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Loading of MHC-I molecules with peptide by the catalytic chaperone tapasin in the peptide loading complex plays a critical role in antigen presentation and immune recognition. Mechanistic insight has been hampered by the lack of detailed structural information concerning tapasin–MHC-I. We present here crystal structures of human tapasin complexed with the MHC-I molecule HLA-B*44:05, and with each of two anti-tapasin antibodies. The tapasin-stabilized peptide-receptive state of HLA-B*44:05 is characterized by distortion of the peptide binding groove and destabilization of the β2-microglobulin interaction, leading to release of peptide. Movements of the membrane proximal Ig-like domains of tapasin, HLA-B*44:05, and β2-microglobulin accompany the transition to a peptide-receptive state. Together this ensemble of crystal structures provides insights into a distinct mechanism of tapasin-mediated peptide exchange.

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.

Immune recognition by T lymphocytes and natural killer (NK) cells is in large part dependent on the identification of cell surface MHC molecules bearing peptides generated from either endogenous (MHC I) or exogenous (MHC II) dependent pathways. This review focuses on MHC I molecules that coordinately fold to bind self or foreign peptides for such surface display. Peptide loading occurs in an antigen presentation pathway that includes either the multimolecular peptide loading complex (PLC) or a single chain chaperone/catalyst, TAP binding protein, related, TAPBPR, that mimics a key component of the PLC, tapasin. Recent structural and dynamic studies of TAPBPR reveal details of its function and reflect on mechanisms common to tapasin. Regions of structural conservation among species suggest that TAPBPR and tapasin have evolved to satisfy functional complexities demanded by the enormous polymorphism of MHC I molecules. Recent studies suggest that these two chaperone/catalysts exploit structural flexibility and dynamics to stabilize MHC molecules and facilitate peptide loading.

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.

Recognition of foreign and dysregulated antigens by the cellular innate and adaptive immune systems is in large part dependent on the cell surface display of peptide/MHC (pMHC) complexes. The formation of such complexes requires the generation of antigenic peptides, proper folding of MHC molecules, loading of peptides onto MHC molecules, glycosylation, and transport to the plasma membrane. This complex series of biosynthetic, biochemical, and cell biological reactions is known as "antigen processing and presentation". Here, we summarize recent work, focused on the structural and functional characterization of the key MHC-I-dedicated chaperones, tapasin, and TAPBPR. The mechanisms reflect the ability of conformationally flexible molecules to adapt to their ligands, and are comparable to similar processes that are exploited in peptide antigen loading in the MHC-II pathway.

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.

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.

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.

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

Chaperones TAPBPR and tapasin associate with class-I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR, and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product, and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, where the lifetime and structural features of transiently bound peptides controls the regulation of a conformational switch, located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex., Graphical abstract

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.

Major histocompatibility class I (MHC-I) molecules bind peptides derived from cellular synthesis and display them at the cell surface for recognition by receptors on T lymphocytes (TCR) or natural killer (NK) cells. Such recognition provides a crucial step in autoimmunity, identification of bacterial and viral pathogens, and anti-tumor responses. Understanding the mechanism by which such antigenic peptides in the ER are loaded and exchanged for higher affinity peptides onto MHC molecules has recently been clarified by cryo-EM and X-ray studies of the multimolecular peptide loading complex (PLC) and a unimolecular tapasin-like chaperone designated TAPBPR. Insights from these structural studies and complementary solution NMR experiments provide a basis for understanding mechanisms related to immune antigen presentation.

