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Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy., Highlights • We report the crystal structure of human PD-1 and the mapping of the PD-1 ligand binding site. • A high-affinity variant was identified and used to engineer a soluble PD-1 Ig fusion protein (HA PD-1 Ig) for immunotherapy. • HA PD-1 Ig enhanced T cell activation and strongly synergized with RT to control tumor growth in a lung carcinoma model. PD-1 is an inhibitory immune receptor that dampens T cell responses. PD-1 blockade is a successful strategy for cancer immunotherapy. We report the crystal structure of human PD-1 and mapping of its ligand binding site. A high-affinity mutant (HA PD-1) was identified and used to engineer a blocking Ig fusion protein, expected to disrupt the endogenous PD-L/PD-1 pathway. HA PD-1 Ig increased lymphocyte proliferation and cytokine production. Moreover, in combination with radiation therapy it enhanced control of tumor growth in a Lewis lung carcinoma model. This study offers an alternative strategy to manipulate the PD-1 pathway for tumor immunotherapy.

LIGHT initiates intracellular signaling via engagement of the two TNF receptors, HVEM and LTβR. In humans, LIGHT is neutralized by DcR3, a unique soluble member of the TNFR superfamily, which tightly binds LIGHT and inhibits its interactions with HVEM and LTβR. DcR3 also neutralizes two other TNF ligands, FasL and TL1A. Due to its ability to neutralize three distinct different ligands, DcR3 contributes to a wide range of biological and pathological processes, including cancer and autoimmune diseases. However, the mechanisms which support the broad specificity of DcR3 remain to be fully defined. We report the structures of LIGHT and the LIGHT:DcR3 complex, which reveal the structural basis for the DcR3-mediated neutralization of LIGHT and afford new insights into DcR3 function and binding promiscuity. Based on these structures, we designed novel LIGHT mutants with altered affinities for DcR3 and HVEM, which may represent new mechanistically informative probe reagents.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing pancreatic beta cells. In both humans and the non-obese diabetic (NOD) mouse model of T1D, class II MHC alleles are the primary determinant of disease susceptibility. However, class I MHC genes also influence risk. These findings are consistent with the requirement for both CD4(+) and CD8(+) T cells in the pathogenesis of T1D. Although a large body of work has permitted the identification of multiple mechanisms to explain the diabetes-protective effect of particular class II MHC alleles, studies examining the protective influence of class I alleles are lacking. Here, we explored this question by performing biochemical and structural analyses of the murine class I MHC molecule H-2K(wm7), which exerts a diabetes-protective effect in NOD mice. We have found that H-2K(wm7) molecules are predominantly occupied by the single self-peptide VNDIFERI, derived from the ubiquitous protein histone H2B. This unexpected finding suggests that the inability of H-2K(wm7) to support T1D development could be due, at least in part, to the failure of peptides from critical beta-cell antigens to adequately compete for binding and be presented to T cells. Predominant presentation of a single peptide would also be expected to influence T-cell selection, potentially leading to a reduced ability to select a diabetogenic CD8(+) T-cell repertoire. The report that one of the predominant peptides bound by T1D-protective HLA-A*31 is histone derived suggests the potential translation of our findings to human diabetes-protective class I MHC molecules.

Costimulatory receptors and ligands trigger the signaling pathways that are responsible for modulating the strength, course, and duration of an immune response. High-resolution structures have provided invaluable mechanistic insights by defining the chemical and physical features underlying costimulatory receptor:ligand specificity, affinity, oligomeric state, and valency. Furthermore, these structures revealed general architectural features that are important for the integration of these interactions and their associated signaling pathways into overall cellular physiology. Recent technological advances in structural biology promise unprecedented opportunities for furthering our understanding of the structural features and mechanisms that govern costimulation. In this review, we highlight unique insights that have been revealed by structures of costimulatory molecules from the immunoglobulin and tumor necrosis factor superfamilies and describe a vision for future structural and mechanistic analysis of costimulation. This vision includes simple strategies for the selection of candidate molecules for structure determination and highlights the critical role of structure in the design of mutant costimulatory molecules for the generation of in vivo structure-function correlations in a mammalian model system. This integrated 'atoms-to-animals' paradigm provides a comprehensive approach for defining atomic and molecular mechanisms.

Programmed death-1 (PD-1) is a member of the CD28/B7 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. The high-resolution crystal structure of the complex formed by the complete ectodomains of murine PD-1 and PD-L2 revealed a 1:1 receptor:ligand stoichiometry and displayed a binding interface and overall molecular organization distinct from that observed in the CTLA-4/B7 inhibitory complexes. Furthermore, our structure also provides insights into the association between PD-1 and PD-L1 and highlights differences in the interfaces formed by the two PD-1 ligands (PD-Ls) Mutagenesis studies confirmed the details of the proposed PD-1/PD-L binding interfaces and allowed for the design of a mutant PD-1 receptor with enhanced affinity. These studies define spatial and organizational constraints that control the localization and signaling of PD-1/PD-L complexes within the immunological synapse and provide a basis for manipulating the PD-1 pathways for immunotherapy.

