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Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

The University of Chicago

Here we report a peptide-MHC (pMHC) dodecamer as a "next generation" technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4(+)and CD8(+)αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4(+)CD8(+)double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates. 

Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer.

Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique for studying the conformation dynamics and interactions of individual biomolecules. In this review, we describe the concept and principle of smFRET, illustrate general instrumentation and microscopy settings for experiments, and discuss the methods and algorithms for data analysis. Subsequently, we review applications of smFRET in protein conformational changes, ion channel open-close properties, receptor-ligand interactions, nucleic acid structure regulation, vesicle fusion, and force induced conformational dynamics. Finally, we discuss the main limitations of smFRET in molecular biology.

Single-molecule fluorescence resonance energy transfer in molecular biology.

CD8 T cells and NK cells are both cytotoxic effector cells of the immune system, but the recognition, specificity, sensitivity, and memory mechanisms are drastically different. While many of these topics have been extensively studied in CD8 T cells, very little is known about NK cells. Current cancer immunotherapies mainly focus on CD8 T cells, but have many issues of toxicity and efficacy. Given the heterogeneous nature of cancer, personalized cancer immunotherapy that integrates the power of both CD8 T cells in adaptive immunity and NK cells in innate immunity might be the future direction, along with precision targeting and effective delivery of tumor-specific, memory CD8 T cells and NK cells.

CD8 T Cells and NK Cells: Parallel and Complementary Soldiers of Immunotherapy.

Epigenetic modifications to histones dictate the differentiation of naïve CD4 T cells into different subsets of effector T helper (T) cells. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been implicated in the mechanism regulating the differentiation of T1, T2 and regulatory T (T) cells. However, whether and how EZH2 regulates follicular helper T (T) cell differentiation remain unknown. Using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection, we observed abundant EZH2 expression and associated H3K27me3 modifications preferentially in the early committed virus-specific T cells compared to those in T1 cells. Ablation of EZH2 in LCMV-specific CD4 T cells leads to a selective impairment of early T cell fate commitment, but not late T differentiation or memory T maintenance. Mechanistically, EZH2 specifically stabilizes the chromatin accessibility of a cluster of genes that are important for T fate commitment, particularly B cell lymphoma 6 (Bcl6), and thus directs T cell commitment. Therefore, we identified the chromatin-modifying enzyme EZH2 as a novel regulator of early T differentiation during acute viral infection.

The histone methyltransferase EZH2 primes the early differentiation of follicular helper T cells during acute viral infection.

Mass spectrometry enables global analysis of posttranslationally modified proteoforms from biological samples, yet we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function. Here we describe a proteomic method, Hotspot Thermal Profiling, to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This massively parallel biophysical assay unveiled shifts in overall protein stability in response to site-specific phosphorylation sites, as well as trends related to protein function and structure. This method can detect intrinsic changes to protein structure as well as extrinsic changes to protein-protein and protein-metabolite interactions resulting from phosphorylation. Finally, we show that functional 'hotspot' protein modification sites can be discovered and prioritized for study in a high-throughput and unbiased fashion. This approach is applicable to diverse organisms, cell types and posttranslational modifications.

Jun Huang

Committee on Cancer Biology,
Pritzker School of Molecular Engineering

The University of Chicago

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

Jun Huang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Committee on Cancer Biology,Pritzker School of Molecular Engineering

The University of Chicago

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

Committee on Cancer Biology,
Pritzker School of Molecular Engineering