Single-cell barcoding enables the combined processing and acquisition of multiple individual samples as one. This maximizes assay efficiency and eliminates technical variability in both sample preparation and analysis. Remaining challenges are the barcoding of live, unprocessed cells to increase downstream assay performance combined with the flexibility of the approach towards a broad range of cell types. To that end, we developed a novel antibody-based platform that allows the robust barcoding of live human cells for mass cytometry (CyTOF). By targeting both the MHC class I complex (beta-2-microglobulin) and a broadly expressed sodium-potassium ATPase-subunit (CD298) with platinum-conjugated antibodies, human immune cells, stem cells as well as tumor cells could be multiplexed in the same single-cell assay. In addition, we present a novel palladium-based covalent viability reagent compatible with this barcoding strategy. Altogether, this platform enables mass cytometry-based, live-cell barcoding across a multitude of human sample types and provides a scheme for multiplexed barcoding of human single-cell assays in general.

Efficacy of cancer vaccines remains low and mechanistic understanding of antigen presenting cell function in cancer may improve vaccine design and outcomes. Here, we analyze the transcriptomic and immune-metabolic profiles of Dendritic Cells (DCs) from 35 subjects enrolled in a trial of DC vaccines in late-stage melanoma (NCT01622933). Multiple platforms identify metabolism as an important biomarker of DC function and patient overall survival (OS). We demonstrate multiple immune and metabolic gene expression pathway alterations, a functional decrease in OCR/OXPHOS and increase in ECAR/glycolysis in patient vaccines. To dissect molecular mechanisms, we utilize single cell SCENITH functional profiling and show patient clinical outcomes (OS) correlate with DC metabolic profile, and that metabolism is linked to immune phenotype. With single cell metabolic regulome profiling, we show that MCT1 (monocarboxylate transporter-1), a lactate transporter, is increased in patient DCs, as is glucose uptake and lactate secretion. Importantly, pre-vaccination circulating myeloid cells in patients used as precursors for DC vaccine generation are significantly skewed metabolically as are several DC subsets. Together, we demonstrate that the metabolic profile of DC is tightly associated with the immunostimulatory potential of DC vaccines from cancer patients. We link phenotypic and functional metabolic changes to immune signatures that correspond to suppressed DC differentiation.

Cellular metabolism underpins immune cell functionality, yet our understanding of metabolic influences in human dendritic cell biology and their ability to orchestrate immune responses is poorly developed. Here, we map single-cell metabolic states and immune profiles of inflammatory and tolerogenic monocytic dendritic cells using recently developed multiparametric approaches. Single-cell metabolic pathway activation scores reveal simultaneous engagement of multiple metabolic pathways in distinct monocytic dendritic cell differentiation stages. GM-CSF/IL4-induce rapid reprogramming of glycolytic monocytes and transient co-activation of mitochondrial pathways followed by TLR4-dependent maturation of dendritic cells. Skewing of the mTOR:AMPK phosphorylation balance and upregulation of OXPHOS, glycolytic and fatty acid oxidation metabolism underpin metabolic hyperactivity and an immunosuppressive phenotype of tolerogenic dendritic cells, which exhibit maturation-resistance and a de-differentiated immune phenotype marked by unique immunoregulatory receptor signatures. This single-cell dataset provides important insights into metabolic pathways impacting the immune profiles of human dendritic cells.

The advent of mass cytometry (CyTOF^®) has permitted simultaneous detection of more than 40 antibody parameters at the single-cell level, although a limited number of metal-labeled antibodies are commercially available. Here we present optimized and scalable protocols for conjugation of lanthanide as well as bismuth ions to immunoglobulin (Ig) using a maleimide-functionalized chelating polymer and for characterization of the conjugate. The maleimide functional group is reactive with cysteine sulfhydryl groups generated through partial reduction of the Ig Fc region. Incubation of Ig with polymer pre-loaded with lanthanide ions produces metal-labeled Ig without disrupting antigen specificity. Antibody recovery rates can be determined by UV spectrophotometry and frequently exceeds 60%. Each custom-conjugated antibody is validated using positive and negative cellular control populations and is titrated for optimal staining at concentrations ranging from 0.1 to 10 μg/mL. The preparation of metal-labeled antibodies can be completed in 4.5 h, and titration requires an additional 3-5 h.

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery.

α-Fetoprotein (AFP) is expressed by stem-like and poor outcome hepatocellular cancer tumors and is a clinical tumor biomarker. AFP has been demonstrated to inhibit dendritic cell (DC) differentiation and maturation and to block oxidative phosphorylation. To identify the critical metabolic pathways leading to human DC functional suppression, here, we used two recently described single-cell profiling methods, scMEP (single-cell metabolic profiling) and SCENITH (single-cell energetic metabolism by profiling translation inhibition). Glycolytic capacity and glucose dependence of DCs were significantly increased by tumor-derived, but not normal cord blood-derived, AFP, leading to increased glucose uptake and lactate secretion. Key molecules in the electron transport chain in particular were regulated by tumor-derived AFP. These metabolic changes occurred at mRNA and protein levels, with negative impact on DC stimulatory capacity. Tumor-derived AFP bound significantly more polyunsaturated fatty acids (PUFA) than cord blood-derived AFP. PUFAs bound to AFP increased metabolic skewing and promoted DC functional suppression. PUFAs inhibited DC differentiation in vitro, and ω-6 PUFAs conferred potent immunoregulation when bound to tumor-derived AFP. Together, these findings provide mechanistic insights into how AFP antagonizes the innate immune response to limit antitumor immunity.

Significance

α-Fetoprotein (AFP) is a secreted tumor protein and biomarker with impact on immunity. Fatty acid-bound AFP promotes immune suppression by skewing human dendritic cell metabolism toward glycolysis and reduced immune stimulation.