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.     

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.     

Inflammatory infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection.

Following herpes simplex virus type 1 (HSV-1) infection of the cornea, the virus is transmitted to the trigeminal ganglion, where a brief period of virus replication is followed by establishment of a latent infection in neurons. A possible role of the immune system in regulating virus replication and maintaining latency in the sensory neurons has been suggested. We have investigated the phenotype and cytokine pattern of cells that infiltrate the A/J mouse trigeminal ganglion at various times after HSV-1 corneal infection. HSV antigen expression in the trigeminal ganglion (indicative of the viral lytic cycle) increased until day 3 postinfection (p.i.) and then diminished to undetectable levels by day 7 p.i. The period of declining HSV antigen expression. was associated with a marked increase in Mac-1+ cells. These cells did not appear to coexpress the F4/80+ (macrophage) or the CD8+ (T cell) markers, and none showed polymorphonuclear leukocyte morphology, suggesting a possible early infiltration of natural killer cells. There was also a significant increase in the trigeminal ganglion of cells expressing the gamma delta T-cell receptor, and these cells were found almost exclusively in very close association with neurons. This period was also characterized by a rapid and equivalent increase in cells expressing gamma interferon and interleukin-4. The density of the inflammatory infiltrate in the trigeminal ganglion increased until days 12 to 21 p.i., when it was predominated by CD8+, Mac-1+, and tumor necrosis factor-expressing cells, which surrounded many neurons. By day 92 p.i., the inflammatory infiltrate diminished but was heaviest in mice with active periocular skin disease. Our data are consistent with the notion that gamma interferon produced by natural killer cells and/or gamma delta T cells may play an important role in limiting HSV-1 replication in the trigeminal ganglion during the acute stage of infection. In addition, tumor necrosis factor produced by CD8+ T cells and macrophages may function to maintain the virus in a latent state.     

Tuber development phenotypes in adapted and acclimated, drought-stressed Solanum tuberosum ssp. andigena have distinct expression profiles of genes associated with carbon metabolism.

A drought screen identified accessions of Solanum tuberosum ssp. andigena that showed varying degrees of physiological acclimation or adaptation to repeated drought stress. The accessions also showed variable tuber phenotypes from small tubers that failed to develop in an accession that showed photosynthetic adaptation to normal tubers in an accession with a phenotype showing some degree of photosynthetic adaptation and acclimation. Using microarray data, we correlated the expression of genes associated with carbon metabolism with the tuber development phenotypes under drought. Genes associated with sucrose and starch metabolism showed responses consistent with starch deficiency in the adapted accession and normal starch deposition in the intermediate accession. Starch phosphorylase and glycogen bound starch synthase were induced in the adapted accession, which had abnormal tuber development. Genes associated with trehalose were induced in the intermediate accession with normal tuber development. Genes associated with respiration were also induced in the intermediate accession, and a pattern compatible with the existence of a 3PGA recovery pathway was revealed. Expression of thioredoxin genes also correlated with tuber development phenotypes under drought stress. The data suggest differential regulation of starch deposition in accessions of Andigena with different abilities to respond to drought stress.     

Growth measurements of terrestrial microbial species by a continuous-flow technique

A continuous nutrient flow system has been developed to measure microbial activity in soil with various concentrations of added substrate. The system consists of a thin soil layer through which substrate was added continuously over periods up to 4.5 days. Substrate utilization was determined by effluent analysis. Respiration was measured manually by injecting a sample into a gas chromatograph or automatically by coupling the growth chamber to a computer-controlled gas sampling valve. This permitted respiratory CO₂ to be measured by the gas chromatograph at intervals selected by the investigator. Software controlling the valve and gas chromatograph not only automated gas phase sampling, but also provided a scan of CO₂ evolution and a preliminary data summary. This included the date and time of sample, peak height, and percent CO₂ in the gas phase. Data for growth on glucose using a microbial population native to a California annual grassland soil demonstrated that the direct cell count and respiratory techniques for biomass estimation give comparable results. This procedure provides the potential for detailed analyses of substrate utilization in studies of the growth and maintenance of soil microorganisms.     

