack both T- and B-lymphocytes. In RAG2-deficient mice, locomotor recovery was associated with a greater number of monoaminergic axons caudal to the injury site which the authors attributed to regeneration (Wu et al., 2012). Although this observation may be due to an enhanced regenerative response in the absence of lymphocytes, it could also be due to a reduction in immune-mediated tissue injury when T- and B-cells are removed. Less tissue damage at the injury site would allow greater numbers of monoaminergic axons to survive and sprout in distal spinal segments. Similar results were observed in a model of peripheral nerve injury in

79

RAG-deficient mice reconstituted with B-cells (Serpe et al., 2003). Collectively, these data suggest that T- and B-cells contribute to postinjury tissue pathology, although their relative contributions cannot be determined from these studies. To evaluate the specific role of B-cells in post-injury neuropathology, Ankeny et al. (2009) used B-cell knockout mice (BCKO) mice that lack mature B-cells, but have a normal repertoire of T-cells. Following moderate contusion SCI, BCKO mice had improved Basso Mouse Scale (BMS) locomotor scores (Basso et al., 2006) associated with decreased lesion volume and lower levels of antibodies (immunoglobulin (Ig)M and IgG) in the cerebrospinal fluid. The presence of B220 + IgG + B-cells in the spinal cord of injured wild-type mice indicates a population of activated, mature B effector cells that had not differentiated into plasma cells (Ankeny et al., 2009). Results of this study are consistent with the studies in RAG-deficient mice described above and specifically implicate B-cells as effectors of pathology. Ankeny et al. (2009) demonstrated that the attenuation of lesion pathology and functional impairment in BCKO mice was due in part to antibodies binding to either complement receptor 3 (CR3) or Fc gamma receptor (FcγR), suggesting that the mechanism by which B-cells exert their pathogenic effects is either via activation of complement or the recruitment and activation of cells (e.g., macrophages) that express receptors for Igs (Ankeny et al., 2009). Indeed, it may be both. Complement components are present within the chronically injured spinal cord environment and contribute to pathology (Beck et al., 2010), and ligation of FcγRs modulates CR3-mediated phagocytosis (Huang et al., 2011b); also see Peterson et al., 2014-in this issue). The interaction of these two receptors likely plays an important role in modulation of the adaptive immune response. Pharmacologic suppression of adaptive immunity Several studies have demonstrated beneficial effects of Cyclosporin A (CsA) and tacrolimus (FK506) on locomotor recovery after SCI (Ibarra et al., 2003; Lopez-Vales et al., 2005; Lu et al., 2010; Madsen et al., 1998; McMahon et al., 2009; Nottingham et al., 2002). These agents have documented neuroprotective effects in experimental models of peripheral nerve and CNS injury, although the precise mechanisms are not well understood (Toll et al., 2011). In experimental SCI, improved functional recovery following CsA treatment was associated with increased survival of motor neurons in the spinal cord (Lu et al., 2010) and brainstem (Ibarra et al., 2003), suggesting a direct effect on neuronal survival. Cyclosporin A and FK506 also act as immunosuppressive agents that can inhibit T-cell proliferation via inhibitory effects on calcineurin (Fruman et al., 1992). Following SCI, treatment with CsA reduced T-cell infiltration into the CNS which occurred in parallel with a reduction in macrophage activation; this is possibly due to decreased T-cell cytokines in the injury site, although this was not specifically evaluated (Lu et al., 2010). Importantly, treatment with CsA improved outcomes whether given prophylactically or therapeutically, although earlier treatment seemed to provide greater benefit (Ibarra et al., 2003; McMahon et al., 2009). Inhibition of T-cell function via antibody-mediated blockade of CD25, the high-affinity α chain of the interleukin (IL)-2 receptor, beginning at six weeks post-injury improved functional recovery in contused mice, suggesting that the inflammatory environment within the chronically injured spinal cord may have negative effects on any reparative processes that are initiated within the site (Arnold and Hagg, 2011). CD25 is expressed on recently activated CD4+ T effector cells and naturally occurring CD4+ regulatory T-cells (nTregs) which endows them with the ability to respond to IL-2 signaling, a requirement for subsequent antigen-specific proliferation and differentiation. Because CD25 is constitutively expressed by Tregs and is required for their survival and proliferation (Furtado et al., 2002; Littman and Rudensky, 2010), anti-CD25 treatment is generally considered to act via downregulation or inactivation of Tregs (Arnold and Hagg, 2011). However, it is also

80


possible that anti-CD25 treatment inhibited early activation of CD4+ T effector cells or promoted immunoregulatory functions in NK cells (Bielekova et al., 2006; Rommer et al., 2014; Wiendl and Gross, 2013). Indeed, such a mechanism was postulated to provide local immune regulation underlying T-cell-mediated neuroprotection in a model of optic nerve crush injury (Shaked et al., 2004). It is also possible that anti-CD25 treatment altered CD4 + T-cell trafficking into the CNS by reducing levels of CNS-derived IL-2. Mice that are deficient in brain IL-2 but have wild-type peripheral immune cells, have increased trafficking of CD4+ T-cells into the brain, suggesting that CNS-derived IL-2 normally limits T-cell entry into the brain (Huang et al., 2011a). Pharmacologic modulation of lymphocyte trafficking into the injured CNS Fingolimod (FTY720) is a sphingosine receptor modulator currently undergoing phase III clinical trials in multiple sclerosis (MS). Fingolimod eliminates the sphingosine-1-phosphate signal required to allow egress of lymphocytes from peripheral lymphoid tissues; thus preventing the migration of T-cells into the CNS. In a SCI model, treatment with FTY720 improved locomotor recovery (Lee et al., 2009; Norimatsu et al., 2012; Wang et al., 2009). Functional improvements were accompanied by reduced T-cell infiltration into the injury site and increased residual myelin at and caudal to the epicenter (Lee et al., 2009). Electrophysiological recordings demonstrated increased mean amplitude with shorter latency of somatosensory evoked potentials, indicative of enhanced axonal conduction along the somatosensory tracts in FTY720treated rats (Wang et al., 2009). Similar beneficial effects have been observed in experimental models of cerebral ischemia (Wei et al., 2011) and oral fingolimod reduced indices of disease progression in MS patients in a phase II placebo-controlled clinical trial (Kappos et al., 2010). These data are consistent with a role of T-cells in immunemediated CNS pathology, although both immune-dependent and immune-independent mechanisms of FTY720-mediated protection have been described (Norimatsu et al., 2012). Trauma-induced autoimmunity (TIA) SCI activates CNS-specific lymphocytes There is evidence from both human and animal studies demonstrating that trauma activates an endogenous repertoire of CNS-reactive lymphocytes. The frequency of T lymphocytes reactive to myelin proteins (e.g., myelin basic protein (MBP)) is increased in the serum of SCI patients (Kil et al., 1999; Olsson et al., 1993; Wucherpfennig et al., 1994; Zajarias-Fainsod et al., 2012) and SCI patients have chronically elevated titers of serum autoantibodies (Davies et al., 2007; Hayes et al., 2002; Mizrachi et al., 1983; Zajarias-Fainsod et al., 2012). Collectively, these data indicate the functional activation of autoreactive T- and B-cells, a phenomenon that we previously designated as traumainduced autoimmunity, or TIA (Popovich and Jones, 2003; Popovich et al., 1996). Trauma-induced autoimmunity has also been reported in various models of experimental SCI (Ankeny et al., 2006; Jones et al., 2002; Palladini et al., 1987; Popovich et al., 1996; Rudehill et al., 2006; Willenborg et al., 1977). In mice, serum IgM was preferentially increased early after injury, followed by a significant increase in IgG1 and IgG2a from 14 dpi to 42 dpi. These results indicate that experimental SCI is associated with B-cell activation and isotype switching, events that require functional T–B interactions in the periphery (Ankeny et al., 2006). In human SCI, both IgM and IgG isotypes have been identified in the serum, confirming that similar functional T–B interactions occur in human SCI patients (Davies et al., 2007). This is also supported by recent data demonstrating the upregulation of B-cell maturation antigen (BCMA), a proliferation-inducing ligand (APRIL), and B-cell activating factor (BAFF) expression in the peripheral blood of SCI patients (Saltzman et al., 2013). These mediators coordinate various aspects of

B-cell development, activation, and proliferation, and have been implicated in the development of several autoimmune disorders, including systemic lupus erythematosus and MS (Mackay et al., 2007). Their expression in the periphery of chronically injured patients is indicative of ongoing adaptive immune activation following spinal cord trauma. Investigation into the antigen specificity of the autoreactive lymphocytes revealed the presence of T-cells reactive with MBP (T-MBP) in SCI patients and animals (Ibarra et al., 2007; Kil et al., 1999; Popovich et al., 1996; Wucherpfennig et al., 1994; Zajarias-Fainsod et al., 2012). Whether activation of T-cells with reactivity to other myelin proteins (e.g., myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp)) also occurs after SCI has not been determined. Evaluation of serum revealed antibodies reactive with multiple nonmyelin CNS proteins, including anti-DNA, anti-glutamate receptor (GluR), and anti-nuclear antibodies (Ankeny et al., 2006, 2009). Several studies have reported the presence of antibodies reactive to brain gangliosides in human SCI (e.g., anti-GM1; (Davies et al., 2007; Hayes et al., 2002; Mizrachi et al., 1983) and in human and experimental traumatic brain injury (TBI) (Prochazka et al., 1971; Rudehill et al., 2006). Although previously reported in SCI patients (Kil et al., 1999; Wucherpfennig et al., 1994; Zajarias-Fainsod et al., 2012), there was no indication of anti-MBP antibodies generated in SCI mice (Ankeny et al., 2006) or rats (Ibarra et al., 2000). Functional impact of TIA The pathological impact of TIA is unclear and somewhat controversial (Popovich and Jones, 2003). As discussed above, autoreactive lymphocytes are activated by spinal cord trauma, yet these cells do not appear to cause overt signs of autoimmune disease in SCI individuals. This may be due to the induction of regulatory and/or reparative mechanisms activated in parallel with neurodestructive mechanisms. It is also possible that TIA limits or prevents spontaneous neurological recovery; any deleterious effects of TIA would be masked by the manifestation of other converging symptoms in the SCI patient. An additional possibility is that TIA manifests as pathologic changes in other areas of the body (i.e., outside the spinal cord) that are generally considered to be secondary consequences of the functional deficits caused by SCI. For example, cardiovascular disease, kidney dysfunction, and reproductive deficits are all considered consequences of post-injury sequelae, yet these could be caused by or exacerbated in response to TIA. Indeed, antiprostacyclin receptor antibodies are generated in response to SCI and could contribute to the cardiovascular deficits observed in SCI individuals (Kahn et al., 2005). Whether or not autoimmune disease develops is dependent on interactions between antigen-specific (effector) CD4 + T-cells and Tregs specific for the same antigen (Fig. 1). Tregs act via several mechanisms to maintain effector cells in a non-responsive state; when the balance between regulation and activation of autoreactivity is disrupted and/or when a certain threshold of activation is exceeded, autoimmune disease develops (Ankeny and Popovich, 2010). There is evidence that TIA has pathological consequences, suggesting that SCI is sufficient to exceed this threshold. For example, autoreactive T lymphocytes isolated from spinal injured rats cause neuropathology and transient paralysis reminiscent of the autoimmune demyelinating disorder, experimental autoimmune encephalomyelitis (EAE), when injected into naïve rats (Popovich et al., 1996). Transgenic mice in which the majority of CD4+ T-cells are specific for MBP exhibit exacerbated locomotor deficits and enhanced lesion pathology relative to SCI mice with normal T-cell repertoires (Jones et al., 2002, 2005). Autoantibodies from SCI patients block sprouting of neurites when overlaid onto dorsal root ganglion neurons in vitro (Mizrachi et al., 1983). Moreover, autoantibodies from SCI mice cause intraspinal pathology in an Fc and complementdependent manner when injected into naïve spinal cord (Ankeny et al., 2009) and neuronal death when injected into the hippocampus of naïve animals (Ankeny et al., 2006).