Major histocompatibility complex encoded class I (MHC-I) molecules bind a broad spectrum of peptides generated in the cytoplasm and encountered during protein folding and maturation in the endoplasmic reticulum (ER). For cell surface expression and recognition by T cell receptors (TCR) and natural killer (NK) receptors, MHC-I require loading with high affinity peptides. Peptide optimization is catalyzed by either of two pathways. The first is via the peptide-loading complex (PLC) which consists of the transporter associated with antigen processing (TAP)1/TAP2 heterodimer, tapasin (an ER resident chaperone, also known as TAP-binding protein (TAPBP)), ERp57 (an oxidoreductase), and calreticulin (a sugar-binding chaperone) [1]. The second pathway depends on TAP-binding protein, related (TAPBPR), a PLC-independent chaperone, that is similar in amino acid sequence and structure to tapasin [2]. Until recently, mechanistic understanding of how the PLC or TAPBPR influences MHC-I peptide loading has been hampered by a lack of detailed structural information on the modification of the MHC-I peptide-binding site by chaperone interactions. Here we review recent functional, structural, and computational dynamic studies of tapasin and TAPBPR that contribute to a vivid description of the molecular changes in MHC-I molecules that accompany tapasin or TAPBPR interaction.

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by ) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using -deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence , independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression. Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells (“missing-self”). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

Molecules encoded by the Major Histocompatibility Complex (MHC) bind self or foreign peptides and display these at the cell surface for recognition by receptors on T lymphocytes (designated T cell receptors-TCR) or on natural killer (NK) cells. These ligand/receptor interactions govern T cell and NK cell development as well as activation of T memory and effector cells. Such cells participate in immunological processes that regulate immunity to various pathogens, resistance and susceptibility to cancer, and autoimmunity. The past few decades have witnessed the accumulation of a huge knowledge base of the molecular structures of MHC molecules bound to numerous peptides, of TCRs with specificity for many different peptide/MHC (pMHC) complexes, of NK cell receptors (NKR), of MHC-like viral immunoevasins, and of pMHC/TCR and pMHC/NKR complexes. This chapter reviews the structural principles that govern peptide/MHC (pMHC), pMHC/TCR, and pMHC/NKR interactions, for both MHC class I (MHC-I) and MHC class II (MHC-II) molecules. In addition, we discuss the structures of several representative MHC-like molecules. These include host molecules that have distinct biological functions, as well as virus-encoded molecules that contribute to the evasion of the immune response.

Natural Killer (NK) cells are innate lymphocytes involved in the first line of immune defense and T cell adaptive immunity against viral infection and cancer, including metastasis. While on patrol, NK cells contact other cells and recognize MHC-I Ags via stochastically expressed MHC-I–specific inhibitory receptors (Ly49s in mice and KIRs in humans) that prevent NK cell activation via cytoplasmic ITIM. The binding site on MHC-Class-I for Ly49 inhibitory receptors is distinct from that for TCRs. The loss of MHC-I expression on tumor cells (“missing self”) abrogates inhibitory signals, resulting in NK activation. Global inhibition of the NK inhibitory receptor interactions in vivo by a pan-anti-MHC-I monoclonal antibody markedly activated IFNg-producing NK cells, independent of Fc receptors. NK cell-derived IFNg, along with other cytokines (MCSF & FLT3L), primed APC to induce IL-12/-15/-18/-21 cytokine cascades and enhanced levels of MHC-I and MHC-II expression that further drove the proliferation of NK cells and memory phenotype (MP) T cells. The global disruption of NK cell/MHC-I interactions significantly enhanced Th1 type signature transcription factors (Tbet & Eomes), cytokines (IFNg & Granzyme B), and chemokine receptors (CXCR3) on NK and MP T cells. Administration of pan-anti-MHC-I to unmanipulated mice profoundly augmented innate and adaptive immunity against viral infection, PD1-resistant transplanted tumors, and successfully constrained lung and liver metastasis, and protected the animals from tumor burden. Moreover, in vitro enhancement of human NK cell proliferation by a pan-anti-HLA Mab suggests that pan-anti-HLA could be a promising therapeutic strategy against chronic viral infection and cancer metastasis.

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.

Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS.

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.

