PKCθ and Itk functionally interact during primary mouse CD3+ T cell activation

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

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The T cell CD6 receptor operates a multitask signalosome with opposite functions in T cell activation

Journal of Experimental Medicine ◽

10.1084/jem.20201011 ◽

2020 ◽

Vol 218 (2) ◽

Cited By ~ 1

Author(s):

Daiki Mori ◽

Claude Grégoire ◽

Guillaume Voisinne ◽

Javier Celis-Gutierrez ◽

Rudy Aussel ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Signal Propagation ◽

Nucleotide Polymorphisms ◽

Transmembrane Receptors ◽

Promising Target ◽

Primary Mouse ◽

Respective Contribution

To determine the respective contribution of the LAT transmembrane adaptor and CD5 and CD6 transmembrane receptors to early TCR signal propagation, diversification, and termination, we describe a CRISPR/Cas9–based platform that uses primary mouse T cells and permits establishment of the composition of their LAT, CD5, and CD6 signalosomes in only 4 mo using quantitative mass spectrometry. We confirmed that positive and negative functions can be solely assigned to the LAT and CD5 signalosomes, respectively. In contrast, the TCR-inducible CD6 signalosome comprised both positive (SLP-76, ZAP70, VAV1) and negative (UBASH3A/STS-2) regulators of T cell activation. Moreover, CD6 associated independently of TCR engagement to proteins that support its implication in inflammatory pathologies necessitating T cell transendothelial migration. The multifaceted role of CD6 unveiled here accounts for past difficulties in classifying it as a coinhibitor or costimulator. Congruent with our identification of UBASH3A within the CD6 signalosome and the view that CD6 constitutes a promising target for autoimmune disease treatment, single-nucleotide polymorphisms associated with human autoimmune diseases have been found in the Cd6 and Ubash3a genes.

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Macropinocytosis drives T cell growth by sustaining the activation of mTORC1

Nature Communications ◽

10.1038/s41467-019-13997-3 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 6

Author(s):

John C. Charpentier ◽

Di Chen ◽

Philip E. Lapinski ◽

Jackson Turner ◽

Irina Grigorova ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Growth ◽

T Cell Activation ◽

Mammalian Cells ◽

Large Scale ◽

Cell Activation ◽

Phase Form ◽

Primary Mouse ◽

T Cell Growth

AbstractMacropinocytosis is an evolutionarily-conserved, large-scale, fluid-phase form of endocytosis that has been ascribed different functions including antigen presentation in macrophages and dendritic cells, regulation of receptor density in neurons, and regulation of tumor growth under nutrient-limiting conditions. However, whether macropinocytosis regulates the expansion of non-transformed mammalian cells is unknown. Here we show that primary mouse and human T cells engage in macropinocytosis that increases in magnitude upon T cell activation to support T cell growth even under amino acid (AA) replete conditions. Mechanistically, macropinocytosis in T cells provides access of extracellular AA to an endolysosomal compartment to sustain activation of the mechanistic target of rapamycin complex 1 (mTORC1) that promotes T cell growth. Our results thus implicate a function of macropinocytosis in mammalian cell growth beyond Ras-transformed tumor cells via sustained mTORC1 activation.

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