IgG3 regulates tissue-like memory B cells in HIV-infected individuals.

Immunoglobulin G3 (IgG3) has an uncertain role in the response to infection with and vaccination against human immunodeficiency virus (HIV). Here we describe a regulatory role for IgG3 in dampening the immune system-activating effects of chronic HIV viremia on B cells. Secreted IgG3 was bound to IgM-expressing B cells in vivo in HIV-infected chronically viremic individuals but not in early-viremic or aviremic individuals. Tissue-like memory (TLM) B cells, a population expanded by persistent HIV viremia, bound large amounts of IgG3. IgG3 induced clustering of B cell antigen receptors (BCRs) on the IgM+ B cells, which was mediated by direct interactions between soluble IgG3 and membrane IgM of the BCR (IgM-BCR). The inhibitory IgG receptor CD32b (FcγRIIb), complement component C1q and inflammatory biomarker CRP contributed to the binding of secreted IgG3 onto IgM-expressing B cells of HIV-infected individuals. Notably, IgG3-bound TLM B cells were refractory to IgM-BCR stimulation, thus demonstrating that IgG3 can regulate B cells during chronic activation of the immune system.

HLA-F: A New Kid Licensed for Peptide Presentation.

HLA-F, a non-classical MHC molecule, is not known to present peptides. Dulberger et al. (2017) show that HLA-F contains a distinct peptide-binding groove and can present a diverse array of peptides. LIR1, however, recognized HLA-F away from bound peptide, leaving open whether peptide-HLA-F-specific T and NK receptors exist.

Identification and structural characterization of a mutant KRAS-G12V specific TCR restricted by HLA-A3.

Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T-cell therapy in metastatic diseases. To expand mutant KRAS-specific immunotherapies, it is critical to identify additional HLA-I allotypes that can present KRAS neoantigens and their cognate T-cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS-G12V neoantigen (7VVVGAVGVGK16) using a vaccination approach with transgenic mice expressing HLA-A*03:01 (HLA-A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA-A3(G12V). G12V-TCR CDR3ß and CDR1ß formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA-A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA-A3 (G12V) but did not sufficiently improve T-cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS-G12V mutations.

High-affinity oligoclonal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D.

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR-HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2ß and CDR3α. This allowed CDR3ß variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.

Developing a secretory AcGFP1-based IRES expression system for efficient production of mammalian recombinant proteins.

To generate stable cell lines that express high levels of recombinant genes often requires screening of a large number of transfected cells using ELISA. The most widely used alternative to ELISA screening is to use an intracellularly expressed GFP reporter construct which allows sorting of recombinant gene expression cells based on GFP fluorescence intensity. The disadvantage of cell sorting, however, is that the resulting population will be polyclonal with the danger of instability and overgrowth of low producers. In addition, GFP or its variants can be toxic to host cells at high concentrations, and thus may reduce growth and robustness of high producer cells or even cause them to become apoptotic. We have developed a new mammalian expression system in which a recombinant protein and a fluorescence protein, AcGFP1, are expressed on the same plasmid separated by an internal ribosome entry site (IRES). A signal peptide was incorporated upstream of AcGFP1 so that the fluorescent protein is secreted from cells, preventing cellular toxicity from intracellular accumulation and enabling convenient and accurate measurement of the protein. Expression tests of Ebola viral envelope GP1 and HIV gp120 proteins using this expression system in 293-H cells showed recombinant protein expression levels were closely correlated with AcGFP1 yield. Therefore, AcGFP1 can serve as an accurate reporter for recombinant protein expression and measuring AcGFP1 concentration provides a convenient, product independent and universal way for efficient clone screening.

Structure of MHC-Independent TCRs and Their Recognition of Native Antigen CD155.

