Byron B. Au-Yeung

ZAP-70: An Essential Kinase in T-cell Signaling

ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical role in the events involved in initiating T-cell responses by the antigen receptor. Here we review the structure of ZAP-70, its regulation, its role in development and in disease. We also describe a model experimental system in which ZAP-70 function can be interrupted by a small chemical inhibitor.

The structure, regulation, and function of ZAP-70.

Summary:  The tyrosine ZAP‐70 (ζ‐associated protein of 70 kDa) kinase plays a critical role in activating many downstream signal transduction pathways in T cells following T‐cell receptor (TCR) engagement. The importance of ZAP‐70 is evidenced by the severe combined immunodeficiency that occurs in ZAP‐70‐deficient mice and humans. In this review, we describe recent analyses of the ZAP‐70 crystal structure, revealing a complex regulatory mechanism of ZAP‐70 activity, the differential requirements for ZAP‐70 and spleen tyrosine kinase (SyK) in early T‐cell development, as well as the role of ZAP‐70 in chronic lymphocytic leukemia and autoimmunity. Thus, the critical importance of ZAP‐70 in TCR signaling and its predominantly T‐cell‐restricted expression pattern make ZAP‐70 an attractive drug target for the inhibition of pathological T‐cell responses in disease.

A genetically selective inhibitor demonstrates a function for the kinase Zap70 in regulatory T cells independent of its catalytic activity

To investigate the role of the kinase Zap70 in T cells, we generated mice expressing a Zap70 mutant whose catalytic activity can be selectively blocked by a small-molecule inhibitor. We found that conventional naive, effector and memory T cells were dependent on the kinase activity of Zap70 for their activation, which demonstrated a nonredundant role for Zap70 in signals induced by the T cell antigen receptor (TCR). In contrast, the catalytic activity of Zap70 was not required for activation of the GTPase Rap1 and inside-out signals that promote integrin adhesion. This Zap70 kinase–independent pathway was sufficient for the suppressive activity of regulatory T cells (Treg cells), which was unperturbed by inhibition of the catalytic activity of Zap70. Our results indicate Zap70 is a likely therapeutic target.

Extrathymic Aire-Expressing Cells Are a Distinct Bone Marrow-Derived Population that Induce Functional Inactivation of CD4+ T Cells

The autoimmune regulator (Aire) is essential for prevention of autoimmunity; its role is best understood in the thymus, where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)(hi), CD80(lo), CD86(lo), epithelial cell adhesion molecule (EpCAM)(hi), CD45(lo) bone marrow-derived peripheral antigen-presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4⁺ T cells through a mechanism that does not require regulatory T cells (Treg) and is resistant to innate inflammatory stimuli. Together, these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.

Cutting Edge: Itk-Dependent Signals Required for CD4+ T Cells to Exert, but Not Gain, Th2 Effector Function

The TCR signals for the release of CD4 effector function are poorly understood. Itk plays an essential role in Th2, but not Th1, responses. However, when Itk is required during Th2 development is unclear. We followed the fate of Itk-deficient T cells during Th2 development in vitro and in vivo using an IL-4/GFP reporter. Surprisingly, a similar frequency of itk−/− CD4+ cells differentiated and committed to the Th2 lineage as wild-type cells. However, Itk-deficient Th2 cells failed to exert effector function upon TCR triggering. Loss of function was marked by defective transcriptional enhancement of Th2 cytokines and GATA3. IL-4 production in itk−/− Th2s could be rescued by the expression of kinase-active Itk. Thus, Itk is necessary for the release, but not gain, of Th2 function. We suggest that the liberation of effector function is tightly controlled through qualitative changes in TCR signals, facilitating postdifferentiation regulation of cytokine responses.

A Key Role for Itk in Both IFNγ and IL-4 Production by NKT Cells

NKT cells rapidly secrete cytokines upon TCR stimulation and thus may modulate the acquired immune response. Recent studies suggest that signaling for development and effector function in NKT cells may differ from conventional T cells. The tyrosine kinase Itk is activated downstream of the TCR, and its absence in CD4+ T cells results in impaired Th2, but not Th1 responses. In this study, we investigated NKT cell function in the absence of Itk as impaired type 2 responses in vivo could be manifest through IL-4 defects in a number of cell types. We show that Itk-deficient NKT cells up-regulate IL-4 mRNA in the thymus and express constitutive IL-4 and IFN-γ transcripts in peripheral organs. Thus, Itk is not required for the developmental activation of cytokine loci in NKT cells. Nevertheless, Itk-deficient NKT cells are severely impaired in IL-4 protein production. Strikingly, unlike conventional CD4+ T cells, Itk-deficient NKT cells also have profound defects in IFN-γ production. Furthermore, both IL-4 and IFN-γ production were markedly impaired following in vivo challenge with α-galactosyl ceramide. Function can be restored in Itk-deficient NKT cells by provision of calcium signals using ionomycin. These results suggest that NKT cells are highly dependent on Itk for IL-4- and IFN-γ-mediated effector function. Thus, the pattern of cytokine genes that are affected by Itk deficiency appears to be cell lineage-specific, likely reflecting differences in activation threshold between immune effectors. The severe defect in NKT cell function may underlie a number of the Th1 and Th2 immune defects in Itk-deficient mice.