81

Fig. 1. Mechanisms of TIA. Whether autoimmune disease develops after SCI is determined by the balance of activation of antigen-specific CD4+ T effector cells and CD4+ regulatory T-cells (Tregs). In the absence of injury, antigen-specific effector cells are functionally suppressed by Tregs. This occurs by a variety of mechanisms, including the production of antiinflammatory mediators such as IL-10 and/or TGF-β that suppress the activation of antigen-specific T-cells or through indirect effects on DCs. IDO is an immunoregulatory molecule which inhibits the maturation and function of DCs, thus preventing the DC from activating antigen-specific effector T-cells. SCI disrupts the balance between effector and regulatory cells; the loss of Treg suppressive ability allows the antigen-specific CD4+ T-cells to become activated and contribute to pathology. Mechanisms by which effector cells contribute to pathology include the production of proinflammatory cytokines (PICs) and chemokines, the promotion of a pro-inflammatory phenotype in macrophages, and ligation of death receptors (i.e., Fas) resulting in apoptosis of neurons and glia. Antigen-specific T-cells activate B-cells specific for the same antigen and promote their proliferation and differentiation into autoantibody-secreting plasma cells. Antigen-specific B-cells also produce cytokines that activate effector cells, thus maintaining their active state. Green shading indicates “functionally active” T-cell.

There are several possible mechanisms by which TIA can contribute to SCI pathology (Fig. 1). T-cells can exert direct effects on neurons or glia (Antel et al., 1994; Giuliani et al., 2003; Nitsch et al., 2004; Yarom et al., 1983) or indirect effects on other CNS cells by producing proinflammatory cytokines (e.g., interferon (IFN)γ, tumor necrosis factor (TNF)α, IL-1β, IL-12) (Jones et al., 2002, 2004), chemokines (e.g., CXCL10, CCL5, CCL2) (Jones et al., 2005), or by activation of microglia (Gimsa et al., 2000). Apoptosis of CNS neurons and glia also occurs via ligation of death receptors which may be mediated by activated T-cells (Aktas et al., 2005; Yu and Fehlings, 2011). It is also clear that autoreactive B-cells play an important role in modulating T-cell functions, thus may participate in promoting tissue injury indirectly via the release of cytokines that activate autoimmune T-cells (Lund and Randall, 2010) and directly via generation of pathogenic autoantibodies (Ankeny and Popovich, 2010). Historically, a loss of peripheral tolerance resulting in the activation of autoreactive lymphocytes was viewed as an ‘attack against self’ with only deleterious consequences recognized as possible outcomes. More recently, this view has been challenged and autoimmune recognition has come to be seen as having a physiological role in modifying the immune response based on the needs of the tissue (Matzinger, 2002; Schwartz and Kipnis, 2005). There is convincing evidence supporting a role for self-recognition and T-cell receptor (TCR) engagement in maintaining the sensitivity of naïve T-cells to antigens, thus ensuring their capacity to respond optimally in situations where the density of ligands may be low (Stefanova et al., 2002). Thus, despite the ability of autoreactive lymphocytes to initiate and perpetuate pathological processes within the CNS as discussed above, it has been proposed that autoreactive T-cells have reparative functions in the injured CNS (Schwartz, 2001).

One mechanism proposed for autoimmune lymphocyte-mediated tissue repair is the production of neurotrophins (e.g., brain-derived neurotrophic factor (BDNF)) (Hammarberg et al., 2000; Moalem et al., 2000) and/or anti-inflammatory cytokines (e.g., IL-10, transforming growth factor (TGF)-β) (Tyor et al., 2002; Zhou et al., 2009a). However, the cell type(s) responsible for the production of neurotrophins and anti-inflammatory mediators within the injured CNS, i.e., whether they are effector cells or induced T regs (iTregs) that bind CNS proteins, or even non-antigen-specific bystander cells recruited to the injury site, has not been definitively established. CNS-specific T-cells produce neurotrophins when stimulated in vitro (Hammarberg et al., 2000) and within the injured CNS, the induction of neurotrophins following transfer of T-MBP has been demonstrated in optic nerve crush (Barouch and Schwartz, 2002) and ventral root avulsion injury models (Hammarberg et al., 2000). However, intraspinal BDNF and neurotrophin (NT)-3 were not increased following SCI in transgenic mice in which ~95% of the endogenous CD4+ T-cell repertoire is specific for MBP (Jones et al., 2002), thus it is unlikely that effector T-cells are a primary source of neurotrophins in the traumatically injured spinal cord. These data are consistent with reports in an EAE model demonstrating that bystander T-cells, rather than autoreactive T-cells, are the main cell type producing neurotrophins (Muhallab et al., 2002). Still others have shown that the level of neurotrophin expression by immune cells during the acute stage of CNS disease is limited and primarily restricted to B-cells, thus calling into question the capacity of T-cells to effectively mediate CNS tissue repair (Edling et al., 2004). This is supported by data in a model of facial nerve axotomy showing that BDNF, although produced by T-cells, was dispensable for CD4+ T-cellmediated neuroprotection (Xin et al., 2012). Collectively, these data call into question whether immune cell-mediated production of

82


neurotrophins mediates CNS tissue repair. The role of IL-10 in mediating tissue repair is discussed in detail below. Autoimmune T-cell-mediated neuroprotection has also been proposed to occur via the induction of CD86 on microglia (Butovsky et al., 2001) and/or the recruitment into the injury site of peripherallyderived macrophages that express an anti-inflammatory phenotype (Shechter et al., 2009). Within the injured spinal cord, macrophages exert neurotoxic or neuroregenerative effects, depending on the context in which they are activated and the resulting phenotype (i.e., M1 or M2) (Gensel et al., 2009; Kigerl et al., 2009). Promotion of an antiinflammatory, M2 phenotype, could promote tissue repair by attenuating the production of neurotoxic mediators that induce cavitation (Fitch et al., 1999), or by enhancing axonal elongation, either directly via secreted products (Kigerl et al., 2009), or indirectly by blocking the generation of inhibitory molecules (Fitch and Silver, 2008). Experimental manipulation of CNS autoimmune lymphocytes in SCI Autoimmune-based vaccinations have been proposed as potential therapies for SCI and a variety of other CNS disorders, including glaucoma, optic nerve damage, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and more recently, cognitive decline and depression (Schwartz and Ziv, 2008). Table 1 provides a summary of published studies that demonstrated beneficial effects of autoimmune vaccination in experimental SCI. These studies and others are described below. Passive immunization with myelin-reactive T-cells Early studies demonstrating the neuroprotective potential of T-MBP utilized two different CNS injury models: optic nerve crush (Moalem et al., 1999) and SCI (Hauben et al., 2000a, 2000b). In the SCI model, post-injury immunization with T-MBP resulted in improvements in functional recovery that were associated with increased survival of rubrospinal neurons (Hauben et al., 2000a, 2000b). However, in a study designed to independently replicate these experiments, no evidence of improved function or neuroprotection was observed; instead, immunized rats exhibited impaired locomotor recovery and

exacerbation of tissue damage, including greater loss of rubrospinal neurons (Jones et al., 2004). The reason for these differences is not entirely clear; however, two findings are of particular interest: first, the pathogenic potential of T-MBP only occurred following severe, but not moderate SCI; second, exacerbation of pathology was greater with passive transfer of T-MBP cells compared with active MBP immunization (Jones et al., 2004). Active immunization elicits a heterogeneous T-cell response in which only a small population of activated cells are myelin-specific (Steinman, 1996). These findings suggest that a severe injury overwhelms any neuroprotective effect of T-cells. No empirical data have defined a specific mechanism; however, it is intriguing to speculate that high concentrations of CNS antigens released by severe injury might increase the number of T-MBP effector cells beyond a critical threshold and overwhelm the regulatory ability of Tregs (Jones et al., 2004). The encephalitogenic potential of the cells prior to transfer was demonstrated by their ability to induce transient EAE when injected into naïve animals, leading the authors to conclude that encephalitogenic T-cells mediate “protective autoimmunity” (Moalem et al., 1999). However, these studies failed to evaluate the phenotype of the cells following their transfer into the injured CNS. Recent data from other CNS models suggests that the phenotype of T-cells is not constant, but may be altered depending on the context (Ha et al., 2012; Liu et al., 2006); indeed, several recent studies that evaluated the phenotype of T-cells isolated from the injury site indicate that the encephalitogenic profile of adoptively transferred T-MBP cells is altered by the injured spinal cord. These studies independently demonstrated that protective effects of T-MBP cells adoptively transferred into SCI animals were associated with increased intraspinal IL-10 (Hu et al., 2012; Ishii et al., 2012; Lu et al., 2008), suggesting that the transferred cells either adopted a regulatory phenotype (e.g., via the induction of FoxP3) or activated IL-10producing CD4+ Tregs (Fig. 2). Regulation of encephalitogenicity occurred even when the T-MBP were skewed toward a Th1 phenotype in vitro prior to transfer (Ishii et al., 2012). There are several possible mechanisms by which production of IL-10 within the injury site could be protective (Fig. 2). IL-10 can directly promote neuron survival following SCI via provision of trophic support (Zhou et al., 2009c) and/or activation of anti-apoptotic proteins (Zhou et al., 2009b), or could act

Table 1 Summary of studies demonstrating protective effects of myelin-based vaccination in SCI. Protocol

Timing of vaccine

Main findings

Lymphocyte phenotype

References

T-MBP MBP/IFA T-MBP T-MBP T-p472 (Nogo-A peptide) SCH/CFA A91/CFA Splenocytes from SCI rats activated ex vivo with MBP Myelin/Alum or IFA rNogo-66/rMAG/Alum/IFA DCs pulsed with APL or MBP anti-NgR DNA vaccine A91/CFA T-MBP

Post Pre Post Post Post Pre Post Post

↑ BBB, ↑neuron survival ↑ BBB, ↑neuron survival, ↑ tissue preservation ↑ BBB, ↓ tissue loss ↑ NF, ↓ cavitation and GFAP ↑ BBB ↑ BBB, ↑ neuron survival ↑ BBB ↑ BBB

n.d. n.d. n.d. ↑ CD86 n.d. n.d. n.d. ↑ GATA-3, IL-10, no IFNγ

Hauben et al. (2000a) Hauben et al. (2000a) Hauben et al. (2000b) Butovsky et al. (2001) Hauben et al. (2001b) Hauben et al. (2001a) Hauben et al. (2001a,b) Yoles et al. (2001)

Pre Pre Post Post Post Post

n.d. n.d. n.d. n.d. n.d. ↑ IL-10, IFNγ, no IL-4

Sicotte et al. (2003) Sicotte et al. (2003) Hauben et al. (2003) Yu et al. (2007) Martinon et al. (2007) Lu et al. (2008)

DCs pulsed with SCH or MBP MBP/CFA or A91/CFA T-MBP skewed to Th1 T-MBP A91/CFA COP-1/CFA or A91/CFA DCs pulsed with SCH

Post Pre Post Post Post Post Post

n.d. n.d. ↑ IL-10, M2 Mф ↑ IL-10, IL-13, BDNF, NT-3, NGF, M2 Mф ↑ IL-4/IFNγ ratio, ↑ BDNF n.d. ↑ IFNγ, IL-12, BDNF, NT-3

Liu et al. (2009b) Ibarra et al. (2010) Ishii et al. (2012) Hu et al. (2012) Martinon et al. (2012) Garcia et al. (2012) Wang et al. (2012), (2013)

A91/CFA

Pre

↑ axon regeneration ↑ axon regeneration ↑ BBB, ↓ cavitation ↑ BBB, grid walking, footprint analysis ↑ BBB, ↑ myelin sparing, ↑neuron survival ↑ BBB, ↑ myelin sparing, ↑neuron survival, ↓ lesion volume ↑ BMS ↓ lipid peroxidation ↑ BMS, ↑ inclined plane, ↑ myelin ↑ BBB, ↑ myelin ↑ BBB ↓ iNOS and NO ↑ BMS, footprint analysis, ↑ neuron survival, ↓ lesion volume ↑ BBB, ↑ neuron survival

n.d.

Ibarra et al. (2013)

Abbreviations: BBB—Basso, Beattie, Bresnahan locomotor rating scale; BMS—Basso Mouse Scale; MBP—myelin basic protein; SCH—spinal cord homogenate; NgR—Nogo receptor; CFA— complete Freund's adjuvant; IFA—incomplete Freund's adjuvant; APL—altered peptide ligand; A91 is an altered MBP peptide; DC—dendritic cells; n.d.—not determined; Mф—macrophage; BDNF—brain-derived neurotrophic factor; NT-3—neurotrophin-3; NGF—nerve growth factor; IFNγ—interferon gamma; COP-1—copolymer 1 (glatiramer acetate).


