Suppression of CD80 expression by ICP22 affect HSV-1 replication and CD8 + IFNγ + infiltrates in the eye of infected mice but not latency-reactivation

Previously, we reported that HSV-1 ICP22 binds to the CD80 promoter and suppresses its expression in vitro and in vivo . To better understand the impact of ICP22 binding to CD80 on HSV-1 infectivity and pathogenicity, we mapped the region of ICP22 required to bind the CD80 promoter to a 40 aa region of ICP22. We constructed a recombinant HSV-1 expressing a truncated form of ICP22 that lacks these 40 aa, that does not bind to the CD80 promoter (KOS-ICP22Δ40) and retains the ability to replicate efficiently in rabbit skin cells, in contrast to ICP22-null virus. Replication of this recombinant virus in vitro and in vivo was higher than the ICP22-null virus but virus replication kinetics were lower than WT control virus. Similar to ICP22-null virus, the KOS-ICP22Δ40 mutant virus increased CD80 expression in DCs and IFNγ expression in CD8 + T cells but not CD4 + T cells in infected mouse corneas. In contrast to significantly reduced virus replication in the eyes of ocularly infected mice, levels of latency-reactivation were similar between KOS-ICP22Δ40 virus and WT virus. Thus, blocking ICP22 binding to the CD80 promoter using a recombinant virus expressing a truncated ICP22 that lacks CD80 promoter binding, appears to reduce virus replication and enhance CD8 + IFNγ + infiltrates in corneas of infected mice, with no effect on latency-reactivation. IMPORTANCE Direct binding of HSV-1 ICP22 to the CD80 promoter downregulates expression of the costimulatory molecule CD80, but not CD86. In this study we fine mapped the region of ICP22 required for binding to the CD80 promoter and constructed a recombinant virus containing a deletion in ICP22 that failed to bind to CD80 promoter. This recombinant virus replicated less efficiently in vitro and in vivo than did WT control virus although CD80-expressing CD11c + cells and IFNγ-expressing CD8 + T cells were increased. Interestingly, levels of latency and reactivation in the two viruses were similar, despite lower virus replication in the eyes of infected mice. Therefore, blocking the interaction of ICP22 to the CD80 promoter could be used to temper the immune response.

IL-15/IL-15Rα/CD80-expressing AML cell vaccines eradicate minimal residual disease in leukemic mice

Engineered autologous acute myeloid leukemia (AML) cells present multiple leukemia-associated and patient-specific antigens and as such hold promise as immunotherapeutic vaccines. However, prior vaccines have not reliably induced effective antileukemic immunity, in part because AML blasts have immune inhibitory effects and lack expression of the critical costimulatory molecule CD80. To enhance induction of leukemia-specific cytolytic activity, 32Dp210 murine AML cells were engineered to express either CD80 alone, or the immunostimulatory cytokine interleukin-15 (IL-15) with its receptor α (IL-15Rα), or heterodimeric IL-15/IL-15Rα together with CD80 and tested as irradiated cell vaccines. IL-15 is a γc-chain cytokine, with unique properties suited to stimulating antitumor immunity, including stimulation of both natural killer and CD8+ memory T cells. Coexpression of IL-15 and IL-15Rα markedly increases IL-15 stability and secretion. Non-tumor-bearing mice vaccinated with irradiated 32Dp210-IL-15/IL-15Rα/CD80 and challenged with 32Dp210 leukemia had greater survival than did mice treated with 32Dp210-CD80 or 32Dp210-IL-15/IL-15Rα vaccines, whereas no unvaccinated mice inoculated with leukemia survived. In mice with established leukemia, treatment with 32Dp210-IL-15/IL-15Rα/CD80 vaccination stimulated unprecedented antileukemic immunity enabling 80% survival, an effect that was abrogated by anti-CD8 antibody-mediated depletion in vivo. Because, clinically, AML vaccines are administered as postremission therapy, we established a novel model in which mice with high leukemic burdens were treated with cytotoxic therapy to induce remission (<5% marrow blasts). Postremission vaccination with 32Dp210-IL-15/IL-15Rα/CD80 achieved 50% overall survival in these mice, whereas all unvaccinated mice achieving remission subsequently relapsed. These studies demonstrate that combined expression of IL-15/IL-15Rα and CD80 by syngeneic AML vaccines stimulates effective and long-lasting antileukemic immunity.

Herpes Simplex Virus 1 ICP22 Suppresses CD80 Expression by Murine Dendritic Cells

ABSTRACTHerpes simplex virus type 1 (HSV-1) has the ability to delay its clearance from the eye during ocular infection. Here, we show that ocular infection of mice with HSV-1 suppressed expression of the costimulatory molecule CD80 but not CD86 in the cornea. The presence of neutralizing anti-HSV-1 antibodies did not alleviate this suppression. At the cellular level, HSV-1 consistently downregulated the expression of CD80 by dendritic cells (DCs) but not by other antigen-presenting cells. Furthermore, flow cytometric analysis of HSV-1-infected corneal cells during a 7-day period reduced CD80 expression in DCs but not in B cells, macrophages, or monocytes. This suppression was associated with the presence of virus. Similar results were obtained using infected or transfected spleen cells or bone marrow-derived DCs. A combination of roscovitine treatment, transfection with immediate early genes (IE), and infection with a recombinant HSV-1 lacking the ICP22 gene shows the importance of ICP22 in downregulation of the CD80 promoter but not the CD86 promoterin vitroandin vivo. At the mechanistic level, we show that the HSV-1 immediate early gene ICP22 binds the CD80 promoter and that this interaction is required for HSV-1-mediated suppression of CD80 expression. Conversely, forced expression of CD80 by ocular infection of mice with a recombinant HSV-1 exacerbated corneal scarring in infected mice. Taken together, these studies identify ICP22-mediated suppression of CD80 expression in dendritic cells as central to delayed clearance of the virus and limitation of the cytopathological response to primary infection in the eye.IMPORTANCEHSV-1-induced eye disease is a major public health problem. Eye disease is associated closely with immune responses to the virus and is exacerbated by delayed clearance of the primary infection. The immune system relies on antigen-presenting cells of the innate immune system to activate the T cell response. We found that HSV-1 utilizes a robust and finely targeted mechanism of local immune evasion. It downregulates the expression of the costimulatory molecule CD80 but not CD86 on resident dendritic cells irrespective of the presence of anti-HSV-1 antibodies. The effect is mediated by direct binding of HSV-1 ICP22, the product of an immediate early gene of HSV-1, to the promoter of CD80. This immune evasion mechanism dampens the host immune response and, thus, reduces eye disease in ocularly infected mice. Therefore, ICP22 may be a novel inhibitor of CD80 that could be used to modulate the immune response.

Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors

B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 × 1011/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule CD80 not only on infected CLL cells but also on noninfected bystander leukemia B cells, whereas this effect induced specific proliferation of HLA-matched allogeneic T cells. Vaccination strategies for patients with B-CLL using leukemia cells infected ex vivo by rAAV vectors now seems possible in the near future.