The immune modulatory role of interferon lambda on human B-cell functions

Interferon lambda (IFN-L) mediates a crucial antiviral response to protect the host cells during viral infection, as well as functioning as a potential immune modulator. In this thesis, we investigated the immune modulatory role of IFN-L in B-cells. In the first part, we established an ELISA-based in vitro assays to study IFN-L ligands and its receptor (Interferon lambda receptor 1 - IFNLR1 and IL10R2) interactions. First, we determined the receptor - ligand (IFNLR1 - IFN-L1-3) dissociation constant (KD) as a measure of the ligand and receptor binding affinity. We found that IFN-L1 showed higher binding affinity to IFNLR1 compared to IFN-L2 and IFN-L3. Further, we screened antagonistic peptides, which act to interfere in IFNLR1 - IFN-Ls interactions. The peptides are designed to compete with IFN-Ls at their IFNLR1 binding sites and this experiment thus allowed us to develop a molecular understanding of the interaction. We have also performed the small molecules screen to identify the potential substances targeting for IFN-L signaling, it will allow us the modulation of IFN-L signaling which is an interesting target for a broad range of applications. In the second part, we screened the immune cell populations to understand the direct response to IFN-L, to resolve discrepancies with previously reported data. B-cells and the subpopulations of naïve, class switched and non-class switched memory B-cells were found to directly respond to IFN-Ls. On the other hand, T-cells, NK-cells and monocytes did not show any response to IFN-Ls. Since B-cells showed a response to IFN-L, we performed transcriptomic profiling of sorted B-cell, to examine the immune modulatory role of IFN-L in B-cells. On the basis of B-cell transcriptome analysis and follow up in vitro experiments, the IFN-L increases the mTORC1 (mammalian/mechanistic target of rapamycin complex 1) activity in B-cells, upon B-cell receptor (BCR) cross linking with anti-IgM. The BCR and IFN-L signaling cascade engage the mTORC1 pathway via phosphoinositide 3-kinase (PI3K). However, it needs further evaluation to see if IFN-L increase the mTORC1 activity indirectly via ISGs. IFN-L enhances the BCR-induced cell cycle progress though this mTORC1 and IFN-L alone did not induce any cell proliferation. Consequently IFN-L further boosts the differentiation of naïve B-cells into plasmablasts upon BCR-activation, so the cells gain effector functions such as cytokines release (IL-6, IL-10) and antibody production (IgM). The role of IFN-L in plasmablast differentiation was previously not known. In this study, we have shown how IFN-L functionally binds to B-cells and that it systematically boosts the differentiation of naïve B-cells into plasmablasts via mTORC1 and cell cycle progression in BCR-activated cells

Self-associated molecular patterns mediate cancer immune evasion by engaging Siglecs on T cells

© 2018, American Society for Clinical Investigation. This article has been published in final form at https://doi.org/10.1172/JCI120612First-generation immune checkpoint inhibitors, including anti-CTLA-4 and anti-programmed death 1 (anti-PD-1) antibodies, have led to major clinical progress, yet resistance frequently leads to treatment failure. Thus, new targets acting on T cells are needed. CD33-related sialic acid-binding immunoglobulin-like lectins (Siglecs) are pattern-recognition immune receptors binding to a range of sialoglycan ligands, which appear to function as self-associated molecular patterns (SAMPs) that suppress autoimmune responses. Siglecs are expressed at very low levels on normal T cells, and these receptors were not until recently considered as interesting targets on T cells for cancer immunotherapy. Here, we show an upregulation of Siglecs, including Siglec-9, on tumor-infiltrating T cells from non-small cell lung cancer (NSCLC), colorectal, and ovarian cancer patients. Siglec-9-expressing T cells coexpressed several inhibitory receptors, including PD-1. Targeting of the sialoglycan-SAMP/Siglec pathway in vitro and in vivo resulted in increased anticancer immunity. T cell expression of Siglec-9 in NSCLC patients correlated with reduced survival, and Siglec-9 polymorphisms showed association with the risk of developing lung and colorectal cancer. Our data identify the sialoglycan-SAMP/Siglec pathway as a potential target for improving T cell activation for immunotherapy.Peer reviewe

Antigen Extraction and B Cell Activation Enable Identification of Rare Membrane Antigen Specific Human B Cells

