Major Histocompatibility Complex Ligands Research Articles

Engineering the Polyproline II Propensity of a Class II Major Histocompatibility Complex Ligand Peptide

Our immune system constantly samples peptides found inside the body as a means to detect foreign pathogens, infected cells, and tumorous cells. T cells, which carry out the critical task of distinguishing self from nonself peptides, can only survey peptides that are presented by the major histocompatibility complex protein. We investigated how the secondary structure of a peptide, namely, the polyproline II helix content, influences major histocompatibility complex binding. We synthesized 12 analogues of the wheat gluten derived α-I-gliadin peptide and tested their binding to the celiac disease associated HLA-DQ2 protein. Our analogue library represents a broad spectrum of polyproline II propensities, ranging from random coil structure to high polyproline II helix content. Overall, there was no noticeable correlation between the peptide polyproline II helix content and HLA-DQ2 binding. One analogue peptide, which has low polyproline II helix content, showed a 4.5-fold superior binding compared to native α-I-gliadin.

Computational Resources for MHC Ligand Identification.

Advances in the high-throughput determination of functional modulators of major histocompatibility complex (MHC) and improved computational predictions of MHC ligands have rendered the rational design of immunomodulatory peptides feasible. Proteome-derived peptides and 'reverse vaccinology' by computational means will play a driving role in future vaccine design. Here we review the molecular mechanisms of the MHC mediated immune response, present the computational approaches that have emerged in this area of biotechnology, and provide an overview of publicly available computational resources for predicting and designing new peptidic MHC ligands.

Inhibitory Receptor Signaling Destabilizes Immunological Synapse Formation in Primary NK Cells

Upon engagement of their cognate class I major histocompatibility complex ligands, receptors containing immunotyrosine-based inhibitory motifs (ITIMs) transduce signals that block cytolytic and inflammatory responses. In this manner, ITIM-coupled receptors play a crucial role in maintaining natural killer (NK) cell tolerance toward normal, healthy tissue. A number of studies, mostly using immortalized NK cell lines, have demonstrated that ITIM signaling functions by disrupting the cytolytic immunological synapse formed between an NK cell and its target. However, more recent imaging experiments using primary NK cells have suggested that inhibitory receptor engagement does not antagonize contact formation, casting doubt on the hypothesis that ITIM signals destabilize the synapse. To resolve this issue, we analyzed primary NK cell activation and contact formation on supported lipid bilayers containing controlled combinations of activating and inhibitory ligands. Under these conditions, we observed that ITIM signaling clearly inhibited adhesion, cell arrest, and calcium influx, three hallmarks of synapse formation. These results are consistent with previous reports showing that inhibitory receptors deliver a “reverse stop” signal, and confirm that ITIM signaling functions at least in part by destabilizing cytolytic synapse formation.

Insights into immune structure, recognition, and signaling

In this volume of Immunological Reviews, the articles focus on the structural biology of immunoreceptors and what structure is telling us about function. The issue is unabashedly ‘receptor-centric’ and the proteins covered range from antigen receptors, innate receptors, and cytokine receptors, thereby providing an unusually comprehensive snapshot of the current state of the art in molecular immunology. Of all the major fields in biomedical research, including neurobiology, developmental biology, immunology, microbiology, and genetics, just to name a subset, perhaps structure in the broadest sense has had more of an impact on immunology and played more of a major role in guiding the evolution and maturation of the field than any other. Here, I use the term ‘structure’ to encompass not only direct structural approaches, such as x-ray crystallography and nuclear magnetic resonance spectroscopy (NMR), but also imaging, biophysical, molecular, and mechanistic approaches to interrogate aspects of molecular recognition, oligomerization, and signaling. Perhaps the fact that immunobiology is centered on questions of recognition and discrimination and is largely controlled by receptor-ligand systems has led to an unusually high level of sophistication about structure among immunologists. Personally speaking, having delved into structural questions in neurobiology and developmental biology, I can state without equivocation that the structural and molecular savvy of immunologists has led to greater mechanistic clarity underlying central tenets of the field compared to other disciplines. Dating back to the days of elucidating the first antibody structures and the relation between hypervariable regions and antigen recognition, to the transformative clarity of the first major histocompatibility complex (MHC) structures in explaining MHC restriction, to more recent biophysical and structural investigations of T-cell receptor (TCR) recognition of both classical and non-classical MHC, innate immune receptors such as natural killer (NK) and Toll-like receptors, and cytokine receptors, molecular science has illuminated the path for the experimentalists and assumed a large and highly visible role in immunobiology as a whole.

