Diana Gil

Recruitment of Nck by CD3ϵ Reveals a Ligand-Induced Conformational Change Essential for T Cell Receptor Signaling and Synapse Formation

How membrane receptors initiate signal transduction upon ligand binding is a matter of intense scrutiny. The T cell receptor complex (TCR-CD3) is composed of TCR alpha/beta ligand binding subunits bound to the CD3 subunits responsible for signal transduction. Although it has long been speculated that TCR-CD3 may undergo a conformational change, confirmation is still lacking. We present strong evidence that ligand engagement of TCR-CD3 induces a conformational change that exposes a proline-rich sequence in CD3 epsilon and results in recruitment of the adaptor protein Nck. This occurs earlier than and independently of tyrosine kinase activation. Finally, by interfering with Nck-CD3 epsilon association in vivo, we demonstrate that TCR-CD3 recruitment of Nck is critical for maturation of the immune synapse and for T cell activation.

T cell receptor engagement by peptide–MHC ligands induces a conformational change in the CD3 complex of thymocytes

The T cell receptor (TCR) can recognize a variety of cognate peptide/major histocompatibility complex (pMHC) ligands and translate their affinity into distinct cellular responses. To achieve this, the nonsignaling αβ heterodimer communicates ligand recognition to the CD3 signaling subunits by an unknown mechanism. In thymocytes, we found that both positive- and negative-selecting pMHC ligands expose a cryptic epitope in the CD3 complex upon TCR engagement. This conformational change is induced in vivo and requires the expression of cognate MHC. We conclude that TCR engagement with a cognate pMHC ligand induces a conformational change in the CD3 complex of thymocytes and propose that this marks an initial event during thymic selection that signals the recognition of self-antigen.

Initiation of TCR signaling: regulation within CD3 dimers

The number of possible T cell activation outcomes resulting from T cell receptor (TCR) engagement suggests that the TCR is able to differentially activate a myriad of signaling pathways depending on the nature of the stimulus. The complex structural organization of the TCR itself could underlie this diversity of responses. Assembly and stoichiometric studies have helped us to shed some light on the initiation of TCR signaling. The TCR is composed of TCR and CD3 dimers. Changes in the interaction between CD3 subunits within the CD3 dimers and in the interaction of these dimers with the TCR heterodimer could be the triggering mechanism that initiates the first activation events. One of the hallmarks of these early changes in TCR conformation is the induced recruitment of the adapter protein Nck to a proline‐rich sequence of the cytoplasmic tail of CD3ε, but there may be others. According to our most recent observations, the TCR is organized in pre‐existing clusters within plasma membrane microdomains, exhibiting a complexity above and beyond that of dimer composition complexity. How the presence of TCR in clusters influences TCR avidity and propagation of TCR signals is something that has yet to be investigated.

TCR Engagement Induces Proline-Rich Tyrosine Kinase-2 (Pyk2) Translocation to the T Cell-APC Interface Independently of Pyk2 Activity and in an Immunoreceptor Tyrosine-Based Activation Motif-Mediated Fashion

The relocation of kinases in T lymphocytes during their cognate interaction with APCs is essential for lymphocyte activation. We found that the proline-rich tyrosine kinase-2 (Pyk2) is rapidly translocated to the T cell-APC contact area upon T cell-specific recognition of superantigen-pulsed APCs. Stimulation with anti-CD3-coated latex microspheres was sufficient for Pyk2 reorientation, and the coengagement of CD28 boosted Pyk2 redistribution. Nevertheless, Pyk2 translocation did not result in its recruitment to lipid rafts. Two results support that Pyk2 translocation was independent of its kinase activity. First, Lck activity was required for TCR-induced Pyk2 translocation, but not for TCR-induced Pyk2 activation. Second, a kinase-dead Pyk2 mutant was equally translocated upon TCR triggering. In addition, Lck activity alone was insufficient to induce Pyk2 reorientation and activation, requiring the presence of at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Despite the dependence on functional Lck and on phosphorylated ITAM for Pyk2 translocation, the ITAM-binding tyrosine kinase ζ-associated protein 70 (ZAP-70) was not essential. All these data suggest that, by translocating to the vicinity of the immune synapse, Pyk2 could play an essential role in T cell activation and polarized secretion of cytokines.

