Immune determinants of CAR-T cell expansion in solid tumor patients receiving GD2 CAR-T cell therapy.

Chimeric antigen receptor T cells (CAR-Ts) have remarkable efficacy in liquid tumors, but limited responses in solid tumors. We conducted a Phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.iC9), demonstrating feasibility and safety of administration in children and young adults with osteosarcoma and neuroblastoma. Since CAR-T efficacy requires adequate CAR-T expansion, patients were grouped into good or poor expanders across dose levels. Patient samples were evaluated by multi-dimensional proteomic, transcriptomic, and epigenetic analyses. T cell assessments identified naive T cells in pre-treatment apheresis associated with good expansion, and exhausted T cells in CAR-T products with poor expansion. Myeloid cell assessment identified CXCR3+ monocytes in pre-treatment apheresis associated with good expansion. Longitudinal analysis of post-treatment samples identified increased CXCR3- classical monocytes in all groups as CAR-T numbers waned. Together, our data uncover mediators of CAR-T biology and correlates of expansion that could be utilized to advance immunotherapies for solid tumor patients.

Unravelling human hematopoietic progenitor cell diversity through association with intrinsic regulatory factors.

Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34- cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and in vitro clonal differentiation assays. Overall, we demonstrate the utility of using molecular regulators as surrogates for cellular identity and functional potential, providing a framework for description, prospective isolation, and cross-tissue comparison of HSPCs in humans.

Supervised dimensionality reduction for exploration of single-cell data by HSS-LDA.

Single-cell technologies generate large, high-dimensional datasets encompassing a diversity of omics. Dimensionality reduction captures the structure and heterogeneity of the original dataset, creating low-dimensional visualizations that contribute to the human understanding of data. Existing algorithms are typically unsupervised, using measured features to generate manifolds, disregarding known biological labels such as cell type or experimental time point. We repurpose the classification algorithm, linear discriminant analysis (LDA), for supervised dimensionality reduction of single-cell data. LDA identifies linear combinations of predictors that optimally separate a priori classes, enabling the study of specific aspects of cellular heterogeneity. We implement feature selection by hybrid subset selection (HSS) and demonstrate that this computationally efficient approach generates non-stochastic, interpretable axes amenable to diverse biological processes such as differentiation over time and cell cycle. We benchmark HSS-LDA against several popular dimensionality-reduction algorithms and illustrate its utility and versatility for the exploration of single-cell mass cytometry, transcriptomics, and chromatin accessibility data.

Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment.

Master transcription factors (TFs) directly regulate present and future cell states by binding DNA regulatory elements and driving gene-expression programs. Their abundance influences epigenetic priming to different cell fates at the chromatin level, especially in the context of differentiation. In order to link TF protein abundance to changes in TF motif accessibility and open chromatin, we developed InTAC-seq, a method for simultaneous quantification of genome-wide chromatin accessibility and intracellular protein abundance in fixed cells. Our method produces high-quality data and is a cost-effective alternative to single-cell techniques. We showcase our method by purifying bone marrow (BM) progenitor cells based on GATA-1 protein levels and establish high GATA-1-expressing BM cells as both epigenetically and functionally similar to erythroid-committed progenitors.

Revealing new biology from multiplexed, metal-isotope-tagged, single-cell readouts.

Mass cytometry (MC) is a recent technology that pairs plasma-based ionization of cells in suspension with time-of-flight (TOF) mass spectrometry to sensitively quantify the single-cell abundance of metal-isotope-tagged affinity reagents to key proteins, RNA, and peptides. Given the ability to multiplex readouts (~50 per cell) and capture millions of cells per experiment, MC offers a robust way to assay rare, transitional cell states that are pertinent to human development and disease. Here, we review MC approaches that let us probe the dynamics of cellular regulation across multiple conditions and sample types in a single experiment. Additionally, we discuss current limitations and future extensions of MC as well as computational tools commonly used to extract biological insight from single-cell proteomic datasets.

Single-cell metabolic profiling of human cytotoxic T cells.

