Transfer of cell membrane components via trogocytosis occurs in CD4+ Foxp3+ CD25+ regulatory T-cell contact-dependent suppression.

A key component of the immune system is its ability to establish and maintain peripheral tolerance. Naturally occurring CD4+ CD25+ Foxp3+ regulatory T (nTreg) cells represent an important means by which this is accomplished, through their potent ability to suppress the actions of both CD4+ and CD8+ effector (Teff) cells in vitro and in vivo. We hypothesized that direct contact between nTreg and Teff cells is sufficient for nTreg cell-contact suppression. We first show that nTreg cell suppression is independent of APCs and their derived co-stimulatory signals. We then used a two-colour, lipid dye labelling and quantification approach to formally demonstrate that nTreg cells specifically form cell conjugates with responding T (Tresp) cells only under TCR activating conditions. Strikingly, activated CD4+ nTreg cells undergo progressive trogocytosis, a process by which membrane fragments are transferred from one cell subset to another, with Tresp cells more readily than Teff cells. These results are the first to show that nTreg cell cognate interactions with Tresp cells leads to trogocytosis between the cells, and the first to relate the degree of trogocytosis with the level of nTreg-mediated suppression.

Quantum dot-FRET systems for imaging of neuronal action potentials.

Fluorescent semiconductor quantum dots (QDs) can act as energy donors or acceptors with a wide variety of environmentally-sensitive molecules. Conjugation of a single QD to a select number of the selected molecule can optimize the range of sensitivity for a given application, and the relatively large size of the QDs allows them to be tracked individually in cells. Using QDs as FRET acceptors, we have created first-generation sensors for membrane potential which shows good signal to noise and time resolution, but prohibitive toxicity. The challenges of delivery, calibration, and toxicity and plans for improvement of the sensors are presented, in the context of the eventual aim of monitoring membrane potential in a cultured motor neuron model of amyotrophic lateral sclerosis.

Quantum dots as phototoxic drugs and sensors of specific metabolic processes in living cells.

When conjugated to CdSe/ZnS nanocrystals (quantum dots), the nucleobase adenine and the neurotransmitter dopamine quench fluorescence emission from in a manner strongly dependent upon the size of the quantum dot. The degree of quenching serves to predict the efficiency with which the conjugates are able to enter living cells. Along with quenching, the presence of specific receptors on the cells is necessary for QD binding, entry, and phototoxicity. Toxicity is manifested by opening of large membrane pores and by oxidative DNA damage, and does not require the release of Cd+2. In bacterial cells, light exposure is necessary for uptake, and procedures to reduce toxicity eliminate labeling. In mammalian cells, antioxidants prevent toxicity but not QD uptake, leading to QD-loaded cells that are nonfluorescent before light exposure. These findings provide a general procedure for rational design of nanoparticle-based photosensitizing drugs and for "off-on" fluorescent labels.