Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca2+ influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca2+ channel components to existing and newly forming immunological synapses.

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The Ca2+-activated K+ channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00376.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. C1431-C1439 ◽

Cited By ~ 43

Author(s):

Stella A. Nicolaou ◽

Lisa Neumeier ◽

YouQing Peng ◽

Daniel C. Devor ◽

Laura Conforti

Keyword(s):

T Cells ◽

T Cell ◽

Antigen Presentation ◽

T Cell Activation ◽

Cell Activation ◽

Immunological Synapse ◽

Cell Receptor ◽

K Channel ◽

Activated T Cells ◽

Barr Virus

T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca2+ influx. KCa3.1 channels modulate Ca2+ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3ε, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3ε to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15–30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca2+ influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.

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Sphingomyelin breakdown in T cells: role in activation, effector functions and immunoregulation

Biological Chemistry ◽

10.1515/hsz-2014-0282 ◽

2015 ◽

Vol 396 (6-7) ◽

pp. 749-758 ◽

Cited By ~ 19

Author(s):

Niklas Beyersdorf ◽

Nora Müller

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Cell Activation ◽

Immunological Synapse ◽

Cell Activity ◽

Cell Receptor ◽

Cell Mediated Immunity ◽

Functional Responses

Abstract Host T cell activation, a key step in obtaining adaptive immunity against pathogens, is initiated by the binding of the T cell receptor to a foreign antigenic peptide presented by the major histocompatibility complex on the surface of an antigen-presenting cell and, consequently, formation of an immunological synapse. Within the immunological synapse, the engagement of the T cell receptor in cooperation with simultaneous ligation of co-stimulatory molecules induces a precisely organized cascade of signaling events and pathways that regulate clonal expansion and differentiation of naïve T cells into effector T cells contributing to pathogen clearance. The biochemical changes that underlie T cell activation and differentiation, however, not only involve proteins but also lipids. In particular, catabolic cleavage of sphingomyelin generating ceramide can substantially influence functional responses in cells of the immune system. Changes in sphingomyelin and ceramide content have been reported to directly impact on membrane physiology, thus modifying signal transmission and interfering with diverse aspects of T cell activity. In this review we will focus on sphingomyelin breakdown/ceramide generation in T cells with regard to their function and development of T cell-mediated immunity.

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HIV Envelope gp120 Alters T Cell Receptor Mobilization in the Immunological Synapse of Uninfected CD4 T Cells and Augments T Cell Activation

Journal of Virology ◽

10.1128/jvi.01532-16 ◽

2016 ◽

Vol 90 (23) ◽

pp. 10513-10526 ◽

Cited By ~ 7

Author(s):

Jing Deng ◽

Yu-ya Mitsuki ◽

Guomiao Shen ◽

Jocelyn C. Ray ◽

Claudia Cicala ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Immunological Synapse ◽

Cell Receptor ◽

Activation Process ◽

Hiv Envelope

ABSTRACTHIV is transmitted most efficiently from cell to cell, and productive infection occurs mainly in activated CD4 T cells. It is postulated that HIV exploits immunological synapses formed between CD4 T cells and antigen-presenting cells to facilitate the targeting and infection of activated CD4 T cells. This study sought to evaluate how the presence of the HIV envelope (Env) in the CD4 T cell immunological synapse affects synapse formation and intracellular signaling to impact the downstream T cell activation events. CD4 T cells were applied to supported lipid bilayers that were reconstituted with HIV Env gp120, anti-T cell receptor (anti-TCR) monoclonal antibody, and ICAM-1 to represent the surface of HIV Env-bearing antigen-presenting cells. The results showed that the HIV Env did not disrupt immunological synapse formation. Instead, the HIV Env accumulated with TCR at the center of the synapse, altered the kinetics of TCR recruitment to the synapse and affected synapse morphology over time. The HIV Env also prolonged Lck phosphorylation at the synapse and enhanced TCR-induced CD69 upregulation, interleukin-2 secretion, and proliferation to promote virus infection. These results suggest that HIV uses the immunological synapse as a conduit not only for selective virus transmission to activated CD4 T cells but also for boosting the T cell activation state, thereby increasing its likelihood of undergoing productive replication in targeted CD4 T cells.IMPORTANCEThere are about two million new HIV infections every year. A better understanding of how HIV is transmitted to susceptible cells is critical to devise effective strategies to prevent HIV infection. Activated CD4 T cells are preferentially infected by HIV, although how this is accomplished is not fully understood. This study examined whether HIV co-opts the normal T cell activation process through the so-called immunological synapse. We found that the HIV envelope is recruited to the center of the immunological synapse together with the T cell receptor and enhances the T cell receptor-induced activation of CD4 T cells. Heightened cellular activation promotes the capacity of CD4 T cells to support productive HIV replication. This study provides evidence of the exploitation of the normal immunological synapse and T cell activation process by HIV to boost the activation state of targeted CD4 T cells and promote the infection of these cells.