Structural insights into activation of antiviral NK cell responses

Natural killer (NK) cells are key components of innate immune responses, providing surveillance against cells undergoing tumorigenesis or infection, by viruses or internal pathogens. NK cells can directly eliminate compromised cells and regulate downstream responses of the innate and acquired immune systems through the release of immune modulators (cytokines, interferons). The importance of the role NK cells play in immune defense was demonstrated originally in herpes viral infections, usually mild or localized, which become severe and life threatening in NK-deficient patients . NK cell effector functions are governed by balancing opposing signals from a diverse array of activating and inhibitory receptors. Many NK receptors occur in paired activating and inhibitory isoforms and recognize major histocompatibility complex (MHC) class I proteins with varying degrees of peptide specificity. Structural studies have made considerable inroads into understanding the molecular mechanisms employed to broadly recognize multiple MHC ligands or specific pathogen-associated antigens and the strategies employed by viruses to thwart these defenses. Although many details of NK development, signaling, and integration remain mysterious, it is clear that NK receptors are key components of a system exquisitely tuned to sense any dysregulation in MHC class I expression, or the expression of certain viral antigens, resulting in the elimination of affected cells.

NK Cells—From Bench to Clinic

After decades of mouse and human research, we now know that natural killer (NK) cells have unique properties including memory. Although initially described as major histocompatibility complex (MHC) unrestricted killers, NK cells have several families of receptors that directly recognize MHC including Ly49 receptors in the mouse and killer immunoglobulin-like receptors (KIR) in humans. The strength of this signal is determined by polymorphisms in NK cell inhibitory receptor genes and their MHC ligands inherited on different chromosomes. Inhibitory receptors protect "self"-expressing normal tissue from being killed by NK cells and protecting against autoimmunity. Therefore, for NK cells to kill and produce cytokines, they must encounter activating receptor ligands in the context of "missing self" that occurs with some viral infections and malignant transformation. The second property of inhibitory receptors is to educate or license NK cells to acquire function. This is best demonstrated in the mouse and in humans by enhanced function on self-inhibitory receptor-expressing NK cells when in a host expressing cognate ligate. In contrast, NK cells without inhibitory receptors or with nonself-inhibitory receptors are relatively hyporesponsive. The basic biology of NK cells in response to cytokines, education, and viruses will translate into strategies to manipulate NK cells for therapeutic purposes.

A Role for Differential Variable Gene Pairing in Creating T Cell Receptors Specific for Unique Major Histocompatibility Ligands

A limited set of T cell receptor (TCR) variable (V) gene segments are used to create a repertoire of TCRs that recognize all major histocompatibility complex (MHC) ligands within a species. How individual αβTCRs are constructed to specifically recognize a limited set of MHC ligands is unclear. Here we have identified a role for the differential pairing of particular V gene segments in creating TCRs that recognized MHC class II ligands exclusively, or cross-reacted with classical and nonclassical MHC class I ligands. Biophysical and structural experiments indicated that TCR specificity for MHC ligands is not driven by germline-encoded pairwise interactions.Rather, identical TCRβ chains can have altered peptide-MHC (pMHC) binding modes when paired with different TCRα chains. The ability of TCR chain pairing to modify how V region residues interact with pMHC helps to explain how the same V genes are used to create TCRs specific for unique MHC ligands.

CD4⁺CD25⁺Foxp3⁺ regulatory T cell formation requires more specific recognition of a self-peptide than thymocyte deletion.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are generated during thymocyte development and play a crucial role in preventing the immune system from attacking the body's cells and tissues. However, how the formation of these cells is directed by T-cell receptor (TCR) recognition of self-peptide:major histocompatibility complex (MHC) ligands remains poorly understood. We show that an agonist self-peptide with which a TCR is strongly reactive can induce a combination of thymocyte deletion and CD4(+)CD25(+)Foxp3(+) Treg cell formation in vivo. A weakly cross-reactive partial agonist self-peptide could similarly induce thymocyte deletion, but failed to induce Treg cell formation. These studies indicate that CD4(+)CD25(+)Foxp3(+) Treg cell formation can require highly stringent recognition of an agonist self-peptide by developing thymocytes. They also refine the "avidity" model of thymocyte selection by demonstrating that the quality of the signal mediated by agonist self-peptides, rather than the overall intensity of TCR signaling, can be a critical factor in directing autoreactive thymocytes to undergo CD4(+)CD25(+)Foxp3(+) Treg cell formation and/or deletion during their development.

