Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development

Au-Yeung, Byron B; Melichar, Heather J.; Ross, Jenny O.; Cheng, Debra A.; Zikherman, Julie; Shokat, Kevan M.; Robey, Ellen; Weiss, Arthur

doi:10.1038/ni.2918

Cited by 72 publications

(106 citation statements)

References 37 publications

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“…1E). These data extend and confirm our analysis of the temporal requirement for TCR signaling (21) and indicate that TCR signaling is required at both early and late time points for efficient positive selection.…”

Section: Resultssupporting

confidence: 86%

Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

Ross

Melichar

Au-Yeung

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Positive selection of CD8 T cells in the thymus is thought to be a multistep process lasting 3-4 d; however, the discrete steps involved are poorly understood. Here, we examine phenotypic changes, calcium signaling, and intrathymic migration in a synchronized cohort of MHC class I-specific thymocytes undergoing positive selection in situ. Transient elevations in intracellular calcium concentration ([Ca 2+ ] i ) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca 2+ ] i over time. Changes in chemokine-receptor expression and relocalization from the cortex to medulla occurred between 12 and 24 h after the initial encounter with positive-selecting ligands, a time frame at which the majority of thymocytes retain CD4 and CD8 expression and still require T-cell receptor (TCR) signaling to efficiently complete positive selection. Our results identify distinct phases in the positive selection of MHC class I-specific thymocytes that are distinguished by their TCR-signaling pattern and intrathymic location and provide a framework for understanding the multistep process of positive selection in the thymus.Zap70 | thymic slice | two photon microscopy D eveloping thymocytes test their newly formed T-cell antigen receptors (TCRs) for their ability to bind self-peptide:major histocompatibility complex (MHC) complexes on thymic support cells. This process encompasses both positive and negative selection and ultimately leads to the formation of a functional and self-tolerant mature T-cell repertoire. During positive selection, CD4 + CD8 + [double positive (DP)] thymocytes with TCRs weakly reactive to self-peptide:MHC complexes receive signals to survive, mature, and give rise to CD4 or CD8 single positive (SP) cells. Positive selection requires close contact with thymic stromal cells and occurs over a period of several days (1-4). However, much of our information about T-cell development is based on analyses of steady-state thymocyte populations, and the individual steps that occur during the prolonged process of positive selection remain obscure.Thymocytes are highly motile within three-dimensional thymic tissue environments, and positive selection is tightly linked to thymocyte migration. The initial encounters with positive-selecting ligands on cortical thymic epithelial cells do not induce strong migratory stop signals, but instead occur as brief migratory pauses associated with transient elevations in intracellular calcium concentration ([Ca 2+ ] i ) (5-7). These brief, serial TCR signals are consistent with indications that positive selection is driven by weak recognition of self-peptide:MHC complexes and with the unique ability of DP thymocytes to respond to low-potency ligands (8, 9). However, there are also indications that thymocytes that have already received TCR signals still require further signaling to complete positive selection (10, 11). Thus, although the encounters with positive-selecting ligands last for...

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Section: Resultssupporting

confidence: 86%

Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

Ross

Melichar

Au-Yeung

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…This model is consistent with recent studies in which we demonstrate the existence of a temporal threshold for positive selection of CD4 + CD8 + double positive thymocytes, which correlates with an integration of multiple transient encounters with positively selecting ligands. We further show that interruptions in Zap70-dependent signaling decreased the frequency and duration of these signaling events, impairing the efficiency of positive selection (33).…”

Section: Discussionmentioning

confidence: 65%

“…Zap70(AS) inhibitor HXJ42 has been described previously (33). CD4, CD8, CD25, CD90.2, and CD45.1 antibodies conjugated to PE, PerCPCy5.5, PECy7, APC, or Qdot 605 (BD Biosciences, eBioscences, Life Technologies), Nur77 Ab conjugated to PE (eBiosciences; clone 12.14).…”