The PD-1 costimulatory receptor inhibits T cell receptor signaling upon interacting with its ligands PD-L1 and PD-L2. The PD-1/PD-L pathway is critical in maintaining self-tolerance. In this study, we examined the role of PD-1 in a mouse model of acute infection withHistoplasma capsulatum, a major human pathogenic fungus. In a lethal model of histoplasmosis, all PD-1-deficient mice survived infection, whereas the wild-type mice died with disseminated disease. PD-L expression on macrophages and splenocytes was up-regulated during infection, and macrophages from infected mice inhibitedin vitroT cell activation. Of interest, antibody blocking of PD-1 significantly increased survival of lethally infected wild-type mice. Thus, our studies extend the role of the PD-1/PD-L pathway in regulating antimicrobial immunity to fungal pathogens. The results show that the PD-1/PD-L pathway has a key role in the regulation of antifungal immunity, and suggest that manipulation of this pathway represents a strategy of immunotherapy for histoplasmosis.

Glucocorticoid-induced TNF receptor ligand (GITRL), a recently identified member of the TNF family, binds to its receptor GITR on both effector and regulatory T cells and generates positive costimulatory signals implicated in a wide range of T cell functions. Structural analysis reveals that the human GITRL (hGITRL) ectodomain self-assembles into an atypical expanded homotrimer with sparse monomer–monomer interfaces. Consistent with the small intersubunit interfaces, hGITRL exhibits a relatively weak tendency to trimerize in solution and displays a monomer–trimer equilibrium not reported for other TNF family members. This unique assembly behavior has direct implications for hGITRL–GITR signaling, because enforced trimerization of soluble hGITRL ectodomain results in an ≈100-fold increase in its receptor binding affinity and also in enhanced costimulatory activity. The apparent reduction in affinity that is the consequence of this dynamic equilibrium may represent a mechanism to realize the biologically optimal level of signaling through the hGITRL–GITR pathway, as opposed to the maximal achievable level.

Oligomeric state makes important contributions to the signaling mechanisms of costimulatory molecules. In this study we address the biological relevance of the disulfide-linked dimeric structure of CD28. Fluorescence Resonance Energy Transfer (FRET) demonstrates that removal of the interdomain disulfide bond (C123) does not interfere with the formation of CD28 oligomers on the cell surface. Although the C123S mutant shows 40% lower binding affinity to the ligand B7-1, it is able to costimulate anti-CD3-induced IL-2 production but at a lower level (150%) compared to the wild-type (270%). Interestingly, binding to B7-2 was not affected. Thus, the covalently linked dimeric structure of CD28 represents an important mechanistic determinant for the optimal costimulatory activity in the immunological synapse.

Inducible costimulator (ICOS) ligand (ICOSL), a B7-related transmembrane glycoprotein with extracellular IgV and IgC domains, binds to ICOS on activated T cells and delivers a positive costimulatory signal for optimal T cell function. Toward determining the structural features of ICOSL crucial for its costimulatory function, the present study shows that ICOSL displays a marked oligomerization potential, resembling more like B7-1 than B7-2. Use of ICOSL constructs lacking either the IgC or IgV domain demonstrates that receptor binding is mediated solely by the IgV domain but requires the IgC domain for maintaining the structural integrity of the protein. To map further the receptor recognition surface on ICOSL, a homology-based protein structure model of the ICOS:ICOSL complex was constructed. Based on predictions from the model, a series of mutations were generated targeting the potential receptor binding surface on ICOSL, and the mutants were tested for their biological function in terms of ICOS binding and T cell costimulation ability. The results provide experimental validation of the model and show that the receptor binding site on ICOSL is constituted chiefly by aromatic/hydrophobic residues. Critical ICOSL residues essential for ICOS binding map to the GFCC′C″ β-sheet face of the IgV domain and approximately overlap with the B7-1/B7-2 motif(s) that recognize CD28/CTLA-4. Altogether, similar structural features of ICOSL and B7 isoforms suggest a close evolutionary relationship between these costimulatory ligands, yet differences at the same time explain their unique specificity for the cognate binding partners, ICOS and CD28/CTLA-4, respectively.

B7-1 and B7-2 are homologous costimulatory ligands expressed on the surface of antigen presenting cells (APCs). Binding of these molecules to the T cell costimulatory receptors, CD28 and CTLA-4, is essential for the activation and regulation of T cell immunity. Despite strong structural similarities, B7-1 and B7-2 exhibit different biochemical features, and their binding to the costimulatory receptors results in distinct T cell functional outcomes. Using photobleaching based fluorescence resonance energy transfer (FRET), our previous studies have demonstrated that B7-1 and B7-2 have different cell surface oligomeric states. While B7-1 is present as a dimer, B7-2 exists as a monomer on the cell surface suggesting that the unique cell surface oligomeric states of the costimulatory ligands may play a key role in the regulation of T cell responses. Moreover, signaling via B7-1 and B7-2 in dendritic cells has been reported to be dependent on their simultaneous expression, raising the possibility that their direct interaction or their involvement in synergistic signaling pathways may play a role in the function of antigen presenting cells. We discuss physiological relevance of distinct oligomeric states of B7-1 and B7-2 and address whether these molecules can associate with one another on the cell surface to form hetero-oligomers. Our findings suggest that B7-1 and B7-2 do not form hetero-oligomers, underscoring the biological relevance of dimeric and monomeric state of B7-1 and B7-2, respectively.