Fig. 2. Treg-mediated neuroprotection. The phenotype of T-cells is flexible and subject to change under varying conditions. CD4+ effector cells can be induced to express FoxP3 which endows them with a regulatory phenotype (i.e., produce IL-10 and/or TGFβ). CD4+ effector cells can themselves be induced to produce IL-10, or they can recruit bystander (non-antigen-specific) IL-10-producing CD4+ T-cells. There are several mechanisms by which Treg-secreted IL-10 exerts protective effects. IL-10 promotes the conversion of pro-inflammatory M1 macrophages that produce a variety of neurotoxic mediators, including several PICs and chemokines, into anti-inflammatory M2 macrophages that produce immunoregulatory cytokines, including IL-10, and neurotrophins. IL-10 can feed back to inhibit effector cells.

indirectly via anti-inflammatory effects on macrophages (Bethea et al., 1999). Macrophages with an M2 phenotype are increased in the injured spinal cord following passive transfer of T-MBP (Hu et al., 2012; Ishii et al., 2012); this was demonstrated to be IL-10-dependent (Ishii et al., 2012). The conversion of encephalitogenic T helper (Th) cells to a regulatory phenotype that promotes the conversion of pro-inflammatory M1 to anti-inflammatory M2 macrophages has been documented in other models of CNS injury, including Parkinson's disease (PD) (Ha et al., 2012), EAE (Martinez-Pasamar et al., 2013) and ischemia (Chen and Flies, 2013). There is no guarantee that the encephalitogenic potential of T-cells will be skewed toward regulation within the dynamic setting of the injury microenvironment and is likely to be affected by several variables including the post-injury time interval. Splenocytes isolated from SCI rats, activated ex vivo with MBP, then transferred into other SCI rats enhanced functional recovery, but only if isolated after 7 dpi and not if isolated at earlier time points (Yoles et al., 2001). These data suggest that SCI elicits a protective autoimmune response aimed at counteracting trauma-induced damage (Yoles et al., 2001). However, an alternate interpretation of these data is that SCI activates encephalitogenic T-MBP cells in parallel with regulatory T-MBP cells. Effector T-cells expand more rapidly than Treg cells specific for the same antigen (Korn et al., 2007), which accounts for expression of their

83

encephalitogenic potential in the early period following activation. This interpretation is consistent with data demonstrating that splenocytes isolated from SCI rats prior to 7 dpi proliferate vigorously in response to MBP and produce neurological deficits when injected into naïve animals, but if isolated at later time points post-injury, their encephalitogenic potential is diminished (Popovich et al., 1996). In the study by Yoles et al. (2001), transferred splenocytes that exhibited beneficial effects demonstrated a low proliferative ability when activated ex vivo with MBP, suggesting the presence of antigen-specific Treg cells able to suppress the effector cells. This is also supported by the finding that T-cells isolated from the injured spinal cord expressed GATA-3 and produced IL-10 but not IFNγ (Yoles et al., 2001) and is consistent with regulation of encephalitogenic autoreactive T-cells in EAE (Korn et al., 2007; Martinez-Pasamar et al., 2013). Taken together, these data suggest the possibility that beneficial effects observed following passive transfer of T-MBP may be due to Tregs induced in parallel. In a model of crush injury to the optic nerve, adoptive transfer of T-MBP cells increased the survival of retinal ganglion cells in intact rats, but not in rats that were neonatally thymectomized (Kipnis et al., 2002). In fact, the thymectomized rats developed EAE, which would be expected with the transfer of encephalitogenic cells in the absence of regulatory cells to suppress their pathogenic potential. Indeed, the transfer of an enriched population of CD4+ lymphocytes (which includes Tregs) restored the neuroprotective effect (Kipnis et al., 2002). Similarly, myelin oligodendrocyte glycoprotein (MOG)-specific Th1 cells were neuroprotective when transferred into intact mice, but not when transferred into athymic nude mice (Kipnis et al., 2002). Collectively, these studies suggest that when encephalitogenic cells are transferred into recipients that lack additional CD4 + T-cells (e.g., Tregs) neuroprotection does not occur. Instead, an additional cell population is required—whether this additional cell population is itself protective, or whether it promotes protection by either converting encephalitogenic T-cells into regulatory cells or via bystander suppression (Fig. 2) is not known. Vaccination with antigen-pulsed DCs Another vaccination approach that has been evaluated in SCI employs the use of DCs primed with a CNS-specific antigen. These cells home to the CNS where they function as antigen-presenting cells (APCs) to stimulate antigen-specific immune responses within the injury site. Because the DCs are pre-loaded with antigen, the time required to generate an antigen-specific immune response locally, where it's needed to promote tissue repair, is reduced. Vaccination protocols that utilize DCs pulsed with specific antigen consistently improve overground locomotion, whether the priming antigen is spinal cord homogenate (SCH), MBP peptide, or altered peptide ligand (APL) (Hauben et al., 2003; Liu et al., 2009b; Wang et al., 2012, 2013), although DCs pulsed with MBP produce less robust improvements in Basso–Beattie– Bresnahan (BBB) locomotor scores compared with SCH-DCs (Liu et al., 2009b; Wang et al., 2013). Within the inflamed CNS, DCs have the ability to either promote or restrict the development of autoimmune disease (Ganguly et al., 2013). In general, mature DCs are considered immunogenic and immature DCs tolerogenic; however, this is likely an oversimplification. Recent evidence indicates that the repertoire of costimulatory and other molecules expressed on the DC cell surface, rather than the maturation state per se, dictates whether antigen-specific T-cells adopt an effector or regulatory phenotype in response to antigen recognition (Ebner et al., 2013). This was recently demonstrated in an EAE model in which mature DCs presenting self-myelin peptides (i.e., MOG) to T-cells were tolerogenic rather than immunogenic as expected, and prevented EAE via the induction of antigen-specific Tregs (Yogev et al., 2012). In SCI models, vaccination with either immature DCs (Liu et al., 2009b) or mature DCs (Wang et al., 2012, 2013) was neuroprotective. The putative mechanism for the neuroprotective effect was the

84


induction of neurotrophins (BDNF and NT-3) by T-cells within the injured spinal cord (Wang et al., 2013), a change associated with an increase in Th1 cytokines, including IL-12 and IFNγ (Wang et al., 2012, 2013). The cytokine profile suggests that neuroprotection was the result of an enhanced Th1 cell response rather than the induction of Tregs. However, in response to stimulation with IL-12 and IL-2, Tregs have been shown to produce IFNγ (Lowther and Hafler, 2012). These socalled Th1-Tregs, which “look” like Th1 cells but which are clearly endowed with regulatory properties, are not terminally differentiated cells, providing them with the necessary flexibility to respond in differing conditions (Lowther and Hafler, 2012). Thus, the presence of Th1-associated cytokines is not by itself sufficient to determine the phenotype of the neuroprotective T-cell and further research is needed to clarify the mechanism of neuroprotection. Active immunization with myelin antigens Pre-injury immunization of mice with either myelin or SCH emulsified in incomplete Freund's adjuvant (IFA) was one of the earliest CNSspecific vaccination approaches evaluated in a model of SCI (Huang et al., 1999). This vaccination protocol increased serum titers of CNS-specific IgM and IgG antibodies and promoted long-distance axon regeneration in vivo, an effect presumably mediated by anti-myelin antibody blockade of myelin inhibitory proteins nearby the injury site (Gonzenbach and Schwab, 2008; Huang et al., 1999; Rodriguez et al., 1996; Sicotte et al., 2003). Beneficial effects on axon regeneration and remyelination were also reported when myelin or myelin proteins were emulsified in alum (an FDA-approved adjuvant for use in humans) or after passive immunization with anti-myelin IgM antibodies (Howe et al., 2004; Rodriguez and Lennon, 1990; Rodriguez et al., 1987; Sicotte et al., 2003). Similarly, pre-injury administration of DNA encoding Nogo receptor (NgR), the common receptor for several known myelin inhibitory proteins, increased anti-NgR antibodies and improved functional recovery in a spinal contusion injury model (Yu et al., 2007). Although in vivo axon regeneration was not directly evaluated in that study, antisera from vaccinated rats promoted neurite outgrowth on a substrate of MAG in vitro (Yu et al., 2007). Immunization with MBP in IFA, or with MBP or a Nogo-A-derived peptide (p472) in complete Freund's adjuvant (CFA), enhanced functional recovery and neuronal survival in a spinal contusion model; however, protective effects were attributed to T-cells, rather than antibodies (Hauben et al., 2001a, 2001b). Passive and active vaccination approaches utilizing antibodies or peptides specific to myelin proteins, including Nogo-A, NgR, MAG, and OMgp, have also demonstrated preclinical efficacy in other CNS disorders. In experimental models of stroke, these myelin peptides reduced infarct volume and improved motor recovery (Yu et al., 2013a), and in MOG-induced EAE, immunization with Nogo-A in IFA decreased clinical disease, accompanied by decreased production of IFNγ and increased production of IL-10 and TGF-β by MOG- or Nogo-stimulated splenocytes (Karnezis et al., 2004). Based on its therapeutic efficacy in several experimental models of CNS injury, including SCI, a humanized monoclonal anti-Nogo-A antibody (ATI 355; Novartis) was approved for a multicenter clinical trial on acute (4–14 dpi) para- and tetra-plegic SCI patients. A recent phase I clinical trial has been completed and was without severe side effects (www.ClinicalTrials.gov; NCT00406016). Each of the vaccination approaches discussed above relies on the induction of an adaptive immune response involving both T- and B-cells specific for myelin proteins. Thus, each carries a risk of inducing pathogenic autoimmunity, similar to that which occurred in the AN1792 vaccine clinical trials in AD (Tabira, 2010). This trial, designed to evaluate active immunization with full-length amyloid (Aβ) peptide (a selfantigen) in adjuvant, caused meningoencephalitis in a subset of patients that was subsequently attributed to development of a pathogenic Aβ-specific T-cell response (Delrieu et al., 2012; Meyer-Luehmann et al., 2011). The risk for development of similar pathological responses

in SCI patients certainly exists with any of the vaccination approaches described in the preceding sections as each of these have the potential to activate CNS-reactive T-cells. Indeed, pathological autoimmune responses (e.g., EAE) were observed when rats were immunized with MBP in CFA, confirming this pathogenic potential and highlighting the need for caution when considering any form of self-protein-based immunotherapy (Hauben et al., 2001a). Still, significant progress has been made in the development of safe, effective therapeutic vaccines that avoid the induction of autoreactive T-cell responses and thus minimize the risk of generating pathogenic T-cell responses. Newer vaccine strategies such as those involving the use of DNA epitope chimeras with toxin-derived carriers as adjuvants (Yu et al., 2013b) and the intranasal administration of viral vectors encoding Aβ epitopes (Hara et al., 2011), show great preclinical promise and provide therapeutic benefit via the generation of humoral immunity and induction of non-pathogenic T-cell responses. These second generation immunotherapeutic approaches may prove similarly beneficial in promoting repair of the injured spinal cord. Epitope vaccines using APLs Vaccination with APLs as an alternative to encephalitogenic peptides has been used to avoid the risk of autoimmune disease and has been shown to have beneficial effects on recovery of function after SCI (Table 1). However, data regarding the efficacy of this paradigm for the treatment of experimental SCI are conflicting. In a series of experiments, post-injury immunization with 100–150 μg of various APLs targeting either MBP or NogoA significantly improved BBB locomotor scores whether the injury was of moderate or severe intensity (Hauben et al., 2001a, 2001b; Martinon et al., 2007), although in another study this protocol was only beneficial after moderate, but not severe SCI (Martinon et al., 2012). The beneficial effect of APL administration was abolished and functional recovery was impaired if the dosage of APL was increased to 500 μg or if a “booster” dose was given (Hauben et al., 2001a). However, the opposite effect was observed in a recent study where a single dose of A91/CFA given pre-injury did not improve BBB scores, but administration of a second “booster” dose pre-injury did (Ibarra et al., 2013). Thus, any potential therapeutic benefit of APL administration following SCI appears to be dependent on dose, time of administration, and injury severity. Few studies have attempted to determine the mechanism responsible for protective effects of APL administration in SCI, making it difficult to discern why certain immunization protocols mediate neuroprotection while others not only fail to do so, but actually worsen outcome. In one study, A91/CFA vaccination was associated with an increased IL-4/IFNγ ratio and the production of BDNF by A91-specific T-cells (Martinon et al., 2012). These data implicate immune deviation toward a Th2 phenotype in APL-mediated protection and are consistent with similar findings of Th2-mediated protection in other models of neurologic disease, including AD and MS (Cao et al., 2009; Yong, 2002). In an ALS model, active immunization with a peptide epitope of mutant superoxide dismutase 1 (SOD1) in CFA induced a Th2-biased antibody response that delayed disease onset (Liu et al., 2012). Similarly, vaccination with Ribi, a Th2-biasing adjuvant, in the absence of antigen also generated a Th2-biased antibody response indicated by a high IgG1/ IgG2c ratio and increased IL-4/IFNγ ratio that positively correlated with lifespan (Takeuchi et al., 2010). Vaccination with Copolymer 1 (Cop-1), an APL designed to mimic MBP, has demonstrated protective effects in experimental models of AD, PD, HIV-1 encephalitis, glaucoma, and EAE (Benner et al., 2004; Butovsky et al., 2006; Gorantla et al., 2008; Kala et al., 2010; Schori et al., 2001). Copolymer-1 has also demonstrated neuroprotective effects in several models of nerve injury or motor neuron death, including optic nerve crush, facial nerve axotomy, ventral root avulsion, and ALS (Angelov et al., 2003; Kipnis et al., 2000; Scorisa et al., 2009). It is interesting that this APL has been vastly overlooked with regard to potential


