Chemical Tools for Studying TLR Signaling Dynamics

Therapeutic cell differentiatio

An alternative model for type I interferon induction downstream of human TLR2

Surface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines including type I interferons (IFN-I). Downstream of TLR4, IFNβ secretion is only vigorously triggered in macrophages when the receptor undergoes endocytosis and switches signaling adaptor; surface TLR4 engagement predominantly induces proinflammatory cytokines via the signaling adaptor MyD88. It is unclear if this dichotomy is generally applicable to other TLRs, cell types, or differentiation states. Here, we report that diverse TLR2 ligands induce an IFN-I response in human monocyte-like cells, but not in differentiated macrophages. This TLR2-dependent IFN-I signaling originates from the cell surface and is dependent on MyD88; it involves combined activation of the transcription factors IRF3 and NF-κB, driven by the kinases TBK1 and TAK1-IKKβ, respectively. TLR2-stimulated monocytes produced modest IFNβ levels that caused productive downstream signaling, reflected by STAT1-phosphorylation and expression of numerous interferon-stimulated genes (ISGs). Our findings reveal that the outcome of TLR2 signaling includes an IFN-I response in human monocytes, which is lost upon macrophage differentiation, and differs mechanistically from IFN-I-induction through TLR4. These findings point to molecular mechanisms tailored to the differentiation state of a cell and the nature of receptors activated to control and limit TLR-triggered IFN-I responses.Bio-organic Synthesi

Conditionally controlling human TLR2 activity via Trans-Cyclooctene Caged Ligands

Toll-like receptors (TLRs) are key pathogen sensors of the immune system. Their activation results in the production of cytokines, chemokines, and costimulatory molecules that are crucial for innate and adaptive immune responses. In recent years, specific (sub)-cellular location and timing of TLR activation have emerged as parameters for defining the signaling outcome and magnitude. To study the subtlety of this signaling, we here report a new molecular tool to control the activation of TLR2 via "click-to-release"-chemistry. We conjugated a bioorthogonal trans-cyclooctene (TCO) protecting group via solid support to a critical position within a synthetic TLR2/6 ligand to render the compound unable to initiate signaling. The TCO-group could then be conditionally removed upon addition of a tetrazine, resulting in restored agonist activity and TLR2 activation. This approach was validated on RAW264.7 macrophages and various murine primary immune cells as well as human cell line systems, demonstrating that TCO-caging constitutes a versatile approach for generating chemically controllable TLR2 agonists.Bio-organic Synthesi

Antibody-mediated delivery of viral epitopes to redirect EBV-specific CD8+ T-cell immunity towards cancer cells

Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8+ T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo. Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Comparison of methods generating antibody-epitope conjugates for targeting cancer with virus-specific T cells

Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells.Bio-organic Synthesi

Immune reconstitution inflammatory syndrome in HIV infected late presenters starting integrase inhibitor containing antiretroviral therapy

Background: Integrase inhibitors (INI) induce a rapid decline of HIV-RNA in plasma and CD4+ T-cell recovery in blood. Both characteristics are also associated with immune reconstitution inflammatory syndrome (IRIS). Whether the use of INI-containing combination antiretroviral therapy (cART) increases the risk of IRIS is being questioned. Methods: Study within the Dutch ATHENA HIV observational cohort. HIV-1 infected late presenters initiating cART after March 2009 were included if they had <200 CD4+ T-cells per μL and were diagnosed with an opportunistic infection. IRIS was defined either according to the criteria by French et al. (IRISFRENCH) or by a clinical IRIS diagnosis of the physician (IRISCLINICAL). The primary outcomes were the association between INI and the occurrence of IRISFRENCH and IRISFRENCH+CLINICAL in multivariable logistic regression. Findings: 672 patients with a median CD4+ T-cell count of 35 cells per μL were included. Treatment with INI was independently associated with IRISFRENCH as well as IRISFRENCH+CLINICAL (OR 2·43, 95%CI:1·45–4·07, and OR 2·17, 95%CI:1·45–3·25). When investigating INI separately, raltegravir (RAL) remained significantly associated with IRISFRENCH (OR 4·04 (95%CI:1·99-8·19) as well as IRISFRENCH+CLINICAL (OR 3·07, 95%CI:1·66-5·69), while dolutegravir (DTG) became associated with IRISFRENCH+CLINICAL after it replaced RAL as preferred INI in the cohort after 2015 (OR 4·08, 95%CI:0·99-16·82, p=0·052). Too few patients used elvitegravir to draw meaningful conclusions. Steroid initiation for IRIS was more likely in those who initiated INI versus in those who did not, but no increased hospital (re)admission or mortality rates were observed. Interpretation: In HIV late presenters from a resource rich setting, INI based treatment initiation increased the risk of IRIS. This was observed for RAL and DTG when being initiated as preferential INI in the presence of specific AIDS-conditions, indicative of channeling bias. Although we controlled for all relevant measured confounders, we cannot exclude that the observed association is partially explained by residual confounding. INI use was not associated with mortality nor hospitalization. Therefore, our observation is no reason to avoid INI in late presenters. Funding: Th

