TCR recognition of peptide/MHC class II complexes and superantigens

Major histocompatibility complex (MHC) class II molecules display peptides to the T cell receptor (TCR). The ability of the TCR to discriminate foreign from self-peptides presented by MHC molecules is a requirement of an effective adaptive immune response. Dysregulation of this molecular recognition event often leads to a disease state. Recently, a number of structural studies have provided significant insight into several such dysregulated interactions between peptide/MHC complexes and TCR molecules. These include TCR recognition of self-peptides, which results in autoimmune reactions, and of mutant self-peptides, common in the immunosurveillance of tumors, as well as the engagement of TCRs by superantigens, a family of bacterial toxins responsible for toxic shock syndrome.     

小鼠I-A~kII类分子限制性MAGE-3T表位筛选

为了筛选小鼠MAGE-3抗原来源的MHCII类分子限制性多肽表位。用计算机模拟设计,从MAGE-3中选取得分最高的5个候选表位。根据诱导T淋巴细胞增殖能力、对肿瘤细胞的特异性识别及释放细胞因子能力评价多肽的免疫原性及免疫反应性。结果DC负载MAGE-322-36能够强效诱导T淋巴细胞增殖,DC负载MAGE-322-36诱导的T细胞以MHCII类分子限制性方式、特异性识别表达MAGE-3抗原的肿瘤细胞。DC负载MAGE-322-36诱导的T细胞群主要为CD4+Th1细胞,同时天然免疫系统中的NK、NKT、γ/δT细胞也得到活化。从小鼠MAGE-3抗原中筛选出MHCII类分子限制性多肽表位MAGE-322-36。     

An integrative model of regulation centered on recognition of TCR peptide/MHC complexes

Summary: We have described a T-cell receptor (TCR)-centered model of immune regulation, in which MHC/TCR peptide complexes provide for the activation of regulatory T cells and likewise act as their target structures. In this model, the disease-causing effectors are TCR Vβ8.2⁺ and each of the required CD4 and CD8 regulatory T-cell populations are specific for different conserved regions of the Vβ8.2 chain, in the appropriate MHC context. We have characterized the dominance, the dynamics as well as the TCR usage of both effector and regulatory T cells. We have begun to characterize the essential elements of the regulatory program, including the mechanism of interaction among effector and regulatory T-cell populations. Principles learned in this model have important implications for immune regulation in general.     

MHC class II peptide flanking residues of exogenous antigens influence recognition by autoreactive T cells

Molecular mimicry between exogenous microbial antigens and self-epitopes has been proposed as a triggering mechanism for autoimmune diseases for many years. We reported that a peptide from a protein specific to Chlamydia pneumoniae (Cpn0483) which shares a motif with the dominant encephalitogenic epitope of the self-antigen, rat myelin basic protein (rat68-86), elicits experimental autoimmune encephalomyelitis (EAE) in Lewis rats. We recently observed that rat68-86 utilizes aspartic acid (D) and arginine (R) in the common motif as primary and secondary TCR contacts, respectively. In contrast, the encephalitogenic activity of Cpn0483 is dependent on R and the C-terminal asparagine (N), which flanks the MHC class II-P9 anchor residue. Thus, rat68-86 and Cpn0483 share a common motif, are encephalitogenic and are both restricted by MHC class II RT1.B(l). T cells from rats immunized with the encephalitogenic Cpn0483 peptide proliferated to the priming peptide as well as to the non-encephalitogenic CpnN>A analog. However, CpnN>A-primed T cells did not respond to the native Cpn0483 peptide. We conclude that the MHC-flanking C-terminal asparagine residue markedly influences T cell recognition by the chlamydial peptide.     

An endogenously processed self peptide and the corresponding exogenous peptide bound to the same MHC class II molecule could be distinct ligands for TCR with different kinetic stability

