A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.

Membrane receptor activation mechanisms and transmembrane peptide tools to elucidate them.

Single-pass membrane receptors contain extracellular domains that respond to external stimuli and transmit information to intracellular domains through a single transmembrane (TM) α-helix. Because membrane receptors have various roles in homeostasis, signaling malfunctions of these receptors can cause disease. Despite their importance, there is still much to be understood mechanistically about how single-pass receptors are activated. In general, single-pass receptors respond to extracellular stimuli via alterations in their oligomeric state. The details of this process are still the focus of intense study, and several lines of evidence indicate that the TM domain (TMD) of the receptor plays a central role. We discuss three major mechanistic hypotheses for receptor activation: ligand-induced dimerization, ligand-induced rotation, and receptor clustering. Recent observations suggest that receptors can use a combination of these activation mechanisms and that technical limitations can bias interpretation. Short peptides derived from receptor TMDs, which can be identified by screening or rationally developed on the basis of the structure or sequence of their targets, have provided critical insights into receptor function. Here, we explore recent evidence that, depending on the target receptor, TMD peptides cannot only inhibit but also activate target receptors and can accommodate novel, bifunctional designs. Furthermore, we call for more sharing of negative results to inform the TMD peptide field, which is rapidly transforming into a suite of unique tools with the potential for future therapeutics.

Nanodomains in biological membranes.

Lipid rafts are defined as cholesterol- and sphingomyelin-enriched membrane domains in the plasma membrane of cells that are highly dynamic and cannot be resolved with conventional light microscopy. Membrane proteins that are embedded in the phospholipid matrix can be grouped into raft and non-raft proteins based on their association with detergent-resistant membranes in biochemical assays. Selective lipid-protein interactions not only produce heterogeneity in the membrane, but also cause the spatial compartmentalization of membrane reactions. It has been proposed that lipid rafts function as platforms during cell signalling transduction processes such as T-cell activation (see Chapter 13 (pages 165-175)). It has been proposed that raft association co-localizes specific signalling proteins that may yield the formation of the observed signalling microclusters at the immunological synapses. However, because of the nanometre size and high dynamics of lipid rafts, direct observations have been technically challenging, leading to an ongoing discussion of the lipid raft model and its alternatives. Recent developments in fluorescence imaging techniques have provided new opportunities to investigate the organization of cell membranes with unprecedented spatial resolution. In this chapter, we describe the concept of the lipid raft and alternative models and how new imaging technologies have advanced these concepts.

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse.

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

Visualization of TCR nanoclusters via immunogold labeling, freeze-etching, and surface replication.

T cells show high sensitivity for antigen, even though their T-cell antigen receptor (TCR) has a low affinity for its ligand, a major histocompatibility complex molecule presenting a short pathogen-derived peptide. Over the past few years, it has become clear that these paradoxical properties rely at least in part on the organization of cell surface-expressed TCRs in TCR nanoclusters. We describe a protocol, comprising immunogold labeling, cell surface replica generation, and electron microscopy (EM) analysis that allows nanoscale resolution of the distribution of TCRs and other cell surface molecules of cells grown in suspension. Unlike most of the light microscopy-based single-molecule resolution techniques, this technique permits visualization of these molecules on cell surfaces that do not adhere to an experimental support. Given the potential of adhesion-induced receptor redistributions, our technique is a relevant complement to the substrate adherence-dependent techniques. Furthermore, it does not rely on introduction of fluorescently labeled recombinant molecules and therefore allows direct analysis of nonmanipulated primary cells.

The two-faced T cell epitope: examining the host-microbe interface with JanusMatrix.

Advances in the field of T cell immunology have contributed to the understanding that cross-reactivity is an intrinsic characteristic of the T cell receptor (TCR), and that each TCR can potentially interact with many different T cell epitopes. To better define the potential for TCR cross-reactivity between epitopes derived from the human genome, the human microbiome, and human pathogens, we developed a new immunoinformatics tool, JanusMatrix, that represents an extension of the validated T cell epitope mapping tool, EpiMatrix. Initial explorations, summarized in this synopsis, have uncovered what appear to be important differences in the TCR cross-reactivity of selected regulatory and effector T cell epitopes with other epitopes in the human genome, human microbiome, and selected human pathogens. In addition to exploring the T cell epitope relationships between human self, commensal and pathogen, JanusMatrix may also be useful to explore some aspects of heterologous immunity and to examine T cell epitope relatedness between pathogens to which humans are exposed (Dengue serotypes, or HCV and Influenza, for example). In Hand-Foot-Mouth disease (HFMD) for example, extensive enterovirus and human microbiome cross-reactivity (and limited cross-reactivity with the human genome) seemingly predicts immunodominance. In contrast, more extensive cross-reactivity with proteins contained in the human genome as compared to the human microbiome was observed for selected Treg epitopes. While it may be impossible to predict all immune response influences, the availability of sequence data from the human genome, the human microbiome, and an array of human pathogens and vaccines has made computationally-driven exploration of the effects of T cell epitope cross-reactivity now possible. This is the first description of JanusMatrix, an algorithm that assesses TCR cross-reactivity that may contribute to a means of predicting the phenotype of T cells responding to selected T cell epitopes. Whether used for explorations of T cell phenotype or for evaluating cross-conservation between related viral strains at the TCR face of viral epitopes, further JanusMatrix studies may contribute to developing safer, more effective vaccines.

Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses.

The immunological synapse is a stable adhesive junction between a polarized immune effector cell and an antigen-bearing cell. Immunological synapses are often observed to have a striking radial symmetry in the plane of contact with a prominent central cluster of antigen receptors surrounded by concentric rings of adhesion molecules and actin-rich projections. There is a striking similarity between the radial zones of the immunological synapse and the dynamic actinomyosin modules employed by migrating cells. Breaking the symmetry of an immunological synapse generates a moving adhesive junction that can be defined as a kinapse, which facilitates signal integration by immune cells while moving over the surface of antigen-presenting cells.

Single-molecule microscopy reveals heterogeneous dynamics of lipid raft components upon TCR engagement.

The existence of lipid rafts and their importance for immunoreceptor signaling is highly debated. By non-invasive single molecule imaging, we analyzed the dynamics of the T-cell antigen receptor (TCR), the lipid raft-associated glycosylphosphatidylinositol (GPI) proteins CD48 and CD59 and the major leukocyte phosphatase CD45 in living naive T lymphocytes. TCR triggering induced the immobilization of CD45 and CD48 at different positions within the T-cell interface. The second GPI protein, CD59, did not co-immobilize indicating lipid raft heterogeneity in living T lymphocytes. A novel biochemical approach confirmed that lipid raft components are not associated in the plasma membrane of resting cells, and variably associate with specific receptors to distinct lipid rafts upon activation.

Molecular analysis of TCR and peptide/MHC interaction using P18-I10-derived peptides with a single D-amino acid substitution.

For the structural analysis of T-cell receptor (TCR) and peptide/MHC interaction, a series of peptides with a single amino acid substitution by a corresponding D-amino acid, having the same weight, size, and charge, within P18-I10 (aa318-327: RGPGRAFVTI), an immunodominant epitope of HIV-1 IIIB envelope glycoprotein, restricted by the H-2Dd class I MHC molecule, has been synthesized. Using those peptides, we have observed that the replacement at positions 324F, 325V, 326T, and 327I with each corresponding D-amino acid induced marked reduction of the potency to sensitize targets for P18-I10-specific murine CD8+ cytotoxic T lymphocytes (CTLs), LINE-IIIB, recognition. To analyze further the role of amino acid at position 325, the most critical site for determining epitope specificity, we have developed a CTL line [LINE-IIIB(325D)] and its offspring clones specific for the epitope I-10(325v) having a D-valine (v) at position 325. Taking advantage of two distinct sets of CD8+ CTLs restricted by the same Dd, three-dimensional structural analysis on TCR and peptide/MHC complexes by molecular modeling was performed, which indicates that the critical amino acids within the TCRs for interacting with 325V or 325v appear to belong to the complementarity-determining region 1 but not to the complementarity-determining region 3 of Vbeta chain.

The immunological synapse.

The adaptive immune response is initiated by the interaction of T cell antigen receptors with major histocompatibility complex molecule-peptide complexes in the nanometer scale gap between a T cell and an antigen-presenting cell, referred to as an immunological synapse. In this review we focus on the concept of immunological synapse formation as it relates to membrane structure, T cell polarity, signaling pathways, and the antigen-presenting cell. Membrane domains provide an organizational principle for compartmentalization within the immunological synapse. T cell polarization by chemokines increases T cell sensitivity to antigen. The current model is that signaling and formation of the immunological synapse are tightly interwoven in mature T cells. We also extend this model to natural killer cell activation, where the inhibitory NK synapse provides a striking example in which inhibition of signaling leaves the synapse in its nascent, inverted state. The APC may also play an active role in immunological synapse formation, particularly for activation of naïve T cells.

MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.