Major histocompatibility class I (MHC-I) molecules bind peptides derived from cellular synthesis and display them at the cell surface for recognition by receptors on T lymphocytes (TCR) or natural killer (NK) cells. Such recognition provides a crucial step in autoimmunity, identification of bacterial and viral pathogens, and anti-tumor responses. Understanding the mechanism by which such antigenic peptides in the ER are loaded and exchanged for higher affinity peptides onto MHC molecules has recently been clarified by cryo-EM and X-ray studies of the multimolecular peptide loading complex (PLC) and a unimolecular tapasin-like chaperone designated TAPBPR. Insights from these structural studies and complementary solution NMR experiments provide a basis for understanding mechanisms related to immune antigen presentation.

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.

A critical step in antigen presentation is the degradative processing of peptides by aminopeptidases in the endoplasmic reticulum. It is unclear whether these enzymes act only on free peptides or on those bound to their major histocompatibility complex (MHC)-I-presenting molecules. A recent study examined the structure and biophysics of N-terminally extended peptides in complex with MHC-I, revealing the conformational adjustment of MHC to permit both binding of the peptide core and exposure of the peptide N terminus. These data suggest a mechanism by which aminopeptidase access is determined and offer an explanation for how longer peptides may be displayed at the cell surface.

T cells recognize antigens at the two-dimensional (2D) interface with antigen-presenting cells (APCs), which trigger T-cell effector functions. T-cell functional outcomes correlate with 2D kinetics of membrane-embedded T-cell receptors (TCRs) binding to surface-tethered peptide-major histocompatibility complex molecules (pMHCs). However, most studies have measured TCR-pMHC kinetics for recombinant TCRs in 3D by surface plasmon resonance, which differs drastically from 2D measurements. Here, we compared pMHC dissociation from native TCR on the T-cell surface to recombinant TCR immobilized on glass surface or in solution. Force on TCR-pMHC bonds regulated their lifetimes differently for native than recombinant TCRs. Perturbing the cellular environment suppressed 2D on-rates but had no effect on 2D off-rate regardless of whether force was applied. In contrast, for the TCR interacting with its monoclonal antibody, the 2D on-rate was insensitive to cellular perturbations and the force-dependent off-rates were indistinguishable for native and recombinant TCRs. These data present novel features of TCR-pMHC kinetics that are regulated by the cellular environment, underscoring the limitations of 3D kinetics in predicting T-cell functions and calling for further elucidation of the underlying molecular and cellular mechanisms that regulate 2D kinetics in physiological settings.

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.

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.

The molecular immunologist's dream is to elucidate a fundamental biochemical process that explains the basis of an affliction that affects millions of people, and that, precisely understood, might yield a rational approach to diagnosis, prevention, or therapy. In this issue of JBC, Ting et al. report proteomic, biochemical, and structural analyses that better explain how the antigen-presenting HLA-DR4 molecules bind citrullinated peptides to provoke rheumatoid arthritis (RA), a chronic autoimmune disease that affects 0.5–1% of the population.

Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with class-I major histocompatibility complex (MHC-I) molecules to promote optimization (editing) of peptide cargo. Using solution NMR, we investigate the molecular mechanism of peptide exchange performed by the 90 kDa chaperone protein complex. We identify TAPBPR-induced conformational changes on conserved MHC-I surfaces, consistent with our independently determined X-ray structure of the empty complex. Conformational dynamics present in the empty MHC-I are stabilized by TAPBPR in a peptide-deficient complex, and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized by the chaperoned groove according to the global stability of the final pMHC-I product, and anneal in a native-like conformation. Our results demonstrate an inverse relationship between MHC-I occupancy by peptide and the affinity of TAPBPR for such pMHC-I molecules, where the lifetime of transiently bound peptides controls the dynamic regulation of a conformational switch, located near the TAPBPR binding site, which triggers TAPBPR release. We further discuss the role of protein dynamics in shaping chaperone specificity towards different human and murine class-I MHC allotypes, and present the high-resolution cryoEM structure of a human A*02/TAPBPR complex with novel insights into the antigen editing mechanism. These results suggest a similar mechanism for the editing function of tapasin in the peptide-loading complex, and provide a molecular blueprint for the design of novel chaperones with tailored antigen editing functions.