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

Significance Biochemical signals triggered by the T-cell receptor (TCR) are required for stimulating T cells and can be initiated within seconds. However, a hallmark of T-cell activation, cell division, occurs hours after TCR signaling has begun, implying that T cells require a minimum duration and/or accumulate TCR signaling events to drive proliferation. To visualize the accumulated signaling experienced by T cells, we used a fluorescent reporter gene that is activated by TCR stimulation. This technique showed a threshold between dividing and nondividing T cells for TCR signaling that does not change with stronger or weaker TCR signaling or the presence of growth factor. Together these data may have implications for the development of T-cell–targeted therapies for autoimmunity. 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.

Itk Controls the Spatiotemporal Organization of T Cell Activation

Loss of the kinase Itk in activated T cells disrupts actin accumulation at the immunological synapse, compromising T cell activation. Focused on the Center After T cell receptor (TCR) stimulation by an antigen-presenting cell (APC), an array of proteins are recruited to the T cell side of the interface with the APC (the immunological synapse), where they generate signals that activate the T cell. Singleton et al. provide data showing that interleukin-2 (IL-2)–inducible T cell kinase (Itk) plays a central role in directing “molecular traffic” at the immunological synapse. By comparing the organization of fluorescent sensors of T cell signaling molecules between antigen-activated wild-type and Itk-deficient T cells, the authors found that loss of Itk resulted in the inappropriate spatial organization of many signaling proteins at the T cell–APC interface, which impaired T cell activation. In particular, Itk was required to direct the activation of the guanosine triphosphatase Cdc42 at the center of the synapse, which led to appropriate actin accumulation downstream of TCR activation. Together, these data establish a central role for Itk in the organization of T cell signaling molecules and provide evidence for the dependence of critical T cell functions on the spatiotemporal organization of signaling intermediates. During T cell activation by antigen-presenting cells (APCs), the diverse spatiotemporal organization of components of T cell signaling pathways modulates the efficiency of activation. Here, we found that loss of the tyrosine kinase interleukin-2 (IL-2)–inducible T cell kinase (Itk) in mice altered the spatiotemporal distributions of 14 of 16 sensors of T cell signaling molecules in the region of the interface between the T cell and the APC, which reduced the segregation of signaling intermediates into distinct spatiotemporal patterns. Activation of the Rho family guanosine triphosphatase Cdc42 at the center of the cell-cell interface was impaired, although the total cellular amount of active Cdc42 remained intact. The defect in Cdc42 localization resulted in impaired actin accumulation at the T cell–APC interface in Itk-deficient T cells. Reconstitution of cells with active Cdc42 that was specifically directed to the center of the interface restored actin accumulation in Itk-deficient T cells. Itk also controlled the central localization of the guanine nucleotide exchange factor SLAT [Switch-associated protein 70 (SWAP-70)–like adaptor of T cells], which may contribute to the activation of Cdc42 at the center of the interface. Together, these data illustrate how control of the spatiotemporal organization of T cell signaling controls critical aspects of T cell function.

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

The catalytic activity of Zap70 is crucial for T cell antigen receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap70 catalytic activity in a model of synchronized thymic selection, we showed that CD4+CD8+ thymocytes integrate multiple, transient, Zap70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas 1 h of signaling was sufficient for negative selection. Titration of Zap70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, which revealed heterogeneity, even among CD4+CD8+ thymocytes expressing identical TCRs undergoing positive selection.

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

Significance Developing T cells are positively selected in the thymus to ensure that their antigen receptors can interact with self-MHC. For CD8 T cells, this process takes days to complete, yet the steps involved are poorly understood. We followed a synchronized wave of cells undergoing positive selection within three-dimensional thymic tissue. Surprisingly, migration from the cortex to the medulla occurred before CD4 down-regulation and while thymocytes still required TCR signaling for efficient positive selection. There was a gradual change in the pattern of calcium signaling over time, with an upward shift in basal intracellular calcium correlating with increased speed and brief signaling events. Our data have interesting implications for how positive and negative selection shape the mature CD8 T-cell repertoire. 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 ([Ca2+]i) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca2+]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.