Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαβ transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRβ transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.

The T-cell antigen receptor (TCR) is pre-organised in oligomers, known as nanoclusters. Nanoclusters could provide a framework for inter-TCR cooperativity upon peptide antigen-major histocompatibility complex (pMHC) binding. Here we have used soluble pMHC oligomers in search for cooperativity effects along the plasma membrane plane. We find that initial binding events favour subsequent pMHC binding to additional TCRs, during a narrow temporal window. This behaviour can be explained by a 3-state model of TCR transition from Resting to Active, to a final Inhibited state. By disrupting nanoclusters and hampering the Active conformation, we show that TCR cooperativity is consistent with TCR nanoclusters adopting the Active state in a coordinated manner. Preferential binding of pMHC to the Active TCR at the immunological synapse suggests that there is a transient time frame for signal amplification in the TCR, allowing the T cells to keep track of antigen quantity and binding time.

Stem cells must proliferate and differentiate to generate the lineages that shape mature organs; understanding these 2 processes and their interaction is one of the central themes in current biomedicine. An intriguing aspect is asymmetric division, by which 2 daughter cells with different fates are generated. Several cell fate determinants participate in asymmetric division, with the endocytic adaptor Numb as the best-known example. Here, we have explored the role of asymmetric division in thymocyte development, visualizing the differential segregation of Numb and pre-TCR in thymic precursors. Analysis of mice where Numb had been inhibited by expressing a dominant negative revealed enhanced pre-T-cell receptor (TCR) signaling and a smaller thymus. Conversely, Numb overexpression resulted in loss of asymmetric division and a larger thymus. The conclusion is that Numb determines the levels of pre-TCR signaling in dividing thymocytes and, ultimately, the size of the pool from which mature T lymphocytes are selected. IntroductionThe mammalian thymus contains different cell types originated from initially equivalent precursors that undergo several rounds of division. To ensure correct numbers of mature lineage-specific cells in adult organs, precursors divide asymmetrically, generating 2 sister cells with different fates. 1,2 One of the "cell fate determinants" asymmetrically segregated is Numb, a plasma membrane-associated protein that contains a phosphotyrosine binding (PTB) domain and antagonizes Notch signaling. Numb function was first described in Drosophila sensory organ precursors, 3 where loss of Numb resulted in both daughter cells adopting the fate of the cell that normally inherits Notch, whereas the opposite was caused by Numb overexpression. [4][5][6] In mammalians, asymmetric division has been studied most extensively during neurogenesis. 7 Deletion of Numb and its homologue Numblike resulted in loss of neural progenitors and block of neurogenesis, 8 as a result of progenitor overdifferentiation and death of young neurons. In other systems, Numb binds to integrins 9 and Src family kinases, 10 mediates receptor internalization, 11,12 and participates in clathrin-dependent endosomal association. 13 Thus, Numb plays multiple and important roles in development and signaling; however, little is known about its role in the thymus.T lymphocytes develop from precursors that undergo a series of cell fate decisions, resulting in differentiation into single positive (SP) thymocytes, the immediate precursors of fully functional CD4 and CD8 T lymphocytes. 14,15 The initial CD4 Ϫ CD8 Ϫ double negative (DN) stages are influenced by signals from the pre-T-cell receptor (TCR) [16][17][18] and Notch. 19,20 Only DN thymocytes receiving proper pre-TCR and Notch signals are able to evolve into the CD4 ϩ CD8 ϩ double positive (DP) ␣␤ lineage stage. Pre-TCR internalization and degradation is very important for correct signaling. [21][22][23] As a consequence of pre-TCR signaling, DN thymocytes proliferate, enabling normal nu...

Modulation of TCR signaling upon ligand binding is achieved by changes in the equilibrium between TCR degradation, recycling and synthesis; surprisingly, the molecular mechanism of such an important process is not fully understood. Here, we describe the role of a new player in the mediation of TCR degradation: the endocytic adaptor Numb. Our data show that Numb inhibition leads to abnormal intracellular distribution and defective TCR degradation in mature T lymphocytes. In addition, we find that Numb simultaneously binds to both Cbl and a site within CD3ε that overlaps with the Nck binding site. As a result, Cbl couples specifically to the CD3ε chain to mediate TCR degradation. The present study unveils a novel role of Numb that lies at the heart of TCR signaling initiation and termination.

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Numb asymmetrically segregates at mitosis to control cell fate choices during development. Numb inheritance specifies progenitor over differentiated cell fates, and, paradoxically, also promotes neuronal differentiation, thus indicating that the role of Numb may change during development. Here we report that Numb nuclear localization is restricted to early thymocyte precursors, whereas timed appearance of pre-T-cell receptor (pre-TCR) and activation of protein kinase Cθ promote phosphorylation-dependent Numb nuclear exclusion. Notably, nuclear localization of Numb in early thymocyte precursors favors p53 nuclear stabilization, whereas pre-TCR-dependent Numb nuclear exclusion promotes the p53 downmodulation essential for further differentiation. Accordingly, the persistence of Numb in the nucleus impairs the differentiation and promotes precursor cell death. This study reveals a novel regulatory mechanism for Numb function based on its nucleus–cytosol shuttling, coupling the different roles of Numb with different stages of T-cell development.

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