benefits in a SCI model. A single study evaluating Cop-1 in SCI described protective effects mediated via promotion of an M2 phenotype and a reduction of reactive oxygen species (ROS) during the acute post-injury phase (Garcia et al., 2012). Additional immunological and neuroprotective mechanisms proposed for the beneficial effects of Cop-1 include bystander suppression of T-cells, deviation of T-cells toward a Th2 phenotype, alteration of microglial phenotype, and direct effects on neurons (Aharoni et al., 1998, 2000; Butovsky et al., 2006; Liu et al., 2007; Qian et al., 2013). Moreover, Cop-1 promotes the conversion of CD4 + CD25− effector T-cells into CD4+CD25+ Tregs via IFNγ-mediated induction of FoxP3 (Hong et al., 2005); Cop-1-induced generation of Tregs increased survival of dopaminergic neurons in an experimental model of PD (Benner et al., 2004). A phase II trial evaluating Cop-1 in ALS has been completed and was found to be safe (www.ClinicalTrials.gov; NCT00326625; (Meininger et al., 2009). Cop-1 is approved for clinical use in MS and has demonstrated therapeutic benefit in this model via deviation toward a Th2 phenotype (Yong, 2002). Despite the somewhat contradictory findings described in the preceding sections, there are some conclusions that can be drawn regarding the therapeutic potential of modulating the adaptive immune response to SCI. First, the data highlight the complexities of the immune response to trauma and emphasize the need for any immune-based therapy to precisely regulate specific aspects of this response (e.g., pathogenic potential of autoreactive lymphocytes) so as to avoid the induction of unwanted immune consequences. Second, although the majority of studies documenting evidence of “protective autoimmunity” made no attempt to assess the effect of vaccination on the phenotype of the responding cells, existing data from SCI and other CNS disorders, implicate Tregs as the neuroprotective effector cells (Table 1). Lastly, whether immune-mediated neuroprotection requires induction of neuroantigenspecific immunity is debatable; indeed, the evidence suggests that the relevant cells are those that produce IL-10 (Fujio et al., 2010). Treg-mediated neuroprotection Despite the presence of autoreactive cells in all individuals, a state of homeostasis is maintained because effector T-cells and Tregs are in balance; when that balance is shifted (i.e., due to injury or disease) toward an increase in effector T-cell activation, pathogenic autoimmunity or neurologic disease results (Fig. 1; (Beers et al., 2011; Henkel et al., 2013; Lowther and Hafler, 2012; Saunders et al., 2012). Conversely, increasing the activity of Tregs ameliorates pathology in experimental models of EAE, stroke, HIV encephalitis, PD, and AD (Chen et al., 2013; Ha et al., 2012; Lambracht-Washington and Rosenberg, 2012; Liu et al., 2009a; Lowther and Hafler, 2012). In an experimental model of PD, administration of vasoactive intestinal peptide (VIP), a neuropeptide known to induce Tregs (Delgado et al., 2005), converted a pathogenic effector T-cell response induced via vaccination with a nitrated form of α-synuclein into a protective regulatory phenotype. The beneficial effect was associated with increased IL-10, IFNγ, and IL-13 (Reynolds et al., 2010). In the same experimental model, vaccination with the adjuvant Bacillus Calmette–Guerin (BCG), despite being a potent inducer of IFNγ-producing Th1 cells, also induced FoxP3 + Tregs and was protective (Lacan et al., 2013). Induction of FoxP3+ Tregs by vaccination with an inhibitor of histone deacetylase was protective in experimental ischemia (Liesz et al., 2013). Collectively, these data suggest that activation of a Tregs in parallel with effector T-cell responses is sufficient to prevent effector cells from becoming overactive and inducing pathology (Yong et al., 2011). There are several mechanisms by which Tregs mediate protective effects, including the production of immunoregulatory cytokines (e.g., IL-10 and/or TGFβ), neurotrophins, and through promotion of an M2 microglial/macrophage phenotype (Fig. 2; (Fujio et al., 2010; Schmetterer et al., 2012). There are many types of T-cells with regulatory phenotypes distinct from naturally occurring CD4+CD25+FoxP3+ regulatory T-cells (nTregs); collectively referred to as inducible Tregs

85

(iTregs), these cells are induced by a variety of conditions and frequently exert their effects via the production of IL-10 (Fujio et al., 2010). In experimental models of stroke, depletion of Tregs increased infarct volume and was associated with elevated TNFα, IFNγ, and IL-1β (Liesz et al., 2009). Conversely, tolerization to myelin peptides reduced infarct size and induced T-cells that produce regulatory cytokines, including TGFβ, IL-10, and IL-4, while decreasing IFNγ (Becker et al., 2003; Frenkel et al., 2005). In a model of HIV-1-associated neurodegeneration, transfer of Tregs decreased TNFα and increased BDNF and also was associated with modulation of microglia to an M2 phenotype (Liu et al., 2009a). Similar protective effects of Tregs were demonstrated in an experimental model of PD using a novel vaccination strategy designed to generate an adaptive memory response to α-synuclein prior to the presentation of the immunogen (Sanchez-Guajardo et al., 2013). When α-synuclein was subsequently expressed via immunization with a viral vector, an α-synuclein-specific memory response was activated that was associated with the induction of FoxP3 + Tregs, increased glial-derived neurotrophic factor expression, and polarization of microglia to an anti-inflammatory phenotype. This strategy also evoked a strong humoral immune response with high titers of anti-αsynuclein IgG antibodies (Sanchez-Guajardo et al., 2013). Whether the protective Treg cell is antigen-specific, or is induced and suppresses encephalitogenic cells non-specifically is still an open question. Translational potential of neuroprotective immunity Within the context of SCI, a major factor that must be considered when evaluating the therapeutic potential of any immunotherapy is the high threshold for the detection of therapeutic benefit that exists in a SCI individual who is already paralyzed (Filli and Schwab, 2012); also see Schwab et al., 2014-in this issue). Thus, treatment effects observed in experimental models must be functionally meaningful (Filli and Schwab, 2012). Minor functional improvements obtained in highly controlled experimental conditions are not likely to produce functionally significant changes in a clinical trial. This is especially true for SCI patients with a more severe injury (e.g., AIS A); these individuals would be expected to have an even greater threshold for detection, thus even more robust protective effects are required for these individuals to derive any benefit. With regard to severe experimental SCI, much of the evidence for autoimmune-mediated neuroprotection is based on statistically significant improvements in BBB scores; however, the majority of these studies failed to demonstrate functionally meaningful recovery, i.e., restoration of weight support and stepping ability (Fig. 3). Furthermore, these modest improvements were obtained in genetically homogenous animals in an injury model designed to minimize variability, thus a similarly modest degree of improvement would likely be below the threshold for detection in the heterogeneous human SCI population that exhibits great variability in spontaneous neurological recovery (Kwon et al., 2012). Given the well-documented challenge of translating basic research findings into successful clinical trials for the treatment of SCI (Filli and Schwab, 2012) and other CNS disorders, such as ischemic stroke (Dayan and Wraith, 2008; Stebbings et al., 2009; Yu et al., 2013a) and AD (see below), these issues are not insignificant and must be addressed adequately prior to clinical trials. Early clinical trials of immunization therapies in AD were either associated with severe adverse side effects or were unsuccessful (Lobello et al., 2012; Morgan, 2011). Some of the reasons for the adverse events are now clear. The initial active immunization with full-length Aβ peptide in adjuvant activated Aβ-reactive Th1 cells that caused autoimmune encephalitis (Delrieu et al., 2012; Meyer-Luehmann et al., 2011). The Aβ-specific T-cell response most likely arose because the Aβ peptide contained T-cell epitopes and/or because of adjuvant effects (Lobello et al., 2012; Morgan, 2011). Newer vaccines currently in clinical trials or in preclinical development use either passive anti-Aβ antibody approaches that avoid activation of T-cell responses altogether, or active immunization with N-terminal Aβ peptides that contain B-cell epitopes

86


2013). This approach is attractive because most individuals would be expected to have pre-existing memory T-cells for tetanus toxin as a result of the public health vaccination program (Davtyan et al., 2013). Because the T-cell responses generated in these newer vaccine approaches are against non-self antigens, they are less likely to induce the pathogenic autoreactive T-cell responses that caused the abrupt termination of previous immunotherapy AD trials (Delrieu et al., 2012; Meyer-Luehmann et al., 2011). These novel vaccination strategies, should they prove successful, could provide new opportunities for development of similar immunotherapies in SCI. Conclusions and future directions

Fig. 3. Effects of “protective autoimmunity” on functional recovery after severe SCI. A majority of studies have utilized Basso–Beattie–Bresnahan (BBB) locomotor rating scale scores as a primary outcome measure to evaluate the neuroprotective potential of various vaccination protocols. The data points presented in the figure were compiled from published studies that demonstrated “protective autoimmunity” via improvements in BBB scores (Hauben et al., 2000a, 2001a, 2001b, 2002, 2003; Martinon et al., 2012; Yoles et al., 2001). To facilitate comparison, only studies that employed a severe (50 mm) contusion injury were included. The BBB scale is a non-linear scale that ranges from 21 (normal locomotion) to 0 (complete paralysis). Scores below 8 represent early stages of recovery and are signified by isolated movements of the hind limbs (shaded area). Conversely, scores above 8 represent intermediate stages of recovery characterized by the development of hind limb weight support and stepping ability (non-shaded area). With few exceptions, the protective effect is restricted to changes in the degree of isolated movements of the hind limbs. Thus, although studies demonstrating “protective autoimmunity” have reported statistically significant improvements in BBB scores, it is questionable whether these modest improvements represent functionally meaningful neuroprotection.

but avoid T-cell epitopes; these are then conjugated to Th cell epitopes from other molecules to achieve the necessary T-cell help (Morgan, 2011; Tabira, 2010). Several passive immunization approaches, mostly evaluating anti-Aβ antibodies, are currently in clinical trials for AD (Lobello et al., 2012). However, two phase 3 trials (bapineuzumab; Pfizer/Janssen Alzheimer Immunotherapy, and solanezumab; Eli Lilly) were recently reported to have failed to achieve primary outcome measures (Karran, 2012). Although the reasons for these failures are not yet clear, one possibility is the selection of doses used for the clinical trials. Bapineuzumab demonstrated side effects of vasogenic edema and microhemorrhage in phase II trials that limited the dosages that were used in phase 3 trials (Delrieu et al., 2012). It was not reported how the doses used in the human trials compare with the allometrically scaled minimal effective doses determined in preclinical studies, but if the doses are eventually determined to be below the effective range, the inability to meet primary end points should not be unexpected (Karran, 2012). With regard to solanezumab, there have been no adverse effects reported thus far, and in a secondary analysis it was demonstrated that this antibody may have efficacy in patients with mild cognitive impairment (Karran, 2012). A novel peptide vaccine being tested, CAD106, consists of the N-terminus B-cell epitope of amyloid Aβ1-6 to promote humoral immunity, conjugated to a bacteriophage coat protein to provide the necessary Th cell help (Lambracht-Washington and Rosenberg, 2012). This vaccine produced a positive antibody response with no adverse effects in an initial first-in-man study, and is currently in phase II clinical trials for the treatment of AD (Lambracht-Washington and Rosenberg, 2012). A similar type of conjugate vaccine composed of the B-cell epitope of amyloid Aβ1–12 and Th cell epitopes derived from tetanus toxin was shown to have efficacy in a translational study utilizing mice, guinea pigs, and cynomolgus monkeys (Davtyan et al., 2013). The use of epitopes to tetanus toxin evoked strong non-self memory T helper cell responses associated with the production of IFNγ and IL-4 in conjunction with high anti-Aβ1–42 IgG antibody titers (Davtyan et al.,

