Search

NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I–specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti–MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity (1) . This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies (1, 2) . Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe (3) . Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8+ T cell response to Bacillus Calmette-Guerin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8+ T cells with an rBCG-based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG-Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). We identified two MHC-I (H2-Kd )-restricted epitopes that induce cross-reactive responses to Mtb and other related mycobacteria in both BALB/c (H2d ) and CB6F1 (H2b/d ) mice. The H2-Kd -restricted peptide epitopes elicited polyfunctional CD8+ T cell responses that were also highly cross-reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2-Kd -restricted epitopes elicit distinct CD8+ T cell populations, a result explained by the X-ray structure of the two peptide/H2-Kd complexes. These results suggest that rBCG-Mkan85B vector-based immunization and DNA-Mkan85B boost may enhance CD8+ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

Intracellular loading of major histocompatibility complex class I (MHC-I) molecules occurs within the peptide loading complex and is accomplished through the concerted action of several proteins, including the chaperone/catalyst tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. To gain a mechanistic understanding of tapasin’s function we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSta1 and PaSta2, and investigated their binding to human recombinant soluble tapasin by surface plasmon resonance methods and X-ray crystallography. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. PaSta1 and PaSta2 recognize distinct epitopes as tapasin captured on a surface immobilized with one PaSta Fab binds the other PaSta Fab with nanomolar affinity. However, tapasin captured on a PaSta2 Fab surface is not able to bind peptide/HLA-B*44:05/beta-2 microglobulin complexes while tapasin captured on a PaSta1 Fab surface can, indicating that PaSta2 sterically competes for the MHC-I binding site. This finding was echoed in the structural characterization of PaSta Fab-tapasin complexes by X-ray crystallography. PaSta1 binds to an extended N-terminal region of tapasin, a site accessible even in the tapasin/ERp57 complex. By contrast, PaSta2 binds at the junction of the N-terminal and C-terminal domains of tapasin, where tapasin may interact with HLA-I molecules. Comparison of conformations of tapasin in complex with PaSta1, PaSta2, HLA-B*44:05, and ERp57 reveals the dynamic nature of tapasin, a versatile chaperone and catalyst. Supported by the intramural research program of the NIAID, NIH

MHC-I molecules loaded with peptide for subsequent cell surface expression are critical for adaptive immunity. Tapasin, a component in the Peptide Loading Complex (PLC), serves as an MHC-I chaperone, and it facilitates peptide loading onto MHC-I in the endoplasmic reticulum (ER). Although the structure of TAPBPR (a homolog of tapasin) complexed with MHC-I was reported several years ago, the detailed mechanism of peptide loading by tapasin remains unclear. Here, we report the structure of a complex of tapasin with the MHC-I molecule, HLA-B* 44:05. The model of tapasin/HLA-B*44:05 compared with that of unliganded HLA-B*44:05 and to the structure of tapasin bound to ERp57 reveals dramatic changes that refect both chaperone and catalytic activities. Tapasin cradles the MHC-I molecule through contacts with the N-terminal alpha1 and alpha2 domains, as well as with alpha3 and beta-2 microglobulin interaction via its C-terminal IgC domain. Thus the MHC-I molecule is stabilized in a peptide accessible form. We compare this tapasin/HLA-B*44:05 structure with our previously determined structure of TAPBPR/H2-D(d). Although the general dispositions of tapasin and TAPBPR are the same in their complexes with MHC-I, the structural details of their interactions differ. Analyses of tapasin mutants that disrupt the interaction with MHC-I are consistent with the X-ray crystal structure. Thus, the two MHC-I dedicated chaperones, tapasin and TAPBPR, although acting at distinct stages of the MHC-I loading pathway, show broadly conserved binding modes and similar mechanisms of peptide loading in antigen presentation. Supported by the intramural research program of the NIAID, NIH