Determining antigen specificity is vital for understanding B cell biology and for producing human monoclonal antibodies. We describe here a powerful method for identifying B cells that recognize membrane antigens expressed on cells. The technique depends on two characteristics of the interaction between a B cell and an antigen-expressing cell: antigen-receptor-mediated extraction of antigen from the membrane of the target cell, and B cell activation. We developed the method using influenza hemagglutinin as a model viral membrane antigen, and tested it using acetylcholine receptor (AChR) as a model membrane autoantigen. The technique involves co-culturing B cells with adherent, bioorthogonally labeled cells expressing GFP-tagged antigen, and sorting GFP-capturing, newly activated B cells. Hemagglutinin-specific B cells isolated this way from vaccinated human donors expressed elevated CD20, CD27, CD71, and CD11c, and reduced CD21, and their secreted antibodies blocked hemagglutination and neutralized viral infection. Antibodies cloned from AChR-capturing B cells derived from patients with myasthenia gravis bound specifically to the receptor on cell membrane. The approach is sensitive enough to detect antigen-specific B cells at steady state, and can be adapted for any membrane antigen

Model-based inference of neutralizing antibody avidities against influenza virus

To assess the response to vaccination, quantity (concentration) and quality (avidity) of neutralizing antibodies are the most important parameters. Specifically, an increase in avidity indicates germinal center formation, which is required for establishing long-term protection. For influenza, the classical hemagglutination inhibition (HI) assay, however, quantifies a combination of both, and to separately determine avidity requires high experimental effort. We developed from first principles a biophysical model of hemagglutination inhibition to infer IgG antibody avidities from measured HI titers and IgG concentrations. The model accurately describes the relationship between neutralizing antibody concentration/avidity and HI titer, and explains quantitative aspects of the HI assay, such as robustness to pipetting errors and detection limit. We applied our model to infer avidities against the pandemic 2009 H1N1 influenza virus in vaccinated patients (n = 45) after hematopoietic stem cell transplantation (HSCT) and validated our results with independent avidity measurements using an enzyme-linked immunosorbent assay with urea elution. Avidities inferred by the model correlated with experimentally determined avidities (ρ = 0.54, 95% CI = [0.31, 0.70], P < 10−4). The model predicted that increases in IgG concentration mainly contribute to the observed HI titer increases in HSCT patients and that immunosuppressive treatment is associated with lower baseline avidities. Since our approach requires only easy-to-establish measurements as input, we anticipate that it will help to disentangle causes for poor vaccination outcomes also in larger patient populations. This study demonstrates that biophysical modelling can provide quantitative insights into agglutination assays and complement experimental measurements to refine antibody response analyses.ISSN:1553-7374ISSN:1553-736

Interferon-λ Enhances the Differentiation of Naive B Cells into Plasmablasts via the mTORC1 Pathway

Type III interferon (interferon lambda [IFN-λ]) is known to be a potential immune modulator, but the mechanisms behind its immune-modulatory functions and its impact on plasmablast differentiation in humans remain unknown. Human B cells and their subtypes directly respond to IFN-λ. Using B cell transcriptome profiling, we investigate the immune-modulatory role of IFN-λ in B cells. We find that IFN-λ-induced gene expression in B cells is steady, prolonged, and importantly, cell type specific. Furthermore, IFN-λ enhances the mTORC1 (mammalian/mechanistic target of rapamycin complex 1) pathway in B cells activated by the B cell receptor (BCR/anti-IgM). Engagement of mTORC1 by BCR and IFN-λ induces cell-cycle progress in B cells. Subsequently, IFN-λ boosts the differentiation of naive B cells into plasmablasts upon activation, and the cells gain effector functions such as cytokine release (IL-6 and IL-10) and antibody production. Our study shows how IFN-λ systematically boosts the differentiation of naive B cells into plasmablasts by enhancing the mTORC1 pathway and cell-cycle progression in activated B cells.ISSN:2666-3864ISSN:2211-124

Proteotype profiling unmasks a viral signalling network essential for poxvirus assembly and transcriptional competence

To orchestrate context-dependent signalling programmes, poxviruses encode two dual-specificity enzymes, the F10 kinase and the H1 phosphatase. These signalling mediators are essential for poxvirus production, yet their substrate profiles and systems-level functions remain enigmatic. Using a phosphoproteomic screen of cells infected with wild-type, F10 and H1 mutant vaccinia viruses, we systematically defined the viral signalling network controlled by these enzymes. Quantitative cross-comparison revealed 33 F10 and/or H1 phosphosites within 17 viral proteins. Using this proteotype dataset to inform genotype-phenotype relationships, we found that H1-deficient virions harbour a hidden hypercleavage phenotype driven by reversible phosphorylation of the virus protease I7 (S134). Quantitative phosphoproteomic profiling further revealed that the phosphorylation-dependent activity of the viral early transcription factor, A7 (Y367), underlies the transcription-deficient phenotype of H1 mutant virions. Together, these results highlight the utility of combining quantitative proteotype screens with mutant viruses to uncover proteotype-phenotype-genotype relationships that are masked by classical genetic studies