Vaccination paradigms have been used successfully to minimize (and in some cases eradicate) pathogen-derived diseases and may represent a viable approach to limiting the deleterious consequences and/or enhancing the reparative aspects of the adaptive immune response to spinal cord trauma and other neurological diseases. The data described above suggest that vaccination strategies, especially those aimed at induction of Tregs, may be a viable approach for treating SCI. However, given the considerable flexibility of T-cell phenotypes, a greater understanding of the molecular signaling events involved in various aspects of T-cell biology is necessary for the development of safe, effective vaccines. To successfully harness the therapeutic potential of regulatory T-cell phenotypes without the risk of unwanted side effects, it will be necessary to: 1) identify the role of Tregs specifically as they operate within the context of acute as well as chronic SCI; 2) clarify the conditions that dictate whether effector or regulatory T-cell functions predominate within the injury microenvironment and how these conditions change as a function of time post-injury; and 3) identify specific molecular targets that can be exploited to augment the suppressive functions of Tregs on pathogenic autoreactive lymphocytes that infiltrate the injury site. One approach to achieving this might be to explore the epigenetic status of genes encoding the transcription factors that specify T-cell lineages. This knowledge would further our understanding of the mechanisms that determine phenotypic plasticity of T-cells and could then be used to design more specifically targeted vaccines (Littman and Rudensky, 2010). The development of new molecular biology applications over the past few years, including genomics and proteomics, makes the idea of generating individualized, antigen-specific, therapeutic vaccines based on an individual's unique biological fingerprint an exciting prospect. Trauma to the CNS disrupts the “status quo” and the goal of immunotherapy should be to restore balance. However, given the nature of the immune system as a “double-edged sword” with well-documented roles in tissue destruction and repair, any immunotherapy is likely to be a risky endeavor. Deviation of the immune response too far, even in a putatively beneficial direction could back fire and thus, must be carefully considered and appropriately monitored (Genain et al., 1996; Stebbings et al., 2009). Regardless of the nature of the therapeutic regimen being proposed, the efficacy and safety of any immunotherapeutic vaccine should be rigorously tested in appropriately designed preclinical studies. Suggestions for improving the methodological rigor and quality of preclinical studies for SCI have been highlighted in several recent reviews and should be considered in the design of future studies (Filli and Schwab, 2012; Hawryluk et al., 2008; Kwon et al., 2011, 2012; Rowland et al., 2008; Steward et al., 2012). Attention to these issues may help avoid the pitfalls that have thus far plagued successful translation into clinical trials in other CNS disorders (Lapchak et al., 2013; Lobello et al., 2012). Acknowledgments The author gratefully acknowledges Dr. Kathryn Leyva for her review of the manuscript and to Brent Adrian for help with the illustrations. This work was supported by Midwestern University.


References Aharoni, R., Teitelbaum, D., Sela, M., Arnon, R., 1998. Bystander suppression of experimental autoimmune encephalomyelitis by T cell lines and clones of the Th2 type induced by copolymer 1. J. Neuroimmunol. 91, 135–146. Aharoni, R., Teitelbaum, D., Leitner, O., Meshorer, A., Sela, M., Arnon, R., 2000. Specific Th2 cells accumulate in the central nervous system of mice protected against experimental autoimmune encephalomyelitis by copolymer 1. Proc. Natl. Acad. Sci. U. S. A. 97, 11472–11477. Aktas, O., Smorodchenko, A., Brocke, S., Infante-Duarte, C., Schulze Topphoff, U., Vogt, J., Prozorovski, T., Meier, S., Osmanova, V., Pohl, E., Bechmann, I., Nitsch, R., Zipp, F., 2005. Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL. Neuron 46, 421–432. Angelov, D.N., Waibel, S., Guntinas-Lichius, O., Lenzen, M., Neiss, W.F., Tomov, T.L., Yoles, E., Kipnis, J., Schori, H., Reuter, A., Ludolph, A., Schwartz, M., 2003. Therapeutic vaccine for acute and chronic motor neuron diseases: implications for amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U. S. A. 100, 4790–4795. Ankeny, D.P., Popovich, P.G., 2010. B cells and autoantibodies: complex roles in CNS injury. Trends Immunol. 31, 332–338. Ankeny, D.P., Lucin, K.M., Sanders, V.M., McGaughy, V.M., Popovich, P.G., 2006. Spinal cord injury triggers systemic autoimmunity: evidence for chronic B lymphocyte activation and lupus-like autoantibody synthesis. J. Neurochem. 99, 1073–1087. Ankeny, D.P., Guan, Z., Popovich, P.G., 2009. B cells produce pathogenic antibodies and impair recovery after spinal cord injury in mice. J. Clin. Invest. 119, 2990–2999. Antel, J.P., Williams, K., Blain, M., McRea, E., McLaurin, J., 1994. Oligodendrocyte lysis by CD4+ T cells independent of tumor necrosis factor. Ann. Neurol. 35, 341–348. Arnold, S.A., Hagg, T., 2011. Anti-inflammatory treatments during the chronic phase of spinal cord injury improve locomotor function in adult mice. J. Neurotrauma 28, 1995–2002. Barouch, R., Schwartz, M., 2002. Autoreactive T cells induce neurotrophin production by immune and neural cells in injured rat optic nerve: implications for protective autoimmunity. FASEB J. 16, 1304–1306. Basso, D.M., Fisher, L.C., Anderson, A.J., Jakeman, L.B., McTigue, D.M., Popovich, P.G., 2006. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J. Neurotrauma 23, 635–659. Beck, K.D., Nguyen, H.X., Galvan, M.D., Salazar, D.L., Woodruff, T.M., Anderson, A.J., 2010. Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment. Brain 133, 433–447. Becker, K., Kindrick, D., McCarron, R., Hallenbeck, J., Winn, R., 2003. Adoptive transfer of myelin basic protein-tolerized splenocytes to naive animals reduces infarct size: a role for lymphocytes in ischemic brain injury? Stroke 34, 1809–1815. Beers, D.R., Henkel, J.S., Zhao, W., Wang, J., Huang, A., Wen, S., Liao, B., Appel, S.H., 2011. Endogenous regulatory T lymphocytes ameliorate amyotrophic lateral sclerosis in mice and correlate with disease progression in patients with amyotrophic lateral sclerosis. Brain 134, 1293–1314. Benner, E.J., Mosley, R.L., Destache, C.J., Lewis, T.B., Jackson-Lewis, V., Gorantla, S., Nemachek, C., Green, S.R., Przedborski, S., Gendelman, H.E., 2004. Therapeutic immunization protects dopaminergic neurons in a mouse model of Parkinson's disease. Proc. Natl. Acad. Sci. U. S. A. 101, 9435–9440. Bethea, J.R., Nagashima, H., Acosta, M.C., Briceno, C., Gomez, F., Marcillo, A.E., Loor, K., Green, J., Dietrich, W.D., 1999. Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats. J. Neurotrauma 16, 851–863. Bielekova, B., Catalfamo, M., Reichert-Scrivner, S., Packer, A., Cerna, M., Waldmann, T.A., McFarland, H., Henkart, P.A., Martin, R., 2006. Regulatory CD56(bright) natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis. Proc. Natl. Acad. Sci. U. S. A. 103, 5941–5946. Butovsky, O., Hauben, E., Schwartz, M., 2001. Morphological aspects of spinal cord autoimmune neuroprotection: colocalization of T cells with B7–2 (CD86) and prevention of cyst formation. FASEB J. 15 (6), 1065–1067. http://dx.doi.org/10.1096/fj.000550fje. Butovsky, O., Koronyo-Hamaoui, M., Kunis, G., Ophir, E., Landa, G., Cohen, H., Schwartz, M., 2006. Glatiramer acetate fights against Alzheimer's disease by inducing dendritic-like microglia expressing insulin-like growth factor 1. Proc. Natl. Acad. Sci. U. S. A. 103, 11784–11789. Cao, C., Arendash, G.W., Dickson, A., Mamcarz, M.B., Lin, X., Ethell, D.W., 2009. Abeta-specific Th2 cells provide cognitive and pathological benefits to Alzheimer's mice without infiltrating the CNS. Neurobiol. Dis. 34, 63–70. Chen, L., Flies, D.B., 2013. Molecular mechanisms of T cell co-stimulation and coinhibition. Nat. Rev. Immunol. 13, 227–242. Chen, S., Wu, H., Klebe, D., Hong, Y., Zhang, J., Tang, J., 2013. Regulatory T cell in stroke: a new paradigm for immune regulation. Clin. Dev. Immunol. 2013, 689827. Davies, A.L., Hayes, K.C., Dekaban, G.A., 2007. Clinical correlates of elevated serum concentrations of cytokines and autoantibodies in patients with spinal cord injury. Arch. Phys. Med. Rehabil. 88, 1384–1393. Davtyan, H., Ghochikyan, A., Petrushina, I., Hovakimyan, A., Davtyan, A., Poghosyan, A., Marleau, A.M., Movsesyan, N., Kiyatkin, A., Rasool, S., Larsen, A.K., Madsen, P.J., Wegener, K.M., Ditlevsen, D.K., Cribbs, D.H., Pedersen, L.O., Agadjanyan, M.G., 2013. Immunogenicity, efficacy, safety, and mechanism of action of epitope vaccine (Lu AF20513) for Alzheimer's disease: prelude to a clinical trial. J. Neurosci. 33, 4923–4934. Dayan, C.M., Wraith, D.C., 2008. Preparing for first-in-man studies: the challenges for translational immunology post-TGN1412. Clin. Exp. Immunol. 151, 231–234. Delgado, M., Chorny, A., Gonzalez-Rey, E., Ganea, D., 2005. Vasoactive intestinal peptide generates CD4+CD25+ regulatory T cells in vivo. J. Leukoc. Biol. 78, 1327–1338.

