A Unified Atlas of T cell Glycophysiology.

Glycans are emerging as important regulators of T cell function but remain poorly characterized across the functionally distinct populations that exist . Here, we couple single-cell analysis technologies with soluble lectins and chemical probes to interrogate glycosylation patterns on major T cell populations across multiple mouse and human tissues. Our analysis focused on terminal glycan epitopes with immunomodulatory functions, including sialoglycan ligands for Siglecs.

Engineering the Sialome.

The surface of every eukaryotic cell is coated in a dense layer of structurally diverse glycans that together comprise the glycocalyx, a key interface between intracellular biochemistry and the external environment. Many of the glycans within the glycocalyx terminate in anionic monosaccharides belonging to the sialic acid family. Advances in our understanding of the biological processes mediated by sialic acids at the interfaces between cells have catalyzed interest in metabolic, enzymatic, and chemical strategies to edit the total complement of cellular sialic acids-the sialome.

Sialic Acid Ligands of CD28 Suppress Costimulation of T Cells.

Effector T cells comprise the cellular arm of the adaptive immune system and are essential for mounting immune responses against pathogens and cancer. To reach effector status, costimulation through CD28 is required. Here, we report that sialic acid-containing glycans on the surface of both T cells and APCs are alternative ligands of CD28 that compete with binding to its well-documented activatory ligand CD80 on the APC, resulting in attenuated costimulation.

An Irreversible Inhibitor to Probe the Role of Cysteine Protease SpeB in Evasion of Host Complement Defenses.

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, , based on X-ray crystallographic analysis and structure-activity relationships.

TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry.

Protein synthesis is central to maintaining cellular homeostasis and its study is critical to understanding the function and dysfunction of eukaryotic systems. Here we report L-2-tellurienylalanine (TePhe) as a noncanonical amino acid for direct measurement of protein synthesis. TePhe is synthetically accessible, nontoxic, stable under biological conditions, and the tellurium atom allows its direct detection with mass cytometry, without postexperiment labeling.

Targeted Delivery of Antigen to Activated CD169 Macrophages Induces Bias for Expansion of CD8 T Cells.

Macrophages (MØs) expressing the endocytic sialic acid-binding immunoglobulin-like lectin 1 (siglec-1, CD169, sialoadhesin) are known to be adept at antigen capture-primarily due to their strategic location within lymphatic tissues. Antigen concentrated in these cells can be harnessed to induce potent anti-tumor/anti-pathogen cytotoxic (CD8) T cell responses. Here, we describe a chemical platform that exploits the CD169-mediated antigen capture pathway for biased priming of antigen-specific CD4 or CD8 T cells in vivo.

Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution.

Changes in the oxygenation state of microenvironments within solid tumors are associated with the development of aggressive cancer phenotypes. Factors that influence cellular hypoxia have been characterized; however, methods for measuring the dynamics of oxygenation at a cellular level in vivo have been elusive. We report a series of tellurium-containing isotopologous probes for cellular hypoxia compatible with mass cytometry (MC)-technology that allows for highly parametric interrogation of single cells based on atomic mass spectrometry.

Organotellurium scaffolds for mass cytometry reagent development.

Mass cytometry (MC) is a powerful tool for studying heterogeneous cell populations. In previous work, our laboratory has developed an MC probe for hypoxia bearing a methyl telluride mass tag. The methyl telluride was unoptimized, displaying stability and toxicity limitations.

Identification of hypoxic cells using an organotellurium tag compatible with mass cytometry.

Mass cytometry (MC) offers unparalleled potential for the development of highly parameterized assays for characterization of single cells within heterogeneous populations. Current reagents compatible with MC analysis employ antibody-metal-chelating polymer conjugates to report on the presence of biomarkers. Here, we expand the utility of MC by developing the first activity-based probe designed specifically for use with the technology.

Protecting-group-free synthesis of glycosyl 1-phosphates.

Glycosyl 1-phosphates enriched in the α-anomer are obtained without the use of protecting groups in two steps starting from the free hemiacetal. Condensation of free hemiacetals with toluenesulfonylhydrazide yields a range of glycosylsulfonohydrazide donors which can be oxidized using cupric chloride in the presence of phosphoric acid and the coordinating additive 2-methyl-2-oxazoline to give useful yields of the fully deprotected glycosyl 1-phosphates.