Beta-2 microglobulin (beta2m) is a member of the immunoglobulin-like domain superfamily that is an essential structural subunit of the MHC class I (MHC-I) molecule. beta2m was previously identified as a susceptibility factor for the development of type 1 diabetes (T1D) in NOD mice, whereby transgenic expression of the beta2ma variant, but not the beta2mb variant, restored diabetes susceptibility to normally resistant NOD.beta2mnull mice. Here we report the crystal structures and thermodynamic stabilities of the NOD MHC-I molecule H2-Db containing these two variants. Our results reveal subtle differences in the structures of the beta2m variants, namely in minor loop shifts and in variations in the hydrogen bonding networks at the interfaces between the components of the ternary complex. We also demonstrate that the thermodynamic stabilities of the beta2m variants in isolation differ. However, the conformation of the peptide in the MHC cleft is unchanged in beta2m allelic Db complexes, as are the TCR recognition surfaces. Thus, despite modest structural differences between allelic complexes, the evidence indicates that Db peptide presentation of the representative peptide is unchanged in the context of either beta2m allelic variant. These data suggest that other mechanisms, such as differential association of MHC-I in multiprotein complexes, are likely responsible for the effect of beta2m on T1D development.

CD8 + T cells recognize peptides of eight to nine amino acid residues long in the context of MHC class I molecules on the surface of antigen-presenting cells (APCs). This recognition event is highly sensitive, as evidenced by the fact that T cells can be activated by cognate peptide/MHC complex (pMHC) at extremely low densities (1-50 molecules). High sensitivity is particularly valuable for detection of antigens at low density, such as those derived from tumor cells and intracellular pathogens, which can down-modulate cognate pMHCs from the surface of APCs to evade recognition by the adaptive immune system. T cell activation is only triggered in response to interactions between the T cell receptor (TCR) and the pMHC ligand that reach a specific half-life threshold. However, interactions with excessively long half-lives result in impaired T cell activation. Thus, efficient T cell activation by pMHC on the surface of APCs requires an optimal dwell time of TCR-pMHC interaction. Here, we show that, although this is a requirement at low cognate pMHC density on the APC surface, at high epitope density there is no impairment of T cell activation by extended TCR-pMHC dwell times. This observation was predicted by mathematical simulations for T cell activation by pMHC at different densities and supported by experiments performed on APCs selected for varied expression of cognate pMHC. According to these results, effective T cell activation depends on a complex interplay between inherent TCR-pMHC binding kinetics and the epitope density on the APC.

To assess the roles of serial engagement and kinetic proofreading in T cell receptor (TCR) internalization, we have developed a mathematical model of this process. Our determination of TCR down-regulation for an array of TCR mutants, interpreted in the context of the model, has provided new information about peptide-induced TCR internalization. The amount of TCR down-regulation increases to a maximum value and then declines as a function of the half-life of the bond between the TCR and peptide-major histocompatibility complex (pMHC). The model shows that this behavior, which reflects competition between serial engagement and kinetic proofreading, arises only if it is postulated that activated TCRs remain marked for internalization after dissociation from pMHC. The model also predicts that because of kinetic proofreading, the range of TCR-pMHC-binding half-lives required for T cell activation depends on the concentrations and localization of intracellular signaling molecules. We show here that kinetic proofreading provides an explanation for the different requirements for activation observed in naïve and memory T cells.

Recent studies have highlighted the structural requirements for T cell costimulation and have revealed unusual modes of dimerization for the cytolytic T lymphocyte-associated antigen 4 (CTLA-4) costimulatory receptor and its B7 ligands. These distinctive quaternary structures potentially endow both receptor and ligand with bivalent binding properties, which suggests a number of mechanistic features relevant to signaling. These include the potential to form a highly ordered, alternating network of CTLA-4 and B7 homodimers that may represent the organization of these molecules and their associated signaling partners within the immunological synapse. Primary sequence and structural considerations suggest that some aspects of the organizational and mechanistic features associated with the CTLA-4-B7 complexes may extend to other members of the costimulatory receptor-ligand family. An examination of the signaling mechanisms within the costimulatory receptor-ligand family provides an excellent framework to consider the general principles that are relevant to cell surface receptor-mediated signaling events.

The mouse H13 minor histocompatibility (H) Ag, originally detected as a barrier to allograft transplants, is remarkable in that rejection is a consequence of an extremely subtle interchange, P4Val/Ile, in a nonamer H2-Db-bound peptide. Moreover, H13 peptides lack the canonical P5Asn central anchor residue normally considered important for forming a peptide/MHC complex. To understand how these noncanonical peptide pMHC complexes form physiologically active TCR ligands, crystal structures of allelic H13 pDb complexes and a P5Asn anchored pDb analog were solved to high resolution. The structures show that the basis of TCRs to distinguish self from nonself H13 peptides is their ability to distinguish a single solvent-exposed methyl group. In addition, the structures demonstrate that there is no need for H13 peptides to derive any stabilization from interactions within the central C pocket to generate fully functional pMHC complexes. These results provide a structural explanation for a classical non-MHC-encoded H Ag, and they call into question the requirement for contact between anchor residues and the major MHC binding pockets in vaccine design.

