NKG2D Receptor Signaling Enhances Cytolytic Activity by Virus-Specific CD8+ T Cells: Evidence for a Protective Role in Virus-Induced Encephalitis

Viral infection of the central nervous system (CNS) presents unique challenges to the immune system with regard to controlling and eliminating the invading pathogen. These obstacles include the presence of a blood-brain barrier that provides a physical and physiological barrier that is difficult for cells and molecules to cross, the absence of classic lymphatic drainage that may impair the generation of an adaptive immune response, and the relative absence of major histocompatibility complex (MHC) class I or II expression on resident cells (3, 26, 39, 73). In addition, the CNS is composed of a variety of highly specialized cells, many of which have limited renewal capacity, that represent potential targets of infection by numerous different viruses (34, 81). Therefore, a significant hurdle encountered by infiltrating antigen-specific lymphocytes is the elimination of virus from infected cells while limiting the damage that may have long-term detrimental consequences to the host. Critical to this is the cellular tropism of the virus, as this is important in dictating the effector response by infiltrating lymphocytes. For example, neuronotropic viruses often are eliminated by noncytolytic mechanisms via cytokine-mediated control and/or neutralizing antibodies, whereas the infection of other cell populations can evoke cytolytic mechanisms for control (17, 33, 45, 53, 75, 78, 87, 91). Therefore, characterizing the mechanisms involved in regulating the interaction between immune cells and virus-infected targets is critical for understanding how viral infection of the CNS is controlled and eventually cleared. The inoculation of mouse hepatitis virus (MHV; a positive-strand RNA virus that is a member of the Coronaviridae family), neurotropic strain JHM (JHMV), into the CNS of susceptible strains of mice provides an excellent model in which to examine host response mechanisms responsible for the control of viral replication within distinct cell lineages present in the brain (6). JHMV infection results in an acute encephalomyelitis characterized by wide-spread replication in astrocytes, microglia, and oligodendrocytes, with relatively few neurons being infected (10, 44, 69, 71). In addition, infection is associated with an orchestrated release of cytokines and chemokines that results in the inflammation and recruitment of inflammatory cells (47, 55, 68-70, 85, 90, 93). The control of viral replication during acute disease is primarily due to virus-specific CD8+ T cells, which function by two different effector mechanisms within the CNS: gamma interferon (IFN-γ) secretion is responsible for controlling viral replication in oligodendrocytes, whereas a perforin-dependent mechanism promotes viral clearance from astrocytes and microglia (53, 69). Infiltrating CD4+ T cells provide a supporting role for the maintenance and expansion of cytotoxic T-lymphocytes (CTLs) within the CNS as well as enhancing effector responses (80). Although a robust cell-mediated immune response occurs during acute disease, sterilizing immunity is not achieved, resulting in viral persistence (82). Factors contributing to this include oligodendrocytes becoming a predominant viral reservoir as well as substantial variation in viral RNA sequences (1, 29, 44). Virus-specific CD8+ T cells are retained within the CNS of persistently infected mice, and although lytic activity is muted, these cells retain the capacity to secrete IFN-γ, which affects viral replication in oligodendrocytes (5, 32, 61, 69). Ultimately, virus-specific antibody is critical in suppressing viral recrudescence and leads to the eventual clearance of virus from the CNS (52). Histological features associated with viral persistence include the development of an immune-mediated demyelinating disease that is similar to the human demyelinating disease multiple sclerosis, with both T cells and macrophages being important in amplifying disease severity by contributing to myelin damage (16, 71). Although elegant work previously has been performed with regard to evaluating the functional role of CD8+ T cells and viral replication within the CNS of JHMV-infected mice (7, 62, 80, 83), it is likely that additional signaling molecules associated with CD8+ T cells also contribute to the control of viral replication. NKG2D is a type II transmembrane glycoprotein that is expressed as a disulfide-linked homodimer on the surface of NK cells, CD8+ T cells, and γδ T cells in mice (20, 41). In some situations, NKG2D functions as a costimulatory receptor for CD8+ T cells following an encounter with cells expressing NKG2D ligands, resulting in enhanced effector functions and cell proliferation (35, 41, 60, 64, 74). For example, antiviral effector responses by some human cytomegalovirus antigen-specific CD8+ T clones characterized by cytokine bursts are amplified when cultured with viral peptide-pulsed cells expressing the human NKG2D ligand MICA (35). Treatment of lymphocytic choriomeningitis virus-specific CD8+ T cells with an NKG2D-activating antibody augmented proliferation compared to that of cells treated with control antibody, indicating that NKG2D receptor signaling on CD8+ T cells offers protection against virus-induced disease (41). Ex vivo anti-NKG2D treatment of Mycobacterium tuberculosis antigen-specific CD8+ T cells resulted in decreased IFN-γ secretion and CTL activity when cultured with bacterial peptide-stimulated macrophages compared to treatment with an isotype-matched control antibody, suggesting that NKG2D receptor signaling on CD8+ T cells affects the outcome of disease (74). Finally, blocking NKG2D receptor signaling in mice persistently infected with JHMV results in diminished immune-mediated pathology, suggesting that IFN-γ production from γδ T cells is impaired (18). These studies highlight the potential protective role of NKG2D in host defense following microbial infection by evoking a range of responses in a variety of immune cells. In this study, the functional role of NKG2D receptor signaling on T cells and NK cells during both acute and chronic virus-induced neurological disease was examined. Following JHMV infection of susceptible mice, mRNA transcripts specific for the NKG2D ligands H60, MULT1, and RAE-1 were expressed within the CNS, and the majority of infiltrating NK cells and CD8+ T cells expressed the NKG2D receptor. Blocking NKG2D receptor signaling in JHMV-infected SCID mice (lacking T and B cells, but functional NK cells are intact) did not affect survival or the viral burden within the brain, indicating that NKG2D does not enhance antiviral effector responses by infiltrating NK cells. However, anti-NKG2D treatment of JHMV-infected BALB/c mice resulted in increased mortality and increased viral burden within the brain. Indeed, treatment with NKG2D neutralizing antibody resulted in diminished CTL activity by virus-specific CD8+ T cells, yet IFN-γ expression was not affected, indicating that blocking NKG2D selectively affects antiviral effector function in response to acute viral infection of the CNS. The administration of anti-NKG2D antibody to persistently infected mice did not result in viral recrudescence, modulate IFN-γ levels, or influence the severity of demyelination, indicating that NKG2D receptor signaling does not influence chronic disease. Therefore, the findings presented highlight an important role for NKG2D in CD8+ T-cell antiviral immunity during acute infection of the CNS by enhancing cytolytic activity.