87

Delrieu, J., Ousset, P.J., Caillaud, C., Vellas, B., 2012. ‘Clinical trials in Alzheimer's disease’: immunotherapy approaches. J. Neurochem. 120 (Suppl. 1), 186–193. Ebner, F., Brandt, C., Thiele, P., Richter, D., Schliesser, U., Siffrin, V., Schueler, J., Stubbe, T., Ellinghaus, A., Meisel, C., Sawitzki, B., Nitsch, R., 2013. Microglial activation milieu controls regulatory T cell responses. J. Immunol. 191, 5594–5602. Edling, A.E., Nanavati, T., Johnson, J.M., Tuohy, V.K., 2004. Human and murine lymphocyte neurotrophin expression is confined to B cells. J. Neurosci. Res. 77, 709–717. Filli, L., Schwab, M.E., 2012. The rocky road to translation in spinal cord repair. Ann. Neurol. 72, 491–501. Fitch, M.T., Silver, J., 2008. CNS injury, glial scars, and inflammation: inhibitory extracellular matrices and regeneration failure. Exp. Neurol. 209, 294–301. Frenkel, D., Huang, Z., Maron, R., Koldzic, D.N., Moskowitz, M.A., Weiner, H.L., 2005. Neuroprotection by IL-10-producing MOG CD4+ T cells following ischemic stroke. J. Neurol. Sci. 233, 125–132. Fruman, D.A., Klee, C.B., Bierer, B.E., Burakoff, S.J., 1992. Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proc. Natl. Acad. Sci. U. S. A. 89, 3686–3690. Fujio, K., Okamura, T., Yamamoto, K., 2010. The family of IL-10-secreting CD4+ T cells. Adv. Immunol. 105, 99–130. Furtado, G.C., Curotto de Lafaille, M.A., Kutchukhidze, N., Lafaille, J.J., 2002. Interleukin 2 signaling is required for CD4(+) regulatory T cell function. J. Exp. Med. 196, 851–857. Ganguly, D., Haak, S., Sisirak, V., Reizis, B., 2013. The role of dendritic cells in autoimmunity. Nat. Rev. Immunol. 13, 566–577. Garcia, E., Silva-Garcia, R., Mestre, H., Flores, N., Martinon, S., Calderon-Aranda, E.S., Ibarra, A., 2012. Immunization with A91 peptide or copolymer-1 reduces the production of nitric oxide and inducible nitric oxide synthase gene expression after spinal cord injury. J. Neurosci. Res. 90, 656–663. Genain, C.P., Abel, K., Belmar, N., Villinger, F., Rosenberg, D.P., Linington, C., Raine, C.S., Hauser, S.L., 1996. Late complications of immune deviation therapy in a nonhuman primate. Science 274, 2054–2057. Gensel, J.C., Nakamura, S., Guan, Z., van Rooijen, N., Ankeny, D.P., Popovich, P.G., 2009. Macrophages promote axon regeneration with concurrent neurotoxicity. J. Neurosci. 29, 3956–3968. Gimsa, U., Peter, S.V., Lehmann, K., Bechmann, I., Nitsch, R., 2000. Axonal damage induced by invading T cells in organotypic central nervous system tissue in vitro: involvement of microglial cells. Brain Pathol. 10, 365–377. Giuliani, F., Goodyer, C.G., Antel, J.P., Yong, V.W., 2003. Vulnerability of human neurons to T cell-mediated cytotoxicity. J. Immunol. 171, 368–379. Gonzenbach, R.R., Schwab, M.E., 2008. Disinhibition of neurite growth to repair the injured adult CNS: focusing on Nogo. Cell. Mol. Life Sci. 65, 161–176. Gorantla, S., Liu, J., Wang, T., Holguin, A., Sneller, H.M., Dou, H., Kipnis, J., Poluektova, L., Gendelman, H.E., 2008. Modulation of innate immunity by copolymer-1 leads to neuroprotection in murine HIV-1 encephalitis. Glia 56, 223–232. Ha, D., Stone, D.K., Mosley, R.L., Gendelman, H.E., 2012. Immunization strategies for Parkinson's disease. Parkinsonism Relat. Disord. 18 (Suppl. 1), S218–S221. Hammarberg, H., Lidman, O., Lundberg, C., Eltayeb, S.Y., Gielen, A.W., Muhallab, S., Svenningsson, A., Linda, H., Der Meide, P.H., Cullheim, S., Olsson, T., Piehl, F., 2000. Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. J. Neurosci. 20, 5283–5291. Hara, H., Mouri, A., Yonemitsu, Y., Nabeshima, T., Tabira, T., 2011. Mucosal immunotherapy in an Alzheimer mouse model by recombinant Sendai virus vector carrying Abeta1–43/IL-10 cDNA. Vaccine 29, 7474–7482. Hauben, E., Butovsky, O., Nevo, U., Yoles, E., Moalem, G., Agranov, E., Mor, F., LeibowitzAmit, R., Pevsner, E., Akselrod, S., Neeman, M., Cohen, I.R., Schwartz, M., 2000a. Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion. J. Neurosci. 20, 6421–6430. Hauben, E., Nevo, U., Yoles, E., Moalem, G., Agranov, E., Mor, F., Akselrod, S., Neeman, M., Cohen, I.R., Schwartz, M., 2000b. Autoimmune T cells as potential neuroprotective therapy for spinal cord injury [letter]. Lancet 355, 286–287. Hauben, E., Agranov, E., Gothilf, A., Nevo, U., Cohen, A., Smirnov, I., Steinman, L., Schwartz, M., 2001a. Posttraumatic therapeutic vaccination with modified myelin self-antigen prevents complete paralysis while avoiding autoimmune disease. J. Clin. Invest. 108, 591–599. Hauben, E., Ibarra, A., Mizrahi, T., Barouch, R., Agranov, E., Schwartz, M., 2001b. Vaccination with a Nogo-A-derived peptide after incomplete spinal-cord injury promotes recovery via a T-cell-mediated neuroprotective response: comparison with other myelin antigens. Proc. Natl. Acad. Sci. U. S. A. 98, 15173–15178. Hauben, E., Mizrahi, T., Agranov, E., Schwartz, M., 2002. Sexual dimorphism in the spontaneous recovery from spinal cord injury: a gender gap in beneficial autoimmunity? Eur. J. Neurosci. 16, 1731–1740. Hauben, E., Gothilf, A., Cohen, A., Butovsky, O., Nevo, U., Smirnov, I., Yoles, E., Akselrod, S., Schwartz, M., 2003. Vaccination with dendritic cells pulsed with peptides of myelin basic protein promotes functional recovery from spinal cord injury. J. Neurosci. 23, 8808–8819. Hawryluk, G.W., Rowland, J., Kwon, B.K., Fehlings, M.G., 2008. Protection and repair of the injured spinal cord: a review of completed, ongoing, and planned clinical trials for acute spinal cord injury. Neurosurg. Focus 25, E14. Hayes, K.C., Hull, T.C., Delaney, G.A., Potter, P.J., Sequeira, K.A., Campbell, K., Popovich, P.G., 2002. Elevated serum titers of proinflammatory cytokines and CNS autoantibodies in patients with chronic spinal cord injury. J. Neurotrauma 19, 753–761. Henkel, J.S., Beers, D.R., Wen, S., Rivera, A.L., Toennis, K.M., Appel, J.E., Zhao, W., Moore, D. H., Powell, S.Z., Appel, S.H., 2013. Regulatory T-lymphocytes mediate amyotrophic lateral sclerosis progression and survival. EMBO Mol. Med. 5, 64–79.

88


Hong, J., Li, N., Zhang, X., Zheng, B., Zhang, J.Z., 2005. Induction of CD4+CD25+ regulatory T cells by copolymer-I through activation of transcription factor Foxp3. Proc. Natl. Acad. Sci. U. S. A. 102, 6449–6454. Howe, C.L., Bieber, A.J., Warrington, A.E., Pease, L.R., Rodriguez, M., 2004. Antiapoptotic signaling by a remyelination-promoting human antimyelin antibody. Neurobiol. Dis. 15, 120–131. Hu, J.G., Shen, L., Wang, R., Wang, Q.Y., Zhang, C., Xi, J., Ma, S.F., Zhou, J.S., Lu, H.Z., 2012. Effects of olig2-overexpressing neural stem cells and myelin basic protein-activated T cells on recovery from spinal cord injury. Neurotherapeutics 9, 422–445. Huang, D.W., McKerracher, L., Braun, P.E., David, S., 1999. A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord. Neuron 24, 639–647. Huang, Z., Meola, D., Petitto, J.M., 2011a. Loss of CNS IL-2 gene expression modifies brain T lymphocyte trafficking: response of normal versus autoreactive Treg-deficient T cells. Neurosci. Lett. 499, 213–218. Huang, Z.Y., Hunter, S., Chien, P., Kim, M.K., Han-Kim, T.H., Indik, Z.K., Schreiber, A.D., 2011b. Interaction of two phagocytic host defense systems: Fcgamma receptors and complement receptor 3. J. Biol. Chem. 286, 160–168. Ibarra, A., Martinez, S., Reyes, J., Meza-Lucas, A., Mandujano, A., Grijalva, I., Madrazo, I., Correa, D., 2000. Search for an IgG response against neural antigens in experimental spinal cord injury. Neuroscience 96, 3–5. Ibarra, A., Correa, D., Willms, K., Merchant, M.T., Guizar-Sahagun, G., Grijalva, I., Madrazo, I., 2003. Effects of cyclosporin-A on immune response, tissue protection and motor function of rats subjected to spinal cord injury. Brain Res. 979, 165–178. Ibarra, A., Jimenez, A., Cortes, C., Correa, D., 2007. Influence of the intensity, level and phase of spinal cord injury on the proliferation of T cells and T-cell-dependent antibody reactions in rats. Spinal Cord 45, 380–386. Ibarra, A., García, E., Flores, N., Martiñón, S., Reyes, R., Campos, M.G., Maciel, M., Mestre, H., 2010. Immunization with neural-derived antigens inhibits lipid peroxidation after spinal cord injury. Neurosci. Lett. 476 (2), 62–65. http://dx.doi.org/10.1016/j.neulet. 2010.04.003. Ibarra, A., Sosa, M., Garcia, E., Flores, A., Cruz, Y., Mestre, H., Martinon, S., PinedaRodriguez, B.A., Gutierrez-Ospina, G., 2013. Prophylactic neuroprotection with A91 improves the outcome of spinal cord injured rats. Neurosci. Lett. 554, 59–63. Ishii, H., Jin, X., Ueno, M., Tanabe, S., Kubo, T., Serada, S., Naka, T., Yamashita, T., 2012. Adoptive transfer of Th1-conditioned lymphocytes promotes axonal remodeling and functional recovery after spinal cord injury. Cell Death Dis. 3, e363. Jones, T.B., Basso, D.M., Sodhi, A., Pan, J.Z., Hart, R.P., MacCallum, R.C., Lee, S., Whitacre, C.C., Popovich, P.G., 2002. Pathological CNS autoimmune disease triggered by traumatic spinal cord injury: implications for autoimmune vaccine therapy. J. Neurosci. 22, 2690–2700. Jones, T.B., Ankeny, D.P., Guan, Z., McGaughy, V., Fisher, L.C., Basso, D.M., Popovich, P.G., 2004. Passive or active immunization with myelin basic protein impairs neurological function and exacerbates neuropathology after spinal cord injury in rats. J. Neurosci. 24, 3752–3761. Jones, T.B., Hart, R.P., Popovich, P.G., 2005. Molecular control of physiological and pathological T-cell recruitment after mouse spinal cord injury. J. Neurosci. 25, 6576–6583. Kahn, N.N., Bauman, W.A., Sinha, A.K., 2005. Appearance of a novel prostacyclin receptor antibody and duration of spinal cord injury. J. Spinal Cord Med. 28, 97–102. Kala, M., Rhodes, S.N., Piao, W.H., Shi, F.D., Campagnolo, D.I., Vollmer, T.L., 2010. B cells from glatiramer acetate-treated mice suppress experimental autoimmune encephalomyelitis. Exp. Neurol. 221, 136–145. Kappos, L., Radue, E.W., O'Connor, P., Polman, C., Hohlfeld, R., Calabresi, P., Selmaj, K., Agoropoulou, C., Leyk, M., Zhang-Auberson, L., Burtin, P., Group, F.S., 2010. A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N. Engl. J. Med. 362, 387–401. Karnezis, T., Mandemakers, W., McQualter, J.L., Zheng, B., Ho, P.P., Jordan, K.A., Murray, B.M., Barres, B., Tessier-Lavigne, M., Bernard, C.C., 2004. The neurite outgrowth inhibitor Nogo A is involved in autoimmune-mediated demyelination. Nat. Neurosci. 7, 736–744. Karran, E., 2012. Current status of vaccination therapies in Alzheimer's disease. J. Neurochem. 123, 647–651. Kigerl, K.A., Gensel, J.C., Ankeny, D.P., Alexander, J.K., Donnelly, D.J., Popovich, P.G., 2009. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J. Neurosci. 29, 13435–13444. Kil, K., Zang, Y.C., Yang, D., Markowski, J., Fuoco, G.S., Vendetti, G.C., Rivera, V.M., Zhang, J. Z., 1999. T cell responses to myelin basic protein in patients with spinal cord injury and multiple sclerosis. J. Neuroimmunol. 98, 201–207. Kipnis, J., Yoles, E., Porat, Z., Cohen, A., Mor, F., Sela, M., Cohen, I.R., Schwartz, M., 2000. T cell immunity to copolymer 1 confers neuroprotection on the damaged optic nerve: possible therapy for optic neuropathies. Proc. Natl. Acad. Sci. U. S. A. 97, 7446–7451. Kipnis, J., Yoles, E., Mizrahi, T., Ben Nur, A., Schwartz, M., 2002. Myelin specific Th1 cells are necessary for post-traumatic protective autoimmunity. J. Neuroimmunol. 130, 78. Korn, T., Anderson, A.C., Bettelli, E., Oukka, M., 2007. The dynamics of effector T cells and Foxp3+ regulatory T cells in the promotion and regulation of autoimmune encephalomyelitis. J. Neuroimmunol. 191, 51–60. Kwon, B.K., Okon, E.B., Tsai, E., Beattie, M.S., Bresnahan, J.C., Magnuson, D.K., Reier, P.J., McTigue, D.M., Popovich, P.G., Blight, A.R., Oudega, M., Guest, J.D., Weaver, L.C., Fehlings, M.G., Tetzlaff, W., 2011. A grading system to evaluate objectively the strength of pre-clinical data of acute neuroprotective therapies for clinical translation in spinal cord injury. J. Neurotrauma 28 (8), 1525–1543. Kwon, B.K., Ghag, A., Reichl, L., Dvorak, M.F., Illes, J., Tetzlaff, W., 2012. Opinions on the preclinical evaluation of novel therapies for spinal cord injury: a comparison between researchers and spinal cord-injured individuals. J. Neurotrauma 29, 2367–2374.

