Enzyme-Mediated Intercellular Proximity Labeling for Detecting Cell–Cell Interactions

Ge, Yun; Chen, Long; Liu, Shibo; Zhao, Jingyi; Zhang, Heng; Chen, Peng R.

doi:10.1021/jacs.8b10286

Cited by 82 publications

(72 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A recent report has described a LIPSTIC-based system that relies on a SrtA variant with increased ability to label non-modified endogenous N-terminal glycines on the acceptor cell. This may provide additional flexibility to the system by not requiring engineering of an acceptor mouse strain [65].…”

Section: Linking Cellular Dynamics Microanatomy and DC Phenotypementioning

confidence: 99%

Studying interactions between dendritic cells and T cells in vivo

Chudnovskiy

Pasqual

Victora

2019

Current Opinion in Immunology

View full text Add to dashboard Cite

Antigen presentation is the key first step in the establishment of an antigen-specific T cell response. Among professional antigen presenting cells (APCs), dendritic cells (DCs) are the major population responsible for the priming of both CD4 + and CD8 + naïve T cells. This priming requires physical interaction between the DC and the T cell, during which signals are exchanged that determine both the magnitude and the quality of the ensuing response. The nature of these signals varies widely depending on the nature of the antigen, the anatomical site in which they take place, and the phenotype of the antigen-presenting DC, making the study of the dynamics, microanatomical distribution and phenotypic variation of DCs a key part of our understanding of adaptive immunity. Here, we provide a brief survey of how our view of T cell activation by DCs has evolved over recent years as intravital multiphoton microscopy and other emerging technologies have expanded our ability to study these events in vivo.

show abstract

Section: Linking Cellular Dynamics Microanatomy and DC Phenotypementioning

confidence: 99%

Studying interactions between dendritic cells and T cells in vivo

Chudnovskiy

Pasqual

Victora

2019

Current Opinion in Immunology

View full text Add to dashboard Cite

show abstract

“…In order to test the stability and expression level of SrtA protein, we employed two different SrtA mutants, [22,24] in two different protein fusion configurations, for a total of four lentiviral constructs ( Figure 1A). SrtA-3M is a calcium-sensitive variant (carrying three mutations as compared to wild-type SrtA, Figure 1A) that has previously been used in in vivo studies.…”

Section: Sortase a Protein Design And Testingmentioning

confidence: 99%

“…[24] SrtA-11M is a calcium-independent mutant (11 substitutions away from wild-type SrtA, Figure 1A) recently developed. [22] To facilitate labeling of nearby cells, we fused Figure 1. Characterization of Sortase A-expressing constructs.…”

Section: Sortase a Protein Design And Testingmentioning

confidence: 99%

“…Cell surface proteins carrying an N-terminal glycine are relatively abundant in both human and mouse cells (e.g., MHC-I, MHC-II, VE-Cadherin, CD19, and integrins). [22,23] We genetically encoded SrtA in parental tumor cell lines, which then release SrtA as an EV-bound transmembrane protein. Once a SrtA+ EV interacts with a target cell in the presence of a substrate peptide (conjugated to a reporter molecule), SrtA catalyzes the formation of a covalent bond between the substrate peptide and surface proteins of the EV-binding cell.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cell Surface Labeling by Engineered Extracellular Vesicles

