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Bioengineering tools for probing intracellular events in T lymphocytes

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Abstract T lymphocytes are the central coordinator and executor of many immune functions. The activation and function of T lymphocytes are mediated through the engagement of cell surface receptors and regulated by a myriad of intracellular signaling network. Bioengineering tools, including imaging modalities and fluorescent probes, have been developed and employed to elucidate the cellular events throughout the functional lifespan of T cells. A better understanding of these events can broaden our knowledge in the immune systems biology, as well as accelerate the development of effective diagnostics and immunotherapies. Here we review the commonly used and recently developed techniques and probes for monitoring T lymphocyte intracellular events, following the order of intracellular events in T cells from activation, signaling, metabolism to apoptosis. The techniques introduced here can be broadly applied to other immune cells and cell systems. This article is categorized under: Immune System Diseases > Molecular and Cellular Physiology Immune System Diseases > Biomedical Engineering Infectious Diseases > Biomedical Engineering
Bioengineering approaches to the immunological synapse formation. (a) A schematic plot of an immunological synapse formed between a T cell and an antigen presenting cell (APC). SMAC: supramolecular activation clusters. MTOC: microtubule organization center. (b) T cell interacting with supported lipid bilayer that mimics the APC membrane. (c) Laser tweezer allows for reorienting T cell/APC pairs for direct imaging of the cell–cell interface. Measuring the bond strength between TCR and pMHC with (d) atomic force microscopy (AFM) and (e) fluorescence biomembrane force probe (fBFP). RBC, red blood cell
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Detection of cytokine/chemokine from single T cells. (a) In ELISpot, cells are immobilized (1), cytokines are captured by precoated antibody on nearby substrate (2) for subsequent staining and microscopic detection (3). (b) In intracellular staining, T cells are activated, fixed, permeabilized, stained for intracellular cytokines, and analyzed with flow cytometry. Protein secretion is inhibited by the addition of inhibitors of Golgi transport. (c) In cell surface capture, a chimeric antibody with specificity to both cell surface receptor and cytokine is preloaded on cell surface (1), to capture the cytokine released by individual cells (2). A detection antibody is used to stain the captured cytokines for measurement in flow cytometry or microscopy (3)
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Examples of FRET sensors for detecting T cell signaling. (a) CD3ζ‐CliF reporters have intermolecular FRET when CD3ζ molecules cluster, resulting in a decrease in Venus fluorescence lifetime. (b) A ZIP reporter contains an SH2 domain, which binds to the tyrosine‐phosphorylated ITAMs to cause FRET between eGFP and mCherry, resulting in a decrease in eGFP fluorescence lifetime. (c) In Raichu‐Ras, Ras‐GTP binds to Raf, which results in FRET from CFP to YFP. (d) In a 3FRET system, there are three FRET pairs: mCFP‐mYFP, mCFP‐mCherry, and mYFP‐mCherry. Each cell is imaged with six channel settings, including three FRET channels and three individual fluorophore channels. The corresponding FRET channel, the donor channel and the receptor channel are used to calculate each FRET pair. (e) In GCaMP, when Ca2+ binds to CaM, CaM‐Ca2+ interact with M13, inducing a conformational change in the circular permutated EGFP (cpEGFP), which leads to fluorescence intensity increase
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Intensity‐based, FRET‐based and FILM‐based sensors. (a) Intensity based sensors are usually fluorescently tagged/labeled proteins or molecule that can specifically bind to target molecules, or a recombinant version of the target molecule with a fluorescent protein tag. The readouts are typically the percentage of cells showing signal above a threshold, or the spatial distribution of the fluorescence. (b) FRET‐based sensors display a change in the emission spectrum when the distance between donor and acceptor is changed. The changes in emission are usually detected at a specific emission wavelength or ratiometrically at two wavelengths. (c) FLIM‐based sensors display drastic changes in fluorescence lifetime upon binding to the target molecule. Some FLIM sensors consist of two parts: a substrate portion which binds to the analyte, and a delivery portion which helps the sensor get into cells. The readout is the fluorescence lifetime of the fluorophore
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Principles of imaging techniques and example images. (a) In TIRF, the intensity of the evanescent wave decays exponentially with distance from the interface. Therefore, background noise from none‐surface fluorophores is greatly reduced. (b) In PALM and STORM, a sparse subset of photo‐switchable fluorophores is excited each time and then reconstruct into high‐resolution images with the repeatedly‐taken pictures. (c) In SIM, samples are excited with different patterns of illumination each time and high‐resolution images are reconstructed by overlaying these images. (d) In STED, fluorophores at the center of focal spot are left active while those at the periphery get selectively deactivated to minimize the background. (e) LifeAct stained F‐actin under TIRF (Treanor et al., 2010). (f) TCR microclusters under light sheet dSTORM (Y. S. Hu, Cang, & Lillemeier, 2016). (g) Primary NK cell stimulated on a surface coated with MICA‐Fc and ICAM‐1 under 3D SIM. Green: actin; Red: MTOC (A. C. N. Brown et al., 2011). (h) Phalloidin stained F‐actin under STED (E. M. Mace & Orange, 2014)
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