Receptores de células T y complejo CD3 / T cell receptors and CD3 complex

Tanto las células T como las células B contactan con antígenos a través de moléculas especializadas presentes en su superficie. En las células T, se han descrito dos tipos de estructuras para interactuar con los antígenos, se trata de los TCR alfa-beta y TCR gamma-delta. El primero, es el que se distribuye más ampliamente en las células T periféricas y timocitos portadores de un receptor definido. Se trata de moléculas heterodiméricas compuestas de dos cadenas polipeptídicas unidas entre sí. En una célula se expresan miles de copias de un receptor cuyas especificidades epitópicas son idénticas, sin embargo, la presencia de un tipo de receptor excluye la del otro. Las células T pueden ser, independiente del receptor desplegado, CD8- CD4+ o CD8+ CD4-, cumpliendo, por lo tanto, funciones ayudadoras o citotóxicas, respectivamente. Ambos receptores para efectuar sus actividades requieren de la expresión de un complejo proteico llamado CD3, compuesto por 5 proteínas denominadas alfa, delta, épsilon, dseta y eta cuya misión, en presencia de interacción con el antígeno, es transducir señales a través de la membrana celular del linfocito. El receptor de la célula T reconoce al antígeno asociado a proteínas de la membrana plasmática, conocidas como moléculas de histocompatibilidad clase I o II, dependiendo si se trata de células no inmunológicamente comprometidas, o bien, células presentadoras de antígeno o linfocitos, respectivamente. La estrategia para generar diversidad en los TCR es similar a la utilizada para las inmunoglobulinas. Los genes que codifican para los TCR se generan por medio de recombinaciones somáticas de segmentos génicos durante la etapa germinal de las células T

Estructura y función del receptor de antígeno de linfocitos T y su papel en enfermedades infecciosas / Structure and function of T-cell receptors and their role in infectious diseases

Los linfocitos T reconocen antígenos a través de un receptor llamado RcT, por medio de las moléculas del complejo de histocompatibilidad. Responden lisando las células que portan los antígenos, o bien, liberan citocinas que son los mediadores de la respuesta inmune. Se conocen dos isotipos de RcT: el gama/delta y el alfa/beta, mismos que aparecen en ese orden durante la ontogenia de los linfocitos T. La selección del RcT, durante la ontogenia tímica, se realiza mediante eventos moleculares que participan en los procesos de regulación de la expresión génica del RcT. El propósito de la presente revisión es hacer un análisis molecular, estructural y funcional del RcT, y correlacionar esta información con los eventos extra e intracelulares que regulan su expresión génica en linfocitos T humanos, y asimismo analizar la participación del RcT en las enfermedades infecciosas y autoimunes

T cell receptor (TCR) repertoire in alloimmune responses.

A large number of alloantigenic determinants could be generated by both the direct and indirect alloantigen presentation pathways. Hence, a heterogeneous population of T cells expressing a wide variety of receptors would be expected to respond to this diverse array of alloantigenic determinants. However, T cells expressing highly restricted T cell receptor (TCR) variable genes have been reported in a variety of alloimmune responses. A similar phenomenon has been observed in a wide variety of other immune responses, from those induced by superantigens, to very specific responses induced by a single peptide presented by a single MHC molecule. Given this scenario, the limited number of T cell clones which dominate an allograft rejection response, or for that matter an autoimmune response or a tumor specific response, could be therapeutically targeted by virtue of the selected TCR expression.

Predicting the size of the T-cell receptor and antibody combining region from consideration of efficient self-nonself discrimination.

The binding of antibody to antigen or T-cell receptor to major histocompatibility complex-peptide complex requires that portions of the two structures have complementary shapes that can closely approach each other. The question that we address here is how large should the complementary regions on the two structures be. The interacting regions are by necessity roughly the same size. To estimate the size (number of contact residues) of an optimal receptor combining region, we assume that the immune system over evolutionary time has been presented with a large random set of foreign molecules that occur on common pathogens, which it must recognize, and a smaller random set of self-antigens to which it must fail to respond. Evolutionarily, the receptors and the molecular groups that the immune system recognizes as epitopes are imagined to have coevolved to maximize the probability that this task is performed. The probability of a receptor matching a random antigen is estimated from this condition. Using a simple model for receptor-ligand interaction, we estimate that the optimal size binding region on immunoglobulin or T-cell receptors will contain about 15 contact residues, in agreement with experimental observation.

Transmembrane helical interactions: zeta chain dimerization and functional association with the T cell antigen receptor.