Lacan, G., Dang, H., Middleton, B., Horwitz, M.A., Tian, J., Melega, W.P., Kaufman, D.L., 2013. Bacillus Calmette–Guerin vaccine-mediated neuroprotection is associated with regulatory T-cell induction in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. J. Neurosci. Res. 91, 1292–1302. Lambracht-Washington, D., Rosenberg, R.N., 2012. Active DNA Abeta42 vaccination as immunotherapy for Alzheimer disease. Transl. Neurosci. 3, 307–313. Lapchak, P.A., Zhang, J.H., Noble-Haeusslein, L.J., 2013. RIGOR guidelines: escalating STAIR and STEPS for effective translational research. Transl. Stroke Res. 4, 279–285. Lee, K.D., Chow, W.N., Sato-Bigbee, C., Graf, M.R., Graham, R.S., Colello, R.J., Young, H.F., Mathern, B.E., 2009. FTY720 reduces inflammation and promotes functional recovery after spinal cord injury. J. Neurotrauma 26, 2335–2344. Liesz, A., Suri-Payer, E., Veltkamp, C., Doerr, H., Sommer, C., Rivest, S., Giese, T., Veltkamp, R., 2009. Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat. Med. 15, 192–199. Liesz, A., Zhou, W., Na, S.Y., Hammerling, G.J., Garbi, N., Karcher, S., Mracsko, E., Backs, J., Rivest, S., Veltkamp, R., 2013. Boosting regulatory T cells limits neuroinflammation in permanent cortical stroke. J. Neurosci. 33, 17350–17362. Littman, D.R., Rudensky, A.Y., 2010. Th17 and regulatory T cells in mediating and restraining inflammation. Cell 140, 845–858. Liu, Y., Teige, I., Birnir, B., Issazadeh-Navikas, S., 2006. Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE. Nat. Med. 12, 518–525. Liu, J., Johnson, T.V., Lin, J., Ramirez, S.H., Bronich, T.K., Caplan, S., Persidsky, Y., Gendelman, H.E., Kipnis, J., 2007. T cell independent mechanism for copolymer-1-induced neuroprotection. Eur. J. Immunol. 37, 3143–3154. Liu, J., Gong, N., Huang, X., Reynolds, A.D., Mosley, R.L., Gendelman, H.E., 2009a. Neuromodulatory activities of CD4+CD25+ regulatory T cells in a murine model of HIV-1-associated neurodegeneration. J. Immunol. 182, 3855–3865. Liu, M., Zhao, J., Liang, H., Bian, X., 2009b. Vaccination with dendritic cells pulsed with homogenate protein of spinal cord promotes functional recovery from spinal cord injury in mice. Spinal Cord 47, 360–366. Liu, H.N., Tjostheim, S., Dasilva, K., Taylor, D., Zhao, B., Rakhit, R., Brown, M., Chakrabartty, A., McLaurin, J., Robertson, J., 2012. Targeting of monomer/misfolded SOD1 as a therapeutic strategy for amyotrophic lateral sclerosis. J. Neurosci. 32, 8791–8799. Lobello, K., Ryan, J.M., Liu, E., Rippon, G., Black, R., 2012. Targeting Beta amyloid: a clinical review of immunotherapeutic approaches in Alzheimer's disease. Int. J. Alzheimers Dis. 2012, 628070. Lopez-Vales, R., Garcia-Alias, G., Fores, J., Udina, E., Gold, B.G., Navarro, X., Verdu, E., 2005. FK 506 reduces tissue damage and prevents functional deficit after spinal cord injury in the rat. J. Neurosci. Res. 81, 827–836. Lowther, D.E., Hafler, D.A., 2012. Regulatory T cells in the central nervous system. Immunol. Rev. 248, 156–169. Lu, H.Z., Xu, L., Zou, J., Wang, Y.X., Ma, Z.W., Xu, X.M., Lu, P.H., 2008. Effects of autoimmunity on recovery of function in adult rats following spinal cord injury. Brain Behav. Immun. 22, 1217–1230. Lu, H.Z., Wang, Y.X., Zhou, J.S., Wang, F.C., Hu, J.G., 2010. Cyclosporin A increases recovery after spinal cord injury but does not improve myelination by oligodendrocyte progenitor cell transplantation. BMC Neurosci. 11, 127. Luchetti, S., Beck, K.D., Galvan, M.D., Silva, R., Cummings, B.J., Anderson, A.J., 2010. Comparison of immunopathology and locomotor recovery in C57BL/6, BUB/BnJ, and NOD-SCID mice after contusion spinal cord injury. J. Neurotrauma 27, 411–421. Lund, F.E., Randall, T.D., 2010. Effector and regulatory B cells: modulators of CD4+ T cell immunity. Nat. Rev. Immunol. 10, 236–247. Mackay, F., Silveira, P.A., Brink, R., 2007. B cells and the BAFF/APRIL axis: fast-forward on autoimmunity and signaling. Curr. Opin. Immunol. 19, 327–336. Madsen, J.R., MacDonald, P., Irwin, N., Goldberg, D.E., Yao, G.L., Meiri, K.F., Rimm, I.J., Stieg, P.E., Benowitz, L.I., 1998. Tacrolimus (FK506) increases neuronal expression of GAP-43 and improves functional recovery after spinal cord injury in rats. Exp. Neurol. 154, 673–683. Martinez-Pasamar, S., Abad, E., Moreno, B., Velez de Mendizabal, N., Martinez-Forero, I., Garcia-Ojalvo, J., Villoslada, P., 2013. Dynamic cross-regulation of antigen-specific effector and regulatory T cell subpopulations and microglia in brain autoimmunity. BMC Syst. Biol. 7, 34. Martinon, S., Garcia, E., Flores, N., Gonzalez, I., Ortega, T., Buenrostro, M., Reyes, R., Fernandez-Presas, A.M., Guizar-Sahagun, G., Correa, D., Ibarra, A., 2007. Vaccination with a neural-derived peptide plus administration of glutathione improves the performance of paraplegic rats. Eur. J. Neurosci. 26, 403–412. Martinon, S., Garcia, E., Gutierrez-Ospina, G., Mestre, H., Ibarra, A., 2012. Development of protective autoimmunity by immunization with a neural-derived peptide is ineffective in severe spinal cord injury. PLoS One 7, e32027. Matzinger, P., 2002. The danger model: a renewed sense of self. Science 296, 301–305. McMahon, S.S., Albermann, S., Rooney, G.E., Moran, C., Hynes, J., Garcia, Y., Dockery, P., O'Brien, T., Windebank, A.J., Barry, F.P., 2009. Effect of cyclosporin A on functional recovery in the spinal cord following contusion injury. J. Anat. 215, 267–279. Meininger, V., Drory, V.E., Leigh, P.N., Ludolph, A., Robberecht, W., Silani, V., 2009. Glatiramer acetate has no impact on disease progression in ALS at 40 mg/day: a double- blind, randomized, multicentre, placebo-controlled trial. Amyotroph. Lateral Scler. 10, 378–383. Meyer-Luehmann, M., Mora, J.R., Mielke, M., Spires-Jones, T.L., de Calignon, A., von Andrian, U.H., Hyman, B.T., 2011. T cell mediated cerebral hemorrhages and microhemorrhages during passive Abeta immunization in APPPS1 transgenic mice. Mol. Neurodegeneration 6, 22. Mizrachi, Y., Ohry, A., Aviel, A., Rozin, R., Brooks, M.E., Schwartz, M., 1983. Systemic humoral factors participating in the course of spinal cord injury. Paraplegia 21, 287–293. Moalem, G., Leibowitz-Amit, R., Yoles, E., Mor, F., Cohen, I.R., Schwartz, M., 1999. Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat. Med. 5, 49–55.

T.B. Jones / Experimental Neurology 258 (2014) 78–90 Moalem, G., Gdalyahu, A., Shani, Y., Otten, U., Lazarovici, P., Cohen, I.R., Schwartz, M., 2000. Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity. J. Autoimmun. 15, 331–345. Morgan, D., 2011. Immunotherapy for Alzheimer's disease. J. Intern. Med. 269, 54–63. Muhallab, S., Lundberg, C., Gielen, A.W., Lidman, O., Svenningsson, A., Piehl, F., Olsson, T., 2002. Differential expression of neurotrophic factors and inflammatory cytokines by myelin basic protein-specific and other recruited T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis. Scand. J. Immunol. 55, 264–273. Nitsch, R., Pohl, E.E., Smorodchenko, A., Infante-Duarte, C., Aktas, O., Zipp, F., 2004. Direct impact of T cells on neurons revealed by two-photon microscopy in living brain tissue. J. Neurosci. 24, 2458–2464. Norimatsu, Y., Ohmori, T., Kimura, A., Madoiwa, S., Mimuro, J., Seichi, A., Yatomi, Y., Hoshino, Y., Sakata, Y., 2012. FTY720 improves functional recovery after spinal cord injury by primarily nonimmunomodulatory mechanisms. Am. J. Pathol. 180, 1625–1635. Nottingham, S., Knapp, P., Springer, J., 2002. FK506 treatment inhibits caspase-3 activation and promotes oligodendroglial survival following traumatic spinal cord injury. Exp. Neurol. 177, 242–251. Olsson, T., Sun, J.B., Solders, G., Xiao, B.G., Hojeberg, B., Ekre, H.P., Link, H., 1993. Autoreactive T and B cell responses to myelin antigens after diagnostic sural nerve biopsy. J. Neurol. Sci. 117, 130–139. Palladini, G., Grossi, M., Maleci, A., Lauro, G.M., Guidetti, B., 1987. Immunocomplexes in rat and rabbit spinal cord after injury. Exp. Neurol. 95, 639–651. Peterson, S.L., Anderson, A.J., 2014. Complement and spinal cord injury: Traditional and non-traditional aspects of complement cascade function in the injured spinal cord microenvironment. Exp. Neurol. 258, 35–47 (in this issue). Popovich, P.G., Jones, T.B., 2003. Manipulating neuroinflammatory reactions in the injured spinal cord: back to basics. Trends Pharmacol. Sci. 24, 13–17. Popovich, P.G., Stokes, B.T., Whitacre, C.C., 1996. Concept of autoimmunity following spinal cord injury: possible roles for T lymphocytes in the traumatized central nervous system POPOVICH1996A. J. Neurosci. Res. 45, 349–363. Potas, J.R., Zheng, Y., Moussa, C., Venn, M., Gorrie, C.A., Deng, C., Waite, P.M., 2006. Augmented locomotor recovery after spinal cord injury in the athymic nude rat. J. Neurotrauma 23, 660–673. Prochazka, M., Voltnerova, M., Stefan, J., 1971. Studies of immunologic reactions after brain injury. II. Antibodies against brain tissue lipids after blunt head injury in man. Int. Surg. 55, 322–326. Qian, S., Tang, Y., Cheng, L., Sun, X., Tian, J., Zhou, C., 2013. Interaction of copolymer-1activated T cells and microglia in RGC protection. Clin. Exp. Ophthalmol. 41 (9), 881–890. Reynolds, A.D., Stone, D.K., Hutter, J.A., Benner, E.J., Mosley, R.L., Gendelman, H.E., 2010. Regulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson's disease. J. Immunol. 184, 2261–2271. Rodriguez, M., Lennon, V.A., 1990. Immunoglobulins promote remyelination in the central nervous system. Ann. Neurol. 27, 12–17. Rodriguez, M., Lennon, V.A., Benveniste, E.N., Merrill, J.E., 1987. Remyelination by oligodendrocytes stimulated by antiserum to spinal cord. J. Neuropathol. Exp. Neurol. 46, 84–95. Rodriguez, M., Miller, D.J., Lennon, V.A., 1996. Immunoglobulins reactive with myelin basic protein promote CNS remyelination. Neurology 46, 538–545. Rommer, P., Dudesek, A., Stuve, O., Zettl, U., 2014. Monoclonal antibodies in treatment of multiple sclerosis. Clin. Exp. Immunol. 175 (3), 373–384. Rowland, J.W., Hawryluk, G.W., Kwon, B., Fehlings, M.G., 2008. Current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon. Neurosurg. Focus 25, E2. Rudehill, S., Muhallab, S., Wennersten, A., von Gertten, C., Al Nimer, F., SandbergNordqvist, A.C., Holmin, S., Mathiesen, T., 2006. Autoreactive antibodies against neurons and basal lamina found in serum following experimental brain contusion in rats. Acta Neurochir. 148, 199–205 (discussion 205). Saltzman, J.W., Battaglino, R.A., Salles, L., Jha, P., Sudhakar, S., Garshick, E., Stott, H.L., Zafonte, R., Morse, L.R., 2013. B-cell maturation antigen, a proliferation-inducing ligand, and B-cell activating factor are candidate mediators of spinal cord injuryinduced autoimmunity. J. Neurotrauma 30, 434–440. Sanchez-Guajardo, V., Annibali, A., Jensen, P.H., Romero-Ramos, M., 2013. alpha-Synuclein vaccination prevents the accumulation of parkinson disease-like pathologic inclusions in striatum in association with regulatory T cell recruitment in a rat model. J. Neuropathol. Exp. Neurol. 72, 624–645. Saunders, J.A., Estes, K.A., Kosloski, L.M., Allen, H.E., Dempsey, K.M., Torres-Russotto, D.R., Meza, J.L., Santamaria, P.M., Bertoni, J.M., Murman, D.L., Ali, H.H., Standaert, D.G., Mosley, R.L., Gendelman, H.E., 2012. CD4+ regulatory and effector/memory T cell subsets profile motor dysfunction in Parkinson's disease. J. Neuroimmune Pharmacol. 7, 927–938. Schmetterer, K.G., Neunkirchner, A., Pickl, W.F., 2012. Naturally occurring regulatory T cells: markers, mechanisms, and manipulation. FASEB J. 26, 2253–2276. Schori, H., Kipnis, J., Yoles, E., WoldeMussie, E., Ruiz, G., Wheeler, L.A., Schwartz, M., 2001. Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: implications for glaucoma. Proc. Natl. Acad. Sci. U. S. A. 98, 3398–3403. Schwab, J.M., Zhang, Y., Kopp, M.A., Brommer, B., Popovich, P.G., 2014. The paradox of chronic neuroinflammation, systemic immune suppression, autoimmunity after traumatic chronic spinal cord injury. Exp. Neurol. 258, 121–129 (in this issue). Schwartz, M., 2001. Protective autoimmunity as a T-cell response to central nervous system trauma: prospects for therapeutic vaccines. Prog. Neurobiol. 65, 489–496.

