Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-α, and Amylin by Human Insulin-Degrading Enzyme

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-β, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-α (TGF-α) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-α, amylin, reduced amylin, and amyloid-β by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-α at 2.3 Å and IDE-amylin at 2.9 Å. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.     

Molecular Basis for the Recognition of Long-chain Substrates by Plant α-Glucosidases

Sugar beet α-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher k_cat/K_m values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific α-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 Å resolution. The active site pocket of SBG is formed by a (β/α)₈ barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (β/α)₈ barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.     

The Molecular Basis for Modulation of Human Vγ9Vδ2 T Cell Responses by CD277/Butyrophilin-3 (BTN3A)-specific Antibodies

Human Vγ9Vδ2 T cells are well known for their rapid and potent response to infection and tumorigenesis when in the presence of endogenous or exogenous phosphoisoprenoids. However, the molecular mechanisms behind the activation of this γδ T cell population remains unclear. Evidence pointing to a role for the CD277/butyrophilin-3 (BTN3A) molecules in this response led us to investigate the structures of these molecules and their modifications upon binding to an agonist antibody (20.1) that mimics phosphoisoprenoid-mediated Vγ9Vδ2 activation and an antagonist antibody (103.2) that inhibits this reactivity. We find that the three BTN3A isoforms: BTN3A1, BTN3A2, and BTN3A3, have high structural homology to the B7 superfamily of proteins and exist as V-shaped homodimers in solution, associating through the membrane proximal C-type Ig domain. The 20.1 and 103.2 antibodies bind to separate epitopes on the BTN3A Ig-V domain with high affinity but likely with different valencies based on their binding orientation. These structures directly complement functional studies of this system that demonstrate that BTN3A1 is necessary for Vγ9Vδ2 activation and begin to unravel the extracellular events that occur during stimulation through the Vγ9Vδ2 T cell receptor.     

