Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands

Abstract

Despite recent advances in single-cell genomic, transcriptional, and mass-cytometric profiling, it remains a challenge to collect highly multiplexed measurements of secreted proteins from single cells for comprehensive analysis of functional states. Herein, we combine spatial and spectral encoding with polydimethylsiloxane (PDMS) microchambers for codetection of 42 immune effector proteins secreted from single cells, representing the highest multiplexing recorded to date for a single-cell secretion assay. Using this platform to profile differentiated macrophages stimulated with lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4), reveals previously unobserved deep functional heterogeneity and varying levels of pathogenic activation. Uniquely protein profiling on the same single cells before and after LPS stimulation identified a role for macrophage inhibitory factor (MIF) to potentiate the activation of LPS-induced cytokine production. Advanced clustering analysis identified functional subsets including quiescent, polyfunctional fully activated, partially activated populations with different cytokine profiles. This population architecture is conserved throughout the cell activation process and prevails as it is extended to other TLR ligands and to primary macrophages derived from a healthy donor. This work demonstrates that the phenotypically similar cell population still exhibits a large degree of intrinsic heterogeneity at the functional and cell behavior level. This technology enables fullspectrum dissection of immune functional states in response to pathogenic or environmental stimulation, and opens opportunities to quantify deep functional heterogeneity for more comprehensive and accurate immune monitoring.single-cell analysis | cytokine | immune effector function | cellular heterogeneity | Toll-like receptor activation E merging evidence indicates that cell-to-cell variability can give rise to phenotypic differences within a genetically identical cell population (1, 2). Nongenetic heterogeneity is also emerging as a potential barrier to effective therapeutic intervention (3, 4). Recent advances in single-cell molecular profiling are beginning to address these questions. Single-cell RNA sequencing revealed dynamic and bimodal gene expression (5). Single-cell multicolor flow cytometry (6) and mass cytometry (7) can quantify phenotypic diversity and differential drug response even across the hematopoietic continuum. Although a limited number of signaling proteins can be measured using intracellular staining, most of these technologies measure transcriptional or phenotypic marker expression in single cells. It remains an unmet need to directly measure cellular functional outcomes in a highly multiplexed manner and in single cells. In the immune system, the immune effector functions are largely mediated by a panel of effector proteins (e.g., cytokines and chemokines) secreted from single cells. Due to phenotypic plasticity and functional diversity, immune cells purified for a well-defined phenot...

Highly Multiplexed Single‐Cell Protein Profiling with Large‐Scale Convertible DNA‐Antibody Barcoded Arrays

“…Interestingly, the overall structure has not been shifted much after LPS stimulation. The MIF subpopulation is completely separated from others, and the heterogeneity is not changed significantly after stimulation 6. This data indicate that MIF secretion is conservative function to macrophages.…”

“…For example, by simply designing more barcode ssDNA probes and cDNA‐antibody conjugates, the same architecture of the current technology can measure up to 40 different cytokines. Noticeably, the other barcode chips in our group and those in Fan's group have microchamber volumes at least eight times larger for single‐cell cytokine assays 6. Therefore, without losing much sensitivity, this strategy by expansion of microarray size and microwell volume can further increase multiplexity to hundreds.…”

“…THP‐1 monocytes can be differentiated into macrophages upon phorbol 12‐myristate 13‐acetate (PMA) stimulation, and further challenging with lipopolysaccharide (LPS) induces production of a variant of cytokines 15, 16. The LPS‐stimulated macrophage cell line is an ideal model system for single‐cell secretion analyses because they rapidly produce a large number of effector proteins, and the influence of cell cycle on heterogeneity is minimal due to postmitotic feature of differentiated macrophages 6, 17. The morphology change from suspension phenotype to adherent phenotype indicates the differentiation of monocytes to macrophages (Figures S2 and S3, Supporting Information) 18…”

“…To overcome this hurdle, integration of microarrays into single‐cell microchips conveniently addresses that limitation by achieving multiplexity as high as 42 6. However, this method still does not overcome the limitation of spectrum overlap, and patterning barcode arrays with a pitch < 10 µm in large‐scale is technically challenging.…”

Spatially and temporally controlled immune cell interactions using microscale tools

“…For example, NK cells were shown to eliminate local target cells independent of the neighboring NK cells [14], and experiments with multiple target cells identified a particularly active serial killer subpopulation with faster kill dynamics [15]. Similar co-culture studies in combination with barcoded antibody arrays [41,42 ] also enabled quantifying the influence of paracrine signaling on tumor cell functional states and signaling networks with multiplexed detection of intracellular and secreted proteins [18,20,21].…”