Aldo Borroto

Triggering the TCR Complex Causes the Downregulation of Nonengaged Receptors by a Signal Transduction-Dependent Mechanism

Downregulation of the TCR complex is believed to be intimately tied to T cell activation, allowing serial triggering of receptors and desensitization of stimulated cells. We studied transfected and transgenic T cells expressing CD3zeta chimeras to demonstrate that ligand engagement of the TCR or chimeras causes comodulation of nonengaged receptors. Comodulation required protein tyrosine kinase activity but not trans-phosphorylation of nonengaged receptors. The TCR appears to be downregulated by at least two mechanisms. One mechanism requires direct engagement, independent of signaling. The second requires signaling and downregulates nontriggered receptors. These results shed new light on the process of TCR downregulation and indicate that the number of downregulated TCRs cannot be assumed to equal the number of engaged receptors.

CHARACTERIZATION OF THE REGION INVOLVED IN CD3 PAIRWISE INTERACTIONS WITHIN THE T CELL RECEPTOR COMPLEX

Assembly of the six-chain T cell antigen receptor-CD3 complex takes place by pairwise interactions. Thus, CD3-ε interacts with either CD3-γ or CD3-δ, and these dimers then associate with the TCR heterodimer (α·β or γ·δ) and the CD3-ζ homodimer to constitute a full complex. We have now mapped the site in CD3-ε responsible for the interaction with CD3-γ and CD3-δ by analysis of a series of deletional mutants encompassing the most conserved regions. We found that the highly conserved juxtamembrane domain is mainly responsible for the interaction. Thus, deletion of this 16-amino acid extracellular sequence resulted in the inhibition of up to 95% of the CD3-ε/γ interaction. A highly conserved sequence is also present in both CD3-γ and CD3-δ, suggesting that the domain in these two chains may reciprocally be involved in the interaction with CD3-ε. Indeed, an immobilized synthetic peptide corresponding to the CD3-γ sequence specifically associated to a bacterially expressed CD3-ε protein, suggesting the 16-amino acid domain is sufficient to promote CD3-ε/CD3-γ assembly. The conservation of the motif in the CD3 chains suggest that, in addition to CD3-ε/CD3-γ and CD3-ε/CD3-δ interactions, it may also mediate homotypic interactions. Indeed, it is shown that it mediates the formation of disulfide-linked homodimers and that the formation of homo- and heterodimers are mutually excluded. Finally, this domain contains a Cys-X-X-Cys sequence that resembles that of p56 lck , which is responsible for the interaction with the cytoplasmic tails of CD4 and CD8. Since the replacement of the two cysteines (Cys97 and Cys100) in CD3-ε by alanines strongly inhibited pair formation, the existence of a Cys-X-X-Cys motif involved in protein-protein interactions is suggested.

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 cell : APC 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.

Relevance of Nck–CD3ε Interaction for T Cell Activation In Vivo

On TCR ligation, the adaptor Nck is recruited through its src homology 3.1 domain to a proline-rich sequence (PRS) in CD3ε. We have studied the relevance of this interaction for T cell activation in vitro and in vivo by targeting the interaction sites in both partners. The first approach consisted of studying a knockin (KI) mouse line (KI-PRS) bearing a conservative mutation in the PRS that makes the TCR incompetent to recruit Nck. This deficiency prevents T cell activation by Ag in vitro and inhibited very early TCR signaling events including the tyrosine phosphorylation of CD3ζ. Most important, KI-PRS mice are partly protected against the development of neurological symptoms in an experimental autoimmune encephalitis model, and show a deficient antitumoral response after vaccination. The second approach consisted of using a high-affinity peptide that specifically binds the src homology 3.1 domain and prevents the interaction of Nck with CD3ε. This peptide inhibits T cell proliferation in vitro and in vivo. These data suggest that Nck recruitment to the TCR is fundamental to mount an efficient T cell response in vivo, and that the Nck–CD3ε interaction may represent a target for pharmacological modulation of the immune response.

Nck Recruitment to the TCR Required for ZAP70 Activation during Thymic Development

The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3ε after TCR engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4+CD8+ thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired tyrosine phosphorylation of CD3ζ, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic differences in both knockin mice.

Conformational changes in the T cell receptor differentially determine T cell subset development in mice