Immunization with self peptides often elicits activation of CD4⁺ T cells in vivo. Although such peptides have been suggested to be derived from minor self determinants or self antigens sequestered from the immune system, we found that immunization with Eα peptide (Eα52 – 68), a major self determinant bound to I-A^b molecules, elicits an immune response in Eα-transgenic C57BL/6 (Eα-B6) mice where Eα52 – 68 is endogenously processed and presented by I-A^b molecules in the thymus and periphery. To better understand this response, a panel of T cell hybridomas raised against exogenous Eα52 – 68 were analyzed for their reactivity to spleen cells from Eα-B6 mice. Some hybridomas were stimulated with Eα-B6 spleen cells in the absence of exogenous Eα52 – 68, whereas others were not stimulated with them. The Eα52 – 68/I-A^b complex recognized by the TCR that is expressed on the hybridoma with reactivity to Eα-B6 spleen cells was found to be quite stable, whereas the complex recognized by the TCR on the hybridoma specific for the exogenous Eα52 – 68 lost the stimulation activity by incubation the complex at 37 °C for 10 min. Stimulation experiments using extensively substituted Eα analogue peptides suggested that amino acid residues at positions 57, 58, 60 and 62 of Eα52 – 68 are involved in the interaction with TCR recognizing the Eα52 – 68/I-A^b complex expressed on Eα-B6 spleen cells. While amino acid substitutions at positions 60 and 62 also affected the recognition of TCR specific for exogenous Eα52 – 68, all or many amino acid substitutions were allowed at position 58 or 57, respectively, without impairing the TCR recognition. Taken together, these results suggest that endogenously processed self peptide and the corresponding exogenous peptide bound to the same MHC class II molecule could be distinct TCR ligands with different kinetic stability and probably with different configuration.     

Molecular recognition of antigen involves lattice formation between CD4, MHC class II and TCR molecules

Recent evidence indicates that CD4 stably binds to major histocompatibility complex (MHC) class II only after assuming an oligomeric state: the membrane-distal CD4 D1–D2 module interacts directly with MHC class II, whereas the membrane-proximal CD4 D3–D4 module mediates oligomerization. This results in the formation of aggregates critical for T-cell activation. The T-cell receptor (TCR) regulates specific crosslinking and is itself dependent on lattice formation to trigger physiological T-cell responses. Here, Toshiko Sakihama, Alex Smolyar and Ellis Reinherz discuss the molecular nature of CD4-MHC class II clustering and how, despite each of the component interactions being of low affinity, the molecular matrix renders T-cell recognition extremely specific and sensitive.     

Mapping and restriction of a dominant viral CD4+ T cell core epitope by both MHC class I and MHC class II

Virus-specific CD4(+) T cells contribute to effective virus control through a multiplicity of mechanisms including direct effector functions as well as "help" for B cell and CD8(+) T cell responses. Here, we have used the lymphocytic choriomeningitis virus (LCMV) system to assess the minimal constraints of a dominant antiviral CD4(+) T cell response. We report that the core epitope derived from the LCMV glycoprotein (GP) is 11 amino acids in length and provides optimal recognition by epitope-specific CD4(+) T cells. Surprisingly, this epitope is also recognized by LCMV-specific CD8(+) T cells and thus constitutes a unique viral determinant with dual MHC class I- and II-restriction.     

Regulatory T cells and co-evolution of allele-specific MHC recognition by the TCR

What is the evolutionary mechanism for the TCR-MHC-conserved interaction? We extend Dembic's model (Dembic Z. In, Scand J Immunol e12806, 2019) of thymus positive selection for high-avidity anti–self-MHC Tregs among double (CD4 + CD8+)-positive (DP) developing thymocytes. This model is based on competition for self-MHC (+ Pep) complexes presented on cortical epithelium. Such T cells exit as CD4 + CD25+FoxP3 + thymic-derived Tregs (tTregs). The other positively selected DP T cells are then negatively selected on medulla epithelium removing high-avidity anti–self-MHC + Pep as T cells commit to CD4 + or CD8 + lineages. The process is likened to the competitive selection and affinity maturation in Germinal Centre for the somatic hypermutation (SHM) of rearranged immunoglobulin (Ig) variable region (V[D]Js) of centrocytes bearing antigen-specific B cell receptors (BCR). We now argue that the same direct SHM processes for TCRs occur in post-antigenic Germinal Centres, but now occurring in peripheral pTregs. This model provides a potential solution to a long-standing problem previously recognized by Cohn and others (Cohn M, Anderson CC, Dembic Z. In, Scand J Immunol e12790, 2019) of how co-evolution occurs of species-specific MHC alleles with the repertoire of their germline TCR V counterparts. We suggest this is not by ‘blind’, slow, and random Darwinian natural selection events, but a rapid structured somatic selection vertical transmission process. The pTregs bearing somatic TCR V mutant genes then, on arrival in reproductive tissues, can donate their TCR V sequences via soma-to-germline feedback as discussed in this journal earlier. (Steele EJ, Lindley RA. In, Scand J Immunol e12670, 2018) The high-avidity tTregs also participate in the same process to maintain a biased, high-avidity anti–self-MHC germline V repertoire.     

