Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR alpha/beta-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-gamma enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.     

Role of viral persistence in retaining CD8(+) T cells within the central nervous system

The continued presence of virus-specific CD8(+) T cells within the central nervous system (CNS) following resolution of acute viral encephalomyelitis implicates organ-specific retention. The role of viral persistence in locally maintaining T cells was investigated by infecting mice with either a demyelinating, paralytic (V-1) or nonpathogenic (V-2) variant of a neurotropic mouse hepatitis virus, which differ in the ability to persist within the CNS. Class I tetramer technology revealed more infiltrating virus-specific CD8(+) T cells during acute V-1 compared to V-2 infection. However, both total and virus-specific CD8(+) T cells accumulated at similar peak levels in spinal cords by day 10 postinfection (p.i.). Decreasing viral RNA levels in both brains and spinal cords following initial virus clearance coincided with an overall progressive loss of both total and virus-specific CD8(+) T cells. By 9 weeks p.i., T cells had largely disappeared from brains of both infected groups, consistent with the decline of viral RNA. T cells also completely disappeared from V-2-infected spinal cords coincident with the absence of viral RNA. By contrast, a significant number of CD8(+) T cells which contained detectable viral RNA were recovered from spinal cords of V-1-infected mice. The data indicate that residual virus from a primary CNS infection is a vital component in mediating local retention of both CD8(+) and CD4(+) T cells and that once minimal thresholds of stimuli are lost, T cells within the CNS cannot survive in an autonomous fashion.     

Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) clear infectious virus; nevertheless, virus persists in the CNS as noninfectious RNA, resulting in ongoing primary demyelination. Phenotypic and functional analysis of CNS infiltrating cells during acute infection revealed a potent regional CD8+ T cell response comprising up to 50% virus-specific T cells. The high prevalence of virus-specific T cells correlated with ex vivo cytolytic activity and efficient reduction in viral titers. Progressive viral clearance coincided with the loss of cytolytic activity, but retention of IFN-gamma secretion and increased expression of the early activation marker CD69, indicating differential regulation of effector function. Although the total number of infiltrating T cells declined following clearance of infectious virus, CD8+ T cells, both specific for the dominant viral epitopes and of unknown specificity, were retained within the CNS, suggesting an ongoing T cell response during persistent CNS infection involving a virus-independent component. Reversed immunodominance within the virus-specific CD8+ T cell population further indicated epitope-specific regulation, supporting ongoing T cell activation. Even in the absence of infectious virus, the CNS thus provides an environment that maintains both unspecific and Ag-specific CD8+ T cells with restricted effector function. Chronic T cell stimulation may thus play a role in preventing viral recrudescence, while increasing the risk of pathological conditions, such as demyelination.     

CD8+T细胞在多发性硬化中的致病性作用

多发性硬化是T细胞介导的自身免疫性疾病。先前对它的研究大多集中在CD4 T细胞的致病和调节性作用上,但是,近几年来越来越多的证据表明CD8 T细胞也参与多发性硬化的病理损伤过程。 CD8 T细胞存在于MS病灶部位,髓鞘抗原特异性CD8 T细胞也从MS患者的血液和脑脊液中分离得到,CD8 T细胞通过直接杀伤或释放细胞因子和趋化因子等间接参与MS的病理过程。本文就近几年关于CD8 T细胞在多发性硬化中的致病性作用的研究进展予以介绍。     

A transgenic model of central nervous system autoimmunity mediated by CD4+ and CD8+ T and B cells

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the α- and β-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.     

Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells.

Both CD4+ and CD8+ T cells are required for the clearance of virus from the central nervous system following infection with the JHM strain of mouse hepatitis virus. Development of antiviral antibodies requires the presence of CD4+ T cells but appears to play a minimal role in the reduction of virus. The data presented are consistent with the hypothesis that clearance of JHM virus is mediated by virus-specific CD8+ T cells, which appear to require the presence of CD4+ T cells.     

