Timothy W. Phares

CD4 T Cells Promote CD8 T Cell Immunity at the Priming and Effector Site during Viral Encephalitis

ABSTRACT CD4 T cell activation during peripheral infections not only is essential in inducing protective CD8 T cell memory but also promotes CD8 T cell function and survival. However, the contributions of CD4 T cell help to antiviral CD8 T cell immunity during central nervous system (CNS) infection are not well established. Encephalitis induced by the sublethal coronavirus JHMV was used to identify when CD4 T cells regulate CD8 T cell responses following CNS infection. Peripheral expansion of virus-specific CD8 T cells was impaired when CD4 T cells were ablated prior to infection but not at 4 days postinfection. Delayed CD4 T cell depletion abrogated CD4 T cell recruitment to the CNS but only slightly diminished CD8 T cell recruitment. Nevertheless, the absence of CNS CD4 T cells was associated with reduced gamma interferon (IFN-γ) and granzyme B expression by infiltrating CD8 T cells, increased CD8 T cell apoptosis, and impaired control of infectious virus. CD4 T cell depletion subsequent to CD4 T cell CNS migration restored CD8 T cell activity and virus control. Analysis of γc-dependent cytokine expression indicated interleukin-21 (IL-21) as a primary candidate optimizing CD8 T cell activity within the CNS. These results demonstrate that CD4 T cells play critical roles in both enhancing peripheral activation of CD8 T cells and prolonging their antiviral function within the CNS. The data highlight the necessity for temporally and spatially distinct CD4 T cell helper functions in sustaining CD8 T cell activity during CNS infection.

Target-Dependent B7-H1 Regulation Contributes to Clearance of Central Nervous Sysyem Infection and Dampens Morbidity

The neurotropic coronavirus JHM strain of mouse hepatitis virus persists in oligodendroglia despite the presence of virus-specific CD8 T cells. Expression of programmed death 1 (PD-1) and B7-H1 were studied during acute and persistent infection to examine whether this negative regulatory mechanism contributes to CNS viral persistence. The majority of CNS-infiltrating CD8 T cells expressed PD-1, with the highest levels on virus-specific CD8 T cells. Moreover, despite control of infectious virus, CD8 T cells within the CNS of persistently infected mice maintained high PD-1 expression. Analysis of virus-susceptible target cells in vivo revealed that B7-H1 expression was regulated in a cell type-dependent manner. Oligodendroglia and microglia up-regulated B7-H1 following infection; however, although B7-H1 expression on oligodendroglia was prominent and sustained, it was significantly reduced and transient on microglia. Infection of mice deficient in the IFN-γ or IFN-α/β receptor demonstrated that B7-H1 expression on oligodendroglia is predominantly regulated by IFN-γ. Ab blockade of B7-H1 on oligodendroglia in vitro enhanced IFN-γ secretion by virus-specific CD8 T cells. More efficient virus control within the CNS of B7-H1-deficient mice confirmed inhibition of CD8 T cell function in vivo. Nevertheless, the absence of B7-H1 significantly increased morbidity without altering demyelination. These data are the first to demonstrate glia cell type-dependent B7-H1 regulation in vivo, resulting in adverse effects on antiviral CD8 T cell function. However, the beneficial role of PD-1:B7-H1 interactions in limiting morbidity highlights the need to evaluate tissue-specific intervention strategies.

CXCR3-Dependent Plasma Blast Migration to the Central Nervous System during Viral Encephalomyelitis

ABSTRACT Immunoglobulin in cerebral spinal fluid and antibody secreting cells (ASC) within the central nervous system (CNS) parenchyma are common hallmarks of microbial infections and autoimmune disorders. However, the signals directing ASC migration into the inflamed CNS are poorly characterized. This study demonstrates that CXCR3 mediates CNS accumulation of ASC during neurotropic coronavirus-induced encephalomyelitis. Expansion of CXCR3-expressing ASC in draining lymph nodes prior to accumulation within the CNS was consistent with their recruitment by sustained expression of CXCR3 ligands during viral persistence. Both total and virus-specific ASC were reduced greater than 80% in the CNS of infected CXCR3−/− mice. Similar T cell CNS recruitment and local T cell-dependent antiviral activity further indicated that the ASC migration defect was T cell independent. Furthermore, in contrast to the reduction of ASC in the CNS, neither virus-specific ASC trafficking to bone marrow nor antiviral serum antibody was reduced relative to levels in control mice. Impaired ASC recruitment into the CNS of infected CXCR3−/− mice coincided with elevated levels of persisting viral RNA, sustained infectious virus, increased clinical disease, and mortality. These results demonstrate that CXCR3 ligands are indispensable for recruitment of activated ASC into the inflamed CNS and highlight their local protective role during persistent infection.

IFN-γ Signaling to Astrocytes Protects from Autoimmune Mediated Neurological Disability

Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS). Cells resident within the central nervous system (CNS) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB) integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.