During normal T cell development in the thymus, αß TCRs signal immature thymocytes to differentiate into mature T cells by binding to peptide-MHC ligands together with CD4/CD8 coreceptors. Conversely, in MHC and CD4/CD8 coreceptor-deficient mice, the thymus generates mature T cells expressing MHC-independent TCRs that recognize native conformational epitopes rather than linear antigenic-peptides presented by MHC. To date, no structural information of MHC-independent TCRs is available, and their structural recognition of non-MHC ligand remains unknown. To our knowledge in this study, we determined the first structures of two murine MHC-independent TCRs (A11 and B12A) that bind with high nanomolar affinities to mouse adhesion receptor CD155. Solution binding demonstrated the Vαß-domain is responsible for MHC-independent B12A recognition of its ligand. Analysis of A11 and B12A sequences against various MHC-restricted and -independent TCR sequence repertoires showed that individual V-genes of A11 and B12A did not exhibit preference against MHC-restriction. Likewise, CDR3 alone did not discriminate against MHC binding, suggesting VDJ recombination together with Vα/Vß pairing determine their MHC-independent specificity for CD155. The structures of A11 and B12A TCR are nearly identical to those of MHC-restricted TCR, including the conformations of CDR1 and 2. Mutational analysis, together with negative-staining electron microscopy images, showed that the CDR regions of A11 and B12A recognized epitopes on D1 domain of CD155, a region also involved in CD155 binding to poliovirus and Tactile in human. Taken together, MHC-independent TCRs adopt canonical TCR structures to recognize native Ags, highlighting the importance of thymic selection in determining TCR ligand specificity.

Human NK cell receptor KIR2DS4 detects a conserved bacterial epitope presented by HLA-C.

Natural killer (NK) cells have an important role in immune defense against viruses and cancer. Activation of human NK cell cytotoxicity toward infected or tumor cells is regulated by killer cell immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen class I (HLA-I). Combinations of KIR with HLA-I are genetically associated with susceptibility to disease. KIR2DS4, an activating member of the KIR family with poorly defined ligands, is a receptor of unknown function. Here, we show that KIR2DS4 has a strong preference for rare peptides carrying a Trp at position 8 (p8) of 9-mer peptides bound to HLA-C*05:01. The complex of a peptide bound to HLA-C*05:01 with a Trp at p8 was sufficient for activation of primary KIR2DS4+ NK cells, independent of activation by other receptors and of prior NK cell licensing. HLA-C*05:01+ cells that expressed the peptide epitope triggered KIR2DS4+ NK cell degranulation. We show an inverse correlation of the worldwide allele frequency of functional KIR2DS4 with that of HLA-C*05:01, indicative of functional interaction and balancing selection. We found a highly conserved peptide sequence motif for HLA-C*05:01-restricted activation of human KIR2DS4+ NK cells in bacterial recombinase A (RecA). KIR2DS4+ NK cells were stimulated by RecA epitopes from multiple human pathogens, including Helicobacter, Chlamydia, Brucella, and Campylobacter. We predict that over 1,000 bacterial species could activate NK cells through KIR2DS4, and propose that human NK cells also contribute to immune defense against bacteria through recognition of a conserved RecA epitope presented by HLA-C*05:01.

High affinity binding of SARS-CoV-2 spike protein enhances ACE2 carboxypeptidase activity.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has emerged to a pandemic and caused global public health crisis. Human angiotensin-converting enzyme 2(ACE2) was identified as the entry receptor for SARS-CoV-2. As a carboxypeptidase, ACE2 cleaves many biological substrates besides angiotensin II to control vasodilatation and vascular permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we investigated how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ∼3-10 fold against model peptide substrates, such as caspase-1 substrate and Bradykinin-analog. The enhancement in ACE2 enzymatic function was mediated by the binding of SARS-CoV-2 spike RBD domain. These results highlighted the potential for SARS-CoV-2 infection to enhance ACE2 activity, which may be relevant to the cardiovascular symptoms associated with COVID-19.

αß T cell receptors that do not undergo major histocompatibility complex-specific thymic selection possess antibody-like recognition specificities.

