Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries.

High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow.

An artificial antigen-presenting cell with paracrine delivery of IL-2 impacts the magnitude and direction of the T cell response.

Artificial antigen-presenting cells (aAPCs) are an emerging technology to induce therapeutic cellular immunity without the need for autologous antigen-presenting cells (APCs). To fully replace natural APCs, an optimized aAPC must present antigen (signal 1), provide costimulation (signal 2), and release cytokine (signal 3). Here we demonstrate that the spatial and temporal characteristics of paracrine release of IL-2 from biodegradable polymer aAPCs (now termed paAPCs) can significantly alter the balance in the activation and proliferation of CD8+ and CD4+ T cells.

Biomimetic approaches to modulating the T cell immune response with nano- and micro- particles.

Modulating immune responses to pathogen invasion and even tumors is a major goal in immunotherapy. T cells play a central role in these responses. Progress towards that goal is accomplished by stimulating the antigen-specific T cell immune response in vivo through active immunization, or by re-transfer of large numbers of T cells expanded outside the body in a process called adoptive immunotherapy.

Antigen presentation on artificial acellular substrates: modular systems for flexible, adaptable immunotherapy.

Background: Recent findings on T cells and dendritic cells have elucidated principles that can be used for a bottom-up approach to engineering artificial antigen presentation on synthetic substrates.

Objective/methods: To compare the latest artificial antigen-presenting cell (aAPC) technology, focussing on acellular systems because they offer advantages such as easy tunability and rapid point-of-care application compared with cellular systems. We review acellular aAPC performance and discuss their promise for clinical applications.

Label-free electronic detection of the antigen-specific T-cell immune response.

Detection of antigen-specific T-cells is critical for diagnostic assessment and design of therapeutic strategies for many disease states. Effective monitoring of these cells requires technologies that assess their numbers as well as functional response. Current detection of antigen-specific T-cells involves flow cytometry and functional assays and requires fluorescently labeled, soluble forms of peptide-loaded major histocompatability complexes (MHC).

Enhanced cellular activation with single walled carbon nanotube bundles presenting antibody stimuli.

Efficient immunotherapy can be accomplished by expanding T cells outside the body using single walled carbon nanotube (SWNT) bundles presenting antibody stimuli. Owing to the large surface area of these bundles, which can reach 1560 m (2)/g, T cell stimulating antibodies such as anti-CD3, can be presented at high local concentrations inducing potent activation of T cells. We show that anti-CD3 adsorbed onto SWNT bundles stimulate cells more effectively than equivalent concentrations of soluble anti-CD3.

A comprehensive platform for ex vivo T-cell expansion based on biodegradable polymeric artificial antigen-presenting cells.

Efficient T-cell stimulation and proliferation in response to specific antigens is a goal of immunotherapy against infectious disease and cancer. Manipulation of this response can be accomplished by adoptive immunotherapy involving the infusion of antigen-specific T-cell populations expanded ex vivo with antigen presenting cells. We mimicked physiological antigen presentation on a biodegradable microparticle constructed from poly(lactide-co-glycolide) (PLGA), a polymer system whose safety has been established for use in humans.

Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease.

We report the construction of a cell-based fluorescent reporter for anthrax lethal factor (LF) protease activity using the principle of fluorescence resonance energy transfer (FRET). This was accomplished by engineering an Escherichia coli cell line to express a genetically encoded FRET reporter and LF protease. Both proteins were encoded in two different expression plasmids under the control of different tightly controlled inducible promoters.