Recruitment of the adaptor protein Nck to the T cell receptor complex contributes to the specification of T cells in the thymus. Nck and T Cell Fate in the Thymus Signals generated by ligand binding to the T cell receptor (TCR) complex are important for the development of common precursor cells into T cells of the αβ and γδ lineages in the thymus. In response to a ligand-dependent conformational change in the TCR, the complex component CD3ε undergoes a shift that enables the recruitment of the cytosolic adaptor protein Nck. Noting that the C80G mutation in CD3ε blocks transmission of the conformational change and inhibits the activation of T cell lines in vitro, Blanco et al. generated mice expressing the C80G mutant CD3ε. Whereas the development of αβ T cells in these mice was blocked, some subsets of γδ T cells developed normally. Mice expressing a mutant CD3ε protein defective in binding to Nck had a similar phenotype, which suggests that the TCR conformation–dependent recruitment of Nck to the receptor complex may have differential effects on T cell fate. In the thymus, immature T cells differentiate from common precursors to become T cells expressing either the αβ or γδ T cell receptor (TCR) complex. The CD3ε subunit of the TCR complex is thought to transduce ligand-induced conformational changes in the TCR by recruiting the cytosolic adaptor protein Nck. To investigate the role of conformational changes in the TCR in T cell development, we generated mice with a germline mutation (C80G) in the extracellular domain of CD3ε, which prevents the outside-in transmission of conformational changes in the TCR. The development of αβ T cells in the C80G mice was blocked at an early stage that depends on signaling by a precursor form of the TCR. In contrast, the C80G mutation did not impair the development of some subsets of γδ T cells, including Vγ1.1+ cells; however, development of other γδ T cell subsets was blocked. A similar phenotype was observed in mice with a mutation in the cytoplasmic proline-rich sequence (PRS) of CD3ε, the binding site for Nck. In a genetic complementation test, the PRS CD3ε mutant failed to rescue the wild-type phenotype when expressed in heterozygosity with the C80G mutant. These data suggest that Nck may function as an effector of TCR conformational changes during T cell development. Additional experiments showed differential effects of the C80G mutation on the activation of TCR-dependent signaling pathways, which suggests that there are pathways that are either dependent on or independent of the transmission of conformational change in the receptor.

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

A novel inhibitor of interactions between signaling proteins in T cells demonstrates promising preventive and therapeutic effects in several models of autoimmune disease. Toning down T cell signaling to treat autoimmunity T cells are important for fighting infectious agents, but T cells that recognize the body’s own cells are often central to the development of autoimmune disease, leading Borroto et al. to develop a compound that hampers T cell signaling without completely blocking it. Treatment with this compound prevented or treated autoimmune disease in multiple mouse models, and the compound was demonstrated to skew human T cell differentiation toward less inflammatory subsets. Treatment with the compound did not prevent T cell pathogen responses in mice, suggesting that it would not leave patients susceptible to infection. Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR’s CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low–molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell–mediated autoimmune and inflammatory diseases.

Aurora A drives early signalling and vesicle dynamics during T-cell activation

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.

Recycling of Cell Surface Pro-transforming Growth Factor-α Regulates Epidermal Growth Factor Receptor Activation

Impairments in signal transduction, leading to the regulation of cell proliferation, differentiation, or migration are frequently the cause of cancer. Since the accurate spatial and temporal location of their components is crucial to ensure the correct regulation of these signaling pathways, it could be anticipated that defects in intracellular trafficking are at the base of certain neoplasias. However, the trafficking of many components of pathways frequently up-regulated in cancers, such as the epidermal growth factor receptor (EGFR) pathway, are largely unknown. Here, we show that the pro-transforming growth factor-α (pro-TGF-α), a prototypical EGFR ligand, is endocytosed from the cell surface via a clathrin-dependent pathway. Internalized pro-TGF-α does not progress to the lysosome; instead, it is delivered to the cell surface via recycling endosomes. To analyze the functional meaning of the internalization of pro-TGF-α, we used a deletion construct that is normally transported to the cell surface but is deficiently endocytosed. Due to this impairment, the levels of this construct at the cell surface are dramatically augmented. Consequently, the deletion construct displays a higher EGFR-activating ability, revealing a link between the trafficking of pro-TGF-α and the signaling by the EGFR and opening the possibility that defects in the trafficking of the growth factor may contribute to the development of tumors.

Interaction between the N-terminal SH3 domain of Nckα and CD3ɛ-derived peptides: Non-canonical and canonical recognition motifs

The first SH3 domain (SH3.1) of Nckalpha specifically recognizes the proline-rich region of CD3varepsilon, a subunit of the T cell receptor complex. We have solved the NMR structure of Nckalpha SH3.1 that shows the characteristic SH3 fold consisting of two antiparallel beta-sheets tightly packed against each other. According to chemical shift mapping analysis, a peptide encompassing residues 150-166 of CD3varepsilon binds at the canonical SH3 binding site. An exhaustive comparison with the structures of other SH3 domains able and unable to bind CD3varepsilon reveals that Nckalpha SH3.1 recognises a non-canonical PxxPxxDY motif that orientates at the binding site as a class II ligand. A positively charged residue (K/R) at position -2 relative to the WW sequence at the beginning of strand beta3 is crucial for PxxDY recognition. A 14-mer optimised Nckalpha SH3.1 ligand was found using a multi-substitution approach. Based on NMR data, this improved ligand binds Nckalpha SH3.1 through a PxxPxRDY motif that combines specific stabilising interactions corresponding to both canonical class II, PxxPx(K/R), and non-canonical PxxPxxDY motifs. This explains its higher capacity for Nckalpha SH3.1 binding relative to the wild type sequence.