The interaction between TCRs and peptides presented by MHC molecules determines the specificity of the T cell-mediated immune response. To elucidate the biologically important structural features of this interaction, we generated TCR β-chain transgenic mice using a TCR derived from a T cell clone specific for the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2Kb. We immunized these mice with VSV8 or analogs substituted at TCR contact residues (positions 1, 4, and 6) and analyzed the CDR3α sequences of the elicited T cells. In VSV8-specific CTLs, we observed a highly conserved residue at position 93 of CDR3α and preferred Jα usage, indicating that multiple residues of CDR3α are critical for recognition of the peptide. Certain substitutions at peptide position 4 induced changes at position 93 and in Jα usage, suggesting a potential interaction between CDR3α and position 4. Cross-reactivity data revealed the foremost importance of the Jα region in determining Ag specificity. Surprisingly, substitution at position 6 of VSV8 to a negatively charged residue induced a change at position 93 of CDR3α to a positively charged residue, suggesting that CDR3α may interact with position 6 in certain circumstances. Analogous interactions between the TCR α-chain and residues in the C-terminal half of the peptide have not yet been revealed by the limited number of TCR/peptide-MHC crystal structures reported to date. The transgenic mouse approach allows hundreds of TCR/peptide-MHC interactions to be examined comparatively easily, thus permitting a wide-ranging analysis of the possibilities for Ag recognition in vivo.

T cell responses against hapten-modified peptides play an important role in the pathogenesis of certain diseases, including contact dermatitis and allergy. However, the structural features of TCRs recognizing bulky, potentially mobile hapten groups remain poorly defined. To analyze the structural basis of TCR recognition of defined hapten-modified peptides, the immunodominant octapeptide derived from vesicular stomatitis virus nucleoprotein (VSV8) was modified with a trinitrophenyl (TNP) group at the primary TCR contact residues (position 4 or 6) and used for immunization of mice carrying either the TCR α- or β-chain of a VSV8 (unmodified)/H-2Kb-specific CTL clone as a transgene. Such mice allow independent analysis of one TCR chain by maintaining the other fixed. The TCR V gene usage of the responding T cell population was specifically altered depending upon the presence of the TNP group and its position on the peptide. The CDR3 sequences of the TNP-modified peptide-specific TCRs showed a preferential J region usage in both the CDR3α and β loops, indicating that the J regions of both CDR3s are critical for recognition of TNP-modified peptides. In contrast to our previous observations showing the prime importance of CDR3β residues encoded by D-segment or N-addition nucleotides for recognition of position 6 of unmodified VSV8, our studies of TNP-modified peptides demonstrate the importance of the Jβ region, while the Jα region was crucial for recognizing both TNP-modified and unmodified peptides. These data suggest that different structural strategies are utilized by the CDR3α and β loops to allow interaction with a haptenated peptide.

A broad repertoire of pancreatic β-cell autoreactive T-cells normally contributes to the development of type 1 diabetes in NOD mice. However, it has been unknown if a large reduction in the precursor pool from which autoreactive T-cells are drawn would inhibit the development of type 1 diabetes. To address this issue, we reduced the precursor frequency of autoreactive T-cells in NOD mice through allelic exclusion induced by transgenic expression of an H2-Db class I−restricted T-cell receptor (TCR) specific for a pathologically irrelevant lymphocytic choriomeningitis virus (LCMV) peptide. TCR allelic exclusion greatly reduced the pool of T-cells from which diabetogenic effectors could be derived in these NOD.LCMV TCR Tg mice. Surprisingly, this did not impair their type 1 diabetes susceptibility. Furthermore, a diabetogenic CD8 T-cell population that is prevalent in standard NOD mice was present at essentially equivalent levels in pancreatic islets of NOD.LCMV TCR Tg mice. Other data indicated that the antigenic specificity of these CD8 T-cells is primarily the function of a shared TCR-α chain. Although the percentage of TCR transgenic T-cells decreased in NOD versus B6,D2 control mice, much higher total numbers of both the TCR transgenic and the nontransgenic T-cells accumulated in the NOD strain. This transgenic T-cell accumulation in the absence of the cognate peptide indicated that the NOD genetic background preferentially promotes a highly efficient antigen-independent T-cell expansion. This might allow diabetogenic T-cells in NOD mice to undergo an efficient expansion before encountering antigen, which would represent an important and previously unconsidered aspect of pathogenesis.

Regulation of T-cell activity is dependent on antigen-independent co-stimulatory signals provided by the disulphide-linked homodimeric T-cell surface receptors, CD28 and CTLA-4 (ref. 1). Engagement of CD28 with B7-1 and B7-2 ligands on antigen-presenting cells (APCs) provides a stimulatory signal for T-cell activation, whereas subsequent engagement of CTLA-4 with these same ligands results in attenuation of the response. Given their central function in immune modulation, CTLA-4- and CD28-associated signalling pathways are primary therapeutic targets for preventing autoimmune disease, graft versus host disease, graft rejection and promoting tumour immunity. However, little is known about the cell-surface organization of these receptor/ligand complexes and the structural basis for signal transduction. Here we report the 3.2-A resolution structure of the complex between the disulphide-linked homodimer of human CTLA-4 and the receptor-binding domain of human B7-2. The unusual dimerization properties of both CTLA-4 and B7-2 place their respective ligand-binding sites distal to the dimer interface in each molecule and promote the formation of an alternating arrangement of bivalent CTLA-4 and B7-2 dimers that extends throughout the crystal. Direct observation of this CTLA-4/B7-2 network provides a model for the periodic organization of these molecules within the immunological synapse and suggests a distinct mechanism for signalling by dimeric cell-surface receptors.