Mutations changing the kinetics of class II MHC peptide exchange

IE/DR MHC class II molecules have an extensive H-bonding network under the bound peptide. In IE(k), two alpha chain acidic amino acids in the core of this network were mutated to amides. At low pH, the mutant molecule exchanged peptide much more rapidly than the wild-type. The crystal structure of the mutant IE(k) revealed the loss of a single buried water molecule and a reorganization of the predicted H-bonding network. We suggest that these mutations enhance the transition of MHC class II to an open conformation at low pH allowing the bound peptide to escape. In wild-type IE(k), the need to protonate these amino acids also may be a bottleneck in the return to a closed conformation after peptide binding.     

In vivo biotinylation of the major histocompatibility complex (MHC) class II/peptide complex by coexpression of BirA enzyme for the generation of MHC class II/tetramers

Success in generation of major histocompatibility complex (MHC) tetramer relies on application of a key technique, biotinylation of MHC molecule specifically on a single lysine residue using the BirA enzyme. However, in vitro biotinylation of MHC-BSP (BirA enzyme substrate peptide) fusion protein using BirA enzyme is laborious and is prone to losses of target proteins to unacceptable levels. To circumvent this problem, an in vivo biotinylation strategy was developed where the BirA enzyme was coexpressed with target protein, HLA-DR2BSP/MBP, in an insect cell expression system. Bacterial BirA enzyme expressed in Drosophila melanogaster 2 (D. Mel-2) cell lines was biologically functional and was able to biotinylate secretary target protein (on specific lysine residue present on the BSP tag). Biotinylation efficiency was maximized by providing exogenous d-biotin in the culture medium and optimization of the expression vector ratios for cotransfection. By limiting dilution cloning, a clone was identified where the expressed DR2BSP/MBP protein was completely biotinylated. DR2BSP/MBP protein expressed and purified from such a clone was ready to be tetramerized with streptavidin to be used for staining antigen-specific T cells.     

MHC class II restricted recognition of FMDV peptides by bovine T cells.

A putative synthetic vaccine for foot-and-mouth disease (FMDV15) has proved less successful in a host species, cattle, than predicted by results in a small-animal model. Possible reasons for this include non-recognition by T cells influenced by major histocompatibility complex (MHC)-linked immune response gene control. It is now possible to type for human leucocyte antigen (HLA) DR-like bovine MHC (BoLA) class II polymorphisms with a one-dimensional isoelectric focusing (IEF) technique. Using this method 14 unrelated cattle were selected with eight different BoLA class II IEF types. After immunization with FMDV15, 13 cattle generated a T-cell response to FMDV15. However, the fine specificity and magnitude of the response was related to BoLA class II type. The non-response by one animal and low response by two other animals were associated with two of the BoLA class II types. Response to the region 149-158 was immunodominant and animals which did not respond to this region had low responses to the whole peptide. Using FMDV-specific T-cell lines five BoLA class II types associated with responder animals were able to present FMDV15 in an MHC class II-restricted fashion, indicating that this peptide is capable of binding to different MHC class II molecules and may account for the broad response observed. The restriction patterns of the lines indicated that the IEF method does not distinguish all functional polymorphisms. At least two of the IEF-defined types could each be split into two distinct specificities and revealed that the three sets of animals with identical IEF types in fact expressed distinct restriction elements.     

Differential expression of CLIP: MHC class II and conventional endogenous peptide: MHC class II complexes by thymic epithelial cells and peripheral antigen-presenting cells

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide : MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of Eα52–68: I-A^b and class II-associated invariant chain peptide (CLIP): I-A^b complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of Eα52–68:I-A^b complexes in thymic epithelial cells is approximately 5–10 % of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A^b complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.     

小鼠I-A^k Ⅱ类分子限制性MAGE-3T表位筛选

为了筛选小鼠MAGE-3抗原来源的MHC Ⅱ类分子限制性多肽表位.用计算机模拟设计,从MAGE-3中选取得分最高的5个候选表位.根据诱导T淋巴细胞增殖能力、对肿瘤细胞的特异性识别及释放细胞因子能力评价多肽的免疫原性及免疫反应性.结果DC负载MAGE-3 22-36能够强效诱导T淋巴细胞增殖,DC负载MAGE-3 22-36诱导的T细胞以MHC Ⅱ类分子限制性方式、特异性识别表达MAGE-3抗原的肿瘤细胞.DC负载MAGE-3 22-36诱导的T细胞群主要为CD4+Th1细胞,同时天然免疫系统中的NK、NKT、γ/δ T细胞也得到活化.从小鼠MAGE-3抗原中筛选出MHC Ⅱ类分子限制性多肽表位MAGE-322-36.     

