Sariah J. Allen

The Role of LAT in Increased CD8+ T Cell Exhaustion in Trigeminal Ganglia of Mice Latently Infected with Herpes Simplex Virus 1

ABSTRACT Herpes simplex virus (HSV) infection is a classic example of latent viral infection in humans and experimental animal models. The HSV-1 latency-associated transcript (LAT) plays a major role in the HSV-1 latency reactivation cycle and thus in recurrent disease. Whether the presence of LAT leads to generation of dysfunctional T cell responses in the trigeminal ganglia (TG) of latently infected mice is not known. To address this issue, we used LAT-positive [LAT(+)] and LAT-deficient [LAT(−)] viruses to evaluate the effect of LAT on CD8 T cell exhaustion in TG of latently infected mice. The amount of latency as determined by quantitative reverse transcription-PCR (qRT-PCR) of viral DNA in total TG extracts was 3-fold higher with LAT(+) than with LAT(−) virus. LAT expression and increased latency correlated with increased mRNA levels of CD8, PD-1, and Tim-3. PD-1 is both a marker for exhaustion and a primary factor leading to exhaustion, and Tim-3 can also contribute to exhaustion. These results suggested that LAT(+) TG contain both more CD8+ T cells and more CD8+ T cells expressing the exhaustion markers PD-1 and Tim-3. This was confirmed by flow cytometry analyses of expression of CD3/CD8/PD-1/Tim-3, HSV-1, CD8+ T cell pentamer (specific for a peptide derived from residues 498 to 505 of glycoprotein B [gB498–505]), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α). The functional significance of PD-1 and its ligands in HSV-1 latency was demonstrated by the significantly reduced amount of HSV-1 latency in PD-1- and PD-L1-deficient mice. Together, these results may suggest that both PD-1 and Tim-3 are mediators of CD8+ T cell exhaustion and latency in HSV-1 infection.

Level of Herpes Simplex Virus Type 1 Latency Correlates with Severity of Corneal Scarring and Exhaustion of CD8+ T Cells in Trigeminal Ganglia of Latently Infected Mice

ABSTRACT A hallmark of infection with herpes simplex virus type 1 (HSV-1) is the establishment of latency in ganglia of the infected individual. During the life of the latently infected individual, the virus can occasionally reactivate, travel back to the eye, and cause recurrent disease. Indeed, a major cause of corneal scarring (CS) is the scarring induced by HSV-1 following reactivation from latency. In this study, we evaluated the relationship between the amount of CS and the level of the HSV-1 latency-associated transcript (LAT) in trigeminal ganglia (TG) of latently infected mice. Our results suggested that the amount of CS was not related to the amount of virus replication following primary ocular HSV-1 infection, since replication in the eyes was similar in mice that did not develop CS, mice that developed CS in just one eye, and mice that developed CS in both eyes. In contrast, mice with no CS had significantly less LAT, and thus presumably less latency, in their TG than mice that had CS in both eyes. Higher CS also correlated with higher levels of mRNAs for PD-1, CD4, CD8, F4/80, interleukin-4, gamma interferon, granzyme A, and granzyme B in both cornea and TG. These results suggest that (i) the immunopathology induced by HSV-1 infection does not correlate with primary virus replication in the eye; (ii) increased CS appears to correlate with increased latency in the TG, although the possible cause-and-effect relationship is not known; and (iii) increased latency in mouse TG correlates with higher levels of PD-1 mRNA, suggesting exhaustion of CD8+ T cells.

Use of cytokine immunotherapy to block CNS demyelination induced by a recombinant HSV-1 expressing IL-2.

We previously have described a model of multiple sclerosis (MS) in which constitutive expression of murine interleukin (IL)-2 by herpes simplex virus type 1 (HSV-1) (HSV-IL-2) causes central nervous system (CNS) demyelination in different strains of mice. In the current study, we investigated whether this HSV-IL-2-induced demyelination can be blocked using recombinant viruses expressing different cytokines or by injection of plasmid DNA. We have found that coinfection of HSV-IL-2-infected mice with recombinant viruses expressing IL-12p35, IL-12p40 or IL-12p35+IL-12p40 did not block the CNS demyelination, and that coinfection with a recombinant virus expressing interferon (IFN)-γ exacerbated it. In contrast, coinfection with a recombinant virus expressing IL-4 reduced demyelination, whereas coinfection of HSV-IL-2-infected mice with a recombinant HSV-1 expressing the IL-12 heterodimer (HSV-IL-12p70) blocked the CNS demyelination in a dose-dependent manner. Similarly, injection of IL-12p70 DNA blocked HSV-IL-2-induced CNS demyelination in a dose-dependent manner and injection of IL-35 DNA significantly reduced CNS demyelination. Injection of mice with IL-12p35 DNA, IL-12p40 DNA, IL-12p35+IL-12p40 DNA or IL-23 DNA did not have any effect on HSV-IL-2-induced demyelination, whereas injection of IL-27 DNA increased the severity of the CNS demyelination in the HSV-IL-2-infected mice. This study demonstrates for the first time that IL-12p70 can block HSV-IL-2-induced CNS demyelination and that IL-35 can also reduce this demyelination, whereas IFN-γ and IL-27 exacerbated the demyelination in the CNS of the HSV-IL-2-infected mice. Our results suggest a potential role for IL-12p70 and IL-35 signaling in the inhibition of HSV-IL-2-induced immunopathology by preventing development of autoaggressive T cells.

