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The T cell receptor (TCR) to NF-κB signaling pathway plays a critical role in regulation of proliferation and effector T cell differentiation and function. In naïve T cells, data suggest that most or all key cytoplasmic NF-κB signaling occurs in a TCR-proximal manner at the immunological synapse (IS). However, the subcellular organization of cytoplasmic NF-κB-activating complexes in effector T cells is more complex, involving signaling molecules and regulatory mechanisms beyond those operative in naïve cells. Additionally, in effector T cells, much signaling occurs at cytoplasmic locations distant from the IS. Visualization of these cytoplasmic signaling complexes has provided key insights into the complex and dynamic regulation of NF-κB signal transduction in effector T cells. In this chapter, we provide in-depth protocols for activating and preparing effector T cells for fluorescence imaging, as well as a discussion of the effective application of distinct imaging methodologies, including confocal and super-resolution microscopy and imaging flow cytometry.

T cells are an immune cell lineage that play a central role in protection against pathogen infection. Antigen, in the form of pathogen-derived peptides, stimulates the T-cell receptor (TCR), leading to activation of the transcription factor, nuclear factor kappa B (NF-κB). The subsequent NF-κB-dependent gene expression program drives expansion and effector differentiation of antigen-specific T cells, leading to the adaptive anti-pathogen immune response. The cell surface TCR transmits activating signals to cytosolic NF-κB by a complex signaling cascade, in which the adapter protein Bcl10 plays a key role. We have previously demonstrated that TCR engagement leads to the formation of cytosolic Bcl10 clusters, called POLKADOTS, that provide a platform for the assembly of the terminal signaling complex that ultimately mediates NF-κB activation. In this chapter, we describe the methods utilized to visualize the formation of TCR-induced POLKADOTS and to study the temporal association between POLKADOTS formation and nuclear translocation of the NF-κB subunit, RelA/p65.

Antigen-mediated stimulation of the T cell receptor (TCR) triggers activation of nuclear factor κB (NF-κB), a key transcriptional regulator of T cell proliferation and effector cell differentiation. TCR signaling to NF-κB requires both the Carma1-Bcl10-Malt1 (CBM) complex and the inhibitor of κB (IκB) kinase (IKK) complex; however, the molecular mechanisms connecting the CBM complex to activation of IKK are incompletely defined. We found that the active IKK complex is a component of a TCR-dependent cytosolic Bcl10-Malt1 signalosome containing the adaptor protein p62, which forms in effector T cells. Phosphorylated IκBα and NF-κB were transiently recruited to this signalosome before NF-κB translocated to the nucleus. Inhibiting the activity of the kinase TAK1 or IKK blocked the phosphorylation of IKK, but not the formation of p62-Bcl10-Malt1 clusters, suggesting that activation of IKK occurs after signalosome assembly. Furthermore, analysis of T cells from p62-deficient mice demonstrated that the p62-dependent clustering of signaling components stimulated activation of NF-κB in effector T cells. Thus, TCR-stimulated activation of NF-κB requires the assembly of cytosolic p62-Bcl10-Malt1-IKK signalosomes, which may ensure highly regulated activation of NF-κB in response to TCR engagement., (Copyright © 2014, American Association for the Advancement of Science.)

Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-κB is required for T cell proliferation and differentiation of effector cells. The TCR-to-NF-κB pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modifications, ultimately culminating in the nuclear translocation of NF-κB. However, recent findings suggest a more complex picture in which distinct signalosomes, previously unrecognized proteins, and newly identified regulatory mechanisms play key roles in signal transmission. In this review, we evaluate recent data and suggest areas of future emphasis in the study of this important pathway., (Published by Elsevier Ltd.)

The adaptor protein Bcl10 is a critically important mediator of T cell receptor (TCR)-to-NF-κB signaling. Bcl10 degradation is a poorly understood biological phenomenon suggested to reduce TCR activation of NF-κB. Here we have shown that TCR engagement triggers the degradation of Bcl10 in primary effector T cells but not in naive T cells. TCR engagement promoted K63 polyubiquitination of Bcl10, causing Bcl10 association with the autophagy adaptor p62. Paradoxically, p62 binding was required for both Bcl10 signaling to NF-κB and gradual degradation of Bcl10 by autophagy. Bcl10 autophagy was highly selective, as shown by the fact that it spared Malt1, a direct Bcl10 binding partner. Blockade of Bcl10 autophagy enhanced TCR activation of NF-κB. Together, these data demonstrate that selective autophagy of Bcl10 is a pathway-intrinsic homeostatic mechanism that modulates TCR signaling to NF-κB in effector T cells. This homeostatic process may protect T cells from adverse consequences of unrestrained NF-κB activation, such as cellular senescence., (Copyright © 2012 Elsevier Inc. All rights reserved.)

TCR-mediated activation of the transcription factor NF-κB is required for T cell proliferation, survival, and effector differentiation. Although this pathway is the subject of intense study, it is not known whether TCR signaling to NF-κB is digital (switch-like) or analog in nature. Through analysis of the phosphorylation and degradation of IκBα and the nuclear translocation and phosphorylation of the NF-κB subunit RelA, we show that TCR-directed NF-κB activation is digital. Furthermore, digitization occurs well upstream of the IκB kinase complex, as protein kinase C translocation to the immunologic synapse and activation-associated aggregation of Bcl10 and Malt1 also demonstrate both digital behavior and high correlation with RelA nuclear translocation. Thus, similar to the TCR-to-MAPK signaling cascade, analog Ag inputs are converted to digital activation outputs to NF-κB at an early step downstream of TCR ligation.