Background. Hepatocyte cell lines are beginning to be developed as universal donors for isolated liver cell transplantation, which is a less invasive method than orthotopic liver transplantation for treatment of metabolic liver disease. The immune response to isolated liver cell transplantation and its modification by costimulatory blockade are as yet not well delineated. Methods. Adenovirus expressing CTLA4Ig was used to study blockade of the costimulatory CD28/B7 pathway in murine models of hepatocyte transplantation, and the effects on alloreactive T and B cells were studied. Results. CTLA4Ig delayed rejection of subcutaneously administered C57L-derived murine hepatoma cells in CBA/J recipients for >50 days. Activation and cytokine secretion by allospecific CD4 + and CD8 + T cells were initially blocked by CTLA4Ig; delayed rejection was associated with tumor infiltration by CD8 + T cells that did not secrete interferon-y. CTLA4Ig failed to block transplant rejection in primed mice, indicating that memory effector T cells were resistant to its action. In contrast, CTLA4Ig suppressed both naive and memory alloreactive B cells. High levels of CTLA4Ig mediated acceptance of hepatoma cells delivered directly into the spleen. However, isolated primary C57BL/6 mouse hepatocytes delivered into the spleen were rejected with only moderately delayed kinetics. Conclusions. Transplant antigenicity, transplant site, and CTLA4Ig dose all affected the survival of transplanted liver cells. CD8 + T cells are significant mediators of hepatocyte transplant rejection and are relatively resistant to costimulatory blockade with CTLA4Ig. Strategies to specifically antagonize CD8 + T cells or to modulate MHC class I expression in association with costimulatory blockade by CTLA4Ig may enhance the clinical feasibility of transplanting allogeneic hepatocytes.

The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8–10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the β-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3β loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.

Comparisons of the structures of different mouse MHC class I molecules define how polymorphic residues determine the unique structural motif and atomic anchoring of their bound peptides. Here, Ted Hansen and colleagues speculate that quantitative differences in how class I molecules interact with peptide, β 2 -microglobulin and molecular chaperones that facilitate peptide loading might determine their relative participation in different pathways of antigen presentation.

MHC class I molecules usually bind short peptides of 8–10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/β2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8–11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR Vβ elements. The precise role of the peptide in causing this restricted TCR Vβ expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR Vβ elements responding to each peptide variant. To focus our analysis solely on the TCR β-chain, we created a transgenic mouse expressing exclusively the TCR α-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR Vβ usage consequent to peptide immunization. However, in both C57BL/6 and TCRα transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR Vβ usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-β loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR Vβ usage, which is consistent with the notion that the TCR β-chain interacts in vivo preferentially with this region of the MHC/peptide complex.

In this study, the fine specificity and MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was analyzed. Based on the structure of peptide/Kb complexes and ternary TCR/Ag/MHC complexes, four TNP peptides, two octamers, and two nonamers were chosen for eliciting anti-TNP CTL responses. Hapten was conjugated at position 4 in the octamers and at position 5 in the nonamers, positions which should allow engagement of the hapten by TCRs. Potent CTL activity for each of the TNP peptides was obtained that was highly hapten-specific; however, there were considerable differences in the extent of cross-reactivity with other TNP peptides, with the octamers generating more cross-reactive CTL than the nonamers. MHC restriction analysis suggested that anti-hapten responses were less dependent on MHC recognition than anti-peptide responses. This was evidenced by the relative ease of detecting cross-reactivity to haptenated peptides presented by allo-MHC and by the relative insensitivity of anti-hapten vs anti-peptide CTL to mutations in the Kb molecule at potential TCR interaction sites. One potential explanation for this insensitivity to MHC mutation was the finding that the anti-hapten response appeared to be of higher avidity, since a >100-fold difference in the amount of Ag required to sensitize target cells was found between these two types of Ags.

Vesicular stomatitis virus (VSV) elicits H-2Kb-restricted CTLs specific for the immunodominant VSV octapeptide RGYVYQGL. To study the structural features important for interaction between the TCR β-chain and the peptide/MHC complex, we immunized TCR α-chain transgenic mice with the VSV peptide and raised a panel of anti-VSV CTL clones with identical TCR α-chains. Consistent with our previous analysis of uncloned populations of primary CTLs, the anti-VSV CTL clones were all Vβ13+ and expressed TCR β-chains with highly homologous complementarity-determining region 3 (CDR3) loops. Although the clones expressed similar TCRs, they differed in their ability to cross-react with VSV peptide variants singly substituted at TCR contact positions 4 and 6. These findings allowed us to identify short stretches of amino acids in the C-terminal region of the CDR3β loop that, when altered, modify the cross-reaction capability of the TCR to position 4 and position 6 variant peptides. To further probe the structural correlates of biologic cross-reactivity, we used cross-reactive CTL clones and cell lines expressing point mutations in H-2Kb to investigate the effect of single amino acid changes in the peptide on the pattern of recognition of the TCR for the peptide/MHC complex. Single conservative substitutions in the peptide were sufficient to alter the recognition contacts between a cross-reactive TCR and the MHC molecule, supporting the idea that the TCR can make overall structural adjustments in MHC contacts to accommodate single amino acid changes in the peptide.

