Encephalitogenic and Regulatory CD8 T Cells in Multiple Sclerosis and Its Animal Models.

Multiple sclerosis (MS), a neuroinflammatory disease that affects millions worldwide, is widely thought to be autoimmune in etiology. Historically, research into MS pathogenesis has focused on autoreactive CD4 T cells because of their critical role in the animal model, experimental autoimmune encephalomyelitis, and the association between MS susceptibility and single-nucleotide polymorphisms in the MHC class II region. However, recent studies have revealed prominent clonal expansions of CD8 T cells within the CNS during MS. In this paper, we review the literature on CD8 T cells in MS, with an emphasis on their potential effector and regulatory properties. We discuss the impact of disease modifying therapies, currently prescribed to reduce MS relapse rates, on CD8 T cell frequency and function. A deeper understanding of the role of CD8 T cells in MS may lead to the development of more effective and selective immunomodulatory drugs for particular subsets of patients.

Serine Phosphorylation of the STAT1 Transactivation Domain Promotes Autoreactive B Cell and Systemic Autoimmunity Development.

Although STAT1 tyrosine-701 phosphorylation (designated STAT1-pY701) is indispensable for STAT1 function, the requirement for STAT1 serine-727 phosphorylation (designated STAT1-pS727) during systemic autoimmune and antipathogen responses remains unclear. Using autoimmune-prone B6.Sle1b mice expressing a STAT1-S727A mutant in which serine is replaced by alanine, we report in this study that STAT1-pS727 promotes autoimmune Ab-forming cell (AFC) and germinal center (GC) responses, driving autoantibody production and systemic lupus erythematosus (SLE) development. In contrast, STAT1-pS727 is not required for GC, T follicular helper cell (Tfh), and Ab responses to various foreign Ags, including pathogens. STAT1-pS727 is also not required for gut microbiota and dietary Ag-driven GC and Tfh responses in B6.Sle1b mice. By generating B cell-specific bone marrow chimeras, we demonstrate that STAT1-pS727 plays an important B cell-intrinsic role in promoting autoimmune AFC, GC, and Tfh responses, leading to SLE-associated autoantibody production. Our analysis of the TLR7-accelerated B6.Sle1b.Yaa SLE disease model expressing a STAT1-S727A mutant reveals STAT1-pS727-mediated regulation of autoimmune AFC and GC responses and lupus nephritis development. Together, we identify previously unrecognized differential regulation of systemic autoimmune and antipathogen responses by STAT1-pS727. Our data implicate STAT1-pS727 as a therapeutic target for SLE without overtly affecting STAT1-mediated protection against pathogenic infections.

CD8 T Cells and STAT1 Signaling Are Essential Codeterminants in Protection from Polyomavirus Encephalopathy.

JC polyomavirus (JCPyV), a human-specific virus, causes the aggressive brain-demyelinating disease progressive multifocal leukoencephalopathy (PML) in individuals with depressed immune status. The increasing incidence of PML in patients receiving immunotherapeutic and chemotherapeutic agents creates a pressing clinical need to define biomarkers to stratify PML risk and develop anti-JCPyV interventions. Mouse polyomavirus (MuPyV) CNS infection causes encephalopathology and may provide insight into JCPyV-PML pathogenesis. Type I, II, and III interferons (IFNs), which all signal via the STAT1 transcription factor, mediate innate and adaptive immune defense against a variety of viral infections. We previously reported that type I and II IFNs control MuPyV infection in non-central nervous system (CNS) organs, but their relative contributions to MuPyV control in the brain remain unknown. To this end, mice deficient in type I, II, or III IFN receptors or STAT1 were infected intracerebrally with MuPyV. We found that STAT1, but not type I, II, or III IFNs, mediated viral control during acute and persistent MuPyV encephalitis. Mice deficient in STAT1 also developed severe hydrocephalus, blood-brain barrier permeability, and increased brain infiltration by myeloid cells. CD8 T cell deficiency alone did not increase MuPyV infection and pathology in the brain. In the absence of STAT1 signaling, however, depletion of CD8 T cells resulted in lytic infection of the choroid plexus and ependymal lining, marked meningitis, and 100% mortality within 2 weeks postinfection. Collectively, these findings indicate that STAT1 signaling and CD8 T cells cocontribute to controlling MuPyV infection in the brain and CNS injury.IMPORTANCE A comprehensive understanding of JCPyV-induced PML pathogenesis is needed to define determinants that predispose patients to PML, a goal whose urgency is heightened by the lack of anti-JCPyV agents. A handicap to achieving this goal is the lack of a tractable animal model to study PML pathogenesis. Using intracerebral inoculation with MuPyV, we found that MuPyV encephalitis in wild-type mice causes an encephalopathy, which is markedly exacerbated in mice deficient in STAT1, a molecule involved in transducing signals from type I, II, and III IFN receptors. CD8 T cell deficiency compounded the severity of MuPyV neuropathology and resulted in dramatically elevated virus levels in the CNS. These findings demonstrate that STAT1 signaling and CD8 T cells concomitantly act to mitigate MuPyV-encephalopathy and control viral infection.

To Go or Stay: The Development, Benefit, and Detriment of Tissue-Resident Memory CD8 T Cells during Central Nervous System Viral Infections.

CD8 T cells coordinate immune defenses against viral infections of the central nervous system (CNS). Virus-specific CD8 T cells infiltrate the CNS and differentiate into brain-resident memory CD8 T cells (CD8 bTRM). CD8 bTRM are characterized by a lack of recirculation and expression of phenotypes and transcriptomes distinct from other CD8 T cell memory subsets. CD8 bTRM have been shown to provide durable, autonomous protection against viral reinfection and the resurgence of latent viral infections. CD8 T cells have also been implicated in the development of neural damage following viral infection, which demonstrates that the infiltration of CD8 T cells into the brain can also be pathogenic. In this review, we will explore the residency and maintenance requirements for CD8 bTRM and discuss their roles in controlling viral infections of the brain.