Factors Supporting Intrathecal Humoral Responses following Viral Encephalomyelitis

ABSTRACT Central nervous system (CNS) infections and autoimmune inflammatory disorders are often associated with retention of antibody-secreting cells (ASC). Although beneficial or detrimental contributions of ASC to CNS diseases remain to be defined, virus-specific ASC are crucial in controlling persistent CNS infection following coronavirus-induced encephalomyelitis. This report characterizes expression kinetics of factors associated with ASC homing, differentiation, and survival in the spinal cord, the prominent site of coronavirus persistence. Infection induced a vast, gamma interferon (IFN-γ)-dependent, prolonged increase in chemokine (C-X-C motif) ligand 9 (CXCL9), CXCL10, and CXCL11 mRNA, supporting a role for chemokine (C-X-C motif) receptor 3 (CXCR3)-mediated ASC recruitment. Similarly, CD4 T cell-secreted interleukin-21, a critical regulator of both peripheral activated B cells and CD8 T cells, was sustained during viral persistence. The ASC survival factors B cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferating-inducing ligand (APRIL) were also significantly elevated in the infected CNS, albeit delayed relative to the chemokines. Unlike IFN-γ-dependent BAFF upregulation, APRIL induction was IFN-γ independent. Moreover, both APRIL and BAFF were predominantly localized to astrocytes. Last, the expression kinetics of the APRIL and BAFF receptors coincided with CNS accumulation of ASC. Therefore, the factors associated with ASC migration, differentiation, and survival are all induced during acute viral encephalomyelitis, prior to ASC accumulation in the CNS. Importantly, the CNS expression kinetics implicate rapid establishment, and subsequent maintenance, of an environment capable of supporting differentiation and survival of protective antiviral ASC, recruited as plasmablasts from lymphoid organs.

Enhanced Antiviral T Cell Function in the Absence of B7-H1 Is Insufficient To Prevent Persistence but Exacerbates Axonal Bystander Damage during Viral Encephalomyelitis

The T cell inhibitory ligand B7-H1 hinders T cell-mediated virus control, but also ameliorates clinical disease during autoimmune and virus-induced CNS disease. In mice infected with gliatropic demyelinating coronavirus, B7-H1 expression on oligodendroglia delays virus control, but also dampens clinical disease. To define the mechanisms by which B7-H1 alters pathogenic outcome, virus-infected B7-H1–deficient (B7-H1−/−) mice were analyzed for altered peripheral and CNS immune responses. B7-H1 deficiency did not affect peripheral T or B cell activation or alter the magnitude or composition of CNS-infiltrating cells. However, higher levels of IFN-γ mRNA in CNS-infiltrating virus-specific CD8 T cells as well as CD4 T cells contributed to elevated IFN-γ protein in the B7-H1−/− CNS. Increased effector function at the single-cell level was also evident by elevated granzyme B expression specifically in virus-specific CNS CD8 T cells. Although enhanced T cell activity accelerated virus control, 50% of mice succumbed to infection. Despite enhanced clinical recovery, surviving B7-H1−/− mice still harbored persisting viral mRNA, albeit at reduced levels compared with wild-type mice. B7-H1−/− mice exhibited extensive loss of axonal integrity, although demyelination, a hallmark of virus-induced tissue damage, was not increased. The results suggest that B7-H1 hinders viral control in B7-H1 expressing glia cells, but does not mediate resistance to CD8 T cell-mediated cytolysis. These data are the first, to our knowledge, to demonstrate that B7-H1–mediated protection from viral-induced immune pathology associated with encephalomyelitis resides in limiting T cell-mediated axonal bystander damage rather than direct elimination of infected myelinating cells.

Astrocyte-Derived CXCL10 Drives Accumulation of Antibody-Secreting Cells in the Central Nervous System during Viral Encephalomyelitis

ABSTRACT Microbial infections of the central nervous system (CNS) are often associated with local accumulation of antibody (Ab)-secreting cells (ASC). By providing a source of Ab at the site of infection, CNS-localized ASC play a critical role in acute viral control and in preventing viral recrudescence. Following coronavirus-induced encephalomyelitis, the CNS accumulation of ASC is chemokine (C-X-C motif) receptor 3 (CXCR3) dependent. This study demonstrates that CNS-expressed CXCR3 ligand CXCL10 is the critical chemokine regulating ASC accumulation. Impaired ASC recruitment in CXCL10−/− but not CXCL9−/− mice was consistent with reduced CNS IgG and κ-light chain mRNA and virus-specific Ab. Moreover, the few ASC recruited to the CNS in CXCL10−/− mice were confined to the vasculature, distinct from the parenchymal localization in wild-type and CXCL9−/− mice. However, neither CXCL9 nor CXCL10 deficiency diminished neutralizing serum Ab, supporting a direct role for CXCL10 in ASC migration. T cell accumulation, localization, and effector functions were also not affected in either CXCL9−/− or CXCL10−/− mice, consistent with similar control of infectious virus. There was also no evidence for dysregulation of chemokines or cytokines involved in ASC regulation. The distinct roles of CXCL9 and CXCL10 in ASC accumulation rather coincided with their differential localization. While CXCL10 was predominantly expressed by astrocytes, CXCL9 expression was confined to the vasculature/perivascular spaces. These results suggest that CXCL10 is critical for two phases: recruitment of ASC to the CNS vasculature and ASC entry into the CNS parenchyma.

