Yvonne J. Yamanaka

A high-throughput single-cell analysis of human CD8 + T cell functions reveals discordance for cytokine secretion and cytolysis

CD8+ T cells are a key component of the adaptive immune response to viral infection. An inadequate CD8+ T cell response is thought to be partly responsible for the persistent chronic infection that arises following infection with HIV. It is therefore critical to identify ways to define what constitutes an adequate or inadequate response. IFN-γ production has been used as a measure of T cell function, but the relationship between cytokine production and the ability of a cell to lyse virus-infected cells is not clear. Moreover, the ability to assess multiple CD8+ T cell functions with single-cell resolution using freshly isolated blood samples, and subsequently to recover these cells for further functional analyses, has not been achieved. As described here, to address this need, we have developed a high-throughput, automated assay in 125-pl microwells to simultaneously evaluate the ability of thousands of individual CD8+ T cells from HIV-infected patients to mediate lysis and to produce cytokines. This concurrent, direct analysis enabled us to investigate the correlation between immediate cytotoxic activity and short-term cytokine secretion. The majority of in vivo primed, circulating HIV-specific CD8+ T cells were discordant for cytolysis and cytokine secretion, notably IFN-γ, when encountering cognate antigen presented on defined numbers of cells. Our approach should facilitate determination of signatures of functional variance among individual effector CD8+ T cells, including those from mucosal samples and those induced by vaccines.

Cellular barcodes for efficiently profiling single-cell secretory responses by microengraving.

We present a method that uses fluorescent cellular barcodes to increase the number of unique samples that can be analyzed simultaneously by microengraving, a nanowell array-based technique for quantifying the secretory responses of thousands of single cells in parallel. Using n different fluorescent dyes to generate 2(n) unique cellular barcodes, we achieved a 2(n)-fold reduction in the number of arrays and quantity of reagents required per sample. The utility of this approach was demonstrated in three applications of interest in clinical and experimental immunology. Using barcoded human peripheral blood mononuclear cells and T cells, we constructed dose-response curves, profiled the secretory behavior of cells treated with mechanistically distinct stimuli, and tracked the secretory behaviors of different lineages of CD4(+) T helper cells. In addition to increasing the number of samples analyzed by generating secretory profiles of single cells from multiple populations in a time- and reagent-efficient manner, we expect that cellular barcoding in combination with microengraving will facilitate unique experimental opportunities for quantitatively analyzing interactions among heterogeneous cells isolated in small groups (~2-5 cells).

The dynamic lives of T cells: new approaches and themes

Activated T cells have classically been thought to progress unidirectionally through discrete phenotypic states and differentiate into static lineages. It is increasingly evident, however, that T cells exhibit much more complex and flexible dynamic behaviors than initially appreciated, and that these behaviors influence the efficacy of T cell responses to immunological challenges. In this review, we discuss how new technologies for monitoring the dynamics of T cells are enhancing the resolution of the fine phenotypic and functional heterogeneity within populations of T cells and revealing how individual T cells transition among a continuum of states. Such insights into the dynamic properties of T cells should improve immune monitoring and inform strategies for therapeutic interventions.

Analytical Technologies for Integrated Single-Cell Analysis of Human Immune Responses

The immune system is a network of cells in which the constitutive members interact through dense and sometimes overlapping connections. The extreme complexity of this network poses a significant challenge for monitoring pathological conditions (e.g., food allergies, autoimmunity, and other chronic inflammatory diseases) and for discovering robust signatures of immunological responses that correlate with or predict the efficacy of interventions. The diversity among immune cells found in clinical samples (variations in cellular functions, lineages, and clonotypic breadth) requires approaches for monitoring immune responses with single-cell resolution.In this chapter, we present an engineering approach for integrated single-cell analysis that uses interchangeable modular operations to provide a comprehensive characterization of the phenotypic, functional, and genetic variations for individual cells. We focus on the use of microfabricated devices to isolate and interrogate single cells, and on the analytical components that enable subsequent detection, correlation, and interpretation of multidimensional sets of data. We discuss specific challenges and opportunities in the realization of this concept, and review two examples where it has been implemented. The presented approach should provide a basis for the design and implementation of nonconventional bioanalytical processes for studying specific responses of an immune system.

Abstract C23: Functional characterization of viable circulating tumor cells using nanowells.

Current characterizations of circulating tumor cells (CTCs) are limited to enumeration, genotyping, and gene expression. Because these assays each require fixation or lysis, it has not been possible to evaluate several important characteristics of live CTCs at the single-cell level. We have interrogated viable, single CTCs using arrays of subnanoliter wells (nanowells), performing functional measurements of viability, migration, and secretion of soluble factors. We developed a process using nanowells to isolate and characterize viable CTCs from whole blood. Each poly(dimethylsiloxane) (PDMS) array comprises 84,762 cubic wells of 275 pL each. CTCs were enriched from whole blood by negative selection against CD45 and loaded onto the array to settle into the wells by limiting serial dilution. Because CTCs are rare, the loading biased the occupancy of the wells to single CTCs or CTCs in preformed clusters, allowing comparisons among individual CTCs. The defined address of each well allowed spatiotemporal tracking of single CTCs, and subsequent registration of data for functional measures on each CTC. Using this approach, we made three types of measurements on viable CTCs isolated from prostate cancer patients: (1) immediate and short-term viability of CTCs, (2) migration and invasion potential of CTCs, and (3) secretion of soluble factors. We found a rare subset of CTCs to possess malignant traits indicative of metastatic potential in late-stage, progressing prostate cancer patients. These CTCs were resistant to anoikis after being in the circulation as they stained Calcein AM+/Annexin V−, and remained viable for at least a week in culture. They were invasive in their epithelial state when embedded in Matrigel. They could also secrete proteases capable of cleaving peptide substrates. However, we did not observe such metastatic potential in every CTC, pointing to the presence of heterogeneity within CTCs and suggesting that enumeration of CTCs alone may be insufficient to understand metastasis or stratify patients. By interrogating the functional behavior of individual CTCs, we identified a rare subset of CTCs with phenotypes consistent with more efficient metastasis. The nanowell technology holds the potential to further investigate the cellular characteristics of rare tumor cells and design therapies to counter metastasis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C23. Citation Format: Xiaosai Yao, Atish D. Choudhury, Yvonne J. Yamanaka, William C. Hahn, K Dane Wittrup, J Christopher Love. Functional characterization of viable circulating tumor cells using nanowells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C23.