Rho regulates T cell receptor ITAM-induced lymphocyte spreading in an integrin-independent manner

T cell receptor (TCR) engagement increases integrin‐mediated adhesion to APC, resulting in the stabilization of the T cellu2009:u2009APC interaction and the close apposition of the two cell membranes. Here we show that engagement of either the TCR or CD3 chimeras with immobilized antibodies causes the rapid spreading of T cells in an integrin‐independent fashion. This effect concurs with the polymerization of the actin cytoskeleton and is dependent on the integrity of the immunoreceptor tyrosine‐based activation motifs of the CD3 subunits. Expression of a dominant negative mutant of RhoA, as well as the Rho‐specific inhibitor C3 toxin, abolished TCR‐induced spreading. In contrast, constitutively active or dominant negative forms of Rac and Cdc42 did not affect cell spreading. We conclude that signals emanating from the TCR can directly induce T cell spreading, independently of integrins, and via a Rho‐dependent reorganization of the actin cytoskeleton.

High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.

Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S-transferase–fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.

A co-housing strategy to improve fecundity of mice in timed matings

Timed matings of mice are often carried out to obtain offspring of a precise age when required for a study. Timed matings involve housing male and female mice together for a limited time period, typically overnight. A limitation of this practice is that many mouse pairs fail to mate during the brief co-housing period. The authors co-housed each breeding pair in the same cage but separated by a transparent partition for 3 d before carrying out timed matings. This co-housing strategy resulted in increased copulation during the timed mating period and also significantly increased the average number of pups produced per breeding pair. The authors suggest that co-housing likely permits male urine-borne pheromones to induce female estrus and also enables the expression of male and female mating behaviors.

Robustness and Specificity in Signal Transduction via Physiologic Protein Interaction Networks.

The collective Protein:Protein Interactions (PPI) of a cell are thought to represent a system with emergent network properties that integrate signals from a multiplicity of inputs into coordinated responses. It is hypothesized that the PPI network supplies both specificity for many distinct signals that utilize common intermediate pathways, and also robustness by allowing specific signals to be communicated by alternate routes. Progress with genetic networks points to these concepts, but the extent to which PPI networks possess these properties has not been empirically tested, due to lack of quantitative data needed for such assessments. Here, a hypothetical physiologic PPI network is used to illustrate how signaling robustness and specificity could be manifest under conditions of (i) deletion mutation, or (ii) changes in signaling due to variation in environmental conditions or stimuli. It is proposed that advances in technology enabling empirical analysis of PPI network principles will have the potential to significantly impact basic understanding of signaling mechanisms, and contribute to the generation of novel applications in drug screening and pharmacology.

Detection of constant domain of human T cell antigen receptor alpha-chain via novel monoclonal antibody 7F18.

The αβ T cell antigen receptor (TCR) endows T lymphocytes with immune specificity and controls their effector functions. Each person possesses a vast repertoire of TCRs that is generated by the well-studied processes of somatic recombination and thymic selection. While many antibodies specific for TCRβ variable domains are available, antibodies specific for human TCRα are rare. We now report a novel monoclonal antibody, 7F18, which binds to human TCRα constant region, with specificity for a denatured epitope that can be visualized by SDS-PAGE followed by Western blot. Both immature and mature TCR α-chain products can be visualized, making 7F18 potentially applicable to various biochemical assays of multiprotein complex assembly and maturation. This new monoclonal antibody provides a tool that can potentially facilitate the biochemical analysis of comprehensive populations of human αβ TCR complexes that need not be limited to small subsets of the repertoire.

A PCR-Based Method to Genotype Mice Knocked Out for All Four CD3 Subunits, the Standard Recipient Strain for Retrogenic TCR/CD3 Bone Marrow Reconstitution Technology

Abstract The novel T-cell receptor (TCR)/CD3-retrogenic-reconstitution system represents a very useful strategy for studying TCR/CD3 signaling. Two retroviral vectors containing genes for all six subunits of the TCR/CD3 complex are used to transduce bone marrow precursors and reconstitute lethally irradiated recipient mice. Mice used in this system as bone marrow donors lack all four CD3 subunits (CD3γδɛζ−/−). These mice are generated by crossing the strains CD3ζ−/− and CD3γδɛ−/−, the latter resulting from a knockout construct targeted to CD3ɛ that additionally silences the linked genes, CD3γ and CD3δ. Lacking mature T-cell function, CD3γδɛζ−/− mice are immunocompromised animals often produced by heterozygous breeding strategies on the C57BL/6 background. As a more rapid and reliable means to identify CD3γδɛζ−/− mice than previously described Northern and Southern blots, we designed polymerase chain reactions to distinguish knockout from wild-type CD3ɛ and CD3ζ alleles, facilitating the identification of CD3γδɛζ−/− mice.