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-␣ (IFN-␣) upon virus exposure; however, the induction of IFN-␣ by HTLV-1 remains unknown. We demonstrate here that cellfree HTLV-1 generated a pDC innate immune response by producing massive levels of IFN-␣ 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" IFNproducing 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-␣ 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. (Blood. 2010;115:2177-2185) IntroductionHuman T-cell leukemia virus 1 (HTLV-1), the first characterized human retrovirus, 1 has been identified as the causative agent for adult T-cell leukemia/lymphoma (ATLL) 2,3 and HTLV-1-associated myelopathy/tropical spastic paraparesis, 4 uveitis, and infective dermatitis in children. 5 HTLV-1 virions infect CD4 ϩ T cells, which represent the main target for HTLV-1 infection in peripheral blood. HTLV-1-associated diseases occur after long periods of virus latency. 6 For years it has been thought that unlike other retroviruses, free virions were poorly infectious. 7 However, Jones et al 8 recently reported that freshly isolated myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) are efficiently and productively infected by cell-free HTLV-1. Furthermore, infected mDCs and pDCs were able to transfer virions to autologous CD4 ϩ T cells, clearly demonstrating that cell-free HTLV-1 can be infectious and target DCs. 8 pDCs participate in innate and adaptive immunity, 9,10 are located in blood and lymphoid organs, 10,11 and produce up to 1000-fold more interferon-␣ (IFN-␣) than other cell types in response to virus exposure. 12 Three molecules have been characterized for HTLV-1 entry into cells, heparan sulfate proteoglycans 13 and BDCA-4 (also called neuropilin-1) 14 for the initial virus binding to target cells 15 and glucose transporter 1 for the postattachment and the viral fusion. 16,17 Interestingly, BDCA-4 is expressed by mDC and T cells 18,19 but cells expressing the greatest level of BDCA...

We demonstrate that, in response to HIV, pDCs from controller patients produce IFN-α, express membrane TRAIL, and induce apoptosis of T-cell lines.

Antigen persistence in chronic infections

The first step of HIV infection involves the interaction of the gp120 envelope glycoprotein to its receptor CD4, mainly expressed on CD4+ T cells. Besides its role on HIV-1 entry, the gp120 has been shown to be involved in the production of IL-1, IL-6, CCL20 and other innate response cytokines by bystander, uninfected CD4+ T cells and monocytes. However, the gp120 determinants involved in these functions are not completely understood. Whether signalling leading to cytokine production is due to CD4 or other receptors is still unclear. Enhanced chemokine receptor binding and subsequent clustering receptors may lead to cytokine production. By using a comprehensive panel of gp120 mutants, here we show that CD4 binding is mandatory for cytokine outburst in monocytes. Our data suggest that targeting monocytes in HIV-infected patients might decrease systemic inflammation and the potential tissue injury associated with the production of inflammatory cytokines. Understanding how gp120 mediates a cytokine burst in monocytes might help develop new approaches to improve the chronic inflammation that persists in these patients despite effective suppression of viremia by antiretroviral therapy.

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.

No abstract

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 upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects. Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10Rα and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 °C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

One of the major impediments of HIV infection is the abnormally high levels of immune activation that contributes to immune dysregulation and impairs viral clearance. While IFNγ secretion is used as a marker of the functional state of T lymphocytes and NK cells, its role in regulating immune responses is poorly understood. We isolated CD8-depleted PBMCs from HIV infected individuals (n=13). We measured proliferation and cytokine production by HIV-specific CD4 T cells stimulated with HIV Gag in the presence of isotype control, anti-IFNγ and/or anti-PD-L1. We performed apoptosis measurements using Annexing-V binding assays. We also determined the modulatory impact of IFNγ on APCs by measuring expression of PD-1 ligands, HLA-DR, HLA-I and IL-12 secretion. Neutralization of IFNγ produced by Gag stimulated CD4 T cells enhanced proliferation (p=0.0161) and reduced apoptosis of HIV-specific CD4 T cells. IFNγ blockade enhanced IL-13 secretion (p=0.0039) but had no effect on IL-2, IL-10 and TNFα levels. IFNγ induced strong up-regulation of PD-L1, HLA-DR and HLA-I on monocytes but not on B, T and NK cells. IFNγ also induced IL-12 secretion by APCs that acted in a positive feedback loop to further enhance IFNγ secretion. Concurrent blockade of IFNγ and PD-L1 led to a more prominent increase in proliferation of HIV-specific CD4 T cells than blockade of individual pathways. These data provide mechanistic insight on the causal role of IFNγ in T-helper dysregulation in HIV infection.