identification-mediators-t-cell-receptor-signaling-via-screening

Identification of Mediators of T-cell Receptor Signaling <em>via</em> the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

use-single-chain-mhc-technology-to-investigate-co-agonism-human-cd8-t

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

National University of Singapore

Signals generated by the T cell receptor (TCR) result in the death or survival of developing thymocytes (T cell precursors), depending on the affinity of the TCR for peptide ligands bound to the major histocompatibility complex (MHC) that are encountered in the thymus. A new study by Ellen Robey's group in this issue of Science Signaling presents a systematic evaluation of signaling and motility changes in thymocytes that encounter ligands of different affinities in the thymic environment. In contrast to previous in vitro studies, the authors found that low-affinity ligands stimulated infrequent transient mobilization of intracellular Ca(2+), whereas high-affinity ligands triggered sustained Ca(2+) signaling and periods of migratory arrest. For ligands of intermediate affinity, changes in thymocyte motility, rather than in Ca(2+) signaling patterns, provided the best correlation with functional outcomes. These findings suggest that transient signaling events in the absence of strong stop signals are required for thymocyte survival and functional maturation in the thymus. 

Too fast to die.

Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αβ-expressing 'single-positive' thymocytes from CD4(+)CD8αβ(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens. 

Themis sets the signal threshold for positive and negative selection in T-cell development.

T cell development from immature CD4(+)CD8(+) double-positive (DP) thymocytes to the mature CD4 or CD8 single-positive (SP) stage requires proper T cell receptor (TCR) signaling. The current working model of thymocyte development is that the strength of the TCR-mediated signal - from little-or-none, through intermediate, to strong - received by the immature cells determines whether they will undergo death by neglect, positive selection, or negative selection, respectively. In recent years, several developmentally regulated, stage-specifically expressed proteins and miRNAs have been found that act like fine-tuners for signal transduction and propagation downstream of the TCR. This allows them to govern thymocyte positive selection. Here, we summarize recent findings on these molecules and suggest new concepts of TCR positive-selection signaling. 

Fine-tuning T cell receptor signaling to control T cell development.

The earliest molecular events in T-cell recognition have not yet been fully described, and the initial T-cell receptor (TCR)-triggering mechanism remains a subject of controversy. Here, using total internal reflection/Forster resonance energy transfer microscopy, we observe a two-stage interaction between TCR, CD8 and major histocompatibility complex (MHC)-peptide. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ-CD8 interactions require CD8-MHC binding. Lck can be found free or bound to the coreceptor. This work indicates that the initial TCR-triggering event is induced by free Lck. 

Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor.

T cell progenitors are known to arise from the foetal liver in embryos and the bone marrow in adults; however different studies have shown that a pool of T cell progenitors may also exist in the periphery. Here, we identified a lymphoid population resembling peripheral T cell progenitors which transiently seed the epidermis during late embryogenesis in both wild-type and T cell-deficient mice. We named these cells ELCs (Epidermal Lymphoid Cells). ELCs expressed Thy1 and CD2, but lacked CD3 and TCRαβ/γδ at their surface, reminiscent of the phenotype of extra- or intra- thymic T cell progenitors. Similarly to Dendritic Epidermal T Cells (DETCs), ELCs were radioresistant and capable of self-renewal. However, despite their progenitor-like phenotype and expression of T cell lineage markers within the population, ELCs did not differentiate into conventional T cells or DETCs in in vitro, ex vivo or in vivo differentiation assays. Finally, we show that ELC expressed NK markers and secreted IFN-γ upon stimulation. Therefore we report the discovery of a unique population of lymphoid cells within the murine epidermis that appears related to NK cells with as-yet-unidentified functions.

Identification of a novel lymphoid population in the murine epidermis.