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Disc Large Homolog 1 Is Critical for Early T Cell Receptor Micro Cluster Formation and Activation in Human T Cells

Vaccines ◽

10.3390/vaccines9121446 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1446

Author(s):

June Guha ◽

Raj Chari

Keyword(s):

T Cells ◽

T Cell ◽

Cell Signaling ◽

T Cell Receptor ◽

T Cell Activation ◽

Cell Activation ◽

Synapse Formation ◽

Immunological Synapse ◽

Cell Receptor ◽

T Cell Signaling

T cell activation by antigen involves multiple sequential steps, including T cell receptor-microcluster TCR-(MC) formation, immunological synapse formation, and phosphorylation of mediators downstream of the TCR. The adaptor protein, Disc Large Homolog 1 (DLG1), is known to regulate proximal TCR signaling and, in turn, T cell activation, acting as a molecular chaperone that organizes specific kinases downstream of antigen recognition. In this study, we used knockdown and knockout technologies in human primary T cells and a human T cell line to demonstrate the role of DLG1 in proximal T cell signaling. High-end confocal microscopy was used for pictorial representation of T cell micro-clusters and colocalization studies. From all these studies, we could demonstrate that DLG1 functions even earlier than immunological synapse formation, to regulate T cell activation by promoting TCR-MC formation. Moreover, we found that DLG1 can act as a bridge between the TCR-ζ chain and ZAP70 while inhibiting binding of the phosphatase SHP1 to TCR-ζ. Together, these effects drive dysregulation of T cell activation in DLG1-deficient T cells. Overall, the activation and survival status of T cell is a critical determinant of effective vaccine response, and DLG1-mediated T cell signaling events can be a driving factor for improving vaccine-designing strategies.

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T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

Journal of Experimental Medicine ◽

10.1084/jem.178.6.2107 ◽

1993 ◽

Vol 178 (6) ◽

pp. 2107-2113 ◽

Cited By ~ 46

Author(s):

A J da Silva ◽

O Janssen ◽

C E Rudd

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Intracellular Signaling ◽

Cell Activation ◽

Cell Receptor ◽

Sh2 Domain ◽

T Complex

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

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T cells: a dedicated effector kinase pathways for every trait?

Biochemical Journal ◽

10.1042/bcj20210006 ◽

2021 ◽

Vol 478 (6) ◽

pp. 1303-1307

Author(s):

Kriti Bahl ◽

Jeroen P. Roose

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cell Receptor ◽

Spectrometric Analysis ◽

Activated T Cells ◽

Biological Responses ◽

Extracellular Signal ◽

Differentiation And Proliferation

Signaling pathways play critical roles in regulating the activation of T cells. Recognition of foreign peptide presented by MHC to the T cell receptor (TCR) triggers a signaling cascade of proximal kinases and adapter molecules that lead to the activation of Effector kinase pathways. These effector kinase pathways play pivotal roles in T cell activation, differentiation, and proliferation. RNA sequencing-based methods have provided insights into the gene expression programs that support the above-mentioned cell biological responses. The proteome is often overlooked. A recent study by Damasio et al. [Biochem. J. (2021) 478, 79–98. doi:10.1042/BCJ20200661] focuses on characterizing the effect of extracellular signal-regulated kinase (ERK) on the remodeling of the proteome of activated CD8+ T cells using Mass spectrometric analysis. Surprisingly, the Effector kinase ERK pathway is responsible for only a select proportion of the proteome that restructures during T cell activation. The primary targets of ERK signaling are transcription factors, cytokines, and cytokine receptors. In this commentary, we discuss the recent findings by Damasio et al. [Biochem. J. (2021) 478, 79–98. doi:10.1042/BCJ20200661] in the context of different Effector kinase pathways in activated T cells.