Major histocompatibility complex (MHC) restriction is the cardinal feature of T cell antigen recognition and is thought to be intrinsic to αß T cell receptor (TCR) structure because of germline-encoded residues that impose MHC specificity. Here, we analyzed αßTCRs from T cells that had not undergone MHC-specific thymic selection. Instead of recognizing peptide-MHC complexes, the two αßTCRs studied here resembled antibodies in recognizing glycosylation-dependent conformational epitopes on a native self-protein, CD155, and they did so with high affinity independently of MHC molecules. Ligand recognition was via the αßTCR combining site and involved the identical germline-encoded residues that have been thought to uniquely impose MHC specificity, demonstrating that these residues do not only promote MHC binding. This study demonstrates that, without MHC-specific thymic selection, αßTCRs can resemble antibodies in recognizing conformational epitopes on MHC-independent ligands.

High level stable expression of recombinant HIV gp120 in glutamine synthetase gene deficient HEK293T cells.

Due to the important pathological roles of the HIV-1 gp120, the protein has been intensively used in the research of HIV. However, recombinant gp120 preparation has proven to be difficult because of extremely low expression levels. In order to facilitate gp120 expression, previous methods predominantly involved the replacement of native signal peptide with a heterologous one, resulting in very limited improvement. Currently, preparation of recombinant gp120 with native glycans relies solely on transient expression systems, which are not amendable for large scale production. In this work, we employed a different approach for gp120 expression. Besides replacing the native gp120 signal peptide with that of rat serum albumin and optimizing its codon usage, we generated a stable gp120-expressing cell line in a glutamine synthetase knockout HEK293T cell line that we established for the purpose of amplification of recombinant gene expressions. The combined usage of these techniques dramatically increased gp120 expression levels and yielded a functional product with human cell derived glycan. This method may be applicable to large scale preparation of other viral envelope proteins, such as that of the emerging SARS-CoV-2, or other glycoproteins which require the presence of authentic human glycans.

Selection of Plasmodium falciparum cytochrome B mutants by putative PfNDH2 inhibitors.

Malaria control is threatened by a limited pipeline of effective pharmaceuticals against drug-resistant strains of Plasmodium falciparum Components of the mitochondrial electron transport chain (ETC) are attractive targets for drug development, owing to exploitable differences between the parasite and human ETC. Disruption of ETC function interferes with metabolic processes including de novo pyrimidine synthesis, essential for nucleic acid replication. We investigated the effects of ETC inhibitor selection on two distinct P. falciparum clones, Dd2 and 106/1. Compounds CK-2-68 and RYL-552, substituted quinolones reported to block P. falciparum NADH dehydrogenase 2 (PfNDH2; a type II NADH:quinone oxidoreductase), unexpectedly selected mutations at the quinol oxidation (Qo) pocket of P. falciparum cytochrome B (PfCytB). Selection experiments with atovaquone (ATQ) on 106/1 parasites yielded highly resistant PfCytB Y268S mutants seen in clinical infections that fail ATQ-proguanil treatment. In contrast, ATQ pressure on Dd2 yielded moderately resistant parasites carrying a PfCytB M133I or K272R mutation. Strikingly, all ATQ-selected mutants demonstrated little change or slight increase of sensitivity to CK-2-68 or RYL-552. Molecular docking studies demonstrated binding of all three ETC inhibitors to the Qo pocket of PfCytB, where Y268 forms strong van der Waals interactions with the hydroxynaphthoquinone ring of ATQ but not the quinolone ring of CK-2-68 or RYL-552. Our results suggest that combinations of suitable ETC inhibitors may be able to subvert or delay the development of P. falciparum drug resistance.

Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

The transcription factor Helios is expressed in a large subset of Foxp3+ Tregs. We previously proposed that Helios is a marker of thymic derived Treg (tTreg), while Helios- Treg were induced from Foxp3- T conventional (Tconv) cells in the periphery (pTreg). To compare the two Treg subpopulations, we generated Helios-GFP reporter mice and crossed them to Foxp3-RFP reporter mice. The Helios+ Treg population expressed a more activated phenotype, had a slightly higher suppressive capacity in vitro and expressed a more highly demethylated TSDR but were equivalent in their ability to suppress inflammatory bowel disease in vivo. However, Helios+ Treg more effectively inhibited the proliferation of activated, autoreactive splenocytes from scurfy mice. When Helios+ and Helios- Treg were transferred to lymphoreplete mice, both populations maintained comparable Foxp3 expression, but Foxp3 expression was less stable in Helios- Treg when transferred to lymphopenic mice. Gene expression profiling demonstrated a large number of differentially expressed genes and showed that Helios- Treg expressed certain genes normally expressed in CD4+ Foxp3- T cells. TCR repertoire analysis indicated very little overlap between Helios+ and Helios- Treg. Thus, Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

Structural mechanism of high affinity FcγRI recognition of immunoglobulin G.

Antibody-based immunotherapies are becoming powerful means of modern medicine for treating cancers and autoimmune diseases. The increasing popularity of antibody-based treatment demands a better understanding of antibody functions and in particular, their interaction with Fc receptors as effectiveness of antibodies often depends on their ability to activate or avoid effector cell functions through Fc receptors. Until recently, our understanding of antibody recognition by Fc receptors is based on the structures of low affinity Fc receptor in complex with Fc. These structural studies provided significant insights to our understanding of how an IgG antibody generally docks on Fcγ receptor and the requirement of immune complex formation for effector cell activations. They are less informative, however, to the molecular forces underlying the vast different affinities between antibodies and their Fcγ receptors. Recently, the structure of the high affinity FcγRI in complex with IgG-Fc has been determined. This review will focus on the knowledge learned from the high affinity complex structural work and a potential receptor-glycan interaction as an important contribution to the receptor affinity.

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding.

Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop-glycan interaction resulted in an ∼ 20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

Overexpression of Ebola virus envelope GP1 protein.

Ebola virus uses its envelope GP1 and GP2 for viral attachment and entry into host cells. Due to technical difficulty expressing full-length envelope, many structural and functional studies of Ebola envelope protein have been carried out primarily using GP1 lacking its mucin-like domain. As a result, the viral invasion mechanisms involving the mucin-like domain are not fully understood. To elucidate the role of the mucin-like domain of GP1 in Ebola-host attachment and infection and to facilitate vaccine development, we constructed a GP1 expression vector containing the entire attachment region (1-496). Cysteine 53 of GP1, which forms a disulfide bond with GP2, was mutated to serine to avoid potential disulfide bond mispairing. Stable expression clones using codon optimized open reading frame were developed in human 293-H cells with yields reaching ∼25 mg of GP1 protein per liter of spent medium. Purified GP1 was functional and bound to Ebola attachment receptors, DC-SIGN and DC-SIGNR. The over-expression and easy purification characteristic of this system has implications in Ebola research and vaccine development. To further understand the differential expression yields between the codon optimized and native GP1, we analyzed the presence of RNA structural motifs in the first 100 nucleotides of translational initiation AUG site. RNA structural prediction showed the codon optimization removed two potential RNA pseudoknot structures. This methodology is also applicable to the expression of other difficult virus envelope proteins.

Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis.

Serum amyloid A (SAA) represents an evolutionarily conserved family of inflammatory acute-phase proteins. It is also a major constituent of secondary amyloidosis. To understand its function and structural transition to amyloid, we determined a structure of human SAA1.1 in two crystal forms, representing a prototypic member of the family. Native SAA1.1 exists as a hexamer, with subunits displaying a unique four-helix bundle fold stabilized by its long C-terminal tail. Structure-based mutational studies revealed two positive-charge clusters, near the center and apex of the hexamer, that are involved in SAA association with heparin. The binding of high-density lipoprotein involves only the apex region of SAA and can be inhibited by heparin. Peptide amyloid formation assays identified the N-terminal helices 1 and 3 as amyloidogenic peptides of SAA1.1. Both peptides are secluded in the hexameric structure of SAA1.1, suggesting that the native SAA is nonpathogenic. Furthermore, dissociation of the SAA hexamer appears insufficient to initiate amyloidogenic transition, and proteolytic cleavage or removal of the C-terminal tail of SAA resulted in formation of various-sized structural aggregates containing ∼5-nm regular repeating protofibril-like units. The combined structural and functional studies provide mechanistic insights into the pathogenic contribution of glycosaminoglycan in SAA1.1-mediated AA amyloid formation.