Molecular Basis of the Recognition of Nephronectin by Integrin ��8��1

Integrin α8β1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of α8β1 integrin toward a panel of RGD-containing ligands. Integrin α8β1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 ± 0.01 nm, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to α8β1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central “linker” segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to α8β1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of α8β1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was ∼2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to α8β1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to α8β1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.Integrins are a family of adhesion receptors that interact with a variety of extracellular ligands, typically cell-adhesive proteins in the extracellular matrix (ECM).² They play mandatory roles in embryonic development and the maintenance of tissue architectures by providing essential links between cells and the ECM (1). Integrins are composed of two non-covalently associated subunits, termed α and β. In mammals, 18 α and 8 β subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers. Based on their ligand-binding specificities, ECM-binding integrins are classified into three groups, namely laminin-, collagen- and RGD-binding integrins (2, 3), of which the RGD-binding integrins have been most extensively investigated. The RGD-binding integrins include α5β1, α8β1, αIIbβ3, and αV-containing integrins, and have been shown to interact with a variety of ECM ligands, such as fibronectin and vitronectin, with distinct binding specificities.The α8 integrin subunit was originally identified in chick nerves (4). Integrin α8β1 is expressed in the metanephric mesenchyme and plays a crucial role in epithelial-mesenchymal interactions during the early stages of kidney morphogenesis. Disruption of the α8 gene in mice was found to be associated with severe defects in kidney morphogenesis (5) and stereocilia development (6). To date, α8β1 integrin has been shown to bind to fibronectin, vitronectin, osteopontin, latency-associated peptide of transforming growth factor-β1, tenascin-W, and nephronectin (also named POEM) (7–13), among which nephronectin is believed to be an α8β1 integrin ligand involved in kidney development (10).Nephronectin is one of the basement membrane proteins whose expression and localization patterns are restricted in a tissue-specific and developmentally regulated manner (10, 11). Nephronectin consists of five epidermal growth factor-like repeats, a linker segment containing the RGD cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor protein-tyrosine phosphatase μ (MAM) domain (see Fig. 3A). Although the physiological functions of nephronectin remain only poorly understood, it is thought to play a role in epithelial-mesenchymal interactions through binding to α8β1 integrin, thereby transmitting signals from the epithelium to the mesenchyme across the basement membrane (10). Recently, mice deficient in nephronectin expression were produced by homologous recombination (14). These nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype reminiscent of α8 integrin knock-out mice (14), despite the fact that other RGD-containing ligands, including fibronectin and osteopontin, were expressed in the embryonic kidneys (9, 15). The failure of the other RGD-containing ligands to compensate for the deficiency of nephronectin in the developing kidneys suggests that nephronectin is an indispensable α8β1 ligand that plays a mandatory role in epithelial-mesenchymal interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its fragments. A, schematic diagrams of full-length nephronectin (NN) and its fragments. RGD-linker and RGD-linker (GST), the central RGD-containing linker segments expressed in mammalian and bacterial expression systems, respectively; PRGDV, a short RGD-containing peptide modeled after nephronectin and expressed as a GST fusion protein (see Fig. 4A for the peptide sequence). The arrowheads indicate the positions of the RGD motif. B, purified recombinant proteins were analyzed by SDS-PAGE in 7–15% gradient (left and center panels) and 12% (right panels) gels, followed by Coomassie Brilliant Blue (CBB) staining, immunoblotting with an anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for blotting with anti-FLAG and PNA) in the left and center panels;1 μg in the right panel. C, recombinant proteins (10 nm) were coated on microtiter plates and assessed for their binding activities toward α8β1 integrin (10 nm) in the presence of 1 mm Mn²⁺. The backgrounds were subtracted as described in the legend to Fig. 2. The results represent the mean ± S.D. of triplicate determinations. D, titration curves of α8β1 integrin bound to full-length nephronectin (NN, closed squares), the RGD-linker segments expressed in 293F cells (RGD-linker, closed triangles) and E. coli (RGD-linker (GST), open triangles), the MAM domain (MAM, closed diamonds), and the PRGDV peptide expressed as a GST fusion protein in E. coli (PRGDV (GST), open circles). The assays were performed as described in the legend to Fig. 2B. The results represent the means of duplicate determinations.Although ligand recognition by RGD-binding integrins is primarily determined by the RGD motif in the ligands, it is the residues outside the RGD motif that define the binding specificities and affinities toward individual integrins (16, 17). For example, α5β1 integrin specifically binds to fibronectin among the many RGD-containing ligands, and requires not only the RGD motif in the 10th type III repeat but also the so-called “synergy site” within the preceding 9th type III repeat for fibronectin recognition (18). Recently, DiCara et al. (19) demonstrated that the high-affinity binding of αVβ6 integrin to its natural ligands, e.g. foot-and-mouth disease virus, requires the RGD motif immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to align the two conserved hydrophobic residues along the length of the helix. Given the presence of many naturally occurring RGD-containing ligands, it is conceivable that the specificities of the RGD-binding integrins are dictated by the sequences flanking the RGD motif or those in neighboring domains that come into close proximity with the RGD motif in the intact ligand proteins. However, the preferences of α8β1 integrin for RGD-containing ligands and how it secures its high-affinity binding toward its preferred ligands remain unknown.In the present study, we investigated the binding specificities of α8β1 integrin toward a panel of RGD-containing cell-adhesive proteins. Our data reveal that nephronectin is a preferred ligand for α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side of its RGD motif serves as a synergy site to ensure the specific high-affinity binding of nephronectin to α8β1 integrin.     

Production of α-Galactosylceramide by a Prominent Member of the Human Gut Microbiota

While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer_Bf), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer_Bf has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.     

Human Monocyte Recognition of Adenosine-Based Cyclic Dinucleotides Unveils the A2a Gαs Protein-Coupled Receptor Tonic Inhibition of Mitochondrially Induced Cell Death

Cyclic dinucleotides are important messengers for bacteria and protozoa and are well-characterized immunity alarmins for infected mammalian cells through intracellular binding to STING receptors. We sought to investigate their unknown extracellular effects by adding cyclic dinucleotides to the culture medium of freshly isolated human blood cells in vitro. Here we report that adenosine-containing cyclic dinucleotides induce the selective apoptosis of monocytes through a novel apoptotic pathway. We demonstrate that these compounds are inverse agonist ligands of A2a, a G_αs-coupled adenosine receptor selectively expressed by monocytes. Inhibition of monocyte A2a by these ligands induces apoptosis through a mechanism independent of that of the STING receptors. The blockade of basal (adenosine-free) signaling from A2a inhibits protein kinase A (PKA) activity, thereby recruiting cytosolic p53, which opens the mitochondrial permeability transition pore and impairs mitochondrial respiration, resulting in apoptosis. A2a antagonists and inverse agonist ligands induce apoptosis of human monocytes, while A2a agonists are antiapoptotic. In vivo, we used a mock developing human hematopoietic system through NSG mice transplanted with human CD34⁺ cells. Treatment with cyclic di-AMP selectively depleted A2a-expressing monocytes and their precursors via apoptosis. Thus, monocyte recognition of cyclic dinucleotides unravels a novel proapoptotic pathway: the A2a G_αs protein-coupled receptor (GPCR)-driven tonic inhibitory signaling of mitochondrion-induced cell death.     