Impact of flooding on the densities of selected aquatic insects

Data from a four-year study of five aquatic insect species,Hydropsyche betteni, H. morosa, H. bronta, Isonychia bicolor, andEphoron leucon, were utilized to evaluate the impact of a 60-year flood and a few lesser floods. The survey began in August, 1984 and was terminated in October, 1987 with the 60-year flood occurring in November, 1985. Four sampling sites were established on the South River and six quantitative samples were taken each month from each site. Gauging stations on the South River provided accurate discharge data for the sampling sites and useful historical data. Densities for the five species were utilized in the evaluation of the floods. The importance of timing is pointed out, that is, floods that occur very close together or near the end of the life cycle of an insect make it difficult to evaluate floods as disturbances. The importance of life history traits, such as behavior and egg diapause, are discussed in respect to floods. Densities were reduced to less than 50% of their average values immediately after the 60-year flood for the threeHydropsyche spp. and at three sites forI. bicolor. Ephoron leucon showed no response to the 60-year flood. Densities of the four impacted species returned to previous levels in the following generation. The 60-year flood was considered a disturbance in the near term but not for more than one generation.     

Disease swamps molecular signatures of genetic-environmental associations to abiotic factors in Tasmanian devil (Sarcophilus harrisii) populations

Landscape genomics studies focus on identifying candidate genes under selection via spatial variation in abiotic environmental variables, but rarely by biotic factors (i.e., disease). The Tasmanian devil (Sarcophilus harrisii) is found only on the environmentally heterogeneous island of Tasmania and is threatened with extinction by a transmissible cancer, devil facial tumor disease (DFTD). Devils persist in regions of long-term infection despite epidemiological model predictions of species’ extinction, suggesting possible adaptation to DFTD. Here, we test the extent to which spatial variation and genetic diversity are associated with the abiotic environment (i.e., climatic variables, elevation, vegetation cover) and/or DFTD. We employ genetic-environment association analyses using 6886 SNPs from 3287 individuals sampled pre- and post-disease arrival across the devil's geographic range. Pre-disease, we find significant correlations of allele frequencies with environmental variables, including 365 unique loci linked to 71 genes, suggesting local adaptation to abiotic environment. The majority of candidate loci detected pre-DFTD are not detected post-DFTD arrival. Several post-DFTD candidate loci are associated with disease prevalence and were in linkage disequilibrium with genes involved in tumor suppression and immune response. Loss of apparent signal of abiotic local adaptation post-disease suggests swamping by strong selection resulting from the rapid onset of DFTD.     

Latent virus influences the generation and maintenance of CD8+ T cell memory

The influence of latent virus on CD8+ T cell memory is poorly understood. HSV type 1 specifically establishes latency in trigeminal ganglia (TG) after corneal infection of mice. In latently infected TG, IL-15 deprivation reduced the following: 1) accumulation of HSV-specific CD8+ effector T cells (HSV-CD8(eff)), 2) accumulation of CD127(+) putative HSV-CD8 memory precursors, and 3) the size and functionality of the memory (HSV-CD8(mem)) population. Although compromised in IL-15(-/-) mice, the HSV-CD8(mem) pool persisted in latently infected tissue, but not in noninfected tissue of the same mice. Anti-IL-2 treatment also dramatically reduced the size of the HSV-CD8(eff) population in the TG, but did not influence the concomitant generation of the CD127+ putative HSV-CD8(mem) precursor population or the size or functionality of the HSV-CD8(mem) pool. Thus, the size of the memory pool appears to be determined by the size of the CD127+ CD8(mem) precursor population and not by the size of the overall CD8(eff) pool. HSV-CD8(mem) showed a higher basal rate of proliferation in latently infected than noninfected tissue, which was associated with a reduced population of CD4+FoxP3+ regulatory T cells. Thus, the generation, maintenance, and function of memory CD8+ T cells is markedly influenced by latent virus.