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands unprecedented attention. We report four X-ray crystal structures of three synthetic nanobodies (sybodies) (Sb16, Sb45 and Sb68) bind to the receptor-binding domain (RBD) of SARS-CoV-2: binary complexes of Sb16–RBD and Sb45–RBD; a ternary complex of Sb45–RBD–Sb68; and Sb16 unliganded. Sb16 and Sb45 bind the RBD at the ACE2 interface, positioning their CDR2 and CDR3 loops diametrically. Sb16 reveals a large CDR2 shift when binding the RBD. Sb68 interacts peripherally at the ACE2 interface; steric clashes with glycans explain its mechanism of viral neutralization. Superposing these structures onto trimeric spike (S) protein models indicates these sybodies bind conformations of the mature S protein differently, which may aid therapeutic design., One Sentence Summary: X-ray structures of synthetic nanobodies complexed with the receptor-binding domain of the spike protein of SARS-CoV-2 reveal details of CDR loop interactions in recognition of distinct epitopic sites.

Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a β-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.

Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.

Unlike cytosolic processing and presentation of viral antigens by virus-infected cells, antigens first expressed in an infected non-professional antigen-presenting cell, such as CD4(+) T cells in the case of HIV, and then taken up by dendritic cells are cross-presented. This generally requires entry through the endocytic pathway, where endosomal proteases have first access for processing. Thus, understanding virus escape during cross-presentation requires an understanding of resistance to endosomal proteases such as cathepsin S. We have modified HIV-1(MN) gp120 (gp120(MN)) by mutating a key cathepsin S cleavage site T(322)T(323) in the V3 loop of the immunodominant epitope IGPGRAFYTT to IGPGRAFYVV to prevent digestion. We found this mutation to facilitate cross-presentation, and provide evidence from both MHC-binding and X-ray crystallographic structural studies that this results from preservation of the epitope rather than an increased epitope affinity for the class I MHC molecule. In contrast, when the protein is expressed by a vaccinia virus in the cytosol, the wild type protein is immunogenic without this mutation. These results demonstrate proof-of-concept that a virus like HIV, infecting predominantly non-professional presenting cells, can escape T cell recognition by incorporating a cathepsin S cleavage site that leads to destruction of an immunodominant epitope when the antigen undergoes endosomal cross-presentation.

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

Summary A fraction of mature T cells can be activated by peripheral self-antigens, potentially eliciting host autoimmunity. We investigated homeostatic control of self-activated T cells within unperturbed tissue environments by combining high-resolution multiplexed and volumetric imaging with computational modeling. In lymph nodes, self-activated T cells produced interleukin (IL)-2, which enhanced local regulatory T cell (Treg) proliferation and inhibitory functionality. The resulting micro-domains reciprocally constrained inputs required for damaging effector responses, including CD28 co-stimulation and IL-2 signaling, constituting a negative feedback circuit. Due to these local constraints, self-activated T cells underwent transient clonal expansion, followed by rapid death (“pruning”). Computational simulations and experimental manipulations revealed the feedback machinery’s quantitative limits: modest reductions in Treg micro-domain density or functionality produced non-linear breakdowns in control, enabling self-activated T cells to subvert pruning. This fine-tuned, paracrine feedback process not only enforces immune homeostasis but also establishes a sharp boundary between autoimmune and host-protective T cell responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, enters host cells through the receptor angiotensin-converting enzyme 2 (ACE2). Identification of numerous variant strains of SARS-CoV-2 have demanded unprecedented attention in developing rapid diagnostics, effective therapies, and safe vaccines to bring the current pandemic under control. Rapid progress is underway in the characterization of fundamental aspects of the structure and mechanisms of viral adsorption to cells, entry, and replication as well as in studies of the immune response to the virus. Here we report binding studies and crystal structures of the spike protein receptor binding domain (RBD) in complex with two individual synthetic nanobodies (sybodies) (Sb16 and Sb45). These bind the RBD at the previously identified ACE2 interface, positioning their complementarity determining region (CDR)2 and CDR3 in diametrically opposite orientations with large buried surface area. We also solved a structure of the RBD simultaneously bound by two sybodies, Sb45 and Sb68. In this structure, Sb45 binds at the ACE2 interface and Sb68 interacts at a second site. Comparison of structures of Sb16 both free and bound to RBD reveals a large movement of its CDR2 loop. Structural insights gained from these synthetic nanobody complexes will be helpful in designing new vaccines and expand the possibilities of using high affinity antibodies or linked bifunctional antibodies for therapy.