T Cell Factor-1 Controls the Lifetime of CD4+ CD8+ Thymocytes In Vivo and Distal T Cell Receptor α-Chain Rearrangement Required for NKT Cell Development

Natural killer T (NKT) cells are a component of innate and adaptive immune systems implicated in immune, autoimmune responses and in the control of obesity and cancer. NKT cells develop from common CD4+ CD8+ double positive (DP) thymocyte precursors after the rearrangement and expression of T cell receptor (TCR) Vα14-Jα18 gene. Temporal regulation and late appearance of Vα14-Jα18 rearrangement in immature DP thymocytes has been demonstrated. However, the precise control of lifetime of DP thymocytes in vivo that enables distal rearrangements remains incompletely defined. Here we demonstrate that T cell factor (TCF)-1, encoded by the Tcf7 gene, is critical for the extended lifetime of DP thymocytes. TCF-1-deficient DP thymocytes fail to undergo TCR Vα14-Jα18 rearrangement and produce significantly fewer NKT cells. Ectopic expression of Bcl-x_L permits Vα14-Jα18 rearrangement and rescues NKT cell development. We report that TCF-1 regulates expression of RORγt, which regulates DP thymocyte survival by controlling expression of Bcl-x_L. We posit that TCF-1 along with its cofactors controls the lifetime of DP thymocytes in vivo.     

A Serine/Threonine Phosphorylation Site in the Ectodomain of a T Cell Receptor β Chain is Required for Activation by Superantigen

Abstract

The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins suggests that such post‐translational modification may be important in regulation of surface receptor activity. To date, the only cell surface receptor for which such ectodomain phosphorylation has been conclusively demonstrated is the clonally expressed T cell antigen receptor (TCR). Attempts to conclusively identify individual phosphorylated residues in TCR α and β chains and determine their functional significance by biochemical approaches failed due to insufficient quantities of purified molecules. Here we present the results of an alternative approach where survey of phosphorylation sites in the TCR α and β chains was accomplished using site‐directed mutagenesis and retroviral vector expression, as well as in vitro phosphorylation of synthetic peptide substrates. All mutants studied directed the cell surface expression of normal amounts of TCR, and all transfectants could be stimulated to produce IL‐2 in response to substrate‐immobilized antibody to TCR. However, mutation of serine‐88 in the protein kinase A phosphorylation site of the TCR β chain resulted in a complete lack of response to the superantigen staphylococcal enterotoxin B (SEB). In addition, this mutation abolished TCR‐associated tyrosine phosphorylation, consistent with the impairment of cell signaling. Reversion of the serine‐88/alanine mutation with phosphorylatable threonine completely restored the SEB recognition by TCR. These results, interpreted in the context of the known three‐dimensional structure of the complex of SEB and TCR, are consistent with the view that serine‐88 is important for the contact of the TCR β chain with SEB.     

The Molecular Basis of Voltage-gated Ca2+ Channel Diversity: Is It Time for T?

The existence of diversity in the voltage activated Ca²⁺ channel populations of vertebrate cells has been long recognized. More recently, the molecular cloning of a considerable number of Ca²⁺ channel subunits from cDNA libraries has indicated that the range of possible Ca²⁺ channel phenotypes a cell can express may be even greater than was previously appreciated. A challenge of recent years has been to resolve how the properties of recombinant channels correspond with their counterparts experimentally characterized in native cells. In this short review I will outline the properties of both native and recombinant Ca²⁺ channels, and will then describe the current agreements and controversies concerning their relationships to each other. Received: 14 July 1997/Revised: 4 November 1997     

