A Cholesterol-Based Allostery Model of T Cell Receptor Phosphorylation.

Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαß 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 TCRß 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.

SFRP1 modulates astrocyte-to-microglia crosstalk in acute and chronic neuroinflammation.

Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron-microglia-astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases.

CD3ε recruits Numb to promote TCR degradation.

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.

Relevance of Nck-CD3 epsilon 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.

Mutation of the Polyproline Sequence in CD3ε Evidences TCR Signaling Requirements for Differentiation and Function of Pro-Inflammatory Tγδ17 Cells.

Tγδ17 cells have emerged as a key population in the development of inflammatory and autoimmune conditions such as psoriasis. Thus, the therapeutic intervention of Tγδ17 cells can exert protective effects in this type of pathologies. Tγδ cells commit to IL-17 production during thymus development, and upon immune challenge, additional extrathymic signals induce the differentiation of uncommitted Tγδ cells into Tγδ17 effector cells. Despite the interest in Tγδ17 cells during the past 20 years, the role of TCR signaling in the generation and function of Tγδ17 cells has not been completely elucidated. While some studies point to the notion that Tγδ17 differentiation requires weak or no TCR signaling, other works suggest that Tγδ17 require the participation of specific kinases and adaptor molecules downstream of the TCR. Here we have examined the differentiation and pathogenic function of Tγδ17 cells in "knockin" mice bearing conservative mutations in the CD3ε polyproline rich sequence (KI-PRS) with attenuated TCR signaling due to lack of binding of the essential adaptor Nck. KI-PRS mice presented decreased frequency and numbers of Tγδ17 cells in adult thymus and lymph nodes. In the Imiquimod model of skin inflammation, KI-PRS presented attenuated skin inflammation parameters compared to wild-type littermates. Moreover, the generation, expansion and effector function Tγδ17 cells were impaired in KI-PRS mice upon Imiquimod challenge. Thus, we conclude that an intact CD3ε-PRS sequence is required for optimal differentiation and pathogenic function of Tγδ17 cells. These data open new opportunities for therapeutic targeting of specific TCR downstream effectors for treatment of Tγδ17-mediated diseases.

Antigen presentation between T cells drives Th17 polarization under conditions of limiting antigen.

T cells form immunological synapses with professional antigen-presenting cells (APCs) resulting in T cell activation and the acquisition of peptide antigen-MHC (pMHC) complexes from the plasma membrane of the APC. They thus become APCs themselves. We investigate the functional outcome of T-T cell antigen presentation by CD4 T cells and find that the antigen-presenting T cells (Tpres) predominantly differentiate into regulatory T cells (Treg), whereas T cells that have been stimulated by Tpres cells predominantly differentiate into Th17 pro-inflammatory cells. Using mice deficient in pMHC uptake by T cells, we show that T-T antigen presentation is important for the development of experimental autoimmune encephalitis and Th17 cell differentiation in vivo. By varying the professional APC:T cell ratio, we can modulate Treg versus Th17 differentiation in vitro and in vivo, suggesting that T-T antigen presentation underlies proinflammatory responses in conditions of antigen scarcity.

Interaction between the N-terminal SH3 domain of Nck-alpha and CD3-epsilon-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.

Dynamic reorganisation of intermediate filaments coordinates early B-cell activation.

During B-cell activation, the dynamic reorganisation of the cytoskeleton is crucial for multiple cellular responses, such as receptor signalling, cell spreading, antigen internalisation, intracellular trafficking, and antigen presentation. However, the role of intermediate filaments (IFs), which represent a major component of the mammalian cytoskeleton, is not well defined. Here, by using multiple super-resolution microscopy techniques, including direct stochastic optical reconstruction microscopy, we show that IFs in B cells undergo drastic reorganisation immediately upon antigen stimulation and that this reorganisation requires actin and microtubules. Although the loss of vimentin in B cells did not impair B-cell development, receptor signalling, and differentiation, vimentin-deficient B cells exhibit altered positioning of antigen-containing and lysosomal associated membrane protein 1 (LAMP1+) compartments, implying that vimentin may play a role in the fine-tuning of intracellular trafficking. Indeed, vimentin-deficient B cells exhibit impaired antigen presentation and delayed antibody responses in vivo. Thus, our study presents a new perspective on the role of IFs in B-cell activation.

A non-conserved amino acid variant regulates differential signalling between human and mouse CD28.

CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P212 in human vs. A210 in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this Y209APP212 sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside.

Author Correction: Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

The original version of this Article contained an error in the spelling of the author José María González-Granado, which was incorrectly given as José María Gozález-Granado. This has now been corrected in both the PDF and HTML versions of the Article.

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

Bacterial phagocytosis and antigen cross-presentation to activate CD8+ T cells are principal functions of professional antigen presenting cells. However, conventional CD4+ T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8+ T cells, which proliferate and become cytotoxic in response. CD4+ T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8+ T cells with low PD-1 expression. Moreover, transphagocytic CD4+ T cells induce protective anti-tumour immune responses by priming CD8+ T cells, highlighting the potential of CD4+ T cells as a tool for cancer immunotherapy.

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.

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

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.

Crammed signaling motifs in the T-cell receptor.

Although the T cell antigen receptor (TCR) is long known to contain multiple signaling subunits (CD3γ, CD3δ, CD3ɛ and CD3ζ), their role in signal transduction is still not well understood. The presence of at least one immunoreceptor tyrosine-based activation motif (ITAM) in each CD3 subunit has led to the idea that the multiplication of such elements essentially serves to amplify signals. However, the evolutionary conservation of non-ITAM sequences suggests that each CD3 subunit is likely to have specific non-redundant roles at some stage of development or in mature T cell function. The CD3ɛ subunit is paradigmatic because in a relatively short cytoplasmic sequence (∼55 amino acids) it contains several docking sites for proteins involved in intracellular trafficking and signaling, proteins whose relevance in T cell activation is slowly starting to be revealed. In this review we will summarize our current knowledge on the signaling effectors that bind directly to the TCR and we will propose a hierarchy in their response to TCR triggering.

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

In the thymus, immature T cells differentiate from common precursors to become T cells expressing either the αß 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 αß 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.

Recycling of cell surface pro-transforming growth factor-{alpha} 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-alpha (pro-TGF-alpha), a prototypical EGFR ligand, is endocytosed from the cell surface via a clathrin-dependent pathway. Internalized pro-TGF-alpha 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-alpha, 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-alpha 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.

Impaired trafficking and activation of tumor necrosis factor-alpha-converting enzyme in cell mutants defective in protein ectodomain shedding.

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.