Differential regulation of primary and secondary CD8+ T cells in the central nervous system

T cell accumulation and effector function following CNS infection is limited by a paucity of Ag presentation and inhibitory factors characteristic of the CNS environment. Differential susceptibilities of primary and recall CD8+ T cell responses to the inhibitory CNS environment were monitored in naive and CD8+ T cell-immune mice challenged with a neurotropic coronavirus. Accelerated virus clearance and limited spread in immunized mice was associated with a rapid and increased CNS influx of virus-specific secondary CD8+ T cells. CNS-derived secondary CD8+ T cells exhibited increased cytolytic activity and IFN-gamma expression per cell compared with primary CD8+ T cells. However, both Ag-specific primary and secondary CD8+ T cells demonstrated similar contraction rates. Thus, CNS persistence of increased numbers of secondary CD8+ T cells reflected differences in the initial pool size during peak inflammation rather than enhanced survival. Unlike primary CD8+ T cells, persisting secondary CD8+ T cells retained ex vivo cytolytic activity and expressed high levels of IFN-gamma following Ag stimulation. However, both primary and secondary CD8+ T cells exhibited reduced capacity to produce TNF-alpha, differentiating them from effector memory T cells. Activation of primary and secondary CD8+ T cells in the same host using adoptive transfers confirmed similar survival, but enhanced and prolonged effector function of secondary CD8+ T cells in the CNS. These data suggest that an instructional program intrinsic to T cell differentiation, rather than Ag load or factors in the inflamed CNS, prominently regulate CD8+ T cell function.     

Vaccine-induced memory CD8+ T cells cannot prevent central nervous system virus reactivation

Noncytopathic viruses use multiple strategies to evade immune detection, challenging a role for vaccine induced CTL in preventing microbial persistence. Recrudescence of neurotropic coronavirus due to loss of T cell-mediated immune control provided an experimental model to test T cell vaccination efficacy in the absence of Ab. Challenge virus was rapidly controlled in vaccinated Ab-deficient mice coincident with accelerated recruitment of memory CD8+ T cells and enhanced effector function compared with primary CD8+ T cell responses. In contrast to primary effectors, reactivated memory cells persisted in the CNS at higher frequencies and retained ex vivo cytolytic activity. Nevertheless, despite earlier and prolonged T cell-mediated control in the CNS of vaccinated mice, virus ultimately reactivated. Apparent loss of memory CD8+ effector function in vivo was supported by a prominent decline in MHC expression on CNS resident target cells, presumably reflecting diminished IFN-gamma. Severely reduced MHC expression on glial cells at the time of recrudescence suggested that memory T cells, although fully armed to exert antiviral activity upon Ag recognition in vitro, are not responsive in an environment presenting few if any target MHC molecules. Paradoxically, effective clearance of viral Ag thus affords persisting virus a window of opportunity to escape from immune surveillance. These studies demonstrate that vaccine-induced T cell memory alone is unable to control persisting virus in a tissue with strict IFN-dependent MHC regulation, as evident in immune privileged sites.     

Bystander CD8 T cell-mediated demyelination after viral infection of the central nervous system

Multiple sclerosis, a chronic inflammatory disease of the CNS, is characterized by immune-mediated demyelination. Many patients have a remitting-relapsing course of disease with exacerbations often following unrelated microbial illnesses. The relationship between the two events remains obscure. One possibility is that T cells specific for the inciting microbial pathogen are able to effect demyelination at a site of ongoing inflammation within the CNS. This possibility was examined in mice infected with mouse hepatitis virus, a well-described model of virus-induced demyelination. Using transgenic TCR/recombination activation gene 2(-/-) mice with only non-mouse hepatitis virus-specific T cells, we show that CD8 T cells are able to cause demyelination in the absence of cognate Ag in the CNS, but only if specifically activated. These findings demonstrate a novel mechanism for immune-mediated neuropathology and show that activated CD8 T cells may serve as important mediators of bystander demyelination during times of infection, including in patients with multiple sclerosis.     