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.

Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

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.

Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized byStaphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.

Understanding the molecular details of peptide loading in the MHC-I pathway is crucial to approaches to manipulate recognition of peptide/MHC-I complexes in treating autoimmunity, tumors, and for vaccine design. Recent insights have been provided by two X-ray structures of TAPBPR, in complex with MHC-I molecules. Although structures determined in the two laboratories are remarkably similar (pdb ID: 5WER and 5OPI), the structural models based on X-ray data obtained at similar resolution, 3.4 and 3.3 Å respectively, reveal a discrepancy in the disposition of the TAPBPR 22–36 loop. This loop was not modeled in 5WER due to a lack of electron density, whereas it was modeled as a 3,10 helix in 5OPI and ascribed a significant function in peptide loading. In an attempt to resolve this structural discrepancy, we reanalyzed 5OPI based on the authors’ deposited structure factors. We calculated the refined average B-factor, an indication of positional uncertainty, as 247 Å2 and 153 Å2 for the 22–36 loop and the entire TAPBPR/MHC-I complex, respectively. Prompted by the unusually high B-factors we reexamined the 22–36 loop by calculating composite omit-simulated annealing maps from deposited structure factors of 5OPI as well as of 5WER. When model bias was eliminated in this way, continuous electron density was not visible in this loop region. Thus, we conclude that although the 22–36 loop of TAPBPR may play a role in peptide exchange, there presently is no reliable crystallographic evidence that this loop is a 3,10 helix and is positioned as modeled in 5OPI. Further structural studies of TAPBPR and of tapasin should help to resolve in detail the mechanism by which these molecules facilitate MHC-I stabilization and peptide exchange.

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. We have explored the interaction between recombinant m06 and both full-length and truncated forms of the MHC-I molecule H2-Ld using in vitro binding assays. Previous work using solution NMR has mapped the interaction footprint of the m06 protein on MHC-I, revealing that the targeted surface area overlaps with that of the MHC-I chaperone molecule Tapasin. Furthermore, we observe enhanced binding between m06 and peptide-free forms of MHC-I, suggesting a possible chaperone-like interaction between m06 and MHC-I. Here, we express recombinant m06 in mammalian cells to explore the interplay between the viral protein and its potential targets through immunofluorescence localization experiments as well as well as binding assays. 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.

Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with major histocompatibility complex class I (MHC-I) to promote the loading of antigenic peptides through a poorly understood mechanism. Here, we use solution Nuclear Magnetic Resonance (NMR) spectroscopy to probe TAPBPR-induced changes on MHC-I. Dynamic motions present in the empty MHC groove become progressively dampened with increasing peptide occupancy, while allosteric communication between the A- and F-pockets regulates a conformational switch located near the TAPBPR binding site, which is crucial for chaperone release from the complex. Our analysis of NMR data recorded for a range of TAPBPR complexes prepared with both murine H2 and human HLA alleles complements the recent X-ray structures to provide atomic-resolution mechanistic insights into the selection of optimal peptide sequences for the displayed antigen repertoire. In particular, our results show that negative allosteric coupling between the MHC groove and chaperone binding sites allows TAPBPR to proofread MHC molecules containing a range of different peptides. Since the affinity of incoming peptides for the empty groove is greatly reduced in the chaperone complex, (micromolar range, relative to nanomolar for the free MHC), these interactions can provide a mechanism for optimizing the peptide repertoire, where only the highest-affinity peptides can drive chaperone release. Finally, our results suggest that TAPBPR may promote the dissociation of tightly bound peptides from MHC molecules, thereby further scrutinizing the displayed repertoire. These findings imply a similar mechanism for the specificity and editing function of tapasin in the peptide-loading complex.

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8+ cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8+ T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2Dd better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8+ T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.