Association of host factors with antibody response to seasonal influenza vaccination in allogeneic hematopoietic stem cell transplant (HSCT) patients

BACKGROUND Influenza vaccination efficacy is reduced after hematopoietic stem cell transplantation (HSCT) and patient factors determining vaccination outcomes are still poorly understood. METHODS We investigated the antibody response to seasonal influenza vaccination in 135 HSCT patients and 69 healthy volunteers (HVs) in a prospective observational multicenter cohort study. We identified patient factors associated with hemagglutination inhibition titers against A/California/2009/H1N1, A/Texas/2012/H3N2, and B/Massachusetts/2012 by multivariable regression on the observed titer levels and on seroconversion/seroprotection categories for comparison. RESULTS Both regression approaches yield consistent results but regression on titers estimated associations with higher precision. HSCT patients required two vaccine doses to achieve average responses comparable to a single dose in HVs. Pre-vaccination titers were positively associated with time after transplantation, confirming that HSCT patients can elicit potent antibody responses. However, an unrelated donor, absolute lymphocyte counts below the normal range and treatment with calcineurin inhibitors lower the odds of responding. CONCLUSIONS HSCT patients show a highly heterogeneous vaccine response, but overall, patients benefited from the booster shot and can acquire seroprotective antibodies over the years after transplantation. Several common patient factors lower the odds of responding, urging to identify additional preventive strategies in the poorly responding groups

IL-28B is a Key Regulator of B- and T-Cell Vaccine Responses against Influenza

Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 p = 0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses

Characterising the epidemic spread of influenza A/H3N2 within a city through phylogenetics

Infecting large portions of the global population, seasonal influenza is a major burden on societies around the globe. While the global source sink dynamics of the different seasonal influenza viruses have been studied intensively, its local spread remains less clear. In order to improve our understanding of how influenza is transmitted on a city scale, we collected an extremely densely sampled set of influenza sequences alongside patient metadata. To do so, we sequenced influenza viruses isolated from patients of two different hospitals, as well as private practitioners in Basel, Switzerland during the 2016/2017 influenza season. The genetic sequences reveal that repeated introductions into the city drove the influenza season. We then reconstruct how the effective reproduction number changed over the course of the season. While we did not find that transmission dynamics in Basel correlate with humidity or school closures, we did find some evidence that it may positively correlated with temperature. Alongside the genetic sequence data that allows us to see how individual cases are connected, we gathered patient information, such as the age or household status. Zooming into the local transmission outbreaks suggests that the elderly were to a large extent infected within their own transmission network. In the remaining transmission network, our analyses suggest that school-aged children likely play a more central role than pre-school aged children. These patterns will be valuable to plan interventions combating the spread of respiratory diseases within cities given that similar patterns are observed for other influenza seasons and cities

Recombinant IL-28B inhibits Influenza H1N1-induced Th2 response and B cell activation in transplant recipients.

<p>(A) Analysis of H1N1-stimulated Th1 cytokine release in transplant recipients (n = 36) from post–vaccine samples in relation to IL-28B pre-treatment. Peripheral blood mononuclear cells (PBMCs) were pre-treated with recombinant IL-28B (100 ng/mL) for two hours prior to overnight stimulation with inactivated Influenza A H1N1 (0.3 µg/mL hemagglutinin). The expression profile of 17 cytokines was determined using a luminex-based platform. Key representative Th1 cytokines are shown. Wilcoxon matched-pairs signed rank (WCR)-test determined statistically significant differences between groups. Before-after plots are shown where each dot is a different patient. (B) PBMCs from transplant recipients were pre-treated with recombinant IL-28B (100 ng/mL) for two hours prior to overnight stimulation. Frequencies of H1N1-specific IL-4-producing CD4⁺ T-cells were measured by intracellular flow cytometry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004556#s4" target="_blank">Methods</a>. Data from 47 transplant recipients are shown in PBMC collected pre- and post-vaccine. (C) PBMCs from transplant (Tx) recipients were pre-treated with recombinant IL-28B (100 ng/mL) for two hours prior to 5-day stimulation with H1N1. The production of H1N1-induced IgG is shown according to pre-treatment groups. Data from 38 transplant recipients are shown.</p