Endoplasmic reticulum aminopeptidase associated with antigen processing regulates quality of processed peptides presented by MHC class I molecules

Effective immune surveillance by CD8 T cells depends on the presentation of diverse peptides by MHC class I (pMHC I) molecules on the cell surface. The pMHC I repertoire is shaped in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP). The ERAAP activity is required for producing peptides of appropriate length for generating optimal pMHC I. Paradoxically, ERAAP also inhibits generation of certain peptides such as the SVL9 (SSVVGVWYL) peptide encoded by the H13(a) histocompatibility gene and presented by D(b) MHC by an unknown mechanism. In this study, we show that the presentation of the SVL9-D(b) complex is inhibited when other peptides compete for binding D(b). Conversely, improving the binding of SVL9 peptide to D(b) suppresses the inhibition. Interestingly, the inhibitory effect of competitor peptides is observed only when ERAAP is expressed in the same cells. Thus, ERAAP, in concert with MHC I molecules, regulates the quality of processed peptides presented on the cell surface.     

Generation of peptide-MHC class I complexes through UV-mediated ligand exchange

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.     

Poor binding of a HER-2/neu epitope (GP2) to HLA-A2.1 is due to a lack of interactions with the center of the peptide

Class I major histocompatibility complex (MHC) molecules bind short peptides derived from proteins synthesized within the cell. These complexes of peptide and class I MHC (pMHC) are transported from the endoplasmic reticulum to the cell surface. If a clonotypic T cell receptor expressed on a circulating T cell binds to the pMHC complex, the cell presenting the pMHC is killed. In this manner, some tumor cells expressing aberrant proteins are recognized and removed by the immune system. However, not all tumors are recognized efficiently. One reason hypothesized for poor T cell recognition of tumor-associated peptides is poor binding of those peptides to class I MHC molecules. Many peptides, derived from the proto-oncogene HER-2/neu have been shown to be recognized by cytotoxic T cells derived from HLA-A2(+) patients with breast cancer and other adenocarcinomas. Seven of these peptides were found to bind with intermediate to poor affinity. In particular, GP2 (HER-2/neu residues 654-662) binds very poorly even though it is predicted to bind well based upon the presence of the correct HLA-A2.1 peptide-binding motif. Altering the anchor residues to those most favored by HLA-A2.1 did not significantly improve binding affinity. The crystallographic structure shows that unlike other class I-peptide structures, the center of the peptide does not assume one specific conformation and does not make stabilizing contacts with the peptide-binding cleft.     

T cell activity correlates with oligomeric peptide-major histocompatibility complex binding on T cell surface

Recognition of virally infected cells by CD8+ T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 D(b) molecule (D(b)). Recognition of both A2 and D(b) are peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.     

A reliable and safe T cell repertoire based on low-affinity T cell receptors

Antigens are presented to T cells as short peptides bound to MHC molecules on the surface of body cells. The binding between MHC/peptides and T cell receptors (TCRs) has a low affinity and is highly degenerate. Nevertheless, TCR-MHC/peptide recognition results in T cell activation of high specificity. Moreover, the immune system is able to mount a cellular response when only a small fraction of the MHC molecules on an antigen-presenting cell is occupied by foreign peptides, while autoimmunity remains relatively rare. We consider how to reconcile these seemingly contradictory facts using a quantitative model of TCR signalling and T cell activation. Taking into account the statistics of TCR recognition and antigen presentation, we show that thymic selection can produce a working T cell repertoire which will produce safe and effective responses, that is, recognizes foreign antigen presented at physiological levels while tolerating self. We introduce "activation curves" as a useful tool to study the repertoire's statistical activation properties.     

An induced fit hypothesis for antigen recognition by T lymphocytes: a role for specific antigen retention structures on antigen-presenting cells

The nature of T lymphocyte recognition of foreign antigens is not known, despite recent advances in elucidating the cellular structures that may be involved in the specific interactions. The central difficulty in this process is that T cells respond to foreign antigen only in the context of major histocompatibility complex (MHC) antigens expressed by another antigen-presenting cell. In addition, T cells that interact with class II MHC antigens do not bind foreign protein antigens in their native form, but seem to recognize only proteolytic peptide fragments as the relevant antigen. The simplest explanation for these observations is that the class II MHC antigens themselves bind antigenic peptides to form the appropriate determinant that interacts with the antigen-specific T cell receptor. However, to date no such antigenic complex has been found with MHC antigens despite rigorous attempts at their demonstration. One alternative explanation described here is that there is no preexisting foreign antigen-MHC antigen complex prior to interaction with T cells, and it is the T cells that cause the two moieties to become associated for recognition by a single antigen-specific T cell receptor. Central to this mechanism is that foreign antigenic peptides must be associated with specific antigen retention structures (SARS) expressed by antigen-presenting cells which retain and protect the peptide on the cell surface. These SARS, upon interaction with T cell membrane moieties, would subsequently associate with MHC antigens. A hypothesis to describe this mechanism is developed to account for published observations of antigen processing by antigen-presenting cells and T cell antigen recognition, and makes several predictions that are experimentally testable. This mechanism is also generally applicable to other cellular interactions in which soluble peptide mediators may become associated with surface components of one cell type, and this newly formed complex is in turn recognized by a receptor on a second cell type to deliver functional signals.     

Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity

At the interface between T cell and antigen-presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to the T cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. An interesting aspect of this puzzle is that although soluble agonist pMHCs cannot trigger TCR even at high concentrations, the same ligands trigger TCR very efficiently on the surface of APCs. Here, using lipid bilayers or plastic-based artificial APCs with defined components, we identify the critical APC-associated factors that confer agonist pMHCs with such potency. We found that CD4⁺ T cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid lipid bilayers or fixed plastic surfaces, in the absence of any other APC surface molecules. Importantly, on bilayers, plastic surfaces, or real APCs, endogenous pMHCs did not enhance TCR triggering. TCR triggering, however, critically depended upon the adhesiveness of the surface and an intact T cell actin cytoskeleton. Based on these observations, we propose the receptor deformation model of TCR triggering to explain the remarkable sensitivity and specificity of TCR triggering.     

Cowpox virus evades CTL recognition and inhibits the intracellular transport of MHC class I molecules

Orthopoxviruses evade host immune responses by using a number of strategies, including decoy chemokine receptors, regulation of apoptosis, and evasion of complement-mediated lysis. Different from other poxviral subfamilies, however, orthopoxviruses are not known to evade recognition by CTL. In fact, vaccinia virus (VV) is used as a vaccine against smallpox and a vector for eliciting strong T cell responses to foreign Ags. and both human and mouse T cells are readily stimulated by VV-infected APC in vitro. Surprisingly, however, CD8(+) T cells of mice infected with cowpox virus (CPV) or VV recognized APC infected with VV but not APC infected with CPV. Likewise, CD8(+) T cells from vaccinated human subjects could not be activated by CPV-infected targets and CPV prevented the recognition of VV-infected APC upon coinfection. Because CD8(+) T cells recognize viral peptides presented by MHC class I (MHC I), we examined surface expression, total levels, and intracellular maturation of MHC I in CPV- and VV-infected human and mouse cells. Although total levels of MHC I were unchanged, CPV reduced surface levels and inhibited the intracellular transport of MHC I early during infection. CPV did not prevent peptide loading of MHC I but completely inhibited MHC I exit from the endoplasmic reticulum. Because this inhibition was independent of viral replication, we conclude that an early gene product of CPV abrogates MHC I trafficking, thus rendering CPV-infected cells "invisible" to T cells. The absence of this immune evasion mechanism in VV likely limits virulence without compromising immunogenicity.     

The CD8 T cell coreceptor exhibits disproportionate biological activity at extremely low binding affinities

T lymphocytes recognize peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. Recognition specificity is determined by the alphabeta T cell receptor (TCR). The T lymphocyte surface glycoproteins CD8 and CD4 enhance T cell antigen recognition by binding to MHC class I and class II molecules, respectively. Biophysical measurements have determined that equilibrium binding of the TCR with natural agonist peptide-MHC (pMHC) complexes occurs with KD values of 1-50 microm. The pMHCI/CD8 and pMHCII/CD4 interactions are significantly weaker than this (KD >100 microm), and the relative roles of TCR/pMHC and pMHC/coreceptor affinity in T cell activation remain controversial. Here, we engineer mutations in the MHCI heavy chain and beta2-microglobulin that further reduce or abolish the pMHCI/CD8 interaction to probe the significance of pMHC/coreceptor affinity in T cell activation. We demonstrate that the pMHCI/CD8 coreceptor interaction retains the vast majority of its biological activity at affinities that are reduced by over 15-fold (KD > 2 mm). In contrast to previous reports, we observe that the weak interaction between HLA A68 and CD8, which falls within this spectrum of reduced affinities, retains substantial functional activity. These findings are discussed in the context of current concepts of coreceptor dependence and the mechanism by which TCR coreceptors facilitate T cell activation.     

Processing of an endogenous protein can generate MHC class II-restricted T cell determinants distinct from those derived from exogenous antigen.