Free IL-12p40 monomer is a polyfunctional adaptor for generating novel IL-12-like heterodimers extracellularly.

IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23, respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines. Although the p40 subunit alone can also be rapidly secreted in response to inflammatory signals, its biological significance remains unclear. In this article, we show that the secreted p40 monomer can generate de novo IL-12-like activities by combining extracellularly with p35 released from other cells. Surprisingly, an unbiased proteomic analysis reveals multiple such extracellular binding partners for p40 in the serum of mice after an endotoxin challenge. We biochemically validate the binding of one of these novel partners, the CD5 Ag-like glycoprotein, to the p40 monomer. Nevertheless, the assembled p40-CD5L heterodimer does not recapitulate the biological activity of IL-12. These findings underscore the plasticity of secreted free p40 monomer, suggesting that p40 functions as an adaptor that is able to generate multiple de novo composites in combination with other locally available polypeptide partners after secretion.

Pentraxins and Fc receptors.

Pentraxins are innate pattern recognition molecules whose major function is to bind microbial pathogens or cellular debris during infection and inflammation and, by doing so, contribute to the clearance of necrotic cells as well as pathogens through complement activations. Fc receptors are the cellular mediators of antibody functions. Although conceptually separated, both pentraxins and antibodies are important factors in controlling acute and chronic inflammation and infections. In recent years, increasing experimental evidence suggests a direct link between the innate pentraxins and humoral Fc receptors. Specifically, both human and mouse pentraxins recognize major forms of Fc receptors in solution and on cell surfaces with affinities similar to antibodies binding to their low affinity Fc receptors. Like immune complex, pentraxin aggregation and opsonization of pathogen result in Fc receptor and macrophage activation. The recently published crystal structure of human serum amyloid P (SAP) in complex with FcγRIIA further illustrated similarities to antibody recognition. These recent findings implicate a much broader role than complement activation for pentraxins in immunity. This review summarizes the structural and functional work that bridge the innate pentraxins and the adaptive Fc receptor functions. In many ways, pentraxins can be regarded as innate antibodies.

Development of an improved mammalian overexpression method for human CD62L.

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5-30mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4-6months to produce. To shorten the construction time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ∼5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.

Structural recognition and functional activation of FcgammaR by innate pentraxins.

Pentraxins are a family of ancient innate immune mediators conserved throughout evolution. The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein, which are two of the acute-phase proteins synthesized in response to infection. Both recognize microbial pathogens and activate the classical complement pathway through C1q (refs 3 and 4). More recently, members of the pentraxin family were found to interact with cell-surface Fcgamma receptors (FcgammaR) and activate leukocyte-mediated phagocytosis. Here we describe the structural mechanism for pentraxin's binding to FcgammaR and its functional activation of FcgammaR-mediated phagocytosis and cytokine secretion. The complex structure between human SAP and FcgammaRIIa reveals a diagonally bound receptor on each SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. The 1:1 stoichiometry between SAP and FcgammaRIIa infers the requirement for multivalent pathogen binding for receptor aggregation. Mutational and binding studies show that pentraxins are diverse in their binding specificity for FcgammaR isoforms but conserved in their recognition structure. The shared binding site for SAP and IgG results in competition for FcgammaR binding and the inhibition of immune-complex-mediated phagocytosis by soluble pentraxins. These results establish antibody-like functions for pentraxins in the FcgammaR pathway, suggest an evolutionary overlap between the innate and adaptive immune systems, and have new therapeutic implications for autoimmune diseases.