Members of the zeta family of receptor subunits (zeta, eta and gamma) are structurally related proteins found as components of the T cell antigen receptor (TCR) and certain Fc receptors. These proteins share the ability to form disulfide-linked dimers with themselves and with other members of the family. Comparison of the amino acid sequences of zeta and gamma reveals a significant degree of homology, which is highest within their membrane-spanning domains. Analysis of their transmembrane sequences on a helical wheel projection suggests that all of the identical amino acids are clustered on one face of a potential alpha-helix. This face contains the only cysteine residue within zeta, suggesting that this conserved region may function to mediate dimerization. Indeed, replacing the transmembrane domain of the Tac antigen (alpha chain of the interleukin-2 receptor) by that of the zeta chain resulted in the formation of disulfide-linked dimers of Tac. The conserved aspartic acid residue found in the zeta and gamma transmembrane sequences was found to play a role in disulfide linkage. Replacing the aspartic acid with a lysine but not with an alanine or valine residue allowed formation of disulfide-linked dimers. The ability of the aspartic acid residue to support dimerization was dependent upon its position within the helix. Thus, these observations indicate that residues within the zeta transmembrane domain play a critical role in the formation of disulfide-linked dimers. Expression of zeta mutants in zeta-deficient T cells revealed that the zeta transmembrane domain is also responsible for reconstituting transport of functional TCR complexes to the cell surface and differentiated the requirements for disulfide-linked dimerization per se from assembly of the TCR complex.

Structural mutations of C-domains in members of the Ig superfamily. Consequences for the interactions between the T cell antigen receptor and the zeta 2 homodimer.

Several molecules belonging to the Ig superfamily are expressed together with noncovalently associated subunits. This applies for membrane-bound IgM and IgD, some of the FcR, and the Ti dimers of the TCR. The interactions between members of the Ig superfamily and their associated subunits are still not fully understood. We locate critical amino acid residues for TCR assembly in the Ti-alpha and -beta extracellular C-domains. A point mutation (phenylalanine195----valine) in a highly conserved residue in the Ti-alpha chain of the Jurkat variant J79 was identified by DNA sequencing. This mutation did not prevent cytoplasmic association of Ti alpha beta and CD3 gamma delta epsilon, but abolished binding of the zeta 2 homodimer to the rest of the TCR. The consequences of this mutation for TCR assembly were confirmed by transfection of a site-directed mutagenized Ti-alpha chain into a Ti-alpha-deficient Jurkat variant. Computer model analysis showed that the Ti-alpha phenylalanine195 directly contributed to the beta-sheet facing away from the Ti-beta chain, indicating that it could be directly involved in the interactions between one or more of the CD3 chains or the zeta 2 dimer. Site-directed mutagenesis of the corresponding residue in the Ti-beta chain demonstrated that a phenylalanine216----valine substitution had similar effects on TCR assembly as the Ti-alpha mutation, whereas a phenylalanine216----histidine substitution allowed TCR assembly and expression. Whether the consequences for TCR assembly of the Ti-alpha and -beta mutations were due to any direct effects on the interaction between zeta and the Ti alpha beta dimer or to indirect effects are discussed.

[The T-lymphocyte antigen receptor]. / Le récepteur d'antigène des lymphocytes T.

The precise knowledge of the T-cells antigen receptor (TCR) is of paramount importance; it is the first structure involved in the antigen (allergen) recognition, provided this one is presented in the right conditions; that is in the context of HLA molecules present at the surface of macrophages, after being processed inside. The TCR alpha/beta, present on more than 90% of peripheral T cells, is formed of two glyco-protein chains of similar molecular weight. The cytoplasmic end of the TCR is two short to transmit the message of recognition. The signal is transduced by neighbouring molecules forming the CD3 complex. Other membrane proteins such as CD2, LFA1, reinforce adhesion between immuno-competent cells. The presence or absence of CD4 or CD8 surface antigens, permit to distinguish two T cell subpopulations, namely helper and suppressor/cytotoxic lymphocytes. The TCR gene organization is very similar to that of light and heavy chains of immunoglobulins. Their fortuitous rearrangement explains the very large diversity of the T-cell repertoire. The TCR gamma/delta, although first appeared on the thymic cells, is present on less than 5% of peripheral lymphocytes, where its exact role is still unknown.

Structure, assembly and intracellular transport of the T cell receptor for antigen.

The T cell receptor for antigen (TCR) is responsible for the recognition of antigen associated with the major histocompatibility complex (MHC). The TCR expressed on the surface of T cells is associated with an invariant structure, CD3. CD3 is assumed to be responsible for intracellular signaling following occupancy of the TCR by ligand. The TCR/CD3 complex consists of six different polypeptides, and represents a uniquely complex multisubunit assembly problem for the cell. The cell copes with this problem by regulating the intracellular assembly of the complex. Within the endoplasmic reticulum, the newly-synthesised chains assemble into the complete structure prior to transport to the cell surface. There are a series of different isoforms of the receptor involving differential use of the TCR heterodimer (alpha-beta or gamma-delta), zeta-family member, and CD3 gamma or delta chains. These are presumably linked to different TCR functions. Assembly of the TCR/CD3 complex competes with specific degradation of unassembled polypeptides. The fate of the receptor depends on the presence of subtle signals on individual chains which determine pairing and assembly or degradation. The T cell is thus able to select a completely assembled fully functional series of distinct TCR/CD3 complexes for expression at the cell surface.