Class II MHC molecules on the surface of an APC present immunogenic peptides derived mainly from exogenous proteins to CD4+ T cells. During its transport to the cell surface, class II molecules intersect the endocytic pathway where they acquire peptides derived from endocytosed proteins. However, class II-restricted presentation of endogenously derived peptides can also occur. The current studies were undertaken to examine the ability of different types of APC to generate and present four different T cell determinants derived from an endogenous, nonsecreted, truncated form of hen-egg white lysozyme (HEL[1-80]-Kk). This was compared with the ability of these APC to generate the same determinants from exogenous HEL. All the peptides derived from endogenous HEL[1-80]-Kk tested, were presented by B cells to HEL-specific T cell hybridomas with an efficiency similar to presentation of the same determinants from exogenous HEL. In contrast, an I-Ak-bearing rat fibroblast was unable to generate the HEL peptide 25-43 from exogenous HEL, but could efficiently produce it from endogenous HEL[1-80]-Kk. The results indicate first, that peptides derived from an endogenous Ag can be presented by MHC class II molecules with an efficiency comparable to that of the presentation of the exogenous Ag. Second, that Ag-presenting B cells can generate the same repertoire of antigenic peptides from endogenous Ag as those generated from the exogenous protein. And third, that in contrast to B cells, certain "nonprofessional" APC can generate, from an endogenous protein, T cell determinants distinct from those generated after endocytosis of the exogenous protein. These results suggest that processing of exogenous and endogenous Ag by different APC take place in different intracellular compartments.     

A Novel Flow Cytometric High Throughput Assay for a Systematic Study on Molecular Mechanisms Underlying T Cell Receptor-Mediated Integrin Activation

Lymphocyte function-associated antigen 1 (LFA-1), a member of β2-integrin family, exerts multiple roles in host T cell immunity and has been identified as a useful drug-development target for inflammatory and autoimmune diseases. Applying the findings that primary resting T cells absorb nanometric membrane vesicles derived from antigen presenting cells (APC) via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide-major histocompatibility complex (MHC) complex (pMHC) and LFA-1 with its ligand, intercellular adhesion molecule-1 (ICAM-1), and that signaling cascades triggered by TCR/pMHC interaction take a part in the vesicle-absorption, we established a cell-based high throughput assay for systematic investigation, via isolation of small molecules modulating the level of vesicle-absorption, of molecular mechanisms underlying the T cell absorption of APC-derived vesicles, i.e., structural basis of TCR/pMHC and LFA-1/ICAM-1 interactions and TCR-mediated LFA-1 activation. As primary T cells along with physiological ligands expressed in biological membrane are used and also individual cells in assay samples are analyzed by flow cytometry, results obtained using the assay system hold superior physiological and therapeutic relevance as well as statistical precision.     

The role of B7 costimulation in T-cell immunity.

CD4+ T cells are considered to be the major controlling element of the adaptive immune response. They recognize foreign peptides by interaction of the T cell receptor (TCR) with peptide complexed to major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Once activated, CD4+ T cells orchestrate the various phases of the immune response. They are responsible for the production of numerous cytokines, which activate specific immune effector cell populations including B cells, eosinophils, mast cells and macrophages. Not surprisingly, the activation of CD4+ T cells needs to be tightly regulated and is subject to finely tuned control mechanisms. The requirement for a second or 'costimulatory' signal, in addition to the antigenic signal, provides a key element for the exquisite control of T cell activation. One of the major signalling pathways responsible for delivery of this costimulatory signal is induced by interaction of CD28 on T cells with B7 molecules found only on APC. The present review outlines our current understanding of the physiological role of B7 costimulatory signals in regulating CD4+ T cell responses.     

Functional evidence that conserved TCR CDR alpha 3 loop docking governs the cross-recognition of closely related peptide:class I complexes.

The TCR recognizes its peptide:MHC (pMHC) ligand by assuming a diagonal orientation relative to the MHC helices, but it is unclear whether and to what degree individual TCRs exhibit docking variations when contacting similar pMHC complexes. We analyzed monospecific and cross-reactive recognition by diverse TCRs of an immunodominant HVH-1 glycoprotein B epitope (HSV-8p) bound to two closely related MHC class I molecules, H-2K(b) and H-2K(bm8). Previous studies indicated that the pMHC portion likely to vary in conformation between the two complexes resided at the N-terminal part of the complex, adjacent to peptide residues 2-4 and the neighboring MHC side chains. We found that CTL clones sharing TCR beta-chains exhibited disparate recognition patterns, whereas those with drastically different TCRbeta-chains but sharing identical TCRalpha CDR3 loops displayed identical functional specificity. This suggested that the CDRalpha3 loop determines the TCR specificity in our model, the conclusion supported by modeling of the TCR over the actual HSV-8:K(b) crystal structure. Importantly, these results indicate a remarkable conservation in CDRalpha3 positioning, and, therefore, in docking of diverse TCRalphabeta heterodimers onto variant peptide:class I complexes, implying a high degree of determinism in thymic selection and T cell activation.     