A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-α chain with the MHC class II β chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3β loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3α loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

IEDB-3D: structural data within the immune epitope database

IEDB-3D is the 3D structural component of the Immune Epitope Database (IEDB) available via the ‘Browse by 3D Structure’ page at http://www.iedb.org. IEDB-3D catalogs B- and T-cell epitopes and Major Histocompatibility Complex (MHC) ligands for which 3D structures of complexes with antibodies, T-cell receptors or MHC molecules are available in the Protein Data Bank (PDB). Journal articles that are primary citations of PDB structures and that define immune epitopes are curated within IEDB as any other reference along with accompanying functional assays and immunologically relevant information. For each curated structure, IEDB-3D provides calculated data on intermolecular contacts and interface areas and includes an application, EpitopeViewer, to visualize the structures. IEDB-3D is fully embedded within IEDB, thus allowing structural data, both curated and calculated, and all accompanying information to be queried using multiple search interfaces. These include queries for epitopes recognized in different pathogens, eliciting different functional immune responses, and recognized by different components of the immune system. The query results can be downloaded in Microsoft Excel format, or the entire database, together with structural data both curated and calculated, can be downloaded in either XML or MySQL formats.

A discrete affinity-driven elevation of ZAP-70 kinase activity initiates negative selection

Context: Although ZAP-70 is required for T-cell development, it’s unclear how this kinase controls both positive and negative selection.Objective and methods: Using OT-I pre-selection thymocytes and a panel of peptide major histocompatibility complex (pMHC) ligands of defined affinity, the recruitment, phosphorylation and activity of ZAP-70 was determined at the interface with antigen-presenting cells (APCs).Results: pMHC ligands promoting negative selection induce a discrete elevation of ZAP-70 recruitment, phosphorylation and enzymatic activity in the thymocyte:APCs interface.Discussion: The quantity of ZAP-70 kinase activity per cell is a key parameter controlling the fate of a developing thymocyte since partial inhibition of ZAP-70 kinase activity converted negative into positive selection. Surprisingly, the amount of ZAP-70 enzymatic activity observed during negative selection is not controlled by differential phosphorylation of the ZAP-70 protein but rather by the total amount of T-cell receptor and co-associated ZAP-70 recruited to the thymocyte:APC interface.Conclusions: These data provide evidence that a burst of ZAP-70 activity initiates the signaling pathways for negative selection.

Interleukin‐10 Promotes NK Cell Killing of Autologous Macrophages by Stimulating Expression of NKG2D Ligands

Under inflammatory conditions, the pleiotropic cytokine interleukin-10 (IL-10) is released in many tissues. It mediates anti-inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL-10. The expression of most activatory NK receptors is increased after culture in the presence of IL-10. On the other hand, macrophages cultured in the presence of IL-10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1-like molecules (MIC) - A and - B, as well as UL-16 binding proteins (ULBP) - ULBP-1, ULBP-2 and ULBP-3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL-10-induced lysis of macrophages. Our data therefore reveal that IL-10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen-presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses.

Persistent viral infection in humans can drive high frequency low-affinity T-cell expansions.

CD8 T cells that recognize cytomegalovirus (CMV) -encoded peptides can be readily detected by staining with human leucocyte antigen (HLA) -peptide tetramers. These cells are invariably highly differentiated effector memory cells with high avidity T-cell receptors (TCR). In this report we demonstrate an HLA-A*0201 restricted CMV-specific CD8 T-cell response (designated YVL) that represents several percent of the CD8 T-cell subset, yet fails to bind tetrameric major histocompatibility complex (MHC) ligands. However, these tetramer-negative cells are both phenotypically and functionally similar to other CMV-specific CD8 T cells. YVL peptide-specific CD8 T-cell clones were generated and found to be of high avidity in both cytotoxicity and interferon-γ (IFN-γ) assays, and comparable with other CMV peptide-specific CD8 T-cell clones. However, under conditions of CD8 blockade, the response was almost nullified even at very high ligand concentrations. This was also the case in IFN-γ experiments using peripheral blood mononuclear cells stimulated with peptide ex vivo. In contrast, all other CMV specificities (tetramer-positive) displayed minimal or only partial CD8 dependence. This suggests that YVL-specific responses depict a low-affinity TCR-MHC-peptide interaction, that is compensated by substantial CD8 involvement for functional purposes, yet cannot engage multivalent soluble ligands for ex vivo analysis. It is interesting that such a phenomenon is apparent in the face of a persistent virus infection such as CMV, where the responding cells represent an immunodominant response in that individual and may present a highly differentiated effector phenotype.