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion ��-Toxins

The scorpion α-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na_vs) and is inactive at insect Na_vs. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa_v1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved “core domain” (residues of the loops connecting the secondary structure elements of the molecule core), and a variable “NC domain” (five-residue turn and the C-tail) as was reported for other scorpion α-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhαIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe¹⁵ of the core domain in toxin potency at rNa_v1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion α-toxins we were able to eliminate the activity of Lqh2 at rNa_v1.4 (skeletal muscle), hNa_v1.5 (cardiac), and rNa_v1.6 channels, with no hindrance of its activity at Na_v1.1–1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.The pivotal role of voltage-gated sodium channels (Na_vs)⁴ in excitability mark them as major targets for a large variety of toxins that bind at distinct receptor sites and modify their gating (1). These channels are large membrane proteins made of a pore-forming α-subunit of ∼260 kDa and auxiliary β-subunits of ∼30 kDa. The α-subunit is composed of four homologous domains (D1–D4), each consisting of six α-helical transmembrane segments (S1–S6) connected by intracellular and extracellular loops. A key feature in Na_vs function is their ability to rapidly activate and inactivate, leading to transient increase in Na⁺ conductance through the cell membrane. This mechanism is attributed to the ability of the positively charged S4 voltage sensors to move across the membrane in response to changes in membrane potential (1, 2).In mammals, at least nine genes encode a variety of Na_v subtypes (1, 3), whose expression varies greatly in different tissues (Na_v1.1–1.3 mainly in the central nervous system; Na_v1.6 in both central and peripheral neurons; Na_v1.7 in the peripheral nervous system; Na_v1.8 and Na_v1.9 in sensory neurons; Na_v1.4 and Na_v1.5 in skeletal and cardiac muscles, respectively). Na_v subtypes are distributed heterogeneously in the human brain and their expression is regulated under developmental and pathological conditions (1, 3–5). In addition, many disorders in humans result from abnormal function due to mutations in various Na_v genes (6–8). Thus, ligands that show specificity for Na_v subtypes may be used for their identification at various tissues and as leads for design of specific drugs. This requires that the bioactive surfaces of these ligands be resolved along with molecular details that determine their specificity.Among the wide range of Na_v modifiers, those derived from scorpion venoms play an important role in studying channel activation (β-toxins) and inactivation (α-toxins) (9–11). The channel site of interaction with scorpion α-toxins, named neurotoxin receptor site-3 (12), is shared also by structurally unrelated toxins from sea anemone and spider venoms (13, 14), which raises questions as to its architecture and boundaries. Based on the findings that site-3 toxins eliminate a gating charge component associated with the movement of D4/S4 (15, 16), and that this segment plays a critical role in coupling channel inactivation to activation (17), scorpion α-toxins were postulated to inhibit channel inactivation by hindering the outward movement of this segment during depolarization (9).Scorpion α-toxins constitute a class of structurally and functionally related 61–67-residue long polypeptides reticulated by four conserved disulfide bridges. Despite a common βαββ core (10, 18, 19) these toxins are highly diverse in sequence and preference for insect and mammalian Na_vs. Indeed, the α-toxin class is divided to pharmacological groups according to their toxicity in insects and mice brain and ability to compete on binding at insect and mammalian Na_vs (10) (supplemental Fig. S1): (i) classical anti-mammalian toxins, such as Aah2 (from Androctonus australis hector) and Lqh2 (from Leiurus quinquestriatus hebraeus), which bind with high affinity to Na_vs at rat brain synaptosomes and are practically non-toxic to insects; (ii) α-toxins, such as LqhαIT, which strongly affect insect Na_vs and are weak in mammalian brain; and (iii) α-like toxins, such as Lqh3 and BmKM1 (from Buthus martensii Karsch), which are active in both mammalian brain and insects.Efforts to identify α-toxin residues involved in the interaction with the Na_v receptor site-3 revealed a generally common bioactive surface divided to two topologically distinct domains: a conserved “core domain” formed by residues of the loops connecting the secondary structure elements of the molecule core, and a variable “NC domain” formed by the five-residue turn (residues 8–12) and the C-tail (20–23). These analyses raised the hypothesis that a protruding conformation of the NC domain correlates with high activity at insect Na_vs, whereas a flat conformation of this domain appears in α-toxins active at the brain channel rNa_v1.2a (21). The correlation of this structural difference with toxin preference for Na_v subtypes was corroborated by constructing the bioactive surface of LqhαIT on the scaffold of the anti-mammalian α-toxin Aah2 ending up with a chimera (Aah2^{LqhαIT(face)}) active on insects, whose NC domain is in the protruding conformation (21). Despite this result, the molecular requirements that enable high affinity binding of classical α-toxins to mammalian Na_vs have not been clarified, and only initial data about the channel region that constitutes receptor site-3 is available (Refs. 24–26; also see Ref. 10