The intracellular loading of major histocompatibility complex class I (MHC-I) molecules with high affinity peptides is a pre-requisite for their cell surface display as ligands for T and NK cells. Peptide acquisition occurs in the ER within the peptide loading complex (PLC) and is accomplished through the concerted action of several proteins, most notably tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. In order to gain a mechanistic understanding of tapasin function as well as its apparent allelomorph specificity we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSTa 1 and PaSTa 2, and investigated their binding to human recombinant soluble tapasin and ERp57/tapasin by SPR methods. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. Based on these results we developed an MHC-I binding assay employing PaSTa 1-captured tapasin or ERp57/tapasin which reveals affinities in the micromolar range for selected MHC-I alleles. These affinities are notably weaker than those obtained for the structurally related but PLC-independent chaperone, TAPBPR (TAP-binding protein, related), and may reflect distinct evolutionarily derived functions for the two related molecules.

Class I MHC molecules (MHC-I) provide crucial cell surface elements that signal health or status of cells for recognition by T cells via their T cell receptor or natural killer (NK) cells via various NK receptors. MHC-I dependent antigen presentation thus influences TCR and NK recognition in immunity to infection, in cancer, and in autoimmunity. Antigen presentation in the MHC-I-dependent pathway depends, in part, on the loading of peptides, derived from proteolysis, aberrant translation, or protein splicing onto MHC-I molecules in the endoplasmic reticulum (ER). The classical pathway of peptide loading and exchange involves components of the peptide loading complex (PLC): the transporter TAP1/2; a lectin, calreticulin; an oxidoreductase, ERp57; a chaperone, tapasin; as well as the peptide-receptive MHC-I molecule and its light chain, b2-microglobulin (b2m). To understand the mechanism of peptide loading and exchange, several laboratories have explored functional and structural aspects of MHC-I interactions with a tapasin homolog, TAP binding protein, related (TAPBPR). We previously reported the structure of a TAPBPR/MHC-I complex at 3.4 Å resolution. However, details of the direct interaction of tapasin with MHC-I are more desirable. Here we report the X-ray crystal structure of tapasin/MHC-I complexes at higher resolution. We discuss similarities and differences between the tapasin/MHC-I and TAPBPR/MHC-I interactions and their implications for MHC-I dependent T cell and NK cell recognition.

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8(+) T cell response to Bacillus Calmette-Guérin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8(+) T cells with an rBCG-based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG-Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). We identified two MHC-I (H2-K(d))-restricted epitopes which induce cross-reactive responses to Mtb and other related mycobacteria in both BALB/c (H2(d)) and CB6F1 (H2(b/d)) mice. The H2-K(d)-restricted peptide epitopes elicited polyfunctional CD8(+) T cell responses that were also highly cross-reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2-K(d)-restricted epitopes elicit distinct CD8(+) T cell populations, a result explained by the X-ray structure of the two peptide/H2-K(d) complexes. These results suggest that rBCG-Mkan85B vector-based immunization and DNA-Mkan85B boost may enhance CD8(+) T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.

