analysis-t-cell-receptor-induced-calcium-influx-primary-murine-t

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...

University of Colorado-Anschutz School of Medicine

Quantitative mass spectrometry applied to T cell activation reveal key insights into signal transduction pathways. These data identify selective alterations in activated T cell protein concentrations, and detail previously undescribed protein-protein interactions.

TCR signaling: it's all about the numbers.

The Tec kinase IL-2-inducible T cell kinase (ITK) regulates the expression of TCR-induced genes.  T cell responses are impaired but not absent. ITK inhibition prevented colitis disease progression and impaired T cell migration to the colon in mice. To examine the function of ITK in T cell migration to the intestine, we examined the number of gut T cells in  mice and then evaluated their expression of gut-homing receptors. Combined with in vitro murine T cell stimulation and in vivo migration assay using congenic B6 mice, we demonstrated an essential role for ITK in T cell migration to the intestine in mice. Reconstitution of  mouse CD8 T cells with IFN regulatory factor 4 restored gut-homing properties, providing mechanistic insight into the function of ITK-mediated signaling in CD8 T cell migration to the intestinal mucosa in mice.

CD8 T Cells Require ITK-Mediated TCR Signaling for Migration to the Intestine.

TCR signal strength is critical for CD8 T cell clonal expansion after Ag stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through the induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. In this study, we show that Ag potency regulates the kinetics but not the magnitude of NFAT1 activation in single mouse CD8 T cells. Consequently, T cells that transduce weaker TCR signals exhibit a marked delay in  mRNA induction, resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. We further show that the activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of  gene transcription. These findings provide insight into the function of ITK in TCR signal transduction that ultimately regulates IRF4 expression levels in response to variations in TCR signal strength.

Activation of the Tec Kinase ITK Controls Graded IRF4 Expression in Response to Variations in TCR Signal Strength.

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal interleukin-2-inducible T cell kinase (ITK) simultaneously trigger many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions, ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through the unequal activation of distinct signaling pathways, we examined Erk1/2 phosphorylation or nuclear factor of activated T cells (NFAT) and nuclear factor (NF)-κB translocation in naïve OT-I CD8 cell nuclei. We observed the consistent digital activation of NFAT1 and Erk1/2, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength, tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed the dampened induction of AP-1 factors  and , NF-κB response gene transcripts, and survival factor  transcripts. ATAC sequencing analysis also revealed that genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned the expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating the optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variations in TCR signal strength can produce patterns of graded gene expression in activated T cells.

Article	Year
TCR signaling: it's all about the numbers. Nature immunology\| PubMed ID: 31591572	2019
CD8 T Cells Require ITK-Mediated TCR Signaling for Migration to the Intestine. ImmunoHorizons\| PubMed ID: 32034085	2020
Activation of the Tec Kinase ITK Controls Graded IRF4 Expression in Response to Variations in TCR Signal Strength. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 32493815	2020
Hierarchy of signaling thresholds downstream of the T cell receptor and the Tec kinase ITK. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 34452995	2021

Leslie J. Berg

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