Medroxyprogesterone acetate inhibits CD8+ T cell viral-specific effector function and induces herpes simplex virus type 1 reactivation

Clinical research suggests hormonal contraceptive use is associated with increased frequencies of HSV reactivation and shedding. We examined the effects of medroxyprogesterone acetate (MPA), the compound most commonly used for injectable hormonal contraception, on HSV type 1 (HSV-1) reactivation and CD8(+) T cell function in murine trigeminal ganglia (TG). In ex vivo TG cultures, MPA dramatically inhibited canonical CD8(+) T cell effector functions, including IFN-gamma production and lytic granule release, and increased HSV-1 reactivation from latency. In vivo, MPA treatment of latently infected ovariectomized mice inhibited IFN-gamma production and lytic granule release by TG resident CD8(+) T cells stimulated directly ex vivo. RNA specific for the essential immediate early viral gene ICP4 as well as viral genome DNA copy number were increased in mice that received MPA during latency, suggesting that treatment increased in vivo reactivation. The increase in HSV-1 copy number appeared to be the result of a two-tine effect, as MPA induced higher reactivation frequencies from latently infected explanted TG neurons in the presence or absence of CD45(+) cells. Our data suggest hormonal contraceptives that contain MPA may promote increased frequency of HSV reactivation from latency through the combinatory effects of inhibiting protective CD8(+) T cell responses and by a leukocyte-independent effect on infected neurons.     

Influence of galectin-9/Tim-3 interaction on herpes simplex virus-1 latency

After HSV-1 infection, CD8(+) T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3)-expressing CD8(+) T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most K(b)-gB tetramer-specific CD8(+) T cells in the TG of HSV-1-infected mice express Tim-3, a molecule that delivers negative signals to CD8(+) T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8(+) T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8(+) T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8(+) T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8(+) T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency.     

Psychological stress compromises CD8+ T cell control of latent herpes simplex virus type 1 infections

Recurrent HSV-1 ocular disease results from reactivation of latent virus in trigeminal ganglia, often following immunosuppression or exposure to a variety of psychological or physical stressors. HSV-specific CD8+ T cells can block HSV-1 reactivation from latency in ex vivo trigeminal ganglia cultures through production of IFN-gamma. In this study, we establish that either CD8+ T cell depletion or exposure to restraint stress permit HSV-1 to transiently escape from latency in vivo. Restraint stress caused a reduction of TG-resident HSV-specific CD8+ T cells and a functional compromise of those cells that survive. Together, these effects of stress resulted in an approximate 65% reduction of cells capable of producing IFN-gamma in response to reactivating virus. Our findings demonstrate persistent in vivo regulation of latent HSV-1 by CD8+ T cells, and strongly support the concept that stress induces HSV-1 reactivation from latency at least in part by compromising CD8+ T cell surveillance of latently infected neurons.     

Rates of reactivation of latent herpes simplex virus from mouse trigeminal ganglia ex vivo correlate directly with viral load and inversely with number of infiltrating CD8+ T cells

Herpes simplex viruses (HSV) reactivate at rates proportional to the viral loads in latently infected ganglia. However, these rates vary substantially among infected animals. We assessed whether the numbers of HSV-specific CD8(+) T cells infiltrating latently infected ganglia also affect reactivation rates and contribute to their variability. Following corneal infection of mice with HSV type 2 (HSV-2), we quantified the latent viral loads in dissociated trigeminal ganglia by real-time PCR, the numbers of infiltrating CD8(+) T cells by flow cytometry, and the rates of reactivation by the detection of cell-free virus released from ganglion cells cultured in 96-well plates. The reactivation rates correlated directly with the latent viral loads (P = 0.001) but did so more strongly (P = 10(-7)) when cultures were depleted of CD8(+) T cells. Reactivation rates were reduced in a dose-dependent fashion by adding back ganglion CD8(+) T cells to the cultures (P = 0.003). We related the latent viral loads, numbers of CD8(+) T cells, and reactivation rates by mathematical equations. The rates of reactivation predicted from latent viral loads and numbers of infiltrating CD8(+) T cells in dissociated ganglia correlated with the observed rates of reactivation (P = 0.04). The reactivation of HSV-2 from ganglia ex vivo is determined both by the latent viral load and the number of infiltrating CD8(+) T cells.     