To study how the T cell receptor interacts with its cognate ligand, the MHC/peptide complex, we used site directed mutagenesis to generate single point mutants that alter amino acids in the CDR3beta loop of a H-2Kb restricted TCR (N30.7) specific for an immunodominant peptide N52-N59 (VSV8) derived from the vesicular stomatitis virus nucleocapsid. The effect of each mutation on antigen recognition was analyzed using wild type H-2Kb and VSV8 peptide, as well as H-2Kb and VSV8 variants carrying single replacements at residues known to be exposed to the TCR. These analyses revealed that point mutations at some positions in the CDR3beta loop abrogated recognition entirely, while mutations at other CDR3beta positions caused an altered pattern of antigen recognition over a broad area on the MHC/peptide surface. This area included the N-terminus of the peptide, as well as residues of the MHC alpha1 and alpha2 helices flanking this region. Assuming that the N30 TCR docks on the MHC/peptide with an orientation similar to that recently observed in two different TCR-MHC/peptide crystal structures, our findings would suggest that single amino acid alterations within CDR3beta can affect the interaction of the TCR with an MHC surface region distal from the predicted CDR3beta-Kb/VSV8 interface. Such unique recognition capabilities are generated with minimal alterations in the CDR3 loops of the TCR. These observations suggest the hypothesis that extensive changes in the recognition pattern due to small perturbations in the CDR3 structure appears to be a structural strategy for generating a highly diversified TCR repertoire with specificity for a wide variety of antigens.

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1–4] and its peptide–MHC ligand, an octapeptide fragment representing amino acids 52–59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2K b class I MHC molecule. Comparison of the structure of the N15 TCR–VSV8–H-2K b complex with the murine 2C TCR–dEV8–H-2K b [5] and the human A6 TCR–Tax–HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable V α domain overlies the MHC α 2 helix and the V β domain overlies the MHC α 1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR V α and V β domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (β2-microglobulin, β2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and β2m by using purified, recombinant H2-Kb/peptide complexes in a cell-freein vitrosystem. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous β2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and β2m are independent events. The exchange rate of β2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous β2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

The surface residues of the VSV8/Kb complex important for recognition by N15 and N26 alphabeta T cell receptors (TCR) were mapped by mutational analysis and compared to each other and with epitopes of well-characterized Kb specific monoclonal antibodies (mAb). Three features of immune receptor recognition emerge. First, the footprints of the two TCR on VSV8/Kb are similar with more than 80 % overlap between sites. Given that only 8 of 14 surface exposed VSV8/Kb residues identified as critical for TCR interaction are in common, the chemical basis of the N15 and N26 interactions is nevertheless distinct. Second, the cognate peptide is a major focus of TCR recognition: mutation at any of the three exposed side chains (at p1, p4 or p6) abrogates interaction of both TCR as measured by functional T cell activation. Third, in contrast to TCR, mAb bind to discrete segments on the periphery of the alpha1 and/or alpha2 helices without orientational restriction. These findings suggest that unlike soluble antibodies, surface membrane receptor-ligand interactions on opposing cells (i.e. TCR-peptide/ MHC, CD8-MHC) limit the orientational freedom of the TCR in the immune recognition process.

SummaryThe members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. Guided by the Brotherhood approach, we present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

The use of peptides as a vaccine is a potentially powerful immunization strategy. We explored the possibility of inducing an efficient cytotoxic T lymphocyte (CTL) mediated immune response in mice, using in vitro reconstituted major histocompatibility complex (MHC) class I/peptide complexes as the immunogen. Recombinant derived H-2Kb and β2-microglobulin (β2m) were properly folded into an MHC class I complex using the vesicular stomatitis virus (VSV)-8mer from the natural nucleocapsid proteinN52–59 (RGYVYQGL), an immunodominant Kb epitope in C57BL 6 (B6) mice. After immunizing mice with the H-2Kb class I/VSV peptide complex and a subsequent in vitro stimulation with the VSV peptide alone, a specific CTL response was demonstrated. The method was also applicable to other peptides, for example, the Sendai virus (SV) peptideN324–332 (FAPGNYPAL). The CTL response was mediated by CD3 + CD8 + T cells and was shown to be allele specific, as only peptide loaded target cells expressing the H-2Kb allele could be recognized. It is of interest that extremely small amounts of injected MHC class I/peptide complex (i.e. 500 pg) could generate a measurable CTL response. The MHC class I/peptide complex had to be intact and properly folded to elicit an immune response, suggesting that the complex protected the peptide for internalization by antigen presenting cells (APCs) or for delivering to the proper site for peptide exchange on the cell surface of APCs. The described immunizing method can be routinely used to prime a CTL response by employing in vitro folded MHC class I/peptide complexes, without the use of adjuvants. It appears to be efficient, sensitive and specific. By using the recombinant protein system, unlimited amounts of MHC class I/peptide complex can be produced for immunization. Moreover, this protocol permits different in vitro combinations of allelic MHC class I molecules and peptide variants.