CD8+ T cells primed in the periphery provide time-bound immune-surveillance to the central nervous system

After vaccination, memory CD8(+) T cells migrate to different organs to mediate immune surveillance. In most nonlymphoid organs, following an infection, CD8(+) T cells differentiate to become long-lived effector-memory cells, thereby providing long-term protection against a secondary infection. In this study, we demonstrated that Ag-specific CD8(+) T cells that migrate to the mouse brain following a systemic Listeria infection do not display markers reminiscent of long-term memory cells. In contrast to spleen and other nonlymphoid organs, none of the CD8(+) T cells in the brain reverted to a memory phenotype, and all of the cells were gradually eliminated. These nonmemory phenotype CD8(+) T cells were found primarily within the choroid plexus, as well as in the cerebrospinal fluid-filled spaces. Entry of these CD8(+) T cells into the brain was governed primarily by CD49d/VCAM-1, with the majority of entry occurring in the first week postinfection. When CD8(+) T cells were injected directly into the brain parenchyma, cells that remained in the brain retained a highly activated (CD69(hi)) phenotype and were gradually lost, whereas those that migrated out to the spleen were CD69(low) and persisted long-term. These results revealed a mechanism of time-bound immune surveillance to the brain by CD8(+) T cells that do not reside in the parenchyma.     

The induction of arthritis in mice by the cartilage proteoglycan aggrecan: roles of CD4+ and CD8+ T cells.

Peripheral arthritis is produced in BALB/c mice after hyperimmunization with the cartilage proteoglycan aggrecan (PG). Adoptive transfer studies have suggested the roles of T cells including CD8+ T cells in the disease process. To evaluate the roles of CD4+ and CD8+ T cell subsets in vivo in the induction of this disease by immunization, PG-immunized mice were treated with isotype-controlled rat IgG2b monoclonal anti-CD4 or anti-CD8 antibodies, or were left untreated. CD4+ T cell depletion resulted in total inhibition of the disease with markedly decreased anti-PG antibody responses. CD8+ T cell depletion, however, significantly enhanced the severity of the disease without affecting peak anti-PG antibodies, as compared to the control mice. These results demonstrate a crucial role for CD4+ T cells in the pathogenesis of this disease. However, CD8+ T cells do not seem to be required for the induction of arthritis by immunization but instead may play an immunoregulatory role.     

Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells

Infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus produces acute and chronic demyelination. The contributions of perforin-mediated cytolysis and gamma interferon (IFN-gamma) secretion by CD8(+) T cells to the control of infection and the induction of demyelination were examined by adoptive transfer into infected SCID recipients. Untreated SCID mice exhibited uncontrolled virus replication in all CNS cell types but had little or no demyelination. Memory CD8(+) T cells from syngeneic wild-type (wt), perforin-deficient, or IFN-gamma-deficient (GKO) donors all trafficked into the infected CNS in the absence of CD4(+) T cells and localized to similar areas. Although CD8(+) T cells from all three donors suppressed virus replication in the CNS, GKO CD8(+) T cells expressed the least antiviral activity. A distinct viral antigen distribution in specific CNS cell types revealed different mechanisms of viral control. While wt CD8(+) T cells inhibited virus replication in all CNS cell types, cytolytic activity in the absence of IFN-gamma suppressed the infection of astrocytes, but not oligodendroglia. In contrast, cells that secreted IFN-gamma but lacked cytolytic activity inhibited replication in oligodendroglia, but not astrocytes. Demyelination was most severe following viral control by wt CD8(+) T cells but was independent of macrophage infiltration. These data demonstrate the effective control of virus replication by CD8(+) T cells in the absence of CD4(+) T cells and support the necessity for the expression of distinct effector mechanisms in the control of viral replication in distinct CNS glial cell types.     