Interactions between herpesvirus entry mediator (TNFRSF14) and latency-associated transcript during herpes simplex virus 1 latency.

ABSTRACT Herpesvirus entry mediator (HVEM) is one of several cell surface proteins herpes simplex virus (HSV) uses for attachment/entry. HVEM regulates cellular immune responses and can also increase cell survival. Interestingly, latency-associated transcript (LAT), the only viral gene consistently expressed during neuronal latency, enhances latency and reactivation by promoting cell survival and by helping the virus evade the host immune response. However, the mechanisms of these LAT activities are not well understood. We show here for the first time that one mechanism by which LAT enhances latency and reactivation appears to be by upregulating HVEM expression. HSV-1 latency/reactivation was significantly reduced in Hvem −/− mice, indicating that HVEM plays a significant role in HSV-1 latency/reactivation. Furthermore, LAT upregulated HVEM expression during latency in vivo and also when expressed in vitro in the absence of other viral factors. This study suggests a mechanism whereby LAT upregulates HVEM expression potentially through binding of two LAT small noncoding RNAs to the HVEM promoter and that the increased HVEM then leads to downregulation of immune responses in the latent microenvironment and increased survival of latently infected cells. Thus, one of the mechanisms by which LAT enhances latency/reactivation appears to be through increasing expression of HVEM.

Exacerbation of corneal scarring in HSV-1 gK immunized mice correlates with elevation of CD8+CD25+ T cells in corneas of ocularly infected mice

We have shown previously that exacerbation of corneal scarring (CS) in HSV-1 glycoprotein K (gK) immunized mice was associated with CD8+ T cells. In this study, we investigated the type and the nature of the immune responses that are involved in the exacerbation of CS in gK-immunized animals. BALB/c mice were vaccinated with baculovirus expressed gK, gD, or mock-immunized. Twenty-one days after the third immunization, mice were ocularly infected with 2 x 10(5) PFU/eye of virulent HSV-1 strain McKrae. Infiltration of the cornea by CD4+, CD8+, CD25+, CD4+CD25+, CD8+CD25+, CD19+, CD40+, CD40L+, CD62L+, CD95+, B7-1+, B7-2+, MHC-I+, and MHC-II+ cells was monitored by immunohistochemistry, qRT-PCR and FACS at various times post-infection (PI). This study demonstrated for the first time that the presence of CD8+CD25+ T cells in the cornea is correlated with exacerbation of CS in the gK-immunized group.

CD11c Controls Herpes Simplex Virus 1 Responses To Limit Virus Replication during Primary Infection

ABSTRACT CD11c is expressed on the surface of dendritic cells (DCs) and is one of the main markers for identification of DCs. DCs are the effectors of central innate immune responses, but they also affect acquired immune responses to infection. However, how DCs influence the efficacy of adaptive immunity is poorly understood. Here, we show that CD11c+ DCs negatively orchestrate both adaptive and innate immunity against herpes simplex virus type 1 (HSV-1) ocular infection. The effectiveness and quantity of virus-specific CD8+ T cell responses are increased in CD11c-deficient animals. In addition, the levels of CD83, CD11b, alpha interferon (IFN-α), and IFN-β, but not IFN-γ, were significantly increased in CD11c-deficient animals. Higher levels of IFN-α, IFN-β, and CD8+ T cells in the CD11c-deficient mice may have contributed to lower virus replication in the eye and trigeminal ganglia (TG) during the early period of infection than in wild-type mice. However, the absence of CD11c did not influence survival, severity of eye disease, or latency. Our studies provide for the first time evidence that CD11c expression may abrogate the ability to reduce primary virus replication in the eye and TG via higher activities of type 1 interferon and CD8+ T cell responses.