CD8(+) T-cell attenuation of cutaneous herpes simplex virus infection reduces the average viral copy number of the ensuing latent infection

During an initial encounter with herpes simplex virus type 1 (HSV-1) it takes several days for an adaptive immune response to develop and for herpes-specific CD8(+) T cells to infiltrate sites of infection. By this time the virus has firmly established itself within the innervating sensory nervous system where it then persists indefinitely. Preventing the establishment of viral latency would require blocking the skin to nervous system transmission of the virus. We wished to examine if CD8(+) T cells present early during acute HSV-1 infection could block this transmission. We show that effector CD8(+) T cells failed to prevent the establishment of HSV latency even when present prior to infection. This lack of blocking likely reflects the delayed infiltration of the CD8(+) T cells into the infected skin. Examination of the kinetics of HSV-1 infection highlighted the rapidity at which the virus infects the sensory ganglia and singled out early viral replication within the skin as an important factor in determining the magnitude of the ensuing latent infection. Though unable to prevent the establishment of latency, CD8(+) T cells could reduce the average viral copy number of the residual latent infection by dampening the skin infection and thus limiting the skin-to-nerve transmission of virus.     

Defining the herpes simplex virus-specific CD8+ T cell repertoire in C57BL/6 mice

HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (β), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8(+) T cell epitope (gB(498-505)) that is recognized by 50% of both the CD8(+) effector T cells in acutely infected trigeminal ganglia (TG) and the CD8(+) memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8(+) T cell epitopes in C57BL/6 mice, 19 (gB(498-505) and 18 subdominant epitopes) stimulated CD8(+) T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8(+) T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8(+) TCR repertoire. Only 11 of ～84 HSV-1 proteins are recognized by CD8(+) T cells, and most (～80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB(498-505) nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8(+) T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 β and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8(+) T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8(+) T cell precursors.     

Reevaluating the CD8 T-Cell Response to Herpes Simplex Virus Type 1: Involvement of CD8 T Cells Reactive to Subdominant Epitopes

