Vasily Rybakin

Themis controls thymocyte selection through regulation of T cell antigen receptor–mediated signaling

Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre–T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4+CD25+Foxp3+ regulatory and CD62LloCD44hi memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.

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

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.

Coronin 7, the mammalian POD-1 homologue, localizes to the Golgi apparatus

Coronins constitute an evolutionary conserved family of WD‐repeat actin‐binding proteins. Their primary function is thought to be regulating the actin cytoskeleton. Apart from that, several coronins were indirectly shown to participate in vesicular transport, establishment of cell polarity and cytokinesis. Here, we report a novel mammalian protein, coronin 7 (crn7), which is significantly different from other mammalian coronins in its domain architecture. Crn7 possesses two stretches of WD repeats in contrast to the other coronins only having one. The protein is expressed throughout the mouse embryogenesis and is strongly upregulated in brain and developing structures of the immune system in the course of development. In adult animals, both crn7 mRNA and protein are abundantly present in most organs, with significantly higher amounts in brain, kidney, thymus and spleen and lower amounts in muscle. At the subcellular level, the bulk of the protein appears to be present in the cytosol and in large cytosolic complexes. However, a significant portion of the protein is detected on vesicle‐like cytoplasmic structures as well as on the cis‐Golgi. In the Golgi region, crn7 staining appears broader than that of the cis‐Golgi markers Erd2p and β‐COP, still, the trans‐Golgi network appears predominantly crn7‐negative. Importantly, the membrane‐associated form of crn7 protein is phosphorylated on tyrosine residues, whereas the cytosolic form is not. Crn7 is the first coronin protein proven to localize to the Golgi membrane. We conclude that it plays a role in the organization of intracellular membrane compartments and vesicular trafficking rather than in remodeling the cytoskeleton.

Fine-tuning T cell receptor signaling to control 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.

Protein Kinase C η Is Required for T Cell Activation and Homeostatic Proliferation

Studies of knockout mice delineate specific and overlapping roles for different protein kinase C isoforms in T cell biology. PKC Isoform Specificity in T Cells When a T cell encounters an antigen-presenting cell (APC), protein kinase C θ (PKCθ) is recruited to the immunological synapse, the interface between the T cell and the APC, where it is thought to mediate co-receptor signaling during T cell activation. Although PKCθ is the most abundant PKC isoform in T cells, thymocyte development in PKCθ-deficient mice is only mildly affected. Fu et al. investigated a role for PKCη, which, unlike PKCθ, increases in abundance upon T cell activation. The authors found that similar to PKCθ, PKCη was recruited to the immunological synapse upon T cell activation and that both isoforms had some overlapping functions. However, PKCη was required for antigen-dependent activation, and loss of PKCη, but not that of PKCθ, resulted in decreased homeostatic proliferation of T cells. Together, these data suggest that PKCη plays critical roles in T cells. Protein kinase C η (PKCη) is abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell; however, its function in T cells is unknown. We showed that PKCη was required for the activation of mature CD8+ T cells through the T cell receptor. Compared with wild-type T cells, PKCη−/− T cells showed poor proliferation in response to antigen stimulation, a trait shared with T cells deficient in PKCθ, which is the most abundant PKC isoform in T cells and was thought to be the only PKC isoform with a specific role in T cell activation. In contrast, only PKCη-deficient T cells showed defective homeostatic proliferation, which requires self-antigen recognition. PKCη was dispensable for thymocyte development; however, thymocytes from mice doubly deficient in PKCη and PKCθ exhibited poor development, indicating some redundancy between the PKC isoforms. Deficiency in PKCη or PKCθ had opposing effects on the relative numbers of CD4+ and CD8+ T cells. PKCη−/− mice had a higher ratio of CD4+ to CD8+ T cells compared to that of wild-type mice, whereas PKCθ−/− mice had a lower ratio. Mice deficient in both isoforms exhibited normal cell ratios. Together, these data suggest that PKCη shares some redundant roles with PKCθ in T cell biology and also performs nonredundant functions that are required for T cell homeostasis and activation.

Initiation of TCR Phosphorylation and Signal Transduction

Recent data with CD8+ T cells show that the initial phase of T cell receptor (TCR) binding to MHC–peptide (MHCp) is quickly followed by a second, stronger, binding phase representing the binding of CD8 to the MHCp. This second phase requires signaling by a Src-family kinase such as Lck. These data point out two aspects of the initial stage of TCR signaling that have not yet been clearly resolved. Firstly, how and by which Src-family kinase, is the initial phosphorylation of CD3ζ accomplished, given that the Lck associated with the co-receptors (CD4 or CD8) is not yet available. Secondly, what is the mechanism by which the co-receptor is brought close to the bound TCR before the co-receptor binds to MHCp?

Crn7 interacts with AP-1 and is required for the maintenance of Golgi morphology and protein export from the Golgi

Crn7 is a novel cytosolic mammalian WD-repeat protein of unknown function that associates with Golgi membranes. Here, we demonstrate that Crn7 knockdown by small interfering RNA results in dramatic changes in the Golgi morphology and function. First, the Golgi ribbon is disorganized in Crn7 KD cells. Second, the Golgi export of several marker proteins including VSV envelope G glycoprotein is greatly reduced but not the retrograde protein import into the Golgi complex. We further establish that Crn7 co-precipitates with clathrin adaptor AP-1 but is not required for AP-1 targeting to Golgi membranes. We identify tyrosine 288-based motif as part of a canonical YXXΦ sorting signal and a major μ1-adaptin binding site in vitro. This study provides the first insight into the function of mammalian Crn7 protein in the Golgi complex.

Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide–MHC

The requirement for the TCR to interact with coagonists, endogenous MHC–peptide complexes which do not themselves activate the T cell, decreases as the strength of the CD8–class I interaction increases.

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

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.

Molecular mechanism underlying the association of Coronin-7 with Golgi membranes

Abstract.Coronin-7 (Crn7) is a ubiquitous mammalian WD40-repeat protein that localizes to the Golgi complex, interacts with AP-1 adaptor complex via binding of a tyrosine-288-based sorting signal to the μ1-subunit of AP-1, and participates in the maintenance of the Golgi structure and function. Here, we define the requirements for the recruitment of Crn7 from the cytosol to the Golgi. We establish that Src activity is indispensable for the interaction of Crn7 with Golgi membranes. Crn7 binds Src in vivo and can be phosphorylated by recombinant Src in vitro. We demonstrate that tyrosine-758 is the major Src phosphorylation site. Further, to be targeted to membranes Crn7 requires the presence of cargo in the Golgi complex. Finally, downregulation of the μ1-subunit of AP-1 leads to the dispersal of Crn7 from the Golgi membranes. We propose a mechanism whereby sequential events of protein interaction and posttranslational modification result in the membrane targeting of Crn7.