PD-1 Dynamically Regulates Inflammation and Development of Brain-Resident Memory CD8 T Cells During Persistent Viral Encephalitis.

Programmed cell death-1 (PD-1) receptor signaling dampens the functionality of T cells faced with repetitive antigenic stimulation from chronic infections or tumors. Using intracerebral (i.c.) inoculation with mouse polyomavirus (MuPyV), we have shown that CD8 T cells establish a PD-1hi, tissue-resident memory population in the brains (bTRM) of mice with a low-level persistent infection. In MuPyV encephalitis, PD-L1 was expressed on infiltrating myeloid cells, microglia and astrocytes, but not on oligodendrocytes. Engagement of PD-1 on anti-MuPyV CD8 T cells limited their effector activity. NanoString gene expression analysis showed that neuroinflammation was higher in PD-L1-/- than wild type mice at day 8 post-infection, the peak of the MuPyV-specific CD8 response. During the persistent phase of infection, however, the absence of PD-1 signaling was found to be associated with a lower inflammatory response than in wild type mice. Genetic disruption and intracerebroventricular blockade of PD-1 signaling resulted in an increase in number of MuPyV-specific CD8 bTRM and the fraction of these cells expressing CD103, the αE integrin commonly used to define tissue-resident T cells. However, PD-L1-/- mice persistently infected with MuPyV showed impaired virus control upon i.c. re-infection with MuPyV. Collectively, these data reveal a temporal duality in PD-1-mediated regulation of MuPyV-associated neuroinflammation. PD-1 signaling limited the severity of neuroinflammation during acute infection but sustained a level of inflammation during persistent infection for maintaining control of virus re-infection.

CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection.

Tissue-resident memory CD8 T (TRM) cells defend against microbial reinfections at mucosal barriers; determinants driving durable TRM cell responses in non-mucosal tissues, which often harbor opportunistic persistent pathogens, are unknown. JC polyomavirus (JCPyV) is a ubiquitous constituent of the human virome. With altered immunological status, JCPyV can cause the oft-fatal brain demyelinating disease progressive multifocal leukoencephalopathy (PML). JCPyV is a human-only pathogen. Using the mouse polyomavirus (MuPyV) encephalitis model, we demonstrate that CD4 T cells regulate development of functional antiviral brain-resident CD8 T cells (bTRM) and renders their maintenance refractory to systemic CD8 T cell depletion. Acquired CD4 T cell deficiency, modeled by delaying systemic CD4 T cell depletion until MuPyV-specific CD8 T cells have infiltrated the brain, impacted the stability of CD8 bTRM, impaired their effector response to reinfection, and rendered their maintenance dependent on circulating CD8 T cells. This dependence of CD8 bTRM differentiation on CD4 T cells was found to extend to encephalitis caused by vesicular stomatitis virus. Together, these findings reveal an intimate association between CD4 T cells and homeostasis of functional bTRM to CNS viral infection.

Maintenance of PD-1 on brain-resident memory CD8 T cells is antigen independent.

Infection of the central nervous system (CNS) by murine polyomavirus (MuPyV), a persistent natural mouse pathogen, establishes brain-resident memory CD8 T cells (bTRM) that uniformly and chronically express programmed cell death protein 1 (PD-1) irrespective of the expression of αE integrin CD103, a TRM cell marker. In contrast, memory antiviral CD8 T cells in the spleen are PD-1-, despite viral loads being similar in both the brain and spleen during persistent infection. Repetitive antigen engagement is central to sustained PD-1 expression by T cells in chronic viral infections; however, recent evidence indicates that expression of inhibitory receptors, including PD-1, is part of the TRM differentiation program. Here we asked whether PD-1 expression by CD8 bTRM cells during persistent MuPyV encephalitis is antigen dependent. By transferring MuPyV-specific CD8 bTRM cells into the brains of naive mice and mice infected with cognate epitope-sufficient and -deficient MuPyVs, we demonstrate that antigen and inflammation are dispensable for PD-1 maintenance. In vitro and direct ex vivo analyses indicate that CD103- MuPyV-specific CD8 bTRM retain functional competence. We further show that the Pdcd-1 promoter of anti-MuPyV bTRM cells is epigenetically fixed in a demethylated state in the brain. In contrast, the PD-1 promoter of splenic antiviral memory CD8 T cells undergoes remethylation after being demethylated during acute infection. These data show that PD-1 expression is an intrinsic property of brain TRM cells in a persistent CNS viral infection.

LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein.

BACKGROUND: Human Immunodeficiency Virus-1 (HIV-1) associated neurocognitive disorders (HANDs) are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART). While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2) as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. METHODS: We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat) protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i). We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. RESULTS: We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence of Tat-activated microglia, as well as AnnexinV, a phosphatidylserine-binding protein. In addition, LRRK2i decreased brain-specific angiogenesis inhibitor 1 (BAI1) receptor expression on BV-2 cells after Tat-treatment, a key receptor in phosphatidylserine-mediated phagocytosis. CONCLUSION: Taken together, these results implicate LRRK2 as a key player in microglial inflammation and, in particular, in the phagocytosis of neuronal elements. These studies show that LRRK2 kinase inhibition may prove an effective therapeutic strategy for HANDs, as well as other neuroinflammatory conditions.