In C57BL/6 (B6) mice, most herpes simplex virus (HSV)-specific CD8 T cells recognize a strongly immunodominant epitope on glycoprotein B (gB₄₉₈) and can inhibit HSV type 1 (HSV-1) reactivation from latency in trigeminal ganglia (TG). However, half of the CD8 T cells retained in latently infected TG of B6 mice are not gB₄₉₈ specific and have been largely ignored. The following observations from our current study indicate that these gB₄₉₈-nonspecific CD8 T cells are HSV specific and may contribute to the control of HSV-1 latency. First, following corneal infection, OVA₂₅₇-specific OT-1 CD8 T cells do not infiltrate the infected TG unless mice are simultaneously immunized with OVA₂₅₇ peptide, and then they are not retained. Second, 30% of CD8 T cells in acutely infected TG that produce gamma interferon in response to HSV-1 stimulation directly ex vivo are gB₄₉₈ nonspecific, and these cells maintain an activation phenotype during viral latency. Finally, gB₄₉₈-nonspecific CD8 T cells are expanded in ex vivo cultures of latently infected TG and inhibit HSV-1 reactivation from latency in the absence of gB₄₉₈-specific CD8 T cells. We conclude that many of the CD8 T cells that infiltrate and are retained in infected TG are HSV specific and potentially contribute to maintenance of HSV-1 latency. Identification of the viral proteins recognized by these cells will contribute to a better understanding of the dynamics of HSV-1 latency.The generation and maintenance of a CD8 T-cell response represent an important line of defense against many viral pathogens. Such responses are typically initiated when host antigen-presenting cells at the site of infection capture and process viral proteins and transport them to local draining lymph nodes (DLN). There the antigen-presenting cells either directly present viral antigens to naïve CD8 T cells or pass them to a distinct LN-resident dendritic cell (DC) subset for antigen presentation in the context of major histocompatibility complex class I (1). Antigen-specific CD8 T cells then undergo robust division and differentiation into effector populations armed to infiltrate infected tissue and eliminate the invading pathogen. The magnitude of the CD8 T-cell response against different viral epitopes is typically aligned within a defined hierarchy. Those epitopes recognized by the largest portion of the pathogen-specific CD8 T-cell population are referred to as immunodominant, while those inciting lesser responses are referred to as subdominant (17). Manipulation of this hierarchal system by the elimination of an immunodominant epitope often results in the expansion of a normally silent or “cryptic” determinant (2, 17, 21).Although the HSV-1 genome contains at least 84 open reading frames (13), it is estimated that 70 to 95% of the acute CD8 T-cell response in lymphoid organs of B6 mice is directed against the single immunodominant gB₄₉₈ epitope (11, 21, 24, 26, 27). The remaining HSV-specific CD8 T cells are thought to be directed against a subdominant epitope on the viral ribonucleotide reductase (RR1₈₂₂) (16). These conclusions are derived from studies characterizing the specificity of CD8 T cells at the peak of the effector response in lymphoid tissue. Interestingly, a recombinant HSV-1 lacking the immunodominant gB₄₉₈ epitope induced an HSV-specific CD8 T-cell response of normal magnitude, while the RR1₈₂₂ epitope remained subdominant (21), suggesting the emergence of previously unrecognized or cryptic epitopes.Following HSV-1 corneal infection of B6 mice, virus is transmitted to the trigeminal ganglia (TG), where it replicates briefly (up to 6 days postinfection [dpi]) and then establishes a latent infection. CD8 effector T cells accumulate to peak levels in the TG by 8 dpi and then undergo contraction, and then a memory population of constant size is maintained for the life of the animal. While 50% of both the effector and memory CD8 T-cell populations are specific for the immunodominant gB₄₉₈ epitope (11, 18), the remaining TG-resident CD8 T cells are specific for neither the dominant gB₄₉₈ nor the subdominant RR1₈₂₂ epitope. Although the phenotype and function of the gB₄₉₈-specific CD8 T cells in sensory ganglia and their role in maintaining HSV-1 latency have been well characterized (3, 5, 9, 11, 12, 14, 18, 19, 22, 24, 25, 27), the properties of the gB₄₉₈-nonspecific TG-resident CD8 T-cell population and their role in maintaining viral latency remain unexplored. Here we demonstrate that many of the gB₄₉₈-nonspecific CD8 T cells in latently infected TG proliferate and some produce gamma interferon (IFN-γ) when stimulated with HSV-1 antigens directly ex vivo. These cells also persistently exhibit an activation phenotype within latently infected TG, are expanded in ex vivo cultures of latently infected TG, and can block HSV-1 reactivation in TG neurons in the absence of gB₄₉₈-specific CD8 T cells.     

Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia

Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.     

ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency

Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957-5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4(-) mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4(-) virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.     

Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.     

Development and migration of protective CD8+ T cells into the nervous system following ocular herpes simplex virus-1 infection

