Anthony J. St. Leger

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 (gB498–505) 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 (gB498–505 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 gB498–505 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.

E47 controls the developmental integrity and cell cycle quiescence of multipotential hematopoietic progenitors.

Little is known about the transcriptional regulators that control the proliferation of multipotent bone marrow progenitors. Understanding the mechanisms that restrict proliferation is of significant interest since the loss of cell cycle integrity can be associated with hematopoietic exhaustion, bone marrow failure, or even oncogenic transformation. Herein, we show that multipotent LSKs (lineage−Scahighc-kit+) from E47-deficient mice exhibit a striking hyperproliferation associated with a loss of cell cycle quiescence and increased susceptibility to in vivo challenge with a mitotoxic drug. Total LSKs contain long-term self-renewing hematopoietic stem cells and downstream multipotential progenitors (MPPs) that possess very limited or no self-renewal ability. Within total LSKs, we found specific developmental and functional deficits in the MPP subset. E47 knockout mice have grossly normal numbers of self-renewing hematopoietic stem cells but a 50–70% reduction in nonrenewing MPPs and downstream lineage-restricted populations. The residual MPPs in E47 knockout mice fail to fully up-regulate flk2 or initiate V(D)J recombination, hallmarks of normal lymphoid lineage progression. Consistent with the loss of normal cell cycle restraints, we show that E47-deficient LSKs have a 50% decrease in p21, a cell cycle inhibitor and known regulator of LSK proliferation. Moreover, enforced expression studies identify p21 as an E47 target gene in primary bone marrow LSKs. Thus, E47 appears to regulate the developmental and functional integrity of early hematopoietic subsets in part through effects on p21-mediated cell cycle quiescence.

Herpes simplex virus and varicella zoster virus, the house guests who never leave.

Human alphaherpesviruses including herpes simplex viruses (HSV-1, HSV-2) and varicella zoster virus (VZV) establish persistent latent infection in sensory neurons for the life of the host. All three viruses have the potential to reactivate causing recurrent disease. Regardless of the homology between the different virus strains, the three viruses are characterized by varying pathologies. This review will highlight the differences in infection pattern, immune response, and pathogenesis associated with HSV-1 and VZV.

CD8+ T cells patrol HSV-1-infected trigeminal ganglia and prevent viral reactivation

A hallmark of herpes viruses is their capacity to cause recurrent disease. Recurrences of herpes simplex virus (HSV)-1 disease do not result from reinfection from external sources, but rather from reactivation of virus that is maintained in a latent state in sensory neurons and periodically reactivates from latency to cause recurrent disease. Recent findings implicate HSV-specific CD8+ T cells in immune surveillance of HSV-1 latently infected sensory neurons in trigeminal ganglia (TG) and inhibition of HSV-1 reactivation from latency. This review summarizes recent findings regarding the characteristics of the TG-resident CD8+ T cell population and certain unique obstacles that might complicate the development of therapeutic vaccines.

Circulating herpes simplex type 1 (HSV-1)-specific CD8+T cells do not access HSV-1 latently infected trigeminal ganglia

BackgroundTherapeutic vaccines can be designed to enhance existing T cell memory populations for increased protection against re-infection. In the case of herpes simplex virus type 1, recurrent disease results from reactivation of latent virus in sensory ganglia, which is controlled in part by a ganglia-resident HSV-specific memory CD8+ T cell population. Thus, an important goal of a therapeutic HSV-1 vaccine would be to enhance this population.MethodsHSV-1-infected mice were treated with TAK-779 to block CCR5- and CXCR3-mediated CD8+ T cell migration during both acute and latent infections. Additionally, HSV-1-specific CD8+ T cells were transferred into HSV-1 latently infected mice to mimic the effect of a therapeutic vaccine, and their migration into trigeminal ganglia (TG) was traced during steady-state latency, or during recovery of the TG-resident memory CD8+ T cell population following stress-, and corticosterone-induced depletion and HSV-1 reactivation from latency. Bromodeoxy uridine (BrdU) incorporation measured cell proliferation in vivo.ResultsTAK-779 treatment during acute HSV-1 infection reduced the number of infiltrating CD8+ T cells but did not alter the number of viral genome copies. TAK-779 treatment during HSV latency did not affect the size of the TG-resident memory CD8+ T cell population. Transferred HSV-specific CD8+ T cells failed to access latently infected TG during steady-state latency, or during recovery of the TG resident HSV-specific CD8+ T cell population following exposure of latently infected mice to stress and corticosterone. Recovery of the HSV-specific CD8+ T cell population after stress and corticosterone treatment occurred with homeostatic levels of cell division and did not require CD4+ T cell help.ConclusionsOur findings are consistent with the notion that the CD8+ T cells in latently infected TG are a tissue-resident memory (Trm) population that is maintained without replenishment from the periphery, and that when this population is disrupted, it recovers without proliferation or detectable recruitment of HSV-specific CD8+ T cells from the blood. The compartmentalization of the HSV-specific CD8+ memory T cell population in latently infected TG will complicate the design of therapeutic vaccines.

