Local innate and adaptive immune responses regulate inflammatory cell influx into the lungs after vaccination with formalin inactivated RSV

Inactivated respiratory syncytial virus (RSV) vaccines tend to predispose for immune mediated enhanced disease, characterized by Th2 responses and airway hypersensitivity reactions. We show in a C57BL/6 mouse model that the early innate response elicited by the challenge virus (RSV versus influenza virus) influences the outcome of the Th1/Th2 balance in the lung after intramuscular priming with inactivated vaccine. Priming of CD4(+)/IFN-gamma(+) T cells by mature dendritic cells administered intravenously and/or priming of a virus specific CD8(+) T cell response ameliorated the Th2-mediated inflammatory response in the lung, suggesting that vaccination procedures are feasible that prevent vaccine induced immune pathology. (C) 2011 Elsevier Ltd. All rights reserved

A secondary role for hypoxia and HIF1 in the regulation of (IFNγ-induced) PD-L1 expression in melanoma

Cancer cells are able to escape immune surveillance by upregulating programmed death ligand 1 (PD-L1). A key regulator of PD-L1 expression is transcriptional stimulation by the IFNγ/JAK/STAT pathway. Recent studies suggest that hypoxia can induce PD-L1 expression. As hypoxia presents a hallmark of solid tumor development, hypoxic control of PD-L1 expression may affect the efficacy of cancer immunotherapy. This study aims to explore the hypoxic regulation of PD-L1 expression in human melanoma, and its interaction with IFNγ-induced PD-L1 expression. Analysis of the cutaneous melanoma dataset from the cancer genome atlas revealed a significant correlation of the HIF1-signaling geneset signature with PD-L1 mRNA expression. However, this correlation is less pronounced than other key pathways known to control PD-L1 expression, including the IFNγ/JAK/STAT pathway. This secondary role of HIF1 in PD-L1 regulation was confirmed by analyzing single-cell RNA-sequencing data of 33 human melanoma tissues. Interestingly, PD-L1 expression in these melanoma tissues was primarily found in macrophages. However, also in these cells STAT1, and not HIF1, displayed the most pronounced correlation with PD-L1 expression. Moreover, we observed that hypoxia differentially affects PD-L1 expression in human melanoma cell lines. Knockdown of HIF1 expression indicated a minor role for HIF1 in regulating PD-L1 expression. A more pronounced influence of hypoxia was found on IFNγ-induced PD-L1 mRNA expression, which is controlled at a 952 bp PD-L1 promoter fragment. These findings, showing the influence of hypoxia on IFNγ-induced PD-L1 expression, are relevant for immunotherapy, as both IFNγ and hypoxia are frequently present in the tumor microenvironment

Immunophenotypic Analysis Reveals Differences in Circulating Immune Cells in the Peripheral Blood of Patients with Segmental and Nonsegmental Vitiligo

Accumulating studies have indicated immune-based destruction of melanocytes in both segmental vitiligo (SV) and non-SV (NSV). Whereas SV often occurs unilaterally during childhood and stabilizes after an initial period of activity, the disease course of NSV is usually slowly progressive, with new lesions occurring bilaterally during life. This suggests an involvement of distinct pathophysiology pathways, specifically increased systemic immune activation in patients with NSV but not in patients with SV. This research aimed to identify the differences in immune cells in the blood of patients with SV and NSV through immunophenotyping of circulating cells. Regulatory T cells were unaffected in patients with SV compared with that in healthy controls but decreased in patients with NSV. In patients with NSV, the reduction in regulatory T cells was associated with the presence of other systemic autoimmune comorbidities, which were less present in SV. Similarly, the absence of a melanocyte-specific antibody response in patients with SV suggests less involvement of B-cell immunity in SV. These data show that in contrast to patients with NSV, no increased systemic immunity is found in patients with SV, indicating that SV pathogenesis is associated with a localized cytotoxic reaction targeting epidermal melanocytes

Serum antibodies critically affect virus-specific CD4+/CD8+ T cell balance during respiratory syncytial virus infections

