Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation

Balagopalan, Lakshmi; Raychaudhuri, Kumarkrishna; Samelson, Lawrence E.

doi:10.3389/fcell.2020.608530

Cited by 11 publications

(6 citation statements)

References 119 publications

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“…7 ; Su et al, 2016 ). Protein phase separation induced by the LAT-GRB2-SOS1 scaffold stabilizes the recruitment of “client” proteins and organizes them into liquid-like condensates (also known as microclusters; Balagopalan et al, 2021 ). They facilitate downstream signaling events such as actin polymerization and activation of the RAS-MAPK-ERK, NFAT, and NF-κB signaling pathways ( Ditlev et al, 2018 ; Ditlev et al, 2019 ; Huang et al, 2017 ; Su et al, 2016 ).…”

Section: Resultsmentioning

confidence: 99%

Systems-level conservation of the proximal TCR signaling network of mice and humans

Nicolas

Ollier

Mori

et al. 2022

Journal of Experimental Medicine

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We exploited traceable gene tagging in primary human T cells to establish the composition and dynamics of seven canonical TCR-induced protein signaling complexes (signalosomes) using affinity purification coupled with mass spectrometry (AP-MS). It unveiled how the LAT adaptor assembles higher-order molecular condensates and revealed that the proximal TCR-signaling network has a high degree of qualitative and quantitative conservation between human CD4+ and CD8+ T cells. Such systems-level conservation also extended across human and mouse T cells and unexpectedly encompassed protein–protein interaction stoichiometry. Independently of evolutionary considerations, our study suggests that a drug targeting the proximal TCR signaling network should behave similarly when applied to human and mouse T cells. However, considering that signaling differences likely exist between the distal TCR-signaling pathway of human and mouse, our fast-track AP-MS approach should be favored to determine the mechanism of action of drugs targeting human T cell activation. An opportunity is illustrated here using an inhibitor of the LCK protein tyrosine kinase as a proof-of-concept.

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Section: Resultsmentioning

confidence: 99%

Systems-level conservation of the proximal TCR signaling network of mice and humans

Nicolas

Ollier

Mori

et al. 2022

Journal of Experimental Medicine

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“…TCR-mediated signaling iniBates formaBon of sub-micron sized molecular aggregates called microclusters (MC). These molecular structures, now known to be phase-separated condensates, contain transmembrane receptors, co-receptors, membrane-associated and cytoplasmic kinases, enzymes and adapter proteins 2,4,[26][27][28][29] . Complex phosphorylaBon and dephosphorylaBon events, and the spaBal arrangements and stoichiometry of these molecules regulate signaling from these MCs from the iniBaBon of acBvaBon.…”

Section: Discussionmentioning

confidence: 99%

CD28 Shapes T Cell Receptor Signaling by Regulating ZAP70 Activation and Lck Dynamics

Raychaudhuri,

Rangu,

et al. 2024

Preprint

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T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation. TCR microclusters (MC) are submicron-sized molecular condensates and basic signaling units that form immediately after TCR ligation. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in MCs and increased ZAP70 activation. This CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.

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“…Balagopalan and colleagues have recently shown that upon stimulus, the formation of native TCR nanoclusters in the resting T cell membrane improves signal transduction [ 32 ]. Based on this result, we hypothesized that the distinct qualities of preformed receptor assemblies in the unstimulated CAR T cell’s membrane, as well as the diffusional mobility of the chimeric antigen receptors, could affect the formation of the immunological synapse and early activation signaling events upon engagement with the specific tumor target.…”

Section: Discussionmentioning

confidence: 99%

CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

Mezősi-Csaplár

Szöór

Vereb

2023

Cancers

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Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small but highly mobile CD28.41BB.z and large but less mobile .z CAR clusters induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs organized in clusters of intermediate size and mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect.

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Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation

Cited by 11 publications

References 119 publications

Systems-level conservation of the proximal TCR signaling network of mice and humans

Systems-level conservation of the proximal TCR signaling network of mice and humans

CD28 Shapes T Cell Receptor Signaling by Regulating ZAP70 Activation and Lck Dynamics

CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

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