Suboptimal engagement of the T‐cell receptor by a variety of peptide–MHC ligands triggers T‐cell anergy

T cells recognize antigen via the T-cell receptor (TCR) and produce a spectrum of responses that range from activation to anergy or cell death. The variety of outcomes may be dictated by the strength of the signals transmitted upon cognate recognition of the TCR. The physiological outcome of TCR engagement is determined by several factors, including the avidity of the ligand for TCR, the duration of engagement, and the presence and nature of accessory molecules present on antigen-presenting cells (APCs). In this review, we discuss a model of anergy induced by presentation of low densities of peptide-major histocompatibility complex (MHC) ligand in CD4(+) T cells and compare it to anergy induced by altered peptide ligands in an effort to identify a unifying mechanism. We suggest that altered peptide ligand (APL) and low densities of agonist ligands induce anergy by engaging less than optimal numbers of TCRs. The physiological impacts of anergy in memory CD4(+) T cells are discussed.

The Immune Epitope Database 2.0

The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course of 4 years, the data from 180 978 experiments were curated manually from the literature, which covers ∼99% of all publicly available information on peptide epitopes mapped in infectious agents (excluding HIV) and 93% of those mapped in allergens. In addition, data that would otherwise be unavailable to the public from 129 186 experiments were submitted directly by investigators. The curation of epitopes related to autoimmunity is expected to be completed by the end of 2010. The database can be queried by epitope structure, source organism, MHC restriction, assay type or host organism, among other criteria. The database structure, as well as its querying, browsing and reporting interfaces, was completely redesigned for the IEDB 2.0 release, which became publicly available in early 2009.

Systematic Characterisation of Cellular Localisation and Expression Profiles of Proteins Containing MHC Ligands

BackgroundPresentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable.Methodology/Principal FindingIn the present large-scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly abundant mRNA were found to be much more likely to be the source of MHC ligands. Of the 2.5% most abundant mRNAs as much as 41% of the proteins encoded by these mRNAs contained MHC class I ligands. For proteins containing MHC class II ligands, the corresponding percentage was 11%. Furthermore, we found that most proteins containing MHC class I ligands were localised to the intracellular parts of the cell including the cytoplasm and nucleus. MHC class II ligand donors were, on the other hand, mostly membrane proteins.Conclusions/SignificanceThe results contribute to the ongoing debate concerning the nature of MHC ligand-containing proteins and can be used to extend the existing methods for MHC ligand predictions by including the source protein's localisation and expression profile. Improving the current methods is important in the growing quest for epitopes that can be used for vaccine or diagnostic purposes, especially when it comes to large DNA viruses and cancer.

Immune Reconstitution: How It Should Work, What's Broken, and Why It Matters

Of the multiple cell lineages undergoing reconstitution in the setting of hematopoietic stem cell transplantation (HSCT), this overview will focus on reconstitution of the T cell arm of the immune system. In clinical HSCT, the autologous setting is free of constraints imposed by reactions to allogeneic antigens, but markers are lacking to distinguish reconstitution from transferred cells versus stem or progenitor cells left residual in the host. In allogeneic transplants, the converse is true – the stem cell origins of the reconstituted populations can be determined, but the biology of such reconstitution is confounded by concomitant allogeneic responses. Because of these constraints, the basic biology of T cell immune reconstitution was first delineated in mouse models in which the availability of congenic strains allowed stem cell origins of reconstituted cell populations to be investigated without the presence of confounding allogenic reactions. Two pathways of T cell regeneration were identified: the thymic-dependent maturation of new T cells from marrow progenitors, and thymic-independent expansion of mature peripheral T cells. 1 These initial studies also determined that no other pathways existed by which substantial peripheral populations of T cells were generated, and also established a means for investigating T cell reconstitution in humans by analyzing the expression of isoforms of the leukocyte common antigen CD45 as markers of naive and memory populations which correlated with the pathway of regeneration. They also demonstrated that homeostatic expansion of mature T cells occurred in lymphopenic hosts. Studies of such expansion have formed central concepts of homeostasis – that T cell levels are maintained by a balance between cytokine-consuming cells and homeostatic cytokines that support the maturation, proliferative expansion and survival of T cells.2 In translating these murine findings to humans, the recovery of CD4+ T cells in pediatric patients who were severely lymphopenic following chemotherapy treatment were initially tracked (Figure 1). There was a strong correlation between the recovery of total CD4+ T cell numbers and the recovery of naive, CD45RA+CD45RO- CD4+ T cells after as few as six months. 3 Furthermore, recovery of naive CD4+ cells was associated with a marked expansion of thymus volume, consistent with a dynamic regulation of the thymus.