et al. 2020

View full text Add to dashboard Cite

Deoxycytidine [3]). Extracellular vesicles (EVs) are increasingly recognized as a new class of multivalent messengers involved in cell-cell communication, both locally and at distance. [4-7] EVs are membranebound particles released by virtually every cell type, with stressed and transformed cells secreting the highest amounts. [8-11] Investigations of EVs' roles in different pathophysiological conditions are thwarted by biases introduced during isolation and reinfusion procedures. In particular, these biases manifest at different levels: (i) certain EV subsets may be excluded, [6] (ii) operator judgment is involved in deciding how many EVs to reinfuse, and (iii) an assumption that EV concentration is highest in blood, whereas several studies have shown that EV biodistribution occurs primarily via lymph fluid. [12-15] To circumvent these issues, the EV-releasing cells of interest can be genetically engineered to express membrane-bound reporters (or other functional biomolecules) in vitro or in vivo. [16] Although this approach is still in its infancy, it avoids the above limitations and to unbiasedly investigate untouched, endogenously released EVs. Although a detailed description of how EVs signal between cells is still missing, two nonexclusive mechanisms have been proposed: (i) membrane fusion between EVs and target cells, thereby releasing EV cargo (e.g., microRNAs and proteins, including oncogenes) directly in the cytoplasm of targeted cells; and (ii) surface binding with subsequent ligand-receptor type signaling. Membrane fusion (also known as horizontal transfer) has been described using several in vitro models (see Pucci and Pittet [17] and references therein), but proved more challenging to demonstrate in vivo. Recently, horizontal transfer of tumor-derived EVs was tested in mice using a sensitive and stringent approach based on the CRE-Lox reporter system. [15,18] Although CRE recombinase has an efficiency of 55%, [19] horizontal transfer was shown to happen mainly between tumor cells in vivo, but not between tumor cells and host cells. [15,18] These results have been further confirmed using other genetic strategies, [20,21] suggesting that surface binding may represent the main mechanism of EV-mediated tumor-host communications in vivo. A major limitation in the study of native EV signaling to host cells in vivo derives from their small size, which restricts the amount of reporter molecules carried by a single EV. As a consequence, only target cells binding the highest levels of EVs [12,14,15] will accumulate enough reporter molecules to be detected and investigated. Nonetheless, many host cell types Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically eng...

show abstract

“…Although a number of enzyme-based proximity-labeling systems have been developed to profile protein-protein interactions (Branon et al, 2018;Kim and Roux, 2016;Liu et al, 2018;Long et al, 2016;Slavoff et al, 2011), very few can be applied to probe cell-cell interactions (Ge et al, 2019;Liu et al, 2018;Pasqual et al, 2018). To the best of our knowledge, all of these approaches rely on genetic manipulations.…”

Section: Install H Pylori α1-3-fucosyltransferase (Ft) Onto the Cellmentioning

confidence: 99%

Detecting Tumor Specific Antigen-Reactive T cells from Tumor Infiltrating Lymphocytes via Interaction Dependent Fucosyl-biotinylation

Liu

Chen

et al. 2020

Preprint

View full text Add to dashboard Cite

HighlightsInteraction dependent fucosylation enables the detection and isolation of bona fide intratumoral tumor specific antigen-reactive T cells Tumor specific antigen-reactive CD8 + T cells possess capabilities to be expanded and adoptively transferred for tumor control Tumor specific antigen-reactive CD8 + T cells feature oligoclonal expansion and upregulate genes for the steroid biosynthesis and metabolic process Intratumoral bystander CD8 + T cells can be separated into two groups based on PD-1 expression that feature distinct gene modules SummaryRe-activation and clonal expansion of tumor specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL) based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. Here we introduce FucoID as a general platform to detect endogenous antigen-specific T cells and study their biology. Through this interaction dependent labeling approach, TSA-reactive T cells can be detected and separated from bystander T cells in primary tumor digests based on their cell-surface enzymatic fucosylbiotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct TCR repertoire and unique gene features. Though exhibiting a dysfunctional phenotype, this subset of TILs possesses substantial capabilities of proliferation and tumor specific killing. FucoID features genetic manipulation-free procedures and a quick turnover cycle, and therefore should have the potential of accelerating the pace of personalized cancer treatment.

show abstract

Enzyme-Mediated Intercellular Proximity Labeling for Detecting Cell–Cell Interactions

Cited by 82 publications

References 28 publications

Studying interactions between dendritic cells and T cells in vivo

Studying interactions between dendritic cells and T cells in vivo

Cell Surface Labeling by Engineered Extracellular Vesicles

Detecting Tumor Specific Antigen-Reactive T cells from Tumor Infiltrating Lymphocytes via Interaction Dependent Fucosyl-biotinylation

Contact Info

Product

Resources

About