IL-2 Suppression of IL-12p70 by a Recombinant HSV-1 Expressing IL-2 Induces T-Cell Auto-Reactivity and CNS Demyelination

To evaluate the role of cellular infiltrates in CNS demyelination in immunocompetent mice, we have used a model of multiple sclerosis (MS) in which different strains of mice are infected with a recombinant HSV-1 expressing IL-2. Histologic examination of the mice infected with HSV-IL-2 demonstrates that natural killer cells, dendritic cells, B cells, and CD25 (IL-2rα) do not play any role in the HSV-IL-2-induced demyelination. T cell depletion, T cell knockout and T cell adoptive transfer experiments suggest that both CD8+ and CD4+ T cells contribute to HSV-IL-2-induced CNS demyelination with CD8+ T cells being the primary inducers. In the adoptive transfer studies, all of the transferred T cells irrespective of their CD25 status at the time of transfer were positive for expression of FoxP3 and depletion of FoxP3 blocked CNS demyelination by HSV-IL-2. The expression levels of IL-12p35 relative to IL-12p40 differed in BM-derived macrophages infected with HSV-IL-2 from those infected with wild-type HSV-1. HSV-IL-2-induced demyelination was blocked by injecting HSV-IL-2-infected mice with IL-12p70 DNA. This study demonstrates that suppression of the IL-12p70 function of macrophages by IL-2 causes T cells to become auto-aggressive. Interruption of this immunoregulatory axis results in demyelination of the optic nerve, the spinal cord and the brain by autoreactive T cells in the HSV-IL-2 mouse model of MS

Inclusion of CD80 in HSV Targets the Recombinant Virus to PD-L1 on DCs and Allows Productive Infection and Robust Immune Responses

CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 infection, we constructed a recombinant HSV-1 virus that expresses two copies of the CD80 gene in place of the latency associated transcript (LAT). This mutant virus (HSV-CD80) expressed high levels of CD80 and had similar virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental virus, this CD80 expressing recombinant virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 infection was mediated by CD80 binding to PD-L1 on DCs. This interaction also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC

Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles

Large oncosomes (LO) are atypically large (1-10 mu m diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep (TM)) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.1182Ysciescopu

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

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

Suppression of IL-12p70 formation by IL-2 or following macrophage depletion causes T-cell autoreactivity leading to CNS demyelination in HSV-1-infected mice.

We have established two mouse models of central nervous system (CNS) demyelination that differ from most other available models of multiple sclerosis (MS) in that they represent a mixture of viral and immune triggers. In the first model, ocular infection of different strains of mice with a recombinant HSV-1 that expresses murine IL-2 constitutively (HSV-IL-2) causes CNS demyelination. In the second model, depletion of macrophages causes CNS demyelination in mice that are ocularly infected with wild-type (WT) HSV-1. In the present study, we found that the demyelination in macrophage-intact mice infected with HSV-IL-2 was blocked by depletion of FoxP3-expressing cells, while concurrent depletion of macrophages restored demyelination. In contrast, demyelination was blocked in the macrophage-depleted mice infected with wild-type HSV-1 following depletion of FoxP3-expressing cells. In macrophage-depleted HSV-IL-2-infected mice, demyelination was associated with the activity of both CD4+ and CD8+ T cells, whereas in macrophage-depleted mice infected with WT HSV-1, demyelination was associated with CD4+ T cells. Macrophage depletion or infection with HSV-IL-2 caused an imbalance of T cells and TH1 responses as well as alterations in IL-12p35 and IL-12p40 but not other members of the IL-12 family or their receptors. Demyelination was blocked by adoptive transfer of macrophages that were infected with HSV-IL-12p70 or HSV-IL-12p40 but not by HSV-IL-12p35. These results indicate that suppression of IL-12p70 formation by IL-2 or following macrophage depletion causes T-cell autoreactivity leading to CNS demyelination in HSV-1-infected mice

Severity of CNS demyelination in macrophage recipient mice.

<p>The LFB-stained sections of the brains, spinal cords, and optic nerves of WT mice that received adoptively transferred macrophages from WT mice and analyzed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006401#ppat.1006401.t002" target="_blank">Table 2</a> were further analyzed in terms of the size of the demyelination plaques in the entire sections of brain, spinal cord, and optic nerves. Data are presented as mean demyelination using a total of 150 sections for brain and spinal cord and 30 sections for optic nerve from 5 mice per group. Arrows indicate no demyelination in the brain, spinal cord and optic nerve of mice received 1 X 10⁷ macrophages infected with HSV-IL-12p70 virus.</p

Role of FoxP3-expressing cells in HSV-induced CNS demyelination.

<p>Female FoxP3^DTR mice were depleted of FoxP3-expressing cells or depleted of both FoxP3-expressing cells and macrophages then ocularly infected with HSV-IL-2 or parental virus as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006401#ppat.1006401.g001" target="_blank">Fig 1</a>. Optic nerve, brain, and spinal cord were collected from euthanized mice on day 14 PI and post-fixed tissue sections were stained with LFB. Representative photomicrographs are shown (Magnification, 20_×; Size bar, 100 μm). Arrows indicate the areas of demyelination. Panels: A) FoxP3 depleted, HSV-IL-2 infected optic nerve; B) FoxP3 depleted, HSV-IL-2 infected brain; C) FoxP3 depleted, HSV-IL-2 infected spinal cord; D) FoxP3 depleted, parental virus infected optic nerve; E) FoxP3 depleted, parental virus infected brain; F) FoxP3 depleted, parental virus infected spinal cord; G) FoxP3 and Macrophage depleted, HSV-IL-2 infected optic nerve; H) FoxP3 and Macrophage depleted, HSV-IL-2 infected brain; I) FoxP3 and Macrophage depleted, HSV-IL-2 infected spinal cord; J) FoxP3 and Macrophage depleted, parental virus infected optic nerve; K) FoxP3 and Macrophage depleted, parental virus infected brain; and L) FoxP3 and Macrophage depleted, parental virus infected spinal cord.</p

Role of macrophages in HSV-induced CNS demyelination.

<p>Female FoxP3^DTR mice were depleted of macrophages or mock-depleted and infected ocularly with HSV-IL-2 or parental virus (dLAT2903) (5 mice per group). Optic nerve, brain, and spinal cord were collected from euthanized mice on day 14 PI and post-fixed tissue sections were stained with LFB. Representative photomicrographs are shown (Magnification, 20_×; Size bar, 100 μm). Arrows indicate the areas of demyelination (a plaque). Panels: A) No depletion, HSV-IL-2 infected optic nerve; B) No depletion, HSV-IL-2 infected brain; C) No depletion, HSV-IL-2 infected spinal cord; D) No depletion, parental virus infected optic nerve; E) No depletion, parental virus infected brain; F) No depletion, parental virus infected spinal cord; G) Macrophage depletion, HSV-IL-2 infected optic nerve; H) Macrophage depletion, HSV-IL-2 infected brain; I) Macrophage depletion, HSV-IL-2 infected spinal cord; J) Macrophage depletion, parental virus infected optic nerve; K) Macrophage depletion, parental virus infected brain; and L) Macrophage depletion, parental virus infected spinal cord.</p