B7 proteins CD80 (B7-1) and CD86 (B7-2) are expressed on most antigen-presenting cells and provide critical co-stimulatory or inhibitory input to T cells via their T-cell-expressed receptors: CD28 and CTLA-4. CD28 is expressed on effector T cells and regulatory T cells (Tregs), and CD28-dependent signals are required for optimum activation of effector T cell functions. CD28 ligation on effector T cells leads to formation of distinct molecular patterns and induction of cytoskeletal rearrangements at the immunological synapse (IS). CD28 plays a critical role in recruitment of protein kinase C (PKC)-θ to the effector T cell IS. CTLA-4 is constitutively expressed on the surface of Tregs, but it is expressed on effector T cells only after activation. As CTLA-4 binds to B7 proteins with significantly higher affinity than CD28, B7 ligand recognition by cells expressing both receptors leads to displacement of CD28 and PKC-θ from the IS. In Tregs, B7 ligand recognition leads to recruitment of CTLA-4 and PKC-η to the IS. CTLA-4 plays a role in regulation of T effector and Treg IS stability and cell motility. Due to their important roles in regulating T-cell-mediated responses, B7 receptors are emerging as important drug targets in oncology. In this review, we present an integrated summary of current knowledge about the role of B7 family receptor-ligand interactions in the regulation of spatial and temporal IS dynamics in effector and Tregs. 

Cell Type-Specific Regulation of Immunological Synapse Dynamics by B7 Ligand Recognition.

Vive la peptide différence!

Thymocyte development and maintenance of peripheral T-cell numbers and functions are critically dependent on T-cell receptor (TCR) signal strength. SHP1 (Src homology region 2 domain-containing phosphatase-1), a tyrosine phosphatase, acts as a negative regulator of TCR signal strength. Moreover, germline SHP1 knockout mice have shown impaired thymic development. However, this has been recently questioned by an analysis of SHP1 conditional knockout mice, which reported normal thymic development of SHP1 deficient thymocytes. Using this SHP1 conditional knockout mice, in this issue of the European Journal of Immunology, Martinez et al. [Eur. J. Immunol. 2016. 46: 2103-2110] show that SHP1 indeed does have a role in the negative regulation of TCR signal strength in positively selected thymocytes, and in the final maturation of single positive thymocytes. They report that thymocyte development in such mice shows loss of mature, post-selection cells. This is due to increased TCR signal transduction in thymocytes immediately post positive-selection, and increased cell death in response to weak TCR ligands. Thus, SHP1-deficiency shows strong similarities to deficiency in the T-cell specific SHP1-associated protein Themis.

SHP1-ing thymic selection.

Thymocyte selection involves the positive and negative selection of the repertoire of T cell receptors (TCRs) such that the organism does not suffer autoimmunity, yet has the benefit of the ability to recognize any invading pathogen. The signal transduced through the TCR is translated into a number of different signaling cascades that result in transcription factor activity in the nucleus and changes to the cytoskeleton and motility. Negative selection involves inducing apoptosis in thymocytes that express strongly self-reactive TCRs, whereas positive selection must induce survival and differentiation programs in cells that are more weakly self-reactive. The TCR recognition event is analog by nature, but the outcome of signaling is not. A large number of molecules regulate the strength of the TCR-derived signal at various points in the cascades. This review discusses the various factors that can regulate the strength of the TCR signal during thymocyte development.

TCR Signal Strength and T Cell Development.

Major histocompatibility complex (MHC) restriction is a unique feature of T cell antigen recognition. Mature T cells respond to antigenic nonself peptides bound to self-MHC molecules, but a sizeable fraction of peripheral T cells can also respond to nonself peptide-MHC (pMHC) complexes in the context of transplantation. MHC specificity of the T cell receptor (TCR) repertoire is shaped during thymic development. Two hypotheses have been proposed to explain MHC specificity of T cells. It has been suggested that MHC specificity is an intrinsic feature of TCR structure, mediated by the germline-encoded regions of the TCR sequence. In support of this model, an estimated 15% to 30% of preselection TCR repertoire is estimated to be MHC-specific. Moreover, structural studies have shown some degree of conserved binding topology for TCR-peptide MHC complexes. However, there is also evidence that MHC restriction can be imposed on the TCR repertoire during thymic development, and it has been proposed that the interaction of the Lck kinase with CD4 or CD8 coreceptors is critical for generation of MHC specificity. This review will discuss recent work on assessment of the preselection of TCR repertoire, molecular evidence for the germline encoded TCR bias for MHC, and for the coreceptor sequestration model in the context of alloreactivity and transplantation.

Thymic Origins of T Cell Receptor Alloreactivity.

Activation of the T cell receptor (TCR) leads to the generation of a network of signaling events critical to the developmental decision making and activation of T cells. Various experimental approaches continue to identify new signaling molecules, adaptor proteins, and other regulators of TCR signaling. We propose a screening strategy for the identification of small molecules affecting TCR signaling based on the uncoupling of TCR stimulation from cellular responses in developing thymocytes. We demonstrate that this strategy successfully identifies inhibitors of kinases already shown to act downstream of TCR engagement, as well as new inhibitors. The proposed strategy is easily scalable for high throughput screening and will contribute to the identification of new druggable targets in T cell activation.

Development of a screening strategy for new modulators of T cell receptor signaling and T cell activation.

Foreign antigens are presented by antigen-presenting cells in the presence of abundant endogenous peptides that are nonstimulatory to the T cell. In mouse T cells, endogenous, nonstimulatory peptides have been shown to enhance responses to specific peptide antigens, a phenomenon termed coagonism. However, whether coagonism also occurs in human T cells is unclear, and the molecular mechanism of coagonism is still under debate since CD4 and CD8 coagonism requires different interactions. Here we show that the nonstimulatory, HIV-derived peptide GAG enhances a specific human cytotoxic T lymphocyte response to HBV-derived epitopes presented by HLA-A*02:01. Coagonism in human T cells requires the CD8 coreceptor, but not T-cell receptor (TCR) binding to the nonstimulatory peptide-MHC. Coagonists enhance the phosphorylation and recruitment of several molecules involved in the TCR-proximal signaling pathway, suggesting that coagonists promote T-cell responses to antigenic pMHC by amplifying TCR-proximal signaling.

Nonstimulatory peptide-MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling.

CD8 T cells have commitment issues.

Thymocyte-expressed molecule involved in selection (Themis) has been shown to be important for T cell selection by setting the threshold for positive versus negative selection. Themis interacts with the protein tyrosine phosphatase (PTP) Src-homology domain containing phosphatase-1 (Shp1), a negative regulator of the T cell receptor (TCR) signaling cascade. However, how Themis regulates Shp1 is still not clear. Here, using a very sensitive phosphatase assay on ex vivo thymocytes, we have found that Themis enhances Shp1 phosphatase activity by increasing its phosphorylation. This positive regulation of Shp1 activity by Themis is found in thymocytes, but not in peripheral T cells. Shp1 activity is modulated by different affinity peptide MHC ligand binding in thymocytes. Themis is also associated with phosphatase activity, due to its constitutive interaction with Shp1. In the absence of Shp1 in thymocytes, Themis interacts with Shp2, which leads to almost normal thymic development in Shp1 conditional knockout (cKO) mice. Double deletion of both Themis and Shp1 leads to a thymic phenotype similar to that of Themis KO. These findings demonstrate unequivocally that Themis positively regulates Shp1 phosphatase activity in TCR-mediated signaling in developing thymocytes.

ArticleTotal : 2	Year
Publication title Cited by 2	2019
Publication title Cited by 6	2019

Article	Year
Too fast to die. Science signaling\| PubMed ID: 24129699	2013
Themis sets the signal threshold for positive and negative selection in T-cell development. Nature\| PubMed ID: 24226767	2013
Fine-tuning T cell receptor signaling to control T cell development. Trends in immunology\| PubMed ID: 24951034	2014
Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor. Nature communications\| PubMed ID: 25427562	2014
Identification of a novel lymphoid population in the murine epidermis. Scientific reports\| PubMed ID: 26223192	2015
Cell Type-Specific Regulation of Immunological Synapse Dynamics by B7 Ligand Recognition. Frontiers in immunology\| PubMed ID: 26870040	2016
Vive la peptide différence! Nature immunology\| PubMed ID: 27434001	2016
SHP1-ing thymic selection. European journal of immunology\| PubMed ID: 27600672	2016
TCR Signal Strength and T Cell Development. Annual review of cell and developmental biology\| PubMed ID: 27712102	2016
Thymic Origins of T Cell Receptor Alloreactivity. Transplantation\| PubMed ID: 28114168	2017
Development of a screening strategy for new modulators of T cell receptor signaling and T cell activation. Scientific reports\| PubMed ID: 29968737	2018
Nonstimulatory peptide-MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling. Nature communications\| PubMed ID: 30006605	2018
CD8 T cells have commitment issues. Nature immunology\| PubMed ID: 30013145	2018
Themis-associated phosphatase activity controls signaling in T cell development. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30413615	2018

Joanna Brzostek

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