After infection of epithelial surfaces, HSV-1 elicits a multifaceted antiviral response that controls the virus and limits it to latency in sensory ganglia. That response encompasses the CD8(+) T cells, whose precise role(s) is still being defined; immune surveillance in the ganglia and control of viral spread to the brain were proposed as the key roles. We tracked the kinetics of the CD8(+) T cell response across lymphoid and extralymphoid tissues after ocular infection. HSV-1-specific CD8(+) T cells first appeared in the draining (submandibular) lymph node on day 5 and were detectable in both nondraining lymphoid and extralymphoid tissues starting on day 6. However, although lymphoid organs contained both resting (CD43(low)CFSE(high)) and virus-specific cells at different stages of proliferation and activation, extralymphoid sites (eye, trigeminal ganglion, and brain) contained only activated cells that underwent more than eight proliferations (CD43(high)CFSE(neg)) and promptly secreted IFN-gamma upon contact with viral Ags. Regardless of the state of activation, these cells appeared too late to prevent HSV-1 spread, which was seen in the eye (from day 1), trigeminal ganglia (from day 2), and brain (from day 3) well before the onset of a detectable CD8(+) T cell response. However, CD8(+) T cells were critical in reducing viral replication starting on day 6 and for its abrogation between days 8 and 10; CD8-deficient animals failed to control the virus, exhibited persisting high viral titers in the brain after day 6, and died of viral encephalitis between days 7 and 12. Thus, CD8(+) T cells do not control HSV-1 spread from primary to tertiary tissues, but, rather, attack the virus in infected organs and control its replication in situ.     

Oligoadenylate synthetase/protein kinase R pathways and alphabeta TCR+ T cells are required for adenovirus vector: IFN-gamma inhibition of herpes simplex virus-1 in cornea

An adenoviral (Ad) vector containing the murine IFN-gamma transgene (Ad:IFN-gamma) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad:IFN-gamma potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-gamma receptor. Moreover, Ad:IFN-gamma was effective when delivered -72 and -24 h before infection as well as 24 h postinfection. Associated with antiviral opposition, TG from Ad:IFN-gamma-transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-gamma was effective but attenuated in TG from alphabeta TCR-deficient mice. In corneas, alphabeta TCR(+) T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid antiviral efficacy of Ad:IFN-gamma was further investigated because types I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-gamma inhibited viral reproduction in corneas and TG from alphabeta IFNR-deficient (CD118(-/-)) mice, although viral titers were 2- to 3-fold higher in cornea and TG compared with wild-type mice. The absence of IFN-stimulated antiviral proteins, 2'-5' oligoadenylate synthetase/RNase L, and dsRNA-dependent protein kinase R completely eliminated the antiviral effectiveness of Ad:IFN-gamma. Collectively, the results demonstrate the following: 1) nonexistence of type I IFN receptor does not abolish defense of Ad:IFN-gamma against HSV-1; 2) antiviral pathways oligoadenylate synthetase-RNase L and protein kinase R are mandatory; and 3) alphabeta TCR(+) T cells are compulsory for Ad:IFN-gamma effectiveness against HSV-1 in cornea but not in TG.     

CD154 signaling regulates the Th1 response to herpes simplex virus-1 and inflammation in infected corneas

Approximately 7 days after HSV-1 corneal infection, BALB/c mice develop tissue-destructive inflammation in the cornea termed herpes stromal keratitis (HSK), as well as periocular skin lesions that are characterized by vesicles, edema, and fur loss. CD4(+) T cells and Th1 cytokines contribute to both the immunopathology in the cornea and the eradication of viral replication in the skin. We demonstrate that disruption of CD40/CD154 signaling does not impact the initial expansion of CD4(+) T cells in the draining lymph nodes, but dramatically reduces the persistence and Th1 polarization of these cells. Despite the reduced Th1 response, CD154(-/-) mice developed HSK and periocular skin disease with similar kinetics and severity (as assessed by clinical examination) as wild-type (WT) mice. However, when the composition of the inflammatory infiltrate was examined by flow cytometric analysis, CD154(-/-) mice exhibited significantly fewer CD4(+) and CD8(+) T cells and neutrophils than WT mice at the peak of HSK. Moreover, CD4(+) T cells from infected corneas of CD154(-/-) mice produced significantly less IFN-gamma than those of WT mice when stimulated with viral Ags in vitro. The IFN-gamma production of cells from infected corneas of WT mice was not affected by addition of anti-CD154 mAb to the stimulation cultures. This suggests that CD154 signaling is required at the inductive phase, but not at the effector phase, of the Th1 response within the infected cornea. We conclude that local disruption of CD40/CD154 signaling is not likely to be a useful therapy for HSK.