Optimization of differential filtration-based mitochondrial isolation for mitochondrial transplant to cerebral organoids.

BACKGROUND: Mitochondrial dysfunction is involved in several diseases ranging from genetic mitochondrial disorders to chronic metabolic diseases. An emerging approach to potentially treat mitochondrial dysfunction is the transplantation of autologous live mitochondria to promote cell regeneration. We tested the differential filtration-based mitochondrial isolation protocol established by the McCully laboratory for use in cellular models but found whole cell contaminants in the mitochondrial isolate. METHODS: Therefore, we explored alternative types of 5-µm filters (filters A and B) for isolation of mitochondria from multiple cell lines including HEK293 cells and induced pluripotent stem cells (iPSCs). MitoTracker™ staining combined with flow cytometry was used to quantify the concentration of viable mitochondria. A proof-of-principle mitochondrial transplant was performed using mitoDsRed2-tagged mitochondria into a H9-derived cerebral organoid. RESULTS: We found that filter B provided the highest quality mitochondria as compared to the 5-µm filter used in the original protocol. Using this method, mitochondria were also successfully isolated from induced pluripotent stem cells. To test for viability, mitoDsRed2-tagged mitochondria were isolated and transplanted into H9-derived cerebral organoids and observed that mitochondria were engulfed as indicated by immunofluorescent co-localization of TOMM20 and MAP2. CONCLUSIONS: Thus, use of filter B in a differential filtration approach is ideal for isolating pure and viable mitochondria from cells, allowing us to begin evaluating long-term integration and safety of mitochondrial transplant using cellular sources.

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. 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.

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. Here, we review strategies for altering the composition of the sialome with particular focus on glycan structures and state-of-the-art tools.

An Irreversible Inhibitor to Probe the Role of Streptococcus pyogenes 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 Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, 2S-alkyne, based on X-ray crystallographic analysis and structure-activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.

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. 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.

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. In the absence of a toll-like receptor (TLR) agonist, antigen delivery through CD169 produced robust CD4+ T cell priming only. However, simultaneous treatment with targeted antigen and a TLR7 agonist induced CD8+ T cell priming, with concomitant suppression of the CD4+ T cell response. We exploited these observations to manipulate the activation ratio of CD4+/CD8+ T cells in the same animal. These findings represent a unique chemical strategy for targeting CD169+ macrophages to modulate antigen-specific T cell immunity.

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. Sequential labeling with the isotopologous probes (SLIP) in pancreatic tumor xenograft models revealed changes in cellular oxygenation over time which correlated with the distance from vasculature, the proliferation of cell populations, and proximity to necrosis. SLIP allows for capture of spatial and temporal dynamics in vivo using enzyme activated probes.

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. 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.

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. 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.

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.