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SLFN2 protection of tRNAs from stress-induced cleavage is essential for T cell–mediated immunity

Science ◽

10.1126/science.aba4220 ◽

2021 ◽

Vol 372 (6543) ◽

pp. eaba4220 ◽

Cited By ~ 1

Author(s):

Tao Yue ◽

Xiaoming Zhan ◽

Duanwu Zhang ◽

Ruchi Jain ◽

Kuan-wen Wang ◽

...

Keyword(s):

Oxidative Stress ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Interleukin 2 ◽

Cell Receptor ◽

Cell Mediated Immunity ◽

Activated T Cells ◽

Granule Formation

Reactive oxygen species (ROS) increase in activated T cells because of metabolic activity induced to support T cell proliferation and differentiation. We show that these ROS trigger an oxidative stress response that leads to translation repression. This response is countered by Schlafen 2 (SLFN2), which directly binds transfer RNAs (tRNAs) to protect them from cleavage by the ribonuclease angiogenin. T cell–specific SLFN2 deficiency results in the accumulation of tRNA fragments, which inhibit translation and promote stress-granule formation. Interleukin-2 receptor β (IL-2Rβ) and IL-2Rγ fail to be translationally up-regulated after T cell receptor stimulation, rendering SLFN2-deficient T cells insensitive to interleukin-2’s mitogenic effects. SLFN2 confers resistance against the ROS-mediated translation-inhibitory effects of oxidative stress normally induced by T cell activation, permitting the robust protein synthesis necessary for T cell expansion and immunity.

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Stomatin-like Protein 2 Links Mitochondria to T-Cell Receptor Signalosomes at the Immunological Synapse and Enhances T-Cell Activation

Nature Precedings ◽

10.1038/npre.2007.1068.1 ◽

2007 ◽

Author(s):

Mark Kirchhof ◽

Luan Chau ◽

Caitlin Lemke ◽

Santosh Vardhana ◽

Peter Darlington ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Cell Activation ◽

Immunological Synapse ◽

Cell Receptor

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Signaling via LAT (linker for T-cell activation) and Syk/ZAP70 is required for ERK activation and NFAT transcriptional activation following CD2 stimulation

Blood ◽

10.1182/blood.v96.6.2181 ◽

2000 ◽

Vol 96 (6) ◽

pp. 2181-2190 ◽

Cited By ~ 34

Author(s):

Maria Paola Martelli ◽

Huamao Lin ◽

Weiguo Zhang ◽

Lawrence E. Samelson ◽

Barbara E. Bierer

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Transcriptional Activation ◽

Cell Activation ◽

Interleukin 2 ◽

Cell Receptor ◽

Adapter Protein ◽

Activated T Cells

Abstract Activation of T cells can be initiated through cell surface molecules in addition to the T-cell receptor-CD3 (TCR-CD3) complex. In human T cells, ligation of the CD2 molecule by mitogenic pairs of anti-CD2 monoclonal antibodies activates T cells via biochemical signaling pathways similar but not identical to those elicited on TCR engagement. This study describes a key role for the p36/38 membrane adapter protein linker for T cell activation (LAT) in CD2-mediated T-cell activation. Following ligation of CD2 on the surface of the Jurkat T-cell line and human purified T cells, LAT was tyrosine phosphorylated and shown to associate in vivo with a number of other tyrosine phosphorylated proteins including PLCγ-1, Grb-2, and SLP-76. Using Jurkat cell lines deficient in ZAP70/Syk (P116) or LAT (ANJ3) expression, CD2-dependent PLCγ-1 and SLP-76 tyrosine phosphorylation required expression both of ZAP70 or Syk and of LAT. As predicted, the absence of either LAT or ZAP70/Syk kinases correlated with a defect in the induction of nuclear factor of activated T cells (NFAT) transcriptional activity, activation of the interleukin-2 promoter, and ERK phosphorylation following CD2 stimulation. These data suggest that LAT is an adapter protein important for the regulation of CD2-mediated T-cell activation.

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