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.

The Src Family kinase Lck sets a critical threshold for T cell activation because it phosphorylates the TCR complex and the Zap70 kinase. How a T cell controls the abundance of active Lck molecules remains poorly understood. We have identified an unappreciated role for a phosphosite, Y192, within the Lck SH2 domain that profoundly affects the amount of active Lck in cells. Notably, mutation of Y192 blocks critical TCR-proximal signaling events and impairs thymocyte development in retrogenic mice. We determined that these defects are caused by hyperphosphorylation of the inhibitory C-terminal tail of Lck. Our findings reveal that modification of Y192 inhibits the ability of CD45 to associate with Lck in cells and dephosphorylate the C-terminal tail of Lck, which prevents its adoption of an active open conformation. These results suggest a negative feedback loop that responds to signaling events that tune active Lck amounts and TCR sensitivity.

T cell receptor (TCR) activation leads to a dramatic reorganisation of both membranes and receptors as the immunological synapse forms. Using a genetic model to rapidly inhibit Zap70 catalytic activity we examined synapse formation between cytotoxic T lymphocytes and their targets. In the absence of Zap70 catalytic activity Vav-1 activation occurs and synapse formation is arrested at a stage with actin and integrin rich interdigitations forming the interface between the two cells. The membranes at the synapse are unable to flatten to provide extended contact, and Lck does not cluster to form the central supramolecular activation cluster (cSMAC). Centrosome polarisation is initiated but aborts before reaching the synapse and the granules do not polarise. Our findings reveal distinct roles for Zap70 as a structural protein regulating integrin-mediated control of actin vs its catalytic activity that regulates TCR-mediated control of actin and membrane remodelling during formation of the immunological synapse. DOI: http://dx.doi.org/10.7554/eLife.01310.001.

Lymphocytes integrate Ag and cytokine receptor signals to make cell fate decisions. Using a specific reporter of TCR signaling that is insensitive to cytokine signaling, Nur77-eGFP, we identify a sharp, minimal threshold of cumulative TCR signaling required for proliferation in CD4 and CD8 T cells that is independent of both Ag concentration and affinity. Unexpectedly, IL-2 reduces this threshold in CD8 but not CD4 T cells, suggesting that integration of multiple mitogenic inputs may alter the minimal requirement for TCR signaling in CD8 T cells. Neither naive CD4 nor naive CD8 T cells are responsive to low doses of IL-2. We show that activated CD8 T cells become responsive to low doses of IL-2 more quickly than CD4 T cells, and propose that this relative delay in turn accounts for the differential effects of IL-2 on the minimal TCR signaling threshold for proliferation in these populations. In contrast to Nur77-eGFP, c-Myc protein expression integrates mitogenic signals downstream of both IL-2 and the TCR, yet marks an invariant minimal threshold of cumulative mitogenic stimulation required for cell division. Our work provides a conceptual framework for understanding the regulation of clonal expansion of CD8 T cells by subthreshold TCR signaling in the context of mitogenic IL-2 signals, thereby rendering CD8 T cells exquisitely dependent upon environmental cues. Conversely, CD4 T cell proliferation requires an invariant minimal intensity of TCR signaling that is not modulated by IL-2, thereby restricting responses to low-affinity or low-abundance self-antigens even in the context of an inflammatory milieu.

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation.DOI: http://dx.doi.org/10.7554/eLife.02270.001.

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.

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.

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.