Inducible gene expression with the Tet-on system in CD4+ T cells and thymocytes of mice

CD4+ T cells with their growing list of effector and regulatory subpopulations have vital functions within the immunohematopoietic system. We report here on the first mouse lines that allow temporally and quantitatively controlled expression of transgenes specifically in CD4+ thymocytes and T cells. These were constructed using the Tet-on system. The rtTA2(S)-M2 version of the reverse tetracycline-dependent transactivator was placed under control of all known CD4 regulatory elements. Reporter transgene expression in mice expressing these constructs is highly specific for CD4+ cells, is strictly dependent on the tetracycline derivative doxycycline, and can be regulated by up to five logs depending on the doxycycline concentration. Moreover, we demonstrate that these mice can be used for noninvasive in vivo imaging of a coexpressed luciferase reporter. These new mouse lines should be highly valuable for studying and manipulating numerous aspects of CD4+ T cell development, biology, and function.     

Contributions of CD8+ T cells and viral spread to demyelinating disease

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.     

Gene expression in antigen-specific CD8+ T cells during viral infection

Following infection with intracellular pathogens, Ag-specific CD8(+) T cells become activated and begin to proliferate. As these cells become activated, they elaborate effector functions including cytokine production and cytolysis. After the infection has been cleared, the immune system returns to homeostasis through apoptosis of the majority of the Ag-specific effector cells. The surviving memory cells can persist for extended periods and provide protection against reinfection. Little is known about the changes in gene expression as Ag-specific cells progress through these stages of development, i.e., naive to effector to memory. Using recombinant MHC class I tetramers, we isolated Ag-specific CD8(+) T cells from mice infected with lymphocytic choriomeningitis virus at various time points and performed semiquantitative RT-PCR. We examined expression of: 1) genes involved in cell cycle control, 2) effector and regulatory functions, and 3) susceptibility to apoptosis. We found that Ag-specific CD8(+) memory T cells contain high steady-state levels of Bcl-2, BAX:, IFN-gamma, and lung Kruppel-like factor (LKLF), and decreased levels of p21 and p27 mRNA. Moreover, the pattern of gene expression between naive and memory cells is distinct and suggests that these two cell types control susceptibility to apoptosis through different mechanisms.     

Functional expression of chemokine receptor CCR5 on CD4(+) T cells during virus-induced central nervous system disease

Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). CD4(+) T cells are important in amplifying demyelination by attracting macrophages into the central nervous system (CNS) following viral infection; however, the mechanisms governing the entry of these cells into the CNS are poorly understood. The role of chemokine receptor CCR5 in trafficking of virus-specific CD4(+) T cells into the CNS of MHV-infected mice was investigated. CD4(+) T cells from immunized CCR5(+/+) and CCR5(-/-) mice were expanded in the presence of the immunodominant epitope present in the MHV transmembrane (M) protein encompassing amino acids 133 to 147 (M133-147). Adoptive transfer of CCR5(+/+)-derived CD4(+) T cells to MHV-infected RAG1(-/-) mice resulted in CD4(+)-T-cell entry into the CNS and clearance of virus from the brain. These mice also displayed robust demyelination correlating with macrophage accumulation within the CNS. Conversely, CD4(+) T cells from CCR5(-/-) mice displayed an impaired ability to traffic into the CNS of MHV-infected RAG1(-/-) recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. Analysis of chemokine receptor mRNA expression by M133-147-expanded CCR5(-/-)-derived CD4(+) T cells revealed reduced expression of CCR1, CCR2, and CXCR3, indicating that CCR5 signaling is important in increased expression of these receptors, which aid in trafficking of CD4(+) T cells into the CNS. Collectively these results demonstrate that CCR5 signaling is important to migration of CD4(+) T cells to the CNS following MHV infection.     

Human CD4+ T cells displaying viral epitopes elicit a functional virus-specific memory CD8+ T cell response