Malt1 is a multi-domain cytosolic signaling molecule that was originally identified as the target of recurrent translocations in a large fraction of MALT lymphomas. The product of this translocation is a chimeric protein in which the N-terminus is contributed by the apoptosis inhibitor, cIAP2, and the C-terminus is contributed by Malt1. Early studies suggested that Malt1 is an essential intermediate in antigen receptor activation of NF-kappaB, and that the juxtaposition of the cIAP2 N-terminus and the Malt1 C-terminus results in deregulation of Malt1 NF-kappaB stimulatory activity. Initial experimental data further suggested that the molecular mechanisms of Malt1- and cIAP-Malt1-mediated NF-kappaB activation were quite similar. However, a number of more recent studies of both Malt1 and cIAP2-Malt1 now reveal that these proteins influence NF-kappaB activation by multiple distinct mechanisms, several of which are non-overlapping. Currently available data suggest a revised model in which cIAP2-Malt1 induces NF-kappaB activation via a mechanism that depends equally on domains contributed by cIAP2 and Malt1, which confer spontaneous oligomerization activity, polyubiquitin binding, proteolytic activity, and association with and activation of TRAF2 and TRAF6 at several independent binding sites. By contrast, emerging data suggest that the wild-type Malt1 protein uniquely contributes to NF-kappaB activation primarily through the control of two proteolytic cleavage mechanisms. Firstly, Malt1 directly cleaves and inactivates A20, a negative regulator of the antigen receptor-to-NF-kappaB pathway. Secondly, Malt1 interacts with caspase-8, inducing caspase-8 cleavage of c-FLIP(L), initiating a pathway that contributes to activation of the I kappaB kinase (IKK) complex. Furthermore, data suggest that Malt1 plays a more limited and focused role in antigen receptor activation of NF-kappaB, serving to augment weak antigen signals and stimulate a defined subset of NF-kappaB dependent responses. Thus, the potent activation of NF-kappaB by cIAP2-Malt1 contrasts with the more subtle role of Malt1 in regulating specific NF-kappaB responses downstream of antigen receptor ligation.

The cytosolic proteins protein kinase Ctheta (PKCtheta), Bcl10, and Malt1 play critical roles in TCR signaling to the transcription factor NF-kappaB. Our data confirm that CD4(+) T cells from PKCtheta, Bcl10, and Malt1 knockout mice show severe impairment of proliferation in response to TCR stimulation. Unexpectedly, we find that knockout CD8(+) T cells proliferate to a similar extent as wild-type cells in response to strong TCR signals, although a survival defect prevents their accumulation. Both CD4(+) and CD8(+) knockout T cells express activation markers, including CD25, following TCR stimulation. Addition of exogenous IL-2 rescues survival of knockout CD4(+) and CD8(+) T cells, but fails to overcome the proliferation defect of CD4(+) T cells. CD4(+) T cells from knockout mice are extremely deficient in TCR-induced NF-kappaB activation, whereas NF-kappaB activation is only partially impaired in CD8(+) T cells. Overall, our results suggest that defects in TCR signaling through PKCtheta, Bcl10, and Malt1 predominantly impair NF-kappaB activation and downstream functional responses of CD4(+) T cells. In contrast, CD8(+) T cells maintain substantial NF-kappaB signaling, implying the existence of a significant TCR-regulated NF-kappaB activation pathway in CD8(+) T cells that is independent of PKCtheta, Bcl10, and Malt1.

Stimulation of the T-cell receptor (TCR) results in the activation of several transcription factors, including NF-kappaB, that are crucial for T-cell proliferation and gain of effector functions. On TCR engagement, several proteins within the TCR-directed NF-kappaB signaling pathway undergo dynamic spatial redistribution, but the significance of these redistribution events is largely unknown. We have previously described TCR-induced cytoplasmic structures called POLKADOTS (punctate and oligomeric killing or activating domains transducing signals) that are enriched in the NF-kappaB signaling intermediate, Bcl10. We now show that these structures are formed only under conditions that promote efficient NF-kappaB activation. Furthermore, POLKADOTS formation is dependent on functional domains of specific NF-kappaB signal transducers. Through use of a photoactivatable GFP, we demonstrate that POLKADOTS contain both a highly stable and a rapidly equilibrating protein component. FRET analyses show that POLKADOTS are sites of enriched interactions between Bcl10 and partner signaling proteins. These observations strongly suggest that POLKADOTS are focal sites of dynamic information exchange between cytosolic intermediates in the process of TCR activation of NF-kappaB.

The central zone of the supramolecular activation cluster (c-SMAC) is a zone of T cell receptor (TCR) enrichment that forms at a T cell/antigen-presenting cell (APC) junction in response to antigen stimulation. We demonstrate that there is a surprisingly complex relocalization process that brings PKC and Bcl10, two intermediates in TCR activation of NF-kappaB, to the cytoplasmic face of the c-SMAC. TCR activation causes enrichment of PKC at the c-SMAC, followed by Bcl10 relocalization to punctate cytoplasmic structures, often at sites distant from the c-SMAC. These Bcl10 structures then undergo further relocalization, becoming enriched at the c-SMAC. TCR activation of NF-kappaB therefore involves the dynamic relocalization of multiple signaling intermediates, with distinct phases proximal to and distant from the c-SMAC.