in-vitro-assay-to-evaluate-impact-immunoregulatory-pathways-on-hiv

<em>In Vitro</em> Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

The Ragon Institute of MGH, MIT and Harvard

Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM)

A recent report demonstrated that free human T-cell leukemia virus 1 (HTLV-1) could infect plasmacytoid dendritic cells (pDCs). The major role of pDCs is to secrete massive levels of interferon-alpha (IFN-alpha) upon virus exposure; however, the induction of IFN-alpha by HTLV-1 remains unknown. We demonstrate here that cell-free HTLV-1 generated a pDC innate immune response by producing massive levels of IFN-alpha that were inhibited by anti-HTLV-1 antibodies. HTLV-1 induced costimulatory molecules and rapid expression of the apoptotic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Furthermore, HTLV-1 stimulated pDC-induced apoptosis of CD4(+) T cells expressing DR5, transforming pDCs into IFN-producing killer pDCs. We also observed that an endosomal acidification inhibitor and a Toll-like receptor-7 (TLR7)-specific blocker drastically inhibited pDC response to HTLV-1. Three-dimensional microscopy analysis revealed that unstimulated pDCs were "dormant" IFN-producing killer pDCs with high levels of intracellular TRAIL that could be rapidly mobilized to the surface in response to TLR7 activation. Inhibition of viral degradation in endosomes by chloroquine maintained viral integrity, allowing virus detection by 3-dimensional microscopy. We demonstrate that pDCs respond to cell-free HTLV-1 by producing high levels of IFN-alpha and by mobilizing TRAIL on cell surface after TLR7 triggering. This is the first demonstration of an innate immune response induced by free HTLV-1.

Free HTLV-1 induces TLR7-dependent innate immune response and TRAIL relocalization in killer plasmacytoid dendritic cells.

Activation-induced cell death is a natural process that prevents tissue damages from over-activated immune cells. TNF-Related apoptosis ligand (TRAIL), a TNF family member, induces apoptosis of infected and tumor cells by binding to one of its two death receptors, DR4 or DR5. TRAIL was reported to be secreted by phytohemagglutinin (PHA)-stimulated CD4(+) T cells in microvesicles.We investigate here TRAIL and DR5 regulation by activated primary CD4(+) T cells and its consequence on cell death. We observed that PHA induced CD4(+) T cell apoptosis in a dose-dependent manner. Thus, we investigated molecules involved in PHA-mediated cell death and demonstrated that TRAIL and DR5 were over-expressed on the plasma membrane of PHA-stimulated CD4(+) T cells. Surprisingly, DR5 was constitutively expressed in naive CD4(+) T cells at messenger RNA (mRNA) and protein levels. Thus, using 3 dimensional microscopy and intracellular staining assays, we show that DR5 is constitutively expressed in CD4(+) T cells and is pre-stocked in the cytoplasm. When cells are stimulated by PHA, DR5 is relocalized from cytoplasm to plasma membrane. Small interference RNA (siRNA) and blocking antibody assays demonstrate that TRAIL/DR5 interaction is mainly responsible for PHA-mediated CD4(+) T cell apoptosis. Thus, membrane DR5 expression leading to TRAIL-mediated apoptosis may represent one of the pathways responsible for eradication of over-activated CD4(+) T cells during immune responses.

Three-dimensional microscopy characterization of death receptor 5 expression by over-activated human primary CD4+ T cells and apoptosis.

Human immunodeficiency virus (HIV) controllers spontaneously control viremia and CD4 T-cell depletion in contrast to viremic patients. After HIV exposure, plasmacytoid dendritic cells (pDCs) produce high levels of interferon alpha (IFN-Î±) and express the apoptotic ligand TRAIL (tumor necrosis factor-related apoptosis inducing ligand). Simian models have shown that prolonged high levels of IFN-Î± production could be responsible for AIDS progression.

Lucie Barblu

The Ragon Institute of MGH, MIT and Harvard

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function