A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells

Abstract

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (~1 × 104 cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen–specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen–specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.

Polyfunctional responses by human T cells result from sequential release of cytokines

“…Indeed, the production of both IL-2 and IFN-γ by CD4 + T cells in vivo has been shown to begin within hours of stimulation and wane after 16-18 h (13, 14). It has not been possible, however, to determine whether cells release multiple cytokines simultaneously, or sequentially, in time because techniques such as intracellular cytokine staining (ICS) and multiparametric ELISpot provide only integrative, endpoint measures (15)(16)(17)(18). Therefore, resolving when activated T cells initiate the release of cytokines, and how their responses evolve in time, should provide fundamental insight into how individual cells dynamically modulate intercellular signals to affect population-level responses toward pathological conditions or clinical interventions.…”

A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

“…However, it provides an attractive combination of multiplexing (up to 50 using the platform described here), miniaturization, reproducibility, sensitivity, cost, and throughput. This combination of characteristics makes it enabling for several exciting fundamental and clinical applications ranging from single cell proteomics 13,15,28 to high resolution tissue engineering. 30 …”

“…barcode chip (SCBC) to perform a comprehensive functional analysis of rare cells from clinical specimens. 13 This chip was composed of ∼1000 separate microchambers into which single cells or small, defined cell colonies were isolated. Each microchamber contained two duplicate copies of an antibody array.…”

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry

“…Single-cell analysis has become increasingly important in biology, especially in neuroscience research, because neurons are specialized cells with a huge diversity of shapes, connections, and anatomical, electrophysiological, and biochemical properties (13)(14)(15)(16)(17). Single-cell chemical profiling could further the current understanding of biological variability and differential susceptibility to disease and treatment, as well as heterogeneity among similar cells (18,19). Most of currently available single-cell techniques require the chemical of interest (usually large molecules, such as DNA, RNA, and proteins) to be labeled, amplified, or electrochemically detectable (4,6,10,12).…”