Antigen presentation is a cellular process that involves a number of steps, beginning with the production of peptides by proteolysis or aberrant synthesis and the delivery of peptides to cellular compartments where they are loaded on MHC class I (MHC-I) or MHC class II (MHC-II) molecules. The selective loading and editing of high-affinity immunodominant antigens is orchestrated by molecular chaperones: tapasin/TAP-binding protein, related for MHC-I and HLA-DM for MHC-II. Once peptide/MHC (pMHC) complexes are assembled, following various steps of quality control, they are delivered to the cell surface, where they are available for identification by αβ receptors on CD8+ or CD4+ T lymphocytes. In addition, recognition of cell surface peptide/MHC-I complexes by natural killer cell receptors plays a regulatory role in some aspects of the innate immune response. Many of the components of the pathways of antigen processing and presentation and of T cell receptor (TCR)-mediated signaling have been studied extensively by biochemical, genetic, immunological, and structural approaches over the past several decades. Until recently, however, dynamic aspects of the interactions of peptide with MHC, MHC with molecular chaperones, or of pMHC with TCR have been difficult to address experimentally, although computational approaches such as molecular dynamics (MD) simulations have been illuminating. Studies exploiting X-ray crystallography, cryo-electron microscopy, and multidimensional nuclear magnetic resonance (NMR) spectroscopy are beginning to reveal the importance of molecular flexibility as it pertains to peptide loading onto MHC molecules, the interactions between pMHC and TCR, and subsequent TCR-mediated signals. In addition, recent structural and dynamic insights into how molecular chaperones define peptide selection and fine-tune the MHC displayed antigen repertoire are discussed. Here, we offer a review of current knowledge that highlights experimental data obtained by X-ray crystallography and multidimensional NMR methodologies. Collectively, these findings strongly support a multifaceted role for protein plasticity and conformational dynamics throughout the antigen processing and presentation pathway in dictating antigen selection and recognition.

Central to CD8 + T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

The molecular mechanism through which the interaction of a clonotypic αβ T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd). In addition to conformational changes in complementarity-determining regions (CDRs) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β-chain dynamics reveals significant chemical shift effects in sites removed from the MHC-binding site. Remodelling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests a possible role for an allosteric mechanism in TCR signalling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T-cell functional assays., Binding of T cell receptors (TCR) to peptide-loaded major histocompatibility complexes (p/MHC) leads to T-cell activation. Here the authors give structural insights into T-cell signalling and show that p/MHC binding induces conformational changes at the membrane-proximal site of the TCR.

In recent years, optical evanescent wave biosensors have been used to characterize protein-protein interactions, including determination of equilibrium binding constants and bimolecular rate constants. This surface binding technique can provide information about chemical on-rate constants, the lifetimes of complexes formed, and the time course of the signal. This unit provides a thorough, well-illustrated discussion of the principles of optical biosensors, experimental design, ligand immobilization, experimental data analysis, and common obstacles and possible solutions.

Immunoevasins are key proteins used by viruses to subvert host immune responses. Determining their high-resolution structures is key to understanding virus-host interactions toward the design of vaccines and other antiviral therapies. Mouse cytomegalovirus encodes a unique set of immunoevasins, the m02-m06 family, that modulates major histocompatibility complex class I (MHC-I) antigen presentation to CD8+ Tcells and natural killer cells. Notwithstanding the large number of genetic and functional studies, the structural biology of immunoevasins remains incompletely understood, largely because of crystallization bottlenecks. Here we implement atechnology using sparse nuclear magnetic resonance data and integrative Rosetta modeling to determine the structure of the m04/gp34 immunoevasin extracellular domain. The structure reveals a β fold that is representative of the m02-m06 family of viral proteins, several of which are known to bind MHC-I molecules and interfere with antigen presentation, suggesting its role as a diversified immune regulation module.

