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.

Simplified Monopalmitoyl Toll-like Receptor 2 Ligand Mini-UPam for Self-Adjuvanting Neoantigen-Based Synthetic Cancer Vaccines.

Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3 CysSK4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.

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, IFNß 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 whether 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 depends on MyD88; it involves combined activation of the transcription factors IRF3 and NF-κB, driven by the kinases TBK1 and TAK1-IKKß, respectively. TLR2-stimulated monocytes produced modest IFNß levels that caused productive downstream signaling, reflected by STAT1 phosphorylation and expression of numerous interferon-stimulated genes. 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.

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.

Immunoproteasome Inhibitor-Doxorubicin Conjugates Target Multiple Myeloma Cells and Release Doxorubicin upon Low-Dose Photon Irradiation.

Proteasome inhibitors are established therapeutic agents for the treatment of hematological cancers, as are anthracyclines such as doxorubicin. We here present a new drug targeting approach that combines both drug classes into a single molecule. Doxorubicin was conjugated to an immunoproteasome-selective inhibitor via light-cleavable linkers, yielding peptide epoxyketone-doxorubicin prodrugs that remained selective and active toward immunoproteasomes. Upon cellular uptake and immunoproteasome inhibition, doxorubicin is released from the immunoproteasome inhibitor through photoirradiation. Multiple myeloma cells in this way take a double hit: immunoproteasome inhibition and doxorubicin-induced toxicity. Our strategy, which entails targeting of a cytotoxic agent, through a covalent enzyme inhibitor that is detrimental to tumor tissue in its own right, may find use in the search for improved anticancer drugs.

Peptides conjugated to 2-alkoxy-8-oxo-adenine as potential synthetic vaccines triggering TLR7.

Covalent linking of immunogenic oligopeptides with synthetic Toll-like receptor ligands is a useful approach to develop self-adjuvanting vaccines. In particular, small-molecule based agonists of Toll-like receptor 7 (TLR7) that are derived from 8-oxo-adenine core are potentially promising because these chemically robust TLR7 ligands can be connected to peptide T-cell epitopes via straightforward solid-phase peptide synthesis. In this contribution we present the synthesis of a Boc-protected 9-benzyl-2-alkoxy-8-oxo-adenine building block and its application in the online solid phase synthesis of three peptide conjugates that differ in the position of the TLR7 ligand within the peptide. The conjugates are able to induce dendritic cell maturation and T cell proliferation while the position of the ligand impacts T cell proliferation potency.

Chemical Tools for Studying TLR Signaling Dynamics.

The detection of infectious pathogens is essential for the induction of antimicrobial immune responses. The innate immune system detects a wide array of microbes using a limited set of pattern-recognition receptors (PRRs). One family of PRRs with a central role in innate immunity are the Toll-like receptors (TLRs). Upon ligation, these receptors initiate signaling pathways culminating in the release of pro-inflammatory cytokines and/or type I interferons (IFN-I). In recent years, it has become evident that the specific subcellular location and timing of TLR activation affect signaling outcome. The subtlety of this signaling has led to a growing demand for chemical tools that provide the ability to conditionally control TLR activation. In this review, we survey current models for TLR signaling in time and space, discuss how chemical tools have contributed to our understanding of TLR ligands, and describe how they can aid further elucidation of the dynamic aspects of TLR signaling.

The Optimization of Bioorthogonal Epitope Ligation within MHC-I Complexes.

Antigen recognition followed by the activation of cytotoxic T-cells (CTLs) is a key step in adaptive immunity, resulting in clearance of viruses and cancers. The repertoire of peptides that have the ability to bind to the major histocompatibility type-I (MHC-I) is enormous, but the approaches available for studying the diversity of the peptide repertoire on a cell are limited. Here, we explore the use of bioorthogonal chemistry to quantify specific peptide-MHC-I complexes (pMHC-I) on cells. We show that modifying epitope peptides with bioorthogonal groups in surface accessible positions allows wild-type-like MHC-I binding and bioorthogonal ligation using fluorogenic chromophores in combination with a Cu(I)-catalyzed Huisgen cycloaddition reaction. We expect that this approach will make a powerful addition to the antigen presentation toolkit as for the first time it allows quantification of antigenic peptides for which no detection tools exist.