Section: Zap70mentioning

confidence: 99%

A sharp T-cell antigen receptor signaling threshold for T-cell proliferation

Au-Yeung¹,

Zikherman²,

Mueller³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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T-cell antigen receptor (TCR) signaling is essential for activation, proliferation, and effector function of T cells. Modulation of both intensity and duration of TCR signaling can regulate these events. However, it remains unclear how individual T cells integrate such signals over time to make critical cell-fate decisions. We have previously developed an engineered mutant allele of the critical T-cell kinase zeta-chain-associated protein kinase 70 kDa (Zap70) that is catalytically inhibited by a small molecule inhibitor, thereby blocking TCR signaling specifically and efficiently. We have also characterized a fluorescent reporter Nur77-eGFP transgenic mouse line in which T cells up-regulate GFP uniquely in response to TCR stimulation. The combination of these technologies unmasked a sharp TCR signaling threshold for commitment to cell division both in vitro and in vivo. Further, we demonstrate that this threshold is independent of both the magnitude of the TCR stimulus and Interleukin 2. Similarly, we identify a temporal threshold of TCR signaling that is required for commitment to proliferation, after which T cells are able to proliferate in a Zap70 kinase-independent manner. Taken together, our studies reveal a sharp threshold for the magnitude and duration of TCR signaling required for commitment of T cells to proliferation. These results have important implications for understanding T-cell responses to infection and optimizing strategies for immunomodulatory drug delivery.

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“…Additionally, a reporter mouse model in which GFP levels reflect TCR signal strength revealed that nTregs show higher levels of sustained TCR signaling than do Tconvs in the thymus (19). The observations that different temporal TCR signaling patterns, namely sustained and transient signals, are associated with positive and negative selections (40)(41)(42)(43)(44)(45)(46)(47) underpin the relevance of the dynamics of TCR stimulation for thymocyte selection. Therefore, the history of TCR stimulation has to be taken into account for a model of thymic selection.…”

mentioning

confidence: 97%

“…The disruption of thymocyte-APC interactions or blockade of TCR signaling at different selection stages prevents successful positive selection, implying a requirement for sustained signals (38)(39)(40). In contrast to positive selection, negative selection is a fast process and requires a strong TCR signaling event (41). Negative selection is preceded by a strong transient increase of TCR-mediated signals (42).…”

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confidence: 99%

A Signal Integration Model of Thymic Selection and Natural Regulatory T Cell Commitment

Khailaie

Robert

Toker

et al. 2014

The Journal of Immunology

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The extent of TCR self-reactivity is the basis for selection of a functional and self-tolerant T cell repertoire and is quantified by repeated engagement of TCRs with a diverse pool of self-peptides complexed with self-MHC molecules. The strength of a TCR signal depends on the binding properties of a TCR to the peptide and the MHC, but it is not clear how the specificity to both components drives fate decisions. In this study, we propose a TCR signal-integration model of thymic selection that describes how thymocytes decide among distinct fates, not only based on a single TCR–ligand interaction, but taking into account the TCR stimulation history. These fates are separated based on sustained accumulated signals for positive selection and transient peak signals for negative selection. This spans up the cells into a two-dimensional space where they are either neglected, positively selected, negatively selected, or selected as natural regulatory T cells (nTregs). We show that the dynamics of the integrated signal can serve as a successful basis for extracting specificity of thymocytes to MHC and detecting the existence of cognate self-peptide-MHC. It allows to select a self-MHC–biased and self-peptide–tolerant T cell repertoire. Furthermore, nTregs in the model are enriched with MHC-specific TCRs. This allows nTregs to be more sensitive to activation and more cross-reactive than conventional T cells. This study provides a mechanistic model showing that time integration of TCR-mediated signals, as opposed to single-cell interaction events, is needed to gain a full view on the properties emerging from thymic selection.

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Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development

Cited by 72 publications

References 37 publications

Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

A sharp T-cell antigen receptor signaling threshold for T-cell proliferation

A Signal Integration Model of Thymic Selection and Natural Regulatory T Cell Commitment

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Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development

Cited by 72 publications

References 37 publications

Distinct phases in the positive selection of CD8+T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

Distinct phases in the positive selection of CD8+T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

A sharp T-cell antigen receptor signaling threshold for T-cell proliferation

A Signal Integration Model of Thymic Selection and Natural Regulatory T Cell Commitment

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Product

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Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

Distinct phases in the positive selection of CD8⁺T cells distinguished by intrathymic migration and T-cell receptor signaling patterns