Cell surface major histocompatibility complex class I (MHC-I) molecules play a crucial role in immunity to microbes and tumors by presenting intracellular peptides for recognition by CD8+ T cells. Since the repertoire of bound peptides is crucial to MHC-I stability as well as to T cell and NK cell recognition, the molecular mechanism by which peptides are selectively loaded onto MHC-I molecules is of considerable interest. A key role is played by the well-characterized chaperone tapasin as indicated by reduced cell surface MHC-I levels and altered peptide repertoires in tapasin deficient cell lines and mice. Despite extensive studies of the functional role of tapasin in shaping peptide repertoires, direct structural information on its interaction with MHC-I is lacking. Following the recent biochemical and crystallographic studies of the interaction of the tapasin homolog, TAPBPR, with MHC-I, we have engineered soluble versions of tapasin for comparison with TAPBPR. Using surface plasmon resonance assays of purified, untethered proteins, we observe that tapasin binds directly to an MHC-I molecule loaded with truncated peptides but with lower apparent affinity than TAPBPR. Similarly, using fluorescence polarization, we find that tapasin facilitates peptide exchange onto MHC-I with a lower efficiency than TAPBPR. These experiments permit direct comparisons of tapasin/MHC-I and TAPBPR/MHC-I interactions and help elucidate the functional relationship of these two distinct chaperones in shaping the MHC-I peptide repertoire.

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8(+) T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I–restricted CD8+ T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 310 helix that flips from an exposed “open” position in the PR transition state to a “closed” position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46–53 of murine H-2Ld MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.

We examined TCR:MHC/peptide interactions and in vivo epitope availability to explore the Th1- or Th2-like phenotype of autoimmune disease in two TCR Tg mouse models of autoimmune gastritis (AIG). The TCR of strains A23 and A51 recognize distinct IA(d)-restricted peptides from the gastric parietal cell H/K-ATPase. Both peptides form extremely stable MHC/peptide (MHC/p) complexes. All A23 animals develop a Th1-like aggressive, inflammatory AIG early in life, while A51 mice develop indolent Th2-like AIG at 6-8 wk with incomplete penetrance. A51 T cells were more sensitive than A23 to low doses of soluble antigen and to MHC/p complexes. Staining with IA(d)/peptide tetramers was only detectable on previously activated T cells from A51. Thus, despite inducing a milder AIG, the A51 TCR displays a higher avidity for its cognate IA(d)/peptide. Nonetheless, in vivo proliferation of adoptively transferred A51 CFSE-labeled T cells in the gastric lymph node was relatively poor compared with A23 T cells. Also, DC from WT gastric lymph node, presenting processed antigen available in vivo, stimulated proliferation of A23 T cells better than A51. Thus, the autoimmune potential of these TCR in their respective Tg lines is strongly influenced by the availability of the peptide epitope, rather than by differential avidity for their respective MHC/p complexes.

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L(d) by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L(d), "mini-H2-L(d)," consisting of only the α1α2 platform domain. Mini-H2-L(d) refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β2-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β2-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L(d) MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.

A wide variety of genetically determined autoimmune phenomena and adverse drug reactions (ADR) are linked to specific HLA class I or class II genes. A particularly severe ADR is caused by the anti-HIV drug abacavir and is associated with HLA-B*57:01 expression. We have produced several transgenic mouse lines on the C57BL/6 background expressing HLA-B*57:01 as: 1) a full length HLA-B*57:01 protein; 2) a chimeric version in which the α3, transmembrane (TM) and cytoplasmic (CY) domains derived from H2-Dd; and 3) a single-chain molecule consisting of human β2-microglobulin linked to a chimeric version, HLA-B*57:01α1α2 and the H2-Dbα3, TM, and CY domains. Flow cytometry showed cell surface expression on all lymphoid cell populations tested. Expression of the transgenes on the H2-Kb−Db− background was significantly higher than in MHC-I sufficient mice. Immunization with an HLA-B*57:01-restricted peptide induced a CD8+ T-cell response, indicating the biological antigen presentation function of the transgenically expressed protein, and abacavir inhibited the peptide specific response. Furthermore, in vivo administration of abacavir followed by in vitro culture induced a CD8+ T-cell response and production of proinflammatory cytokines in the transgenics and not in controls. Thus, transgenic strains expressing HLA-B*57:01 present a specific HLA-B*57:01-restricted peptide, presentation is inhibited by abacavir, and abacavir alone can elicit CD8+ T cell responses. These observations support the view that HLA-B* 57:01 expression uniquely confers the abacavir sensitive phenotype. Such transgenic mouse strains provide useful models for exploring the mechanisms of HLA-B*57:01-linked adverse drug hypersensitivity reactions.