Broadening the Repertoire of Functional Herpes Simplex Virus Type 1–Specific CD8+ T Cells Reduces Viral Reactivation from Latency in Sensory Ganglia

A large proportion of the world population harbors HSV type 1 (HSV-1) in a latent state in their trigeminal ganglia (TG). TG-resident CD8+ T cells appear important for preventing HSV-1 reactivation from latency and recurrent herpetic disease. In C57BL/6J mice, half of these cells are specific for an immunodominant epitope on HSV-1 glycoprotein B, whereas the other half are specific for 18 subdominant epitopes. In this study, we show that the CD8+ T cell dominance hierarchy in the TG established during acute infection is maintained during latency. However, CD8+ T cells specific for subdominant epitopes lose functionality, whereas those specific for the immunodominant epitope exhibit increased functionality in latently infected TG. Furthermore, we show that IL-10 produced by 16.4 ± 2.8% of TG-resident CD4+ T cells maintains the immunodominance hierarchy in part through selective inhibition of subdominant CD8+ T cell proliferation. Upon systemic anti–IL-10R Ab treatment, we observed a significant expansion of functional subdominant CD8+ T cells, resulting in significantly improved protection from viral reactivation. In fact, systemic anti–IL-10R Ab treatment prevented viral reactivation in up to 50% of treated mice. Our results not only demonstrate that HSV-1 reactivation from latency can be prevented by expanding the repertoire of functional TG-resident CD8+ T cells, but also that IL-10R blockade might have therapeutic potential to reduce or eliminate recurrent herpetic disease.

Dendritic Cell Activation and Memory Cell Development Are Impaired among Mice Administered Medroxyprogesterone Acetate Prior to Mucosal Herpes Simplex Virus Type 1 Infection

Epidemiological studies indicate that the exogenous sex steroid medroxyprogesterone acetate (MPA) can impair cell-mediated immunity, but mechanisms responsible for this observation are not well defined. In this study, MPA administered to mice 1 wk prior to HSV type 1 (HSV-1) infection of their corneal mucosa impaired initial expansion of viral-specific effector and memory precursor T cells and reduced the number of viral-specific memory T cells found in latently infected mice. MPA treatment also dampened expression of the costimulatory molecules CD40, CD70, and CD80 by dendritic cells (DC) in lymph nodes draining acute infection, whereas coculture of such DC with T cells from uninfected mice dramatically impaired ex vivo T cell proliferation compared with the use of DC from mice that did not receive MPA prior to HSV-1 infection. In addition, T cell expansion was comparable to that seen in untreated controls if MPA-treated mice were administered recombinant soluble CD154 (CD40L) concomitant with their mucosal infection. In contrast, the immunomodulatory effects of MPA were infection site dependent, because MPA-treated mice exhibited normal expansion of virus-specific T cells when infection was systemic rather than mucosal. Taken together, our results reveal that the administration of MPA prior to viral infection of mucosal tissue impairs DC activation, virus-specific T cell expansion, and development of virus-specific immunological memory.