As an approach to determine the structural basis of interactions between T cell receptors (TCRs) and MHC class I/peptide complexes, the fine specificities of a panel of vesicular stomatitis virus (VSV)-specific CTL clones recognizing the antigenic peptide (nucleoprotein 52-59) and the class I (Kb) molecule were correlated with the TCR primary structure. Each TCR showed a distinct interaction pattern with N52-59 and the Kb molecule. The large majority of the TCRs expressed by the panel of CTL clones used V beta 13 gene segments that had randomly recombined with D beta and J beta gene segments. The alpha chains were from randomly assorted V alpha and J alpha gene segments. Thus, the panel was found to be a highly heterogeneous set of TCRs, each member of which appeared to have an unique surface interface area, the recognition site, that interacted with a complementary surface formed by the single peptide bound in the class I antigenic groove.

Nectins are members of the Ig superfamily that mediate cell–cell adhesion through homophilic and heterophilic interactions. We have determined the crystal structure of the nectin-2 homodimer at 1.3 Å resolution. Structural analysis and complementary mutagenesis studies reveal the basis for recognition and selectivity among the nectin family members. Notably, the close proximity of charged residues at the dimer interface is a major determinant of the binding affinities associated with homophilic and heterophilic interactions within the nectin family. Our structural and biochemical data provide a mechanistic basis to explain stronger heterophilic versus weaker homophilic interactions among these family members and also offer insights into nectin-mediated transinteractions between engaging cells.

Generation of soluble T-cell receptor (TCR) molecules by a variety of genetic engineering methods has been hampered by inefficient pairing of alpha and beta subunits in the absence of their respective transmembrane regions and associated CD3 components. To overcome this obstacle, we have added 30-amino acid-long segments to the carboxyl termini of alpha and beta extracellular domains via a cleavable flexible linker. These peptide segments (BASE-p1 for alpha and ACID-p1 for beta) have been previously shown to selectively associate to form a stable heterodimeric coiled coil termed a leucine zipper. Homodimeric structures are not permitted due to electrostatic repulsion among amino acid side chains. Expression of a representative TCR-leucine zipper fusion protein in a baculovirus expression system results in production of alpha beta TCR heterodimer at 0.6-1.4 mg/liter. This yield is 5- to 10-fold greater than that of the TCR expressed in the absence of the synthetic leucine zipper sequence. The structure of the TCR component of the fusion heterodimer was judged to be native when probed with a panel of 17 mAbs specific for alpha and beta constant and variable domains. A mAb specific for the isolated BASE-p1/ACID-p1 coiled coil was also generated and shown to react with the TCR fusion protein. The above technology should be broadly useful in the efficient production and purification of TCRs as well as other heterodimeric proteins.

We have identified two forms of a major histocompatibility complex (MHC) class I molecule, H-2Kb, distinguishable by specific antibodies through a study of a genetically engineered mouse cell line that overexpresses these molecules. One form, a complex associated with beta 2-microglobulin (native, beta 2m+ class I), is detectable by conformation-dependent antibodies. The other form, which remains after preclearing cell lysates of native class I, is only poorly, if at all, associated with beta 2-microglobulin (beta 2m- class I) and is detectable by an antiserum against the cytoplasmic tail region of H-2K molecules. Both forms are also present in normal cell lines. The affinity-purified native class I molecules bind short peptides (8 or 9 residues) and assemble tightly with beta 2-microglobulin. In striking contrast, the beta 2m- class I molecules bind peptides that are longer (> 15 residues) than those bound to native class I molecules. This finding is consistent with the recent evidence that peptides longer than 8-10 amino acid residues are transported into the endoplasmic reticulum and suggests the possibility of a control step for peptide presentation by MHC in which the incompletely processed peptides bind to the heavy chain and a selected fraction undergoes final processing and presentation on the cell surface.

Solution at 2.4 A resolution of the structure of H-2Db with the influenza virus peptide NP366-374 (ASNEN-METM) and comparison with the H-2Kb-VSV (RGY-VYQGL) structure allow description of the molecular details of MHC class I peptide binding interactions for mice of the H-2b haplotype, revealing a strategy that maximizes the repertoire of peptides than can be presented. The H-2Db cleft has a mouse-specific hydrophobic ridge that causes a compensatory arch in the backbone of the peptide, exposing the arch residues to TCR contact and requiring the peptide to be at least 9 residues. This ridge occurs in about 40% of the known murine D and L allelic molecules, classifying them as a structural subgroup.

Unlike the highly polymorphic major histocompatibility complex (MHC) class Ia molecules, which present a wide variety of peptides to T cells, it is generally assumed that the nonpolymorphic MHC class Ib molecules may have evolved to function as highly specialized receptors for the presentation of structurally unique peptides. However, a thorough biochemical analysis of one class Ib molecule, the soluble isoform of Qa-2 antigen (H-2SQ7b), has revealed that it binds a diverse array of structurally similar peptides derived from intracellular proteins in much the same manner as the classical antigen-presenting molecules. Specifically, we find that SQ7b molecules are heterodimers of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. These peptides contain a conserved hydrophobic residue at the COOH terminus and a combination of one or more conserved residue(s) at P7 (histidine), P2 (glutamine/leucine), and/or P3 (leucine/asparagine) as anchors for binding SQ7b. 2 of 18 sequenced peptides matched cytosolic proteins (cofilin and L19 ribosomal protein), suggesting an intracellular source of the SQ7b ligands. Minimal estimates of the peptide repertoire revealed that at least 200 different naturally processed self-peptides can bind SQ7b molecules. Since Qa-2 molecules associate with a diverse array of peptides, we suggest that they function as effective presenting molecules of endogenously synthesized proteins like the class Ia molecules.

