TCR activation kinetics and feedback regulation in primary human T cells.

BACKGROUND: Signaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses. RESULTS: Here, we have analyzed signaling dynamics correlating with either unresponsiveness or proliferation induced upon TCR/CD28 ligation in primary human T cells. We used two widely employed methods to stimulate T cells in vitro, antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs). A comparative analysis of the signaling properties of iAbs and sAbs revealed that, under proliferation-inducing conditions, feedback regulation is markedly different from that leading to an unresponsive state. In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling. We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated. CONCLUSIONS: In summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.

Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation.

Signaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses. Here, we have analyzed signaling dynamics induced upon TCR ligation in primary human T cells. We used CD3 antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs), two widely employed methods to stimulate T cells in vitro. We show that classical sAbs stimulation induces a transient and abortive response, whereas iAbs induce sustained TCR-mediated signaling, resulting in productive T-cell responses previously observed only in antigen-specific murine systems. In summary, our analysis documents TCR signaling kinetics and suggests that iAbs are better suited for studying TCR-mediated signaling as they mimic antigen specific systems.

TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells.

Sos proteins are ubiquitously expressed activators of Ras. Lymphoid cells also express RasGRP1, another Ras activator. Sos and RasGRP1 are thought to cooperatively control full Ras activation upon T-cell receptor triggering. Using RNA interference, we evaluated whether this mechanism operates in primary human T cells. We found that T-cell antigen receptor (TCR)-mediated Erk activation requires RasGRP1, but not Grb2/Sos. Conversely, Grb2/Sos­but not RasGRP1­are required for IL2-mediated Erk activation. Thus, RasGRP1 and Grb2/Sos are insulators of signals that lead to Ras activation induced by different stimuli, rather than cooperating downstream of the TCR.

Control of lymphocyte development and activation by negative regulatory transmembrane adapter proteins.

Signals emanating from antigen receptors critically regulate immune cell activation, survival, and differentiation. Transmembrane adapter proteins (TRAPs), a group of molecules that organize signaling complexes at the plasma membrane, play a pivotal role in propagating and fine-tuning antigen receptor-mediated signaling. During the last years, it has been demonstrated that most of the TRAPs possess inhibitory functions, including linker for activation of T cells (LAT), the best characterized adapter that links the T-cell receptor (TCR) to Ca(2+) flux and mitogen-activated protein kinase activation. Indeed, it appears that LAT may assemble inhibitory complexes that trigger negative feedback loops, thus terminating T-cell activation. Additionally, recent data demonstrate that SIT [Src homology 2 domain-containing phosphatase 2 (SHP2)-interacting TRAP] fine-tunes TCR-mediated signaling events and negatively regulates T-cell development and homeostasis. The experimental evidence suggests that TRAPs play a crucial role also in establishing tolerance. In fact, loss of SIT, LAX, or NTAL (non-T cell activation linker)/linker for activation of B cells (LAB) resulted in the spontaneous development of autoimmune diseases. Moreover, we recently showed that in addition to the inhibition of Src-family kinases, PAG (phosphoprotein associated with glycosphingolipid-enriched domains) is also involved in the negative regulation of Ras activation. Collectively, these data demonstrate that TRAPs are important modulators of immune cell activation and function. Finally, it appears that TRAPs possess redundant yet not completely overlapping functions.

Regulation of T cell homeostasis by the transmembrane adaptor protein SIT.

The transmembrane adaptor protein SIT is a negative regulator of TCR-mediated signaling. However, little is known about the functional role of SIT in mature T cells. In this study, we show that mice deficient for SIT display a decreased number of naive CD8(+) T cells and a progressive accumulation of memory-like (CD44(high)) CD8(+) T lymphocytes that resemble cells undergoing homeostatic proliferation. Indeed, when transferred into lymphopenic hosts, SIT(-/-) naive CD8(+) T cells undergo enhanced homeostatic proliferation and express a higher level of CD44 in comparison to wild-type T cells. By using class-I-restricted TCR transgenic models with different ligand affinity/avidity, we show that lymphopenia-induced homeostatic proliferation is more pronounced in cells carrying low-affinity TCRs. Strikingly, the loss of SIT induces homeostatic proliferation of HY TCR transgenic cells, which are normally unable to proliferate in lymphopenic mice. Collectively, these data demonstrate that SIT negatively regulates T cell homeostasis. Finally, we show that SIT-deficient T cells develop a mechanism analogous to sensory adaptation as they up-regulate CD5, down-regulate the coreceptor, and display impaired TCR-mediated ZAP-70 activation.

A logical model provides insights into T cell receptor signaling.

Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.