Shifting Hierarchies of Interleukin-10-Producing T Cell Populations in the Central Nervous System during Acute and Persistent Viral Encephalomyelitis

ABSTRACT Interleukin-10 (IL-10) mRNA is rapidly upregulated in the central nervous system (CNS) following infection with neurotropic coronavirus and remains elevated during persistent infection. Infection of transgenic IL-10/green fluorescent protein (GFP) reporter mice revealed that CNS-infiltrating T cells were the major source of IL-10, with minimal IL-10 production by macrophages and resident microglia. The proportions of IL-10-producing cells were initially similar in CD8+ and CD4+ T cells but diminished rapidly in CD8+ T cells as the virus was controlled. Overall, the majority of IL-10-producing CD8+ T cells were specific for the immunodominant major histocompatibility complex (MHC) class I epitope. Unlike CD8+ T cells, a large proportion of CD4+ T cells within the CNS retained IL-10 production throughout persistence. Furthermore, elevated frequencies of IL-10-producing CD4+ T cells in the spinal cord supported preferential maintenance of IL-10 production at the site of viral persistence and tissue damage. IL-10 was produced primarily by the CD25+ CD4+ T cell subset during acute infection but prevailed in CD25− CD4+ T cells during the transition to persistent infection and thereafter. Overall, these data demonstrate significant fluidity in the T-cell-mediated IL-10 response during viral encephalitis and persistence. While IL-10 production by CD8+ T cells was limited primarily to the time of acute effector function, CD4+ T cells continued to produce IL-10 throughout infection. Moreover, a shift from predominant IL-10 production by CD25+ CD4+ T cells to CD25− CD4+ T cells suggests that a transition to nonclassical regulatory T cells precedes and is retained during CNS viral persistence.

The Spleen Is the Major Source of Antidonor Antibody-Secreting Cells in Murine Heart Allograft Recipients

Antibody‐mediated allograft rejection is an increasingly recognized problem in clinical transplantation. However, the primary location of donor‐specific alloantibody (DSA)‐producing cells after transplantation have not been identified. The purpose of this study was to test the contribution of allospecific antibody‐secreting cells (ASCs) from different anatomical compartments in a mouse transplantation model. Fully MHC‐mismatched heart allografts were transplanted into three groups of recipients: nonsensitized wild type, alloantigen‐sensitized wild‐type and CCR5−/− mice that have exaggerated alloantibody responses. We found that previous sensitization to donor alloantigens resulted in the development of antidonor alloantibody (alloAb) with accelerated kinetics. Nevertheless, the numbers of alloantibody‐secreting cells and the serum titers of antidonor IgG alloantibody were equivalent in sensitized and nonsensitized recipients 6 weeks after transplantation. Regardless of recipient sensitization status, the spleen contained higher numbers of donor‐reactive ASCs than bone marrow at days 7–21 after transplantation. Furthermore, individual spleen ASCs produced more antidonor IgG alloantibody than bone marrow ASCs. Taken together, our results indicate that the spleen rather than bone marrow is the major source of donor‐reactive alloAb early after transplantation in both sensitized and nonsensitized recipients.

Stability and function of adult vasculature is sustained by Akt/Jagged1 signalling axis in endothelium

The signalling pathways operational in quiescent, post-development vasculature remain enigmatic. Here we show that unlike neovascularization, endothelial Akt signalling in established vasculature is crucial not for endothelial cell (EC) survival, but for sustained interactions with pericytes and vascular smooth muscle cells (VSMCs) regulating vascular stability and function. Inducible endothelial-specific Akt1 deletion in adult global Akt2KO mice triggers progressive VSMC apoptosis. In hearts, this causes a loss of arteries and arterioles and, despite a high capillary density, diminished vascular patency and severe cardiac dysfunction. Similarly, endothelial Akt deletion induces retinal VSMC loss and basement membrane deterioration resulting in vascular regression and retinal atrophy. Mechanistically, the Akt/mTOR axis controls endothelial Jagged1 expression and, thereby, Notch signalling regulating VSMC maintenance. Jagged1 peptide treatment of Akt1ΔEC;Akt2KO mice and Jagged1 re-expression in Akt-deficient endothelium restores VSMC coverage. Thus, sustained endothelial Akt1/2 signalling is critical in maintaining vascular stability and homeostasis, thereby preserving tissue and organ function.