The Ag-specific cellular recall response to herpes virus infections is characterized by a swift recruitment of virus-specific memory T cells. Rapid activation is achieved through formation of the immunological synapse and supramolecular clustering of signal molecules at the site of contact. During the formation of the immunological synapse, epitope-loaded MHC molecules are transferred via trogocytosis from APCs to T cells, enabling the latter to function as Ag-presenting T cells (T-APCs). The contribution of viral epitope expressing T-APCs in the regulation of the herpes virus-specific CD8+ T cell memory response remains unclear. Comparison of CD4+ T-APCs with professional APCs such as Ag-presenting CD40L-activated B cells (CD40B-APCs) demonstrated reduced levels of costimulatory ligands. Despite the observed differences, CD4+ T-APCs are as potent as CD40B-APCs in stimulating herpes virus-specific CD8+ T cells resulting in a greater than 35-fold expansion of CD8+ T cells specific for dominant and subdominant viral epitopes. Virus-specific CD8+ T cells generated by CD4+ T-APCs or CD40B-APCs showed both comparable effector function such as specific lysis of targets and cytokine production and also did not differ in their phenotype after expansion. These results indicate that viral epitope presentation by Ag-specific CD4+ T cells may contribute to the rapid recruitment of virus-specific memory CD8+ T cells during a viral recall response.     

Marek's disease viral interleukin-8 promotes lymphoma formation through targeted recruitment of B cells and CD4+ CD25+ T cells

Marek''s disease virus (MDV) is a cell-associated and highly oncogenic alphaherpesvirus that infects chickens. During lytic and latent MDV infection, a CXC chemokine termed viral interleukin-8 (vIL-8) is expressed. Deletion of the entire vIL-8 open reading frame (ORF) was shown to severely impair disease progression and tumor development; however, it was unclear whether this phenotype was due to loss of secreted vIL-8 or of splice variants that fuse exons II and III of vIL-8 to certain upstream open reading frames, including the viral oncoprotein Meq. To specifically examine the role of secreted vIL-8 in MDV pathogenesis, we constructed a recombinant virus, vΔMetvIL-8, in which we deleted the native start codon from the signal peptide encoding exon I. This mutant lacked secreted vIL-8 but did not affect Meq–vIL-8 splice variants. Loss of secreted vIL-8 resulted in highly reduced disease and tumor incidence in animals infected with vΔMetvIL-8 by the intra-abdominal route. Although vΔMetvIL-8 was still able to spread to naïve animals by the natural route, infection and lymphomagenesis in contact animals were severely impaired. In vitro assays showed that purified recombinant vIL-8 efficiently binds to and induces chemotaxis of B cells, which are the main target for lytic MDV replication, and also interacts with CD4⁺ CD25⁺ T cells, known targets of MDV transformation. Our data provide evidence that vIL-8 attracts B and CD4⁺ CD25⁺ T cells to recruit targets for both lytic and latent infection.     

Therapeutic induction of regulatory, cytotoxic CD8+ T cells in multiple sclerosis

In the setting of autoimmunity, one of the goals of successful therapeutic immune modulation is the induction of peripheral tolerance, a large part of which is mediated by regulatory/suppressor T cells. In this report, we demonstrate a novel immunomodulatory mechanism by an FDA-approved, exogenous peptide-based therapy that incites an HLA class I-restricted, cytotoxic suppressor CD8+ T cell response. We have shown previously that treatment of multiple sclerosis (MS) with glatiramer acetate (GA; Copaxone) induces differential up-regulation of GA-reactive CD8+ T cell responses. We now show that these GA-induced CD8+ T cells are regulatory/suppressor in nature. Untreated patients show overall deficit in CD8+ T cell-mediated suppression, compared with healthy subjects. GA therapy significantly enhances this suppressive ability, which is mediated by cell contact-dependent mechanisms. CD8+ T cells from GA-treated patients and healthy subjects, but not those from untreated patients with MS, exhibit potent, HLA class I-restricted, GA-specific cytotoxicity. We further show that these GA-induced cytotoxic CD8+ T cells can directly kill CD4+ T cells in a GA-specific manner. Killing is enhanced by preactivation of target CD4+ T cells and may depend on presentation of GA through HLA-E. Thus, we demonstrate that GA therapy induces a suppressor/cytotoxic CD8+ T cell response, which is capable of modulating in vivo immune responses during ongoing therapy. These studies not only explain several prior observations relating to the mechanism of this drug but also provide important insights into the natural immune interplay underlying this human immune-mediated disease.