T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex.

T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

Basic and translational applications of engineered MHC class I proteins.

Major histocompatibility complex (MHC) class I molecules can be engineered as single chain trimers (SCTs) that sequentially incorporate all three subunits of the fully assembled proteins, namely peptide, ß2 microglobulin, and heavy chain. SCTs have been made with many different MHC-peptide complexes and are used as novel diagnostic and therapeutic reagents, as well as probes for diverse biological questions. Here, we review the recent and diverse applications of SCTs. These applications include new approaches to enumerate disease-related T cells, DNA vaccines, eliciting responses to pre-assembled MHC-peptide complexes, and unique probes of lymphocyte development and activation. Future applications of SCTs will be driven by their further engineering and the ever-expanding identification of disease-related peptides using chemical, genetic and computational approaches.

Structural engineering of pMHC reagents for T cell vaccines and diagnostics.

MHC class I peptide complexes (pMHC) are routinely used to enumerate T cell populations and are currently being evaluated as vaccines to tumors and specific pathogens. Herein, we describe the structures of three generations of single-chain pMHC progressively designed for the optimal presentation of covalently associated epitopes. Our ultimate design employs a versatile disulfide trap between an invariant MHC residue and a short C-terminal peptide extension. This general strategy is nondisruptive of native pMHC conformation and T cell receptor engagement. Indeed, cell-surface-expressed MHC complexes with disulfide-trapped epitopes are refractory to peptide exchange, suggesting they will make safe and effective vaccines. Furthermore, we find that disulfide-trap stabilized, recombinant pMHC reagents reliably detect polyclonal CD8 T cell populations as proficiently as conventional reagents and are thus well suited to monitor or modulate immune responses during pathogenesis.

Applications of major histocompatibility complex class I molecules expressed as single chains.

Generation of CD8 T-cell responses to pathogens and tumors requires optimal expression of class I major histocompatibility complex/peptide complexes, which, in turn, is dependent on host cellular processing events and subject to interference by pathogens. To create a stable structure that is more immunogenic and resistant to immune evasion pathways, we have engineered class I molecules as single-chain trimers (SCTs), with flexible linkers connecting peptide, beta2m, and heavy chain. Herein we extend our earlier studies with SCTs to the K(b) ligand derived from vesicular stomatitis virus (VSV) to characterize further SCTs as probes of immune function as well as their potential in immunotherapy. The VSVp-beta2m-K(b) SCTs were remarkably stable at the cell surface, and immunization with DNA encoding SCTs elicited complex-specific antibody. In addition, SCTs were detected by cytotoxic T-lymphocytes specific for the native molecule, and the covalently bound peptide was highly resistant to displacement by exogenous peptide. SCTs can also prime CD8 T-cells in vivo that recognize the native molecule. Furthermore, SCTs were resistant to downregulation by the immune evasion protein mK3 of gamma herpesvirus 68. Moreover, owing to their preassembled nature, SCTs should be resistant to other immune evasion proteins that restrict peptide supply. Thus, SCTs possess therapeutic potential both for prophylactic treatment and for the treatment of ongoing infection.

Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide-MHC.

Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide-MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4-MHCII interaction is generally weaker than CD8-MHCI.

A single peptide-MHC complex positively selects a diverse and specific CD8 T cell repertoire.

Pathogen recognition by T cells is dependent on their exquisite specificity for self-major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide-MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.

Human major histocompatibility complex (MHC) class I molecules with disulfide traps secure disease-related antigenic peptides and exclude competitor peptides.

The ongoing discovery of disease-associated epitopes detected by CD8 T cells greatly facilitates peptide-based vaccine approaches and the construction of multimeric soluble recombinant proteins (e.g. tetramers) for isolation and enumeration of antigen-specific CD8 T cells. Related to these outcomes of epitope discovery is the recent demonstration that MHC class I/peptide complexes can be expressed as single chain trimers (SCTs) with peptide, beta(2)m and heavy chain connected by linkers to form a single polypeptide chain. Studies using clinically relevant mouse models of human disease have shown that SCTs expressed by DNA vaccination are potent stimulators of cytotoxic T lymphocytes. Their vaccine efficacy has been attributed to the fact that SCTs contain a preprocessed and preloaded peptide that is stably displayed on the cell surface. Although SCTs of HLA class I/peptide complexes have been previously reported, they have not been characterized for biochemical stability or susceptibility to exogenous peptide binding. Here we demonstrate that human SCTs remain almost exclusively intact when expressed in cells and can incorporate a disulfide trap that dramatically excludes the binding of exogenous peptides. The mechanistic and practical applications of these findings for vaccine development and T cell isolation/enumeration are discussed.

Disulfide bond engineering to trap peptides in the MHC class I binding groove.

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells-particularly when the antigenic peptide has relatively weak affinity for the MHC.

Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells.

We report in this work the expression and characterization of class I molecules expressed as single-chain trimers consisting of an antigenic peptide-spacer-beta(2)-microglobulin-spacer H chain. Our results indicate that these single-chain constructs assemble efficiently, maintain their covalent structure, and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and they are potent simulators of peptide-specific CTL and Abs. Our combined findings suggest that single-chain trimers may have applications as DNA vaccines against virus infection or tumors.

Homology between an alloantigen and a self MHC allele calibrates the avidity of the alloreactive T cell repertoire independent of TCR affinity.

The self-restricted T cell repertoire exhibits a high frequency of alloreactivity. Because these alloreactive T cells are derived from the pool of cells selected on several different self MHC alleles, it is unknown how development of the alloantigenic repertoire is influenced by homology between a self MHC allele and an alloantigen. To address this, we used the 2C transgenic TCR that is selected by K(b), is alloreactive for L(d), and cross-reacts with L(q). L(q) is highly homologous to L(d) and binds several of the same peptide ligands, including p2Ca, the peptide recognized by 2C. We find that L(d)/p2Ca is a high avidity agonist ligand, whereas L(q)/p2Ca is a low avidity agonist ligand for 2C T cells. When mice transgenic for the 2C TCR are bred to L(q)-expressing mice, 2C(+) T cells develop; however, they express lower levels of either the 2C TCR or CD8 and require a higher L(d)/p2Ca ligand density to be activated than 2C(+) T cells selected by K(b). Furthermore, the 2C T cells selected in the presence of L(q) fail to detect L(q)/p2Ca complexes even at high ligand density. Thus, despite possessing the identical TCR, there is a functional avidity difference between 2C(+) T cells selected in the presence of L(q) vs K(b). These data provide evidence that homology between the selecting ligand and an alloantigen can influence the avidity of the T cell repertoire for the alloantigen, and suggest that thymic selection can fine tune T cell avidity independent of intrinsic TCR affinity.

Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide.

Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.