Mandana Zandian

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.

MYC mediates large oncosome-induced fibroblast reprogramming in prostate cancer

Communication between cancer cells and the tumor microenvironment results in the modulation of complex signaling networks that facilitate tumor progression. Here, we describe a new mechanism of intercellular communication originating from large oncosomes (LO), which are cancer cell-derived, atypically large (1-10 μm) extracellular vesicles (EV). We demonstrate that, in the context of prostate cancer, LO harbor sustained AKT1 kinase activity, nominating them as active signaling platforms. Active AKT1 was detected in circulating EV from the plasma of metastatic prostate cancer patients and was LO specific. LO internalization induced reprogramming of human normal prostate fibroblasts as reflected by high levels of α-SMA, IL6, and MMP9. In turn, LO-reprogrammed normal prostate fibroblasts stimulated endothelial tube formation in vitro and promoted tumor growth in mice. Activation of stromal MYC was critical for this reprogramming and for the sustained cellular responses elicited by LO, both in vitro and in vivo in an AKT1-dependent manner. Inhibition of LO internalization prevented activation of MYC and impaired the tumor-supporting properties of fibroblasts. Overall, our data show that prostate cancer-derived LO powerfully promote establishment of a tumor-supportive environment by inducing a novel reprogramming of the stroma. This mechanism offers potential alternative options for patient treatment. Cancer Res; 77(9); 2306-17. ©2017 AACR.

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.

Optic neuritis in different strains of mice by a recombinant HSV-1 expressing murine interleukin-2.

PURPOSE The authors have shown previously that a recombinant HSV-1 that constitutively expresses two copies of murine IL-2 (HSV-IL-2) induces demyelination by activated CD8(+) T cells in the brain and spinal cord of ocularly infected female BALB/c mice. The present study was conducted to determine whether the ocular infection with this recombinant virus induces optic neuritis independent of virus dose, major histocompatibility complex (MHC) background, or sex. METHODS Female BALB/c, C57BL/6, SJL/6, and 129SVE mice and male BALB/c mice were ocularly infected with different doses of recombinant HSV-IL-2 virus. Demyelination of optic nerves in infected mice was monitored histologically using Luxol fast blue staining and by measurement of visual-evoked cortical potentials (VECPs). RESULTS Both focal and diffuse regions of demyelination of the optic nerves were observed in the HSV-IL-2-infected mice as early as day 10 after infection and as late as day 60 after infection (the final experimental time point) in all strains of mice tested. Optic nerve demyelination was not observed in control mice ocularly infected with HSV-IL-4 or wild-type HSV-1. VECP responses were delayed significantly in the HSV-IL-2-infected mice compared with mice infected with control viruses. CONCLUSIONS The results demonstrate for the first time that a combination of viral infection and constitutive expression of IL-2, but not IFN-gamma or IL-4, can result in demyelination and visual impairment in the optic nerves of ocularly 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.

Coregulatory Interactions among CD8α Dendritic Cells, the Latency-Associated Transcript, and Programmed Death 1 Contribute to Higher Levels of Herpes Simplex Virus 1 Latency

ABSTRACT The latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1), CD8α+ dendritic cells (DCs), and programmed death 1 (PD-1) have all been implicated in the HSV-1 latency-reactivation cycle. It is not known, however, whether an interaction between LAT and CD8α+ DCs regulates latency and T-cell exhaustion. To address this question, we used LAT-expressing [LAT(+)] and LAT-negative [LAT(−)] viruses. Depletion of DCs in mice ocularly infected with LAT(+) virus resulted in a reduction in the number of T cells expressing PD-1 in the trigeminal ganglia (TG), whereas depletion of DCs in mice similarly infected with LAT(−) virus did not alter PD-1 expression. CD8α+ DCs, but not CD4+ DCs, infected with LAT(+) virus had higher levels of ICP0, ICP4, thymidine kinase (TK), and PD-1 ligand 1 (PD-L1) transcripts than those infected with LAT(−) virus. Coculture of infected bone marrow (BM)-derived DCs from wild-type (WT) mice, but not infected DCs from CD8α−/− mice, with WT naive T cells contributed to an increase in PD-1 expression. Transfer of bone marrow from WT mice but not CD8α−/− mice to recipient Rag1−/− mice increased the number of latent viral genomes in reconstituted mice infected with the LAT(+) virus. Collectively, these data indicated that a reduction in latency correlated with a decline in the levels of CD8α+ DCs and PD-1 expression. In summary, our results demonstrate an interaction among LAT, PD-1, and CD11c CD8α+ cells that regulates latency in the TG of HSV-1-infected mice. IMPORTANCE Very little is known regarding the interrelationship of LAT, PD-1, and CD8α+ DCs and how such interactions might contribute to relative numbers of latent viral genomes. We show here that (i) in both in vivo and in vitro studies, deficiency of CD8α+ DCs significantly reduced T-cell exhaustion in the presence of LAT(+) virus but not LAT(−) virus; (ii) HSV-1 infectivity was significantly lower in LAT(−)-infected DCs than in their LAT(+)-infected counterparts; and (iii) adoptive transfer of bone marrow (BM) from WT but not CD8α−/− mice to recipient Rag1−/− mice restored latency to the level in WT mice following infection with LAT(+) virus. These studies point to a key role for CD8α+ DCs in T-cell exhaustion in the presence of LAT, which leads to larger numbers of latent viral genomes. Thus, altering this negative function of CD8α+ DCs can potentially be used to generate a more effective vaccine against HSV infection.

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.

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.

Batf3 deficiency is not critical for the generation of CD8α+ dendritic cells

Recently, we have reported that CD8α(+) DCs, rather than CD8(+) T cells, are involved in the establishment and maintenance of HSV-1 latency in the trigeminal ganglia (TG) of ocularly infected mice. In the current study, we investigated whether similar results can be obtained using Batf3(-/-) mice that previously were reported to lack CD8α(+) DCs. However, our results demonstrate that Batf3(-/-) mice, without any known infection, express CD8α(+) DCs. Consequently, due to the presence of CD8α(+) DCs, no differences were detected in the level of HSV-1 latency between Batf3(-/-) mice compared with wild type control mice.