The molecular mechanism through which the interaction of a clonotypic αβ TCR with peptide/MHC (p/MHC) complexes leads to T cell activation is not yet fully understood. Here we exploit a high affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd) by combining crystallographic, NMR, and functional data. In addition to conformational changes in complementarity determining regions (CDR) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β chain dynamics reveals significant chemical shift effects in sites removed from the MHC binding site. In particular, a remodeling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests an allosteric mechanism for TCR signaling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T cell functional assays.

The cell surface expression of MHC-I molecules displaying peptides derived from intracellular proteolytic processing is a critical requirement for T cell-mediated immunity. A complex series of steps, guided by the peptide loading machinery in the endoplasmic reticulum, ensures that only stably folded MHC-I molecules carrying peptides of proper length and high affinity are transported for display at the cell surface. TAP-binding protein, related (TAPBPR), has been recently shown to directly bind to certain MHC-I alleles and to facilitate peptide loading. To elucidate mechanistic details, we have examined the binding interaction between purified recombinant TAPBPR and MHC-I molecules loaded with truncated peptides that leave a portion of the peptide binding groove unoccupied. Such MHC-I molecules interact with TAPBPR with nanomolar affinities as measured by surface plasmon resonance and these TAPBPR/MHC-I complexes dissociate when offered a high affinity peptide. An extensive crystallization screen with purified TAPBPR/MHC-I complexes yielded crystals that diffracted to 3.5 Å. The structure of the TAPBPR/MHC-I complex, solved by molecular replacement, and refined at this resolution readily reveals the binding footprint and further structural details of the interaction. Data will be presented on the binding interaction and structural characterization of the TAPBPR/MHC-I complex that reveal insights into the mechanism of TAPBPR- and tapasin-mediated loading of peptides onto MHC-I molecules. (KN and JJ contributed equally to this work).

MHC class I (MHC-I) molecules fold and bind high-affinity peptides with the help of chaperones in the endoplasmic reticulum before being transported to the cell surface as stable antigen-presenting proteins. In order to understand this process, it may be necessary to artificially stabilize MHC-I molecules to behave manageably in vitro but still retain pre peptide-loaded characteristics. Others have approached this by engineering a disulfide bond in H2-Kb between residues 84 and 139, resulting in a “stitched” peptide binding grove that bypassed cellular checkpoints for misfolded proteins. We have crystallized H2-Ddwith the same Y84C/A139C mutations, as well as with W51C/G175C mutations that stitch the opposite end of the binding groove, and with all four mutations, all with the high-affinity peptide P18-I10. We determined the crystal structure of each peptide-MHC complex at 2.3, 1.7, and 1.65 Å resolution respectively. The electron density shows disulfide linkages at each expected site, and despite the mutations, the α1α2 platform domains all align to WT with an RMSD under 0.25 Å. Further efforts to measure thermal stability of each construct with high- and low-affinity peptides as well as attempts to crystallize each construct with low affinity peptides are underway. This work should contribute to a structural and mechanistic understanding of MHC-I peptide loading.