Using Mitomycin C mutagenesis and negative and positive selection with monoclonal antibodies specific for H-2Kb and H-2Kbm10, respectively, a mutant cell line clone, Mitc-182, was isolated. Direct sequencing of uncloned cDNA as well as PCR based cloning and sequencing of the H-2Kb182 transcript from this mutant revealed a single G-->T transversion resulting in the substitution of Trp167 by cysteine. Serologically, the mutant Kb182 and Kbm10 are almost identical as each has lost at least five Kb specific mAb epitopes and gained several new epitopes. Interestingly, the mutant cell line, Mitc-182, is efficiently recognized by alloreactive CTLs raised in reciprocal combinations, e.g. CB6 anti Cbm10 and Cbm10 anti CB6, indicating that Kb182 contains both Kb and Kbm10 specific epitopes. The mutation has not affected the ability of Kb182 to present Kb restricted antigenic peptides of Sendai and vesicular stomatitis viruses. In addition to underscoring the importance of amino acid residue 167 in alloreactivity, these results indicate a positive correlation between the gain of both an mAb epitope and a defined alloreactive CTL epitope.

Under steady-state conditions, B7-1 is present as a mixed population of noncovalent dimers and monomers on the cell surface. In this study, we examined the physiological significance of this unique dimer–monomer equilibrium state of B7-1. We demonstrate that altering B7-1 to create a uniformly covalent dimeric state results in enhanced CD28-mediated formation of T cell–APC conjugates. The enhanced T cell–APC conjugate formation correlates with persistent concentration of signaling molecules PKC-θ and lck at the immunological synapse. In contrast, T cell acquisition of B7-1 from APCs, an event that occurs as a consequence of CD28 engagement with B7-1/B7-2 and is thought to play a role in the dissociation of T cell–APC conjugates, is highly reduced when B7-1 is present in the covalently dimeric state. The ability of covalently dimeric and wild type B7-1 to costimulate Ag-specific T cell proliferation was also assessed. In contrast to the enhanced ability of dimeric B7-1 to support conjugate formation and early parameters of T cell signaling, sensitivity to competitive inhibition by soluble CTLA-4–Ig indicated that the covalent dimeric form of B7-1 is less efficient in costimulating T cell proliferation. These findings suggest a novel model in which optimal T cell costimulatory function of B7-1 requires high-avidity CD28 engagement by dimeric B7-1, followed by dissociation of these noncovalent B7-1 dimers, facilitating downregulation of CD28 and internalization of B7-1. These events regulate signaling through TCR/CD28 to maximize T cell activation to proliferation.

This study describes serological and biochemical properties of a novel MHC class I molecule. The mutant H-2Ksm1 molecule was discovered in a mouse because of loss of reactivity of its peripheral blood lymphocytes to monoclonal antibodies. This mutation in the H-2Ks molecule is the first in vivo mutation described that has altered an amino acid residue (amino acid 107) distant from the regions generally considered to be peptide or TCR contacts. Cell surface expression of the mutant molecules remains high but the Arg107 to Trp substitution appears to alter the native protein conformation, markedly decreasing cell surface association with beta 2-microglobulin light chains and conferring a loss of recognition by Ks specific antibodies.

Summary The major histocompatibility complex class I molecules are receptors for intracellular peptides, both of self and non-self origin. When non-self peptides (eg., pathogen derived) are bound to the class I molecules, they form ligands for T cell receptors resulting in antigen specific lysis of the infected cells by cytotoxic T lymphocytes. Therefore, an understanding of the process of antigen recognition requires the precise definition of the structural features of the bimolecular complex formed by a single well defined antigenic peptide bound to the class I molecule. A strategy using antibodies was developed to probe the structural features of the H-2K b containing a defined peptide in the antigen cleft. We report that the binding surface area of a K b specific monoclonal antibody (28-13-3s) includes residues in the α1 (Gly56 and Glu58) and α2 (Trp167) helices of K b thus, binding across the antigen binding groove. When cells treated with the antigenic peptide of vesicular stomatitis virus, N52-59, and its alanine substituted analogs were tested for 28-13-3s binding, it was found that position 1 of the peptide also forms a part of the antibody binding site. This finding strongly supports the positioning of the N-terminus of N52-59 proximal to pocket A, thus, assuming an orientation parallel to the a 1 helix.

This study describes a novel MHC class I mouse mutant that was discovered because of loss of reactivity of its cells to monoclonal antibodies. The mutation occurred in the H-2Ks molecule and is the first in vivo mutation described that has a single altered amino acid residue (amino acid 107) distant from the regions considered to be peptide or TCR contacts. Nevertheless, skin grafts from the mutant to the parent are rejected by CD8+ T-cells. In the reciprocal direction, the mutant shows partial tolerance to parental skin grafts, suggesting that the mutant is inefficient in selecting alloreactive T-cells specific for the wild-type Ks molecule.