Cellular metabolism regulates immune cell activation, differentiation and effector functions, but current metabolic approaches lack single-cell resolution and simultaneous characterization of cellular phenotype. In this study, we developed an approach to characterize the metabolic regulome of single cells together with their phenotypic identity. The method, termed single-cell metabolic regulome profiling (scMEP), quantifies proteins that regulate metabolic pathway activity using high-dimensional antibody-based technologies. We employed mass cytometry (cytometry by time of flight, CyTOF) to benchmark scMEP against bulk metabolic assays by reconstructing the metabolic remodeling of in vitro-activated naive and memory CD8+ T cells. We applied the approach to clinical samples and identified tissue-restricted, metabolically repressed cytotoxic T cells in human colorectal carcinoma. Combining our method with multiplexed ion beam imaging by time of flight (MIBI-TOF), we uncovered the spatial organization of metabolic programs in human tissues, which indicated exclusion of metabolically repressed immune cells from the tumor-immune boundary. Overall, our approach enables robust approximation of metabolic and functional states in individual cells.

Single-cell mass cytometry reveals cross-talk between inflammation-dampening and inflammation-amplifying cells in osteoarthritic cartilage.

Aging or injury leads to degradation of the cartilage matrix and the development of osteoarthritis (OA). Because of a paucity of single-cell studies of OA cartilage, little is known about the interpatient variability in its cellular composition and, more importantly, about the cell subpopulations that drive the disease. Here, we profiled healthy and OA cartilage samples using mass cytometry to establish a single-cell atlas, revealing distinct chondrocyte progenitor and inflammation-modulating subpopulations. These rare populations include an inflammation-amplifying (Inf-A) population, marked by interleukin-1 receptor 1 and tumor necrosis factor receptor II, whose inhibition decreased inflammation, and an inflammation-dampening (Inf-D) population, marked by CD24, which is resistant to inflammation. We devised a pharmacological strategy targeting Inf-A and Inf-D cells that significantly decreased inflammation in OA chondrocytes. Using our atlas, we stratified patients with OA in three groups that are distinguished by the relative proportions of inflammatory to regenerative cells, making it possible to devise precision therapeutic approaches.

TRAIL-induced variation of cell signaling states provides nonheritable resistance to apoptosis.

TNFα-related apoptosis-inducing ligand (TRAIL), specifically initiates programmed cell death, but often fails to eradicate all cells, making it an ineffective therapy for cancer. This fractional killing is linked to cellular variation that bulk assays cannot capture. Here, we quantify the diversity in cellular signaling responses to TRAIL, linking it to apoptotic frequency across numerous cell systems with single-cell mass cytometry (CyTOF). Although all cells respond to TRAIL, a variable fraction persists without apoptotic progression. This cell-specific behavior is nonheritable where both the TRAIL-induced signaling responses and frequency of apoptotic resistance remain unaffected by prior exposure. The diversity of signaling states upon exposure is correlated to TRAIL resistance. Concomitantly, constricting the variation in signaling response with kinase inhibitors proportionally decreases TRAIL resistance. Simultaneously, TRAIL-induced de novo translation in resistant cells, when blocked by cycloheximide, abrogated all TRAIL resistance. This work highlights how cell signaling diversity, and subsequent translation response, relates to nonheritable fractional escape from TRAIL-induced apoptosis. This refined view of TRAIL resistance provides new avenues to study death ligands in general.

Parallel analysis of tri-molecular biosynthesis with cell identity and function in single cells.

Cellular products derived from the activity of DNA, RNA, and protein synthesis collectively control cell identity and function. Yet there is little information on how these three biosynthesis activities are coordinated during transient and sparse cellular processes, such as activation and differentiation. Here, we describe Simultaneous Overview of tri-Molecule Biosynthesis (SOM3B), a molecular labeling and simultaneous detection strategy to quantify DNA, RNA, and protein synthesis in individual cells. Comprehensive interrogation of biosynthesis activities during transient cell states, such as progression through cell cycle or cellular differentiation, is achieved by partnering SOM3B with parallel quantification of select biomolecules with conjugated antibody reagents. Here, we investigate differential de novo DNA, RNA, and protein synthesis dynamics in transformed human cell lines, primary activated human immune cells, and across the healthy human hematopoietic continuum, all at a single-cell resolution.