Adverse drug reactions (ADR) are a major obstacle to drug development. Genome-wide association studies identified several human leukocyte antigens (HLA)-class I alleles as risk factors for ADR. The HLA-B*57:01 allele has been found to be associated with the development of abacavir (ABC) hypersensitivity syndrome (AHS) and with the induction of liver injury by the β-lactam antibiotic flucloxacillin. The nucleoside analog ABC, an inhibitor of the HIV reverse transcriptase, can induce severe multi-organ toxicity in >50% of HLA-B*57:01+ patients with HIV infection. In a previous study we showed that ABC induced binding to the HLA-B*57:01 of altered self-peptides containing predominantly isoleucine or leucine residues at the carboxyl terminus. Recognition of these self-peptides drives in vitro activation of cytotoxic CD8+ T cells. However, the early immune events/danger signals required to overcome the immune tolerance that otherwise suppress ADR are still unknown. In order to study HLA-linked drug hypersensitivity in vivo and to understand better drug immune tolerance, HLA-B transgenic mice were generated. Here, we show that ABC activates HLA-B*57:01 transgenic CD8+ T cells in vitro via CD8 and HLA. A systemic, but partial, early response to the drug is also observed in vivo, however, ABC fails to promote skin hypersensitivity in immunocompetent transgenic mice. Instead, depletion of CD4+ T cells breaks immune tolerance to ABC and promotes ADR in HLA-B*57:01 transgenic mice. Supported by the Intramural Research Program of the NIAID, NIH, and CDER, FDA.

We have recently described a mAb, KP15, directed against the MHC-I/peptide molecular complex consisting of H-2Dd and a decamer peptide corresponding to residues 311–320 of the HIV IIIB envelope glycoprotein gp160. When administered at the time of primary immunization with a vaccinia virus vector encoding gp160, the mAb blocks the subsequent appearance of CD8+ CTL with specificity for the immunodominant Ag, P18-I10, presented by H-2Dd. This inhibition is specific for this particular peptide Ag; another H-2Dd-restricted gp160 encoded epitope from a different HIV strain is not affected, and an H-2Ld-restricted epitope encoded by the viral vector is also not affected. Using functional assays and specific immunofluorescent staining with multivalent, labeled H-2Dd/P18-I10 complexes (tetramers), we have enumerated the effects of blocking of priming on the subsequent appearance, avidity, and TCR Vβ usage of Ag-specific CTL. Ab blocking skews the proportion of high avidity cells emerging from immunization. Surprisingly, Vβ7-bearing Ag-specific TCR are predominantly inhibited, while TCR of several other families studied are not affected. The ability of a specific MHC/peptide mAb to inhibit and divert the CD8+ T cell response holds implications for vaccine design and approaches to modulate the immune response in autoimmunity.

The efficient delivery of sample to surface-immobilized sites is a key element in biosensing. For a surface plasmon resonance (SPR) biosensor, this has been addressed by constant flow through a microfluidic system with a sample injection loop (Sjölander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345). The present study describes an alternative mode of sample delivery without constant unidirectional flow. It was implemented on a commercial Biacore X SPR biosensor equipped with a microfluidic cartridge, but with the fluidic handling performed by an externally computer-controlled syringe pump. We demonstrate that sample volumes as low as 2 microL can be reproducibly positioned to cover the sensor surfaces, manipulated in a serial fashion, efficiently mixed by applying an oscillatory flow pattern, and fully recovered. Compared to the traditional continuous unidirectional flow configuration, we found very similar kinetic responses at high analyte concentrations and slightly slower responses at low concentrations, most likely due to depletion of analyte from the small sample volumes due to surface binding. With the antibody-antigen systems tested, binding parameters were obtained that are generally within 10% of those from conventional experiments. In the new configuration, biosensor experiments can be conducted without the usual constraints in the surface contact time that are correlated with sample volume and mass transport rate. This can translate to improved detection limits for slow reactions and can facilitate kinetic and thermodynamic binding studies.

We have used a recombinant, biotinylated form of the mouse NK cell inhibitory receptor, Ly49A, to visualize the expression of MHC class I (MHC-I) ligands on living lymphoid cells. A panel of murine strains, including MHC congenic lines, was examined. We detected binding of Ly49A to cells expressing H-2Dd, H-2Dk, and H-2Dp but not to those expressing other MHC molecules. Cells of the MHC-recombinant strain B10.PL (H-2u) not only bound Ly49A but also inhibited cytolysis by Ly49A+ effector cells, consistent with the correlation of in vitro binding and NK cell function. Binding of Ly49A to H-2Dd-bearing cells of different lymphoid tissues was proportional to the level of H-2Dd expression and was not related to the lineage of the cells examined. These binding results, interpreted in the context of amino acid sequence comparisons and the recently determined three-dimensional structure of the Ly49A/H-2Dd complex, suggest a role for amino acid residues at the amino-terminal end of the α1 helix of the MHC-I molecule for Ly49A interaction. This view is supported by a marked decrease in affinity of an H-2Dd mutant, I52 M, for Ly49A. Thus, allelic variation of MHC-I molecules controls measurable affinity for the NK inhibitory receptor Ly49A and explains differences in functional recognition in different mouse strains.