Following infection with respiratory syncytial virus (RSV), reinfection in healthy individuals is common and presumably due to ineffective memory T cell responses. In peripheral blood of healthy adults, a higher CD4(+)/CD8(+) memory T cell ratio was observed compared with the ratio of virus-specific effector CD4(+)/CD8(+) T cells that we had found in earlier work during primary RSV infections. In mice, we show that an enhanced ratio of RSV-specific neutralizing to nonneutralizing Abs profoundly enhanced the CD4(+) T cell response during RSV infection. Moreover, FcγRs and complement factor C1q contributed to this Ab-mediated enhancement. Therefore, the increase in CD4(+) memory T cell response likely occurs through enhanced endosomal Ag processing dependent on FcγRs. The resulting shift in memory T cell response was likely amplified by suppressed T cell proliferation caused by RSV infection of APCs, a route important for Ag presentation via MHC class I molecules leading to CD8(+) T cell activation. Decreasing memory CD8(+) T cell numbers could explain the inadequate immunity during repeated RSV infections. Understanding this interplay of Ab-mediated CD4(+) memory T cell response enhancement and infection mediated CD8(+) memory T cell suppression is likely critical for development of effective RSV vaccine

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies

<div><p>Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14⁺ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.</p> </div

RSV-specific antibodies inhibit RSV-induced IFN-α production in PBMC, but enhance IFN-α production in CD14⁺ cell depleted PBMC.

<p>(<b>A</b>) Inhibition of RSV infection in pDC. Lineage^neg., MHC-II^high, BDCA-4⁺ pDC are partially infected with rgRSV224 after a period of 20 hours. Infection is blocked after UV inactivation, after neutralization in 10% fresh human serum or in 5µg/ml Palivizumab. Similar results were obtained with sera from all donors used during our studies. IFN-α in supernatant of RSV-A2 exposed PBMC-CD14⁺ cell cultures (<b>B</b>) and rgRSV224 exposed PBMC-CD14⁺ cells or PBMC, (<b>C</b>) was measured after 20 hrs. The role of virus specific antibodies on the cytokine response was tested by removing IgGs from AS with protein G Sepharose^® beads and reconstitution with 2 mg/ml IVIG (<b>B</b>) or 5 µg/ml palivizumab (<b>B</b>, <b>C</b>). Experiments represent the mean ± SEM of experiments performed in 4 different donors and were analyzed using one way ANOVA followed by a Bonferroni post-test, **P <0.01.</p

CD14⁺ monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC.

<p>(<b>A</b>) IFN-α production in CD14⁺ cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking endosomal acidification with 50nM Bafilomycin A₁. (<b>B</b>) Ab-RSV-induced IFN-α production in CD14⁺ cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. (<b>C</b>) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3^neg., CD14^neg., CD19^neg., CD16^neg., CD56^neg., Lin-1), MHC-II^high, BDCA-4⁺ cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. (<b>D</b>) Purified pDC (obtained by negative selection removing CD3⁺, CD19⁺ and CD16⁺ cells from fresh PBMC, followed by FACS purification of the BDCA-4⁺ cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. (<b>E</b>) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). (<b>F</b>) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P< 0.05, **P <0.01. Experiments were performed in 3 different donors with similar results.</p

IFN-α production induced by live RSV in PBMC depends on IFNAR signalling.

<p>PBMC were exposed to live RSV in the presence of interferon-α/β receptor (IFNAR) blocking antibody (5µg/ml), isotype control Ab or no Ab. Levels of IFN-α were determined via intracellular staining (<b>A</b>) or in 20hrs. supernatant by ELISA (<b>B</b>). IFN-α was trapped intracellular by BFA treatment initiated 6 hrs. after RSV infection, or at t=0 for the ODN 2216 control stimulus, because of different kinetics of anti-viral and ODN elicited IFN-α response. For both stimuli, intracellular staining for IFN-α was performed in CD3^neg., CD16^neg., CD19^neg., CD56^neg., MHCII⁺, BDCA-4⁺ cells after 10 hrs. BFA treatment. Experiments were performed in 3 different donors with similar results. Data represent the mean ± SEM of triplicate measurements within 1 donor and were analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, **P <0.01.</p

Virus specific polyclonal antibodies in human serum increase RSV binding to monocytes and B cells.

<p>(<b>A</b>) The effect of autologous serum, IgG-depleted serum and palivizumab in FCS on binding of RSV to monocytes, B cells and NK cells was measured after 1 hour incubation at 4°C. (<b>B</b>) Polyclonal antibodies (IVIG) increase binding of RSV to CD14^+/CD16^neg. cells. Data shown represent the mean ± SEM of triplicate measurements in two different donors (for A and B) and were analyzed using the Kruskal-Wallis test followed by Dunn's Multiple Comparison analysis, *P< 0.05, **P <0.01. Experiments were performed in 3 additional donors with similar results.</p