PD-L1/B7-H1 Regulates the Survival but Not the Function of CD8+ T Cells in Herpes Simplex Virus Type 1 Latently Infected Trigeminal Ganglia

HSV type 1 (HSV-1)–specific CD8+ T cells provide immunosurveillance of trigeminal ganglion (TG) neurons that harbor latent HSV-1. In C57BL/6 mice, the TG-resident CD8+ T cells are HSV specific and maintain a 1:1 ratio of cells recognizing an immunodominant epitope on viral glycoprotein B (gB498–505-Tet+) and cells reactive to subdominant epitopes (gB-Tet−). The gB-Tet− CD8+ T cells maintain their frequency in TG by balancing a higher rate of proliferation with a correspondingly higher rate of apoptosis. The increased apoptosis is associated with higher expression of programmed death-1 (PD-1) on gB-Tet− CD8+ T cells and the interaction with PD-1 ligand (PD-L1/B7-H1). IFN-γ regulated expression of the PD-1 ligand (PD-L1/B7-H1) on neurons bearing higher copies of latent viral genome. In latently infected TG of B7-H1−/− mice, the number and frequency of PD-1+ gB-Tet− CD8+ T cells increases dramatically, but gB-Tet− CD8+ T cells remain largely nonfunctional and do not provide increased protection from HSV-1 reactivation in ex vivo cultures of latently infected TG. Unlike observations in some chronic infection models, B7-H1 blockade did not increase the function of exhausted gB-Tet− CD8 T cells in latently infected TG.

The B lineage transcription factor E2A regulates apoptosis in chronic lymphocytic leukemia (CLL) cells

Chronic lymphocytic leukemia (CLL) is a common malignancy characterized by the accumulation of B lymphocytes with an antigen-experienced activated CD19(+)CD5(+) clonal phenotype. Clinically, ∼50% of cases will behave more aggressively. Here, we investigate the role of the major B-cell transcription factor E2A, a known regulator of B-cell survival and proliferation, to CLL persistence. We show that E2A is elevated at the mRNA and protein levels relative to normal B-cell subsets. E2A silencing in primary CLL cells leads to a significant increase in spontaneous apoptosis in both CD38(+) (aggressive) and CD38(-) (indolent) cases. Moreover, E2A knockdown synergizes with the immunomodulatory drug lenalidomide to reduce CLL viability. E2A is known to restrain the proliferation of primary B and T lymphocytes at multiple stages of maturation and we report that targeted E2A disruption increases the frequency of Ki-67(+) CLL cells in the absence of effects on de novo proliferation. At the molecular level, E2A siRNA-treated CLL cells display reduced expression of key genes associated with survival and cell cycling including p27, p21 and mcl-1, of which the former two are known E2A target genes. Thus, E2A, a key transcription factor associated with the B-cell activation profile, regulates apoptosis in CLL and may contribute to disease pathology.

Interferon Regulator Factor 8 (IRF8) Limits Ocular Pathology during HSV-1 Infection by Restraining the Activation and Expansion of CD8+ T Cells

Interferon Regulatory Factor-8 (IRF8) is constitutively expressed in monocytes and B cell lineages and plays important roles in immunity to pathogens and cancer. Although IRF8 expression is induced in activated T cells, the functional relevance of IRF8 in T cell-mediated immunity is not well understood. In this study, we used mice with targeted deletion of Irf8 in T-cells (IRF8KO) to investigate the role of IRF8 in T cell-mediated responses during herpes simplex virus 1 (HSV-1) infection of the eye. In contrast to wild type mice, HSV-1-infected IRF8KO mice mounted a more robust anti-HSV-1 immune response, which included marked expansion of HSV-1-specific CD8+ T cells, increased infiltration of inflammatory cells into the cornea and trigeminal ganglia (TG) and enhanced elimination of virus within the trigeminal ganglion. However, the consequence of the enhanced immunological response was the development of ocular inflammation, limbitis, and neutrophilic infiltration into the cornea of HSV-1-infected IRF8KO mice. Surprisingly, we observed a marked increase in virus-specific memory precursor effector cells (MPEC) in IRF8KO mice, suggesting that IRF8 might play a role in regulating the differentiation of effector CD8+ T cells to the memory phenotype. Together, our data suggest that IRF8 might play a role in restraining excess lymphocyte proliferation. Thus, modulating IRF8 levels in T cells can be exploited therapeutically to prevent immune-mediated ocular pathology during autoimmune and infectious diseases of the eye.