Natural killer cell function is controlled by interaction of NK receptors with MHC I molecules expressed on target cells. We describe the binding of bacterially expressed Ly49A, the prototype murine NK inhibitory receptor, to similarly engineered H-2Dd. Despite its homology to C-type lectins, Ly49A binds independently of carbohydrate and Ca2+ and shows specificity for MHC I but not bound peptide. The affinity of the Ly49A/H-2Dd interaction as determined by surface plasmon resonance is from 6 to 26 microM at 25 degrees C and is greater by ultracentrifugation at 4 degrees C. Biotinylated Ly49A stains H-2Dd-expressing cells. Competition experiments indicate that the Ly49A and T cell receptor (TCR) binding sites on MHC I are distinct, suggesting complex regulation of cells that bear both TCR and NK cell receptors.

To develop very small antibody-derived recognition units for experimental, medical, and drug design purposes, a heavy chain variable region (VH) single-domain phage-display library was designed and constructed. The scaffold that was used for library construction was a native sequence of a monoclonal antibody with a unique VH/VL interface. There was no need to modify any residues in the VL interface to avoid non-specific binding of VH domain. The library repertoire, consisting of 4x10(8)independent clones, was generated by the randomization of nine amino acid residues in complementary determining region 3. The library was screened by binding to protein antigens, and individual clones were isolated. The VH genes encoding for specific binding clones were rescued and large amounts of soluble and stable single-domain VH protein were made from insoluble inclusion bodies by in vitro refolding and purification. Biochemical and biophysical characterization of the VH protein revealed a highly specific, correctly folded, and stable monomeric molecule. Binding studies demonstrated an affinity of 20 nM. The properties of these molecules make them attractive for clinical, industrial, and research applications, as well as a step toward improvement in the design of small molecules that are based on the hypervariable loops of antibodies.

Cryptococcus neoformans is an encapsulated fungus that causes a life-threatening meningoencephalitis in patients with AIDS. Monoclonal antibodies to the capsular glucuronoxylomannan can modulate the infection in mice, but the epitopes on this complex polysaccharide recognized by protective and non-protective antibodies have not been defined. We have used 2H1, one of our most protective antibodies, to screen phage display peptide libraries for peptide mimotopes that would allow us to explore the fine specificity of anti-cryptococcal polysaccharide antibodies. Hexa- and decapeptides have been identified with sequence homologies that define four motifs: 1, (E)TPXWM/LM/L; 2, W/YXWM/ LYE; 3, DWXDW; and 4, (Ar)WDGQ(Ar). Peptides representing these motifs compete with each other for a shared binding site that overlaps the polysaccharide binding site. Motifs 1 and 2 confer high affinity binding, and PA1, which displays a motif 1 peptide with the sequence LQYTPSWMLV, binds to 2H1 with a Kd of 295 nM. Analysis of the interaction between the 2H1 binding peptides and 24 structurally related anti-polysaccharide antibodies reveals a complex pattern of reactivity that strongly suggests binding to or close to the complementary determining regions. Furthermore, those antibodies that have been shown to have different specificity, and in some cases different protective potential, do not bind any of the peptides selected by the protective 2H1 antibody. This study shows that peptide mimotopes for a complex microbial polysaccharide can be identified by screening phage peptide libraries and demonstrates the usefulness of such peptides in analyzing closely related interactive sites of proteins in general and of antibodies in particular.