The specific T-cell response to antigenic peptides is influenced by bystander peptides

T lymphocytes recognize antigens in the form of peptides presented by major histocompatibility complex (MHC) molecules on the cell surface. Only a small proportion of MHC class I and class II molecules are loaded with foreign antigenic peptides; the vast majority are loaded with thousands of different self peptides. It was suggested that MHC molecules presenting self peptides may serve either to decrease (antagonistic effect) or increase (synergistic effect) the T cell response to a specific antigen. Here, we present our finding that transfected mouse fibroblasts presenting a single antigenic peptide covalently bound to a class II MHC molecule stimulated specific mouse T cell hybridoma cells to an interleukin-2 response less efficiently than fibroblasts presenting a similar amount of antigenic peptide in the presence of class II molecules loaded with heterogenous bystander peptides.     

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.     

Differential CD3 zeta phosphorylation is not required for the induction of T cell antagonism by altered peptide ligands.

T cells recognize foreign Ags in the form of short peptides bound to MHC molecules. Ligation of the TCR:CD3 complex gives rise to the generation of two tyrosine-phosphorylated forms of the CD3 zeta-chain, pp21 and pp23. Replacement of residues in MHC-bound peptides that alter its recognition by the TCR can generate altered peptide ligands (APL) that antagonize T cell responses to the original agonist peptide, leading to altered T cell function and anergy. This biological process has been linked to differential CD3zeta phosphorylation and generation of only the pp21 phospho-species. Here, we show that T cells expressing CD3zeta mutants, which cannot be phosphorylated, exhibit a 5-fold reduction in IL-2 production and a 30-fold reduction in sensitivity following stimulation with an agonist peptide. However, these T cells are still strongly antagonized by APL. These data demonstrate that: 1) the threshold required for an APL to block a response is much lower than for an agonist peptide to induce a response, 2) CD3zeta is required for full agonist but not antagonist responses, and 3) differential CD3zeta phosphorylation is not a prerequisite for T cell antagonism.     

Modulation of immunological synapse by membrane-bound and soluble ligands

An efficient adaptive immune response should prevent pathogen infections and tumor growth without causing significant damage to host constituents. A crucial event determining the balance between tolerance and immunity is antigen recognition by T cells on the surface of antigen presenting cells (APC). Several molecular contacts at the interface between T cells and APCs contribute to define the nature of the adaptive immune response against a particular antigen. Upon TCR engagement by a peptide-MHC complex (pMHC) on the surface of an APC, a specialized supra-molecular structure known as immunological synapse (IS) assembles at the interface between these two cells. This structure involves massive re-distribution of membrane proteins, including TCR and pMHC complexes, as well as co-stimulatory and adhesion molecules. Furthermore, IS assembly leads to several important intracellular events necessary for T cell activation, such as recruitment of signaling molecules and cytoskeleton rearrangements. Because IS assembly leads to major consequences on the function of T cells, several studies have attempted to identify both soluble and membrane-bound molecules that could contribute to modulate the IS function. Here we describe recent literature on the regulation of IS assembly and modulation by TCR/pMHC binding kinetics, chemokines and cytokines focusing on their role at controlling the balance between adaptive immunity and tolerance.     

Cysteinylation of MHC class II ligands: peptide endocytosis and reduction within APC influences T cell recognition

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4(+) T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.     

Recognition of self and altered self by T cells in autoimmunity and allergy

T cell recognition of foreign peptide antigen and tolerance to self peptides is key to the proper function of the immune system. Usually, in the thymus T cells that recognize self MHC+ self peptides are deleted and those with the potential to recognize self MHC+ foreign peptides are selected to mature. However there are exceptions to these rules. Autoimmunity and allergy are two of the most common immune diseases that can be related to recognition of self. Many genes work together to lead to autoimmunity. Of those, particular MHC alleles are the most strongly associated, reflecting the key importance of MHC presentation of self peptides in autoimmunity. T cells specific for combinations of self MHC and self peptides may escape thymus deletion, and thus be able to drive autoimmunity, for several reasons: the relevant self peptide may be presented at low abundance in the thymus but at high level in particular peripheral tissues; the relevant self peptide may bind to MHC in an unusual register, not present in the thymus but apparent elsewhere; finally the relevant self peptide may be post translationally modified in a tissue specific fashion. In some types of allergy, the peptide+ MHC combination may also be fully derived from self. However the combination in question may be modified by the presence of other ligands, such as small drug molecules or metal ions. Thus these types of allergies may act like the post translationally modified peptides involved some types of autoimmunity.