Macrophage IL-12p70 Signaling Prevents HSV-1–Induced CNS Autoimmunity Triggered by Autoaggressive CD4+ Tregs

PURPOSE CD4(+)CD25(+)FoxP3(+) naturally occurring regulatory T cells (Tregs) maintain self-tolerance and function to suppress overly exuberant immune responses. However, it is unclear whether innate immune cells modulate Treg function. Here the authors examined the role of innate immunity in lymphomyeloid homeostasis. METHODS The involvement of B cells, dendritic cells (DCs), macrophages, natural killer (NK) cells, and T cells in central nervous system (CNS) demyelination in different strains of mice infected ocularly with herpes simplex virus type 1 (HSV-1) was investigated. RESULTS The authors found that depletion of macrophages, but not DCs, B cells, NK cells, CD4(+) T cells, or CD8(+) T cells, induced CNS demyelination irrespective of virus or mouse strain. As with macrophage depletion, mice deficient in interleukin (IL)-12p35 or IL-12p40 showed CNS demyelination after HSV-1 infection, whereas demyelination was undetectable in HSV-1-infected, IL-23p19-deficient, or Epstein-Barr virus-induced gene 3-deficient mice. Demyelination could be rescued in macrophage-depleted mice after the injection of IL-12p70 DNA and in IL-12p35(-/-) or IL-12p40(-/-) mice after injection with IL-12p35 or IL-12p40 DNA or with recombinant viruses expressing IL-12p35 or IL-12p40. Using FoxP3-, CD4-, CD8-, or CD25-depletion and gene-deficient mouse approaches, the authors demonstrated that HSV-1-induced demyelination was blocked in the absence of CD4, CD25, or FoxP3 in macrophage-depleted mice. Flow cytometry showed an elevation of CD4(+)CD25(+)FoxP3(+) T cells in the spleens of infected macrophage-depleted mice, and adoptive transfer of CD4(+)CD25(+) T cells to infected macrophage-depleted severe combined immunodeficient mice induced CNS demyelination. CONCLUSIONS The authors demonstrated that macrophage IL-12p70 signaling plays an important role in maintaining immune homeostasis in the CNS by preventing the development of autoaggressive CD4(+) Tregs.

Immunization with Different Viral Antigens Alters the Pattern of T Cell Exhaustion and Latency in Herpes Simplex Virus Type 1-Infected Mice

ABSTRACT We have shown previously that immunization with herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) exacerbated corneal scarring (CS) in ocularly infected mice. In this study, we investigated whether higher levels of CS were correlated with higher levels of latency and T cell exhaustion in gK-immunized mice. BALB/c mice were vaccinated with baculovirus-expressed gK or gD or mock immunized. Twenty-one days after the third immunization, mice were ocularly infected with 2 × 104 PFU/eye of virulent HSV-1 strain McKrae. On day 5 postinfection, virus replication in the eye was measured, and on day 30 postinfection, infiltration of the trigeminal ganglia (TG) by CD4, CD8, programmed death 1 (PD-1), and T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was monitored by immunohistochemistry and quantitative real-time PCR (qRT-PCR). This study demonstrated that higher levels of CS were correlated with higher levels of latency, and this was associated with the presence of significantly higher numbers of CD4+PD-1+ and CD8+PD-1+ cells in the TG of the gK-immunized group than in both the gD- and mock-immunized groups. Levels of exhaustion associated with Tim-3 were the same among gK- and mock-vaccinated groups but higher than levels in the gD-vaccinated group. In this study, we have shown for the first time that both PD-1 and Tim-3 contribute to T cell exhaustion and an increase of latency in the TG of latently infected mice.

CD8α Dendritic Cells Drive Establishment of HSV-1 Latency

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α−/− (lack functional CD8 T cells and CD8α+ DCs), CD8β−/− (have functional CD8α+ T cells and CD8α+ DCs), and β2m−/− (lack functional CD8 T cells but have CD8α+ DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α+ DCs) versus WT C3H (have functional CD8α T cells and CD8α+ DCs) mice. We also determined whether the phenotype of CD8α−/− and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8+ T cells or bone marrow (BM) derived CD8α+ DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences.

Binding of HSV-1 Glycoprotein K (gK) to Signal Peptide Peptidase (SPP) Is Required for Virus Infectivity

Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication in vitro. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also suggest that SPP plays an important role in viral replication and possibly virus pathogenesis. This makes SPP unique in that its function appears to be required by the virus as no other protein can compensate its loss in terms of viral replication.