Single-cell analysis of regions of interest (SCARI) using a photosensitive tag

The functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a tradeoff between spatial resolution and cell profiling depth. Here, we develop a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran) cage coupled to a set of nanobodies allows high-resolution photo-uncaging of different cell types in areas of interest. Single-cell RNA-sequencing of spatially defined CD8+ T cells is used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.ERC Cog KineTic grant 865175Bio-organic Synthesi

Has the Rate of CD4 Cell Count Decline before Initiation of Antiretroviral Therapy Changed over the Course of the Dutch HIV Epidemic among MSM?

Introduction:Studies suggest that the HIV-1 epidemic in the Netherlands may have become more virulent, leading to faster disease progression if untreated. Analysis of CD4 cell count decline before antiretroviral therapy (ART) initiation, a surrogate marker for disease progression, may be hampered by informative censoring as ART initiation is more likely with a steeper CD4 cell count decline.Methods:Development of CD4 cell count from 9 to 48 months after seroconversion was analyzed using a mixed-effects model and 2 models that jointly modeled CD4 cell counts and time to censoring event (start ART

Non-AIDS defining cancers in the D:A:D Study-time trends and predictors of survival : a cohort study

BACKGROUND:Non-AIDS defining cancers (NADC) are an important cause of morbidity and mortality in HIV-positive individuals. Using data from a large international cohort of HIV-positive individuals, we described the incidence of NADC from 2004-2010, and described subsequent mortality and predictors of these.METHODS:Individuals were followed from 1st January 2004/enrolment in study, until the earliest of a new NADC, 1st February 2010, death or six months after the patient's last visit. Incidence rates were estimated for each year of follow-up, overall and stratified by gender, age and mode of HIV acquisition. Cumulative risk of mortality following NADC diagnosis was summarised using Kaplan-Meier methods, with follow-up for these analyses from the date of NADC diagnosis until the patient's death, 1st February 2010 or 6 months after the patient's last visit. Factors associated with mortality following NADC diagnosis were identified using multivariable Cox proportional hazards regression.RESULTS:Over 176,775 person-years (PY), 880 (2.1%) patients developed a new NADC (incidence: 4.98/1000PY [95% confidence interval 4.65, 5.31]). Over a third of these patients (327, 37.2%) had died by 1st February 2010. Time trends for lung cancer, anal cancer and Hodgkin's lymphoma were broadly consistent. Kaplan-Meier cumulative mortality estimates at 1, 3 and 5 years after NADC diagnosis were 28.2% [95% CI 25.1-31.2], 42.0% [38.2-45.8] and 47.3% [42.4-52.2], respectively. Significant predictors of poorer survival after diagnosis of NADC were lung cancer (compared to other cancer types), male gender, non-white ethnicity, and smoking status. Later year of diagnosis and higher CD4 count at NADC diagnosis were associated with improved survival. The incidence of NADC remained stable over the period 2004-2010 in this large observational cohort.CONCLUSIONS:The prognosis after diagnosis of NADC, in particular lung cancer and disseminated cancer, is poor but has improved somewhat over time. Modifiable risk factors, such as smoking and low CD4 counts, were associated with mortality following a diagnosis of NADC

Synergy between bio-based industry and the feed industry through biorefinery

Processing biomass into multi-functional components can contribute to the increasing demand for raw materials for feed and bio-based non-food products. This contribution aims to demonstrate synergy between the bio-based industry and the feed industry through biorefinery of currently used feed ingredients. Illustrating the biorefinery concept, rapeseed was selected as a low priced feed ingredient based on market prices versus crude protein, crude fat and apparent ileal digestible lysine content. In addition it is already used a s an alternative protein source in diets and can b e cultivated in European climate zones. Furthermore, inclusion level of rapeseed meal in pig diet is limited because of its nutritionally ac tive factors. A conceptual process was developed to improve rapeseeds nutritional value and producing other bio-based building blocks simultaneously. Based on the correlation between market prices of feed ingredients and its protein and fat content, the value of refined products was estimated. Finally, a sensitivity analysis, under two profit scenario, shows that the process is economically feasible. This study demonstrates that using biorefinery processes on feed ingredients can improve feed quality. In conjunction, it produces building blocks for a bio-based industry and creates synergy between bio-based and feed industry for more efficient use of biomass.<br/