89

Schwartz, M., Kipnis, J., 2005. Protective autoimmunity and neuroprotection in inflammatory and noninflammatory neurodegenerative diseases. J. Neurol. Sci. 233, 163–166. Schwartz, M., Ziv, Y., 2008. Immunity to self and self-maintenance: a unified theory of brain pathologies. Trends Immunol. 29, 211–219. Scorisa, J.M., Zanon, R.G., Freria, C.M., de Oliveira, A.L., 2009. Glatiramer acetate positively influences spinal motoneuron survival and synaptic plasticity after ventral root avulsion. Neurosci. Lett. 451, 34–39. Serpe, C.J., Coers, S., Sanders, V.M., Jones, K.J., 2003. CD4+ T, but not CD8+ or B, lymphocytes mediate facial motoneuron survival after facial nerve transection. Brain Behav. Immun. 17, 393–402. Shaked, I., Porat, Z., Gersner, R., Kipnis, J., Schwartz, M., 2004. Early activation of microglia as antigen-presenting cells correlates with T cell-mediated protection and repair of the injured central nervous system. J. Neuroimmunol. 146, 84–93. Shechter, R., London, A., Varol, C., Raposo, C., Cusimano, M., Yovel, G., Rolls, A., Mack, M., Pluchino, S., Martino, G., Jung, S., Schwartz, M., 2009. Infiltrating blood-derived macrophages are vital cells playing an anti-inflammatory role in recovery from spinal cord injury in mice. PLoS Med. 6, e1000113. Sicotte, M., Tsatas, O., Jeong, S.Y., Cai, C.Q., He, Z., David, S., 2003. Immunization with myelin or recombinant Nogo-66/MAG in alum promotes axon regeneration and sprouting after corticospinal tract lesions in the spinal cord. Mol. Cell. Neurosci. 23, 251–263. Sroga, J.M., Jones, T.B., Kigerl, K.A., McGaughy, V.M., Popovich, P.G., 2003. Rats and mice exhibit distinct inflammatory reactions after spinal cord injury. J. Comp. Neurol. 462, 223–240. Stebbings, R., Poole, S., Thorpe, R., 2009. Safety of biologics, lessons learnt from TGN1412. Curr. Opin. Biotechnol. 20, 673–677. Stefanova, I., Dorfman, J.R., Germain, R.N., 2002. Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes. Nature 420, 429–434. Steinman, L., 1996. A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry. Proc. Natl. Acad. Sci. U. S. A. 93, 2253–2256. Steward, O., Popovich, P.G., Dietrich, W.D., Kleitman, N., 2012. Replication and reproducibility in spinal cord injury research. Exp. Neurol. 233, 597–605. Tabira, T., 2010. Immunization therapy for Alzheimer disease: a comprehensive review of active immunization strategies. Tohoku J. Exp. Med. 220, 95–106. Takeuchi, S., Fujiwara, N., Ido, A., Oono, M., Takeuchi, Y., Tateno, M., Suzuki, K., Takahashi, R., Tooyama, I., Taniguchi, N., Julien, J.P., Urushitani, M., 2010. Induction of protective immunity by vaccination with wild-type apo superoxide dismutase 1 in mutant SOD1 transgenic mice. J. Neuropathol. Exp. Neurol. 69, 1044–1056. Toll, E.C., Seifalian, A.M., Birchall, M.A., 2011. The role of immunophilin ligands in nerve regeneration. Regen. Med. 6, 635–652. Tyor, W.R., Avgeropoulos, N., Ohlandt, G., Hogan, E.L., 2002. Treatment of spinal cord impact injury in the rat with transforming growth factor-beta. J. Neurol. Sci. 200, 33–41. Vaughn, C.N., Iafrate, J.L., Henley, J.B., Stevenson, E.K., Shlifer, I.G., Jones, T.B., 2013. Cellular neuroinflammation in a lateral forceps compression model of spinal cord injury. Anat. Rec. 296, 1229–1246. Wang, D.D., Zhao, Y.F., Wang, G.Y., Sun, B., Kong, Q.F., Zhao, K., Zhang, Y., Wang, J.H., Liu, Y. M., Mu, L.L., Wang, D.S., Li, H.L., 2009. IL-17 potentiates neuronal injury induced by oxygen-glucose deprivation and affects neuronal IL-17 receptor expression. J. Neuroimmunol. 212, 17–25. Wang, Y., Wang, K., Chao, R., Li, J., Zhou, L., Ma, J., Yan, J., 2012. Neuroprotective effect of vaccination with autoantigen-pulsed dendritic cells after spinal cord injury. J. Surg Res. 176, 281–292. Wang, K., Chao, R., Guo, Q.N., Liu, M.Y., Liang, H.P., Liu, P., Zhao, J.H., 2013. Expressions of some neurotrophins and neurotrophic cytokines at site of spinal cord injury in mice after vaccination with dendritic cells pulsed with homogenate proteins. Neuroimmunomodulation 20, 87–98. Wei, Y., Yemisci, M., Kim, H.H., Yung, L.M., Shin, H.K., Hwang, S.K., Guo, S., Qin, T., Alsharif, N., Brinkmann, V., Liao, J.K., Lo, E.H., Waeber, C., 2011. Fingolimod provides long-term protection in rodent models of cerebral ischemia. Ann. Neurol. 69, 119–129. Wiendl, H., Gross, C.C., 2013. Modulation of IL-2Ralpha with daclizumab for treatment of multiple sclerosis. Nat. Rev. Neurol. 9, 394–404. Willenborg, D.O., Staten, E.A., Eidelberg, E., 1977. Studies on cell-mediated hypersensitivity to neural antigens after experimental spinal cord injury. Exp. Neurol. 54, 383–392. Wu, B., Matic, D., Djogo, N., Szpotowicz, E., Schachner, M., Jakovcevski, I., 2012. Improved regeneration after spinal cord injury in mice lacking functional T- and B-lymphocytes. Exp. Neurol. 237, 274–285. Wucherpfennig, K.W., Zhang, J., Witek, C., Matsui, M., Modabber, Y., Ota, K., Hafler, D.A., 1994. Clonal expansion and persistence of human T cells specific for an immunodominant myelin basic protein peptide. J. Immunol. 152, 5581–5592. Xin, J., Mesnard, N.A., Beahrs, T., Wainwright, D.A., Serpe, C.J., Alexander, T.D., Sanders, V. M., Jones, K.J., 2012. CD4+ T cell-mediated neuroprotection is independent of T cellderived BDNF in a mouse facial nerve axotomy model. Brain Behav. Immun. 26, 886–890. Yarom, Y., Naparstek, Y., Lev-Ram, V., Holoshitz, J., Ben Nun, A., Cohen, I.R., 1983. Immunospecific inhibition of nerve conduction by T lymphocytes reactive to basic protein of myelin. Nature 303, 246–247. Yogev, N., Frommer, F., Lukas, D., Kautz-Neu, K., Karram, K., Ielo, D., von Stebut, E., Probst, H.C., van den Broek, M., Riethmacher, D., Birnberg, T., Blank, T., Reizis, B., Korn, T., Wiendl, H., Jung, S., Prinz, M., Kurschus, F.C., Waisman, A., 2012. Dendritic cells ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1 receptor(+) regulatory T cells. Immunity 37, 264–275.

90


Yoles, E., Hauben, E., Palgi, O., Agranov, E., Gothilf, A., Cohen, A., Kuchroo, V., Cohen, I.R., Weiner, H., Schwartz, M., 2001. Protective autoimmunity is a physiological response to CNS trauma. J. Neurosci. 21, 3740–3748. Yong, V.W., 2002. Differential mechanisms of action of interferon-beta and glatiramer aetate in MS. Neurology 59, 802–808. Yong, J., Lacan, G., Dang, H., Hsieh, T., Middleton, B., Wasserfall, C., Tian, J., Melega, W.P., Kaufman, D.L., 2011. BCG vaccine-induced neuroprotection in a mouse model of Parkinson's disease. PLoS One 6, e16610. Yu, W.R., Fehlings, M.G., 2011. Fas/FasL-mediated apoptosis and inflammation are key features of acute human spinal cord injury: implications for translational, clinical application. Acta Neuropathol. 122, 747–761. Yu, P., Huang, L., Zou, J., Zhu, H., Wang, X., Yu, Z., Xu, X.M., Lu, P.H., 2007. DNA vaccine against NgR promotes functional recovery after spinal cord injury in adult rats. Brain Res. 1147, 66–76. Yu, C.Y., Ng, G., Liao, P., 2013a. Therapeutic antibodies in stroke. Transl. Stroke Res. 4, 477–483. Yu, Y.Z., Wang, S., Bai, J.Y., Zhao, M., Chen, A., Wang, W.B., Chang, Q., Liu, S., Qiu, W.Y., Pang, X.B., Xu, Q., Sun, Z.W., 2013b. Effective DNA epitope chimeric vaccines for

Alzheimer's disease using a toxin-derived carrier protein as a molecular adjuvant. Clin. Immunol. 149, 11–24. Yuseff, M.I., Pierobon, P., Reversat, A., Lennon-Dumenil, A.M., 2013. How B cells capture, process and present antigens: a crucial role for cell polarity. Nat. Rev. Immunol. 13, 475–486. Zajarias-Fainsod, D., Carrillo-Ruiz, J., Mestre, H., Grijalva, I., Madrazo, I., Ibarra, A., 2012. Autoreactivity against myelin basic protein in patients with chronic paraplegia. Eur. Spine J. 21, 964–970. Zhou, Z., Peng, X., Fink, D.J., Mata, M., 2009a. HSV-mediated transfer of artemin overcomes myelin inhibition to improve outcome after spinal cord injury. Mol. Ther. 17, 1173–1179. Zhou, Z., Peng, X., Insolera, R., Fink, D.J., Mata, M., 2009b. IL-10 promotes neuronal survival following spinal cord injury. Exp. Neurol. 220, 183–190. Zhou, Z., Peng, X., Insolera, R., Fink, D.J., Mata, M., 2009c. Interleukin-10 provides direct trophic support to neurons. J. Neurochem. 110, 1617–1627.

The paradox of chronic neuroinflammation, systemic immune suppression, autoimmunity after traumatic chronic spinal cord injury.

Serotonergic transmission after spinal cord injury.

Urethral hypotonicity after suprasacral spinal cord injury.

Percutaneous suprapubic cystostomy after spinal cord injury.

Prevention of thromboembolism after spinal cord injury.

Intrathecal Pressure After Spinal Cord Injury.

Urodynamic patterns after traumatic spinal cord injury.

Electrophysiologic changes in lumbar spinal cord after cervical cord injury.

Trigemino-cervical-spinal reflexes after traumatic spinal cord injury.

Dopamine is produced in the rat spinal cord and regulates micturition reflex after spinal cord injury.

Exercise awareness and barriers after spinal cord injury.

Race-ethnicity and poverty after spinal cord injury.

Neurological and functional recovery after thoracic spinal cord injury.

Depression after spinal cord injury and medication: The journey continues.

Motor unit numbers and contractile properties after spinal cord injury.

Intracranial hypertension after spinal cord injury and suboptimal cervical fusion.

Spinal cord injury: 10 and 15 years after.

Combinatorial strategy facilitates spinal cord plasticity and repair after injury.

Curcumin improves the integrity of blood-spinal cord barrier after compressive spinal cord injury in rats.

Review of Epidural Spinal Cord Stimulation for Augmenting Cough after Spinal Cord Injury.

Spinal cord transplants enhance the recovery of locomotor function after spinal cord injury at birth.

Spinal cord injury and pregnancy.

Adiposity and spinal cord injury.

Spinal cord injury rehabilitation.