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. Here, we investigate three classes of organotelluriums as MC mass tags: methyl tellurides, trifluoromethyl tellurides and 2-alkyl-tellurophenes. NMR was used to compare the stability of these compounds in aqueous and organic solutions and the compounds were analysed for toxicity in Jurkat cells. The methyl tellurides were moderately stable to aerobic oxidation in organic solution under dry ambient conditions. The trifluoromethyl tellurides were stable to aerobic oxidation in organic solution but decomposed in aqueous solution. The 2-alkyl-tellurophenes proved to be stable in both organic and aqueous solutions under ambient conditions and showed limited toxicity (IC50 > 200 μM) in cell based assays. The synthetic feasibility, chemically stability, and limited toxicity of tellurophenes suggests these groups will be good choices for MC reagent development.

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. TePhe labeling is competitive with phenylalanine but not other large and aromatic amino acids, demonstrating its molecular specificity as a phenylalanine mimic; labeling is also abrogated in vitro and in vivo by the protein synthesis inhibitor cycloheximide, validating TePhe as a translation reporter. In vivo, imaging mass cytometry with TePhe visualizes translation dynamics in the mouse gut, brain, and tumor. The strong performance of TePhe as a probe for protein synthesis, coupled with the operational simplicity of its use, suggests TePhe could become a broadly applied molecule for measuring translation in vitro and in vivo. mass cytometry | protein synthesis | tellurium

Macrophages expressing the sialic acidbinding lectin CD169 are involved in mounting antigen-specific T cell responses. Here, Edgar et al. describe a CD169-targeted liposome-based platform able to selectively activate and deliver antigen to these macrophages. This modular platform enabled control over antigen-specific CD4 + :CD8 + T cell ratios in vivo.

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. Removal of sialic acids enhances antigen-mediated activation of naïve T cells and also increases the revival of effector T cells made hypofunctional or exhausted via chronic viral infection. This occurs through a mechanism that is synergistic with antibody blockade of the inhibitory PD-1 axis. These results reveal a previously unrecognized role of sialic acid ligands in attenuation of CD28-mediated costimulation of T cells.

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. A compact MC-detectable telluroether is linked to a bioreductively sensitive 2-nitroimidazole scaffold, thereby generating a probe sensitive to cellular hypoxia. The probe exhibits low toxicity and is able to selectively label O2-deprived cells. A proof-of-concept experiment employing metal-bound DNA intercalators demonstrates that a heterogeneous mixture of cells with differential exposure to O2 can be effectively discriminated by the quantity of tellurium-labeling. The organotellurium reagents described herein provide a general approach to the development of a large toolkit of MC-compatible probes for activity-based profiling of single cells.