In this study, we analyzed the phenotypic and physiological consequences of the interaction of plasmacytoid dendritic cells (pDCs) with human immunodeficiency virus type 1 (HIV-1). pDCs are one cellular target of HIV-1 and respond to the virus by producing alpha/beta interferon (IFN-alpha/beta) and chemokines. The outcome of this interaction, notably on the function of bystander myeloid DC (CD11c+ DCs), remains unclear. We therefore evaluated the effects of HIV-1 exposure on these two DC subsets under various conditions. Blood-purified pDCs and CD11c+ DCs were exposed in vitro to HIV-1, after which maturation markers, cytokine production, migratory capacity, and CD4 T-cell stimulatory capacity were analyzed. pDCs exposed to different strains of infectious or even chemically inactivated, nonreplicating HIV-1 strongly upregulated the expression of maturation markers, such as CD83 and functional CCR7, analogous to exposure to R-848, a synthetic agonist of toll-like receptor-7 and -8. In addition, HIV-1-activated pDCs produced cytokines (IFN-alpha and tumor necrosis factor alpha), migrated in response to CCL19 and, in coculture, matured CD11c+ DCs, which are not directly activated by HIV. pDCs also acquired the ability to stimulate naïve CD4+ T cells, albeit less efficiently than CD11c+ DCs. This HIV-1-induced maturation of both DC subsets may explain their disappearance from the blood of patients with high viral loads and may have important consequences on HIV-1 cellular transmission and HIV-1-specific T-cell responses.
<p>PDF file - 223K, Supplemental Figure 4: Activation of pDC in response to MV-infected tumor cells is independent of MV replication in pDC and infection by CD46. (A) pDC were cultured with IL-3 alone, R848, IL-3/MV-eGFP (MV), IL-3/MV-eGFP with 10mg/ml of anti-CD46 or IL-3/UV-irradiated MV-eGFP (MV*) at MOI=1 during 18hrs. Expression of CD83, CD80 and CD86 by pDC was determined by flow cytometry. (B) pDC were cultured with IL-3, UV-irradiated M18, MV-infected M18 (M18MV) in presence or absence of 10mg/ml of anti-CD46 (Hycult biotech), or MV-infected M18 irradiated by UV before exposition to pDC (M18MV*). Expression of CD83, CD80 and CD86 by pDC (gate on BDCA-4+/HLA-DR+ cells) was determined by flow cytometry. (C) IFN-a production by pDC measured by ELISA. (D) MV-eGFP infection inhibition of M18 by 10mg/ml of anti-CD46 monoclonal antibody after 72hrs of culture.</p>
<p>PDF file - 115K, Supplemental figure 3: Infection of pDC by MV-eGFP or UV-irradiated MV-eGFP: pDC in presence of IL-3 or Meso13 cells were cultured alone (NI) or with MV-eGFP (MV) or UV-irradiated (312nm - 100kj/m�) MV-eGFP (MV*) at MOI=50 during 72hrs (upper panel) or during 2hrs and then cultured during 70hrs (lower panel). Fluorescence was analyzed by flow cytometry.</p>
DCMU [N-(3,4-dichlorophenyl)-N-dimethylurea] or diuron is a widely used herbicide, which can cause adverse effects on human, especially on immune cells, due to their intrinsic properties and wide distribution. These cells are important for fighting not only against virus or bacteria but also against neoplastic cell development. We developed an approach that combines functional studies and miRNA and RNA sequencing data to evaluate the effects of DCMU on the human immune response against cancer, particularly the one carried out by CD8+ T cells. We found that DCMU modulates the expression of miRNA in a dose-dependent manner, leading to a specific pattern of gene expression and consequently to a diminished cytokine and granzyme B secretions. Using mimics or anti-miRs, we identified several miRNA, such as hsa-miR-3135b and hsa-miR-21-5p, that regulate these secretions. All these changes reduce the CD8+ T cells’ cytotoxic activity directed against cancer cells, in vitro and in vivo in a zebrafish model. To conclude, our study suggests that DCMU reduces T-cell abilities, participating thus to the establishment of an environment conducive to cancer development.
We reported previously that a large fraction of melanoma cell lines induced a suboptimal activation of specific CTL clones, characterized by good tumor cell lysis but no detectable IL-2 production. Using synthetic peptides, we demonstrated recently that this was due to expression of subthreshold levels of appropriate MHC-peptide complexes. We measure here by semiquantitative reverse transcription-PCR the expression of two melanoma Ag (NA17-A and Melan-A/MART-1) mRNAs in 13 melanoma cell lines and analyze the responses to these cell lines of specific HLA-A2-restricted CTL clones. In line with the idea that the density of MHC-antigenic peptide complexes on melanoma cells is a direct function of the Ag's mRNA level, we found that CTL lysis was grossly proportional to this level. We also established that a minimal level of transcription is required for melanoma cells to induce IL-2 secretion. Interestingly, all cell lines that expressed the Ag above this minimal level, either spontaneously or after gene transfection, stimulated the secretion by tumor-infiltrating lymphocyte of IL-2 amounts proportional to Ag expression unless they exhibited a defective expression of intracellular adhesion molecule-1 or LFA-3 molecules or a low expression of the restricting HLA element. These results indicate that optimal activation and therefore, doubtless, full functionality of melanoma-specific CTL clones critically depend on the mRNA level of the Ag in tumor cells and also on a minimal expression of the HLA restriction element, intracellular adhesion molecule-1, and LFA-3. These data provide a rationale for a better selection of patients to be included in Ag-specific immunization protocols.
<p>PDF file - 214K, Supplemental Figure 6: Monocyte-derived DC exhibit a similar level of NYESO-1 cross-presentation compared to pDC. (A) on day0, pDC and monocytes from a single donor were purified. pDC were frozen and monocytes cultured with IL-4 and GM-CSF. On day 5, pDC were thawed. pDC and monocyte-dervied DC were cultured alone or with UV-irradiated M18 (M18UV) or MV-infected M18 (M18MV) for 18hrs. On day6, DC were cultured with the NYESO-1 specific CD8+ T cell clone during 6hrs in presence of brefeldinA. Cells were then stained with anti-CD8+ antibody, fixed, permeabilized and stained with IFN-g specific antibody. IFN-g secretion by the the T cell clone was analyzed by flow cytometry. We conclude that level of cross-presentation was somewhat similar between the two cell types. It was a little bit weaker for pDC, but these cells have been frozen, whereas Monocyte-derived DC were not. (B) scatter plot representation of three cross-presentation experiments performed with monocyte-derived DC. Error bars represent standard deviation.</p>