HPLC and MS Analysis for the Identification and Characterisation of Peptides Presented in the Context of the Non-Classical Human Leukocytes Antigen (HLA) Class I Molecule HLA-E

In addition to a variety of other techniques used in T-cell epitope identification, mass spectrometery coupled to liquid chromatography have now become an important and sensitive tool in separation, detection, and sequence analysis of highly complex natural major histocompatibility complex (MHC) ligand mixtures. In this article, we present current strategies for the identification of MHC eluted peptides using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) with a particular recall to those presented in the context of the non classical human leukocytes antigen (HLA) class I molecule HLA-E. In addition we also discuss the advantages and disadvantages of the methods available in the literature to concentrate and fractionate the peptides prior to analysis by mass spectrometry. An application of our method for isolation and characterization of peptides presented in the context of HLA-E is finally reported.

LFA-1-dependent Ca2+ Entry following Suboptimal T Cell Receptor Triggering Proceeds without Mobilization of Intracellular Ca2+

A surge in cytosolic calcium ion concentration by entry of extracellular Ca2+ is a hallmark of T cell activation. According to store-operated Ca2+ entry mechanism, the Ca2+ entry is preceded by activation of phospholipase C-gamma1 (PLC-gamma1) and the consequent mobilization of intracellular Ca2+. Using membrane vesicles expressing the mouse class I major histocompatibility complex, i.e. Ld plus costimulatory ligands, i.e. B7-1 and intercellular adhesion molecule-1 along with 2C T cell receptor transgenic T cells, we investigated the roles of CD28 and LFA-1 (lymphocyte function-associated antigen-1) in the activation of PLC-gamma1 and Ca2+ signaling. Both CD28 and LFA-1 made significant and comparable contributions to the activation of PLC-gamma1 as gauged by the level of its phosphorylation at tyrosine 783. In contrast, their roles in Ca2+ signaling were quite distinct so that LFA-1/intercellular adhesion molecule-1 interaction exerted a determining role, whereas CD28/B7-1 interaction played only a minimal role. In particular, when the T cells were activated by suboptimal T cell receptor stimulation, LFA-1 played an indispensable role in the Ca2+ signaling. Further experiments using Ca2+-free medium demonstrated that the entry of extracellular Ca2+ was not always accompanied by mobilization of intracellular Ca2+. Thus, intracellular Ca2+ mobilization was hardly detected under the condition that LFA-1 played the indispensable role in the entry of extracellular Ca2+, while a distinct level of intracellular Ca2+ mobilization was readily detected under the condition that LFA-1 played only the supporting role. These results ensure the unique role of LFA-1 in T cell Ca2+ signaling and reveal that LFA-1-dependent Ca2+ entry proceeds via a mechanism separate from store-operated Ca2+ entry.

Evidence for balancing selection acting on KIR2DL4 genotypes in rhesus macaques of Indian origin

The interaction of killer-cell immunoglobulin-like receptors (KIR) and their respective major histocompatibility complex (MHC) ligands can alter the activation state of the natural killer (NK) cell. In both humans and rhesus macaques, particular types of non-classical MHC class I molecules are predominantly expressed on the trophoblast. In humans, human leukocyte antigen G has been demonstrated to act as a ligand for KIR2DL4, present on all NK cells, whereas Mamu-AG may execute a similar function in rhesus macaques. During primate evolution, orthologues of KIR2DL4 appear to have been highly conserved, suggesting strong purifying selection. A cohort of 112 related and unrelated rhesus macaques of mostly Indian origin were selected to study their KIR2DL4 genes for the occurrence of polymorphism. Comparison of the proximal region provided evidence for strong conservative selection acting on the exons encoding the Ig domains. As is found in humans, in the Indian rhesus macaque population, two different KIR2DL4 entities are encountered, which differ for their intra-cellular signalling motifs. One genotype contains a complex mutation in the distal region of exon 9, which negates a serine/threonine kinase site. Furthermore, both allelic entities are present in a distribution, which suggests that balancing selection is operating on these two distinct forms of KIR2DL4.