Probing the accessibility of peptide in an MHC class II protein--foreign peptide complex

The peptide antigen OVA323-339 forms a highly stable complex with the I-Ad molecule which when reconstituted into lipid bilayers can stimulate the release of interleukin 2 (IL-2) by specific T cell hybrids. To probe the accessibility of various regions of the peptide in the I-Ad binding site, six analogues of OVA323-339, each with a unique site for fluorescein labeling, were synthesized. OVA peptides modified with fluorescein at the N terminus position 323 and at the epsilon-amino group of lysines at positions 328, 329, 330, 331 and 336 all bind to the I-Ad molecule. These substitutions include four amino acids previously identified as representing a structural motif common to MHC binding peptides. The fluorescein hapten on each analogue was fully accessible to quenching by anti-fluorescein antibody after binding of the peptide to the MHC class II protein, indicating that these regions of the peptide are exposed in the MHC binding site. These data suggest that the MHC class II peptide binding site is remarkably permissive with respect to tolerating bulky substitutions in the peptide antigen. The data further suggest that either the central region (328-331) of the peptide OVA323-339 is oriented in MHC class II binding site such that all of its side chains are exposed, or that the binding of the peptide is conformationally flexible allowing reorientation of the bulky substituent to the outside of the binding site in each case studied.     

Three-dimensional structures of MHC class I-peptide complexes: implications for peptide recognition

Over the last decade, the number of crystal structures of major histocompatibility complex (MHC) class I-peptide complexes has increased rapidly. These studies have provided unique and fascinating insights into the structural basis of MHC-peptide interactions and the specificity of peptide recognition by MHC class I molecules.     

Insights into antigen processing gained by direct analysis of the naturally processed class I MHC associated peptide repertoire

MHC class I molecules are responsible for the presentation of antigenic peptides to CD8+ T lymphocytes. Based on their relatively promiscuous binding of peptides, these molecules display information derived from a large fraction of proteins that are made inside the cell. This review describes our characterization of the peptides comprising this repertoire, with particular attention given to their complexity and quantities, their post-translational modification, and the pathways leading to their expression.     

Supermotif peptide binding and degeneracy of MHC: peptide recognition in an EBV peptide-specific CTL response with highly restricted TCR usage

We have investigated the presentation and CTL recognition of an HLA A*1101-restricted CTL peptide epitope AVFDRKSDAK (AVF)(3), derived from the EBV nuclear antigen (EBNA) 4, in the context of alleles belonging to the A3-supertype, A*0101, 0301, 1101, 3101, 3301, and 6801. The peptide binds to a A*6801 molecule as efficiently as to A*1101. The A*6801:AVF complex is recognized by some A*1101-restricted AVF- specific CTL clones. However, A*6801-positive (A*6801+) EBV-transformed lymphoblastoid cell lines (LCLs) are not killed by the same effectors. Furthermore, two A*6801+ donors did not mount an AVF-specific CTL response in vitro and lacked detectable AVF-specific effectors. Thus, this epitope is either subdominant, or non-immunogenic in the context of A*6801. These characteristics correlate with low stability of this MHC:peptide complex in living cells. We also demonstrate that a highly conserved AVF-specific TCR that dominates the AVF-specific CTL response in the majority of A*1101+ individuals recognizes the A*6801 molecule as a crossreactive alloantigen. Therefore, deletion of AVF-specific T cells may contribute to the non-immunogenicity or subdominance of the peptide in A*6801+ individuals.     

Intracellular transport and peptide loading of MHC class II molecules: regulation by chaperones and motors

Summary: MHC dass II molecules are important in the onset and modulation of cellular immune responses. Studies on the intracellular transport of these molecules has provided insight into the way pathogens are processed and presented at the cell surface and may result in future immunological intervention strategies. Recent reviews have extensively described structural properties and early events in the biosynthesis of MHC class II (1-3). In this review, the focus will be on the function of the dedicated chaperone proteins Ii, DM and DO in the class II assembly, transport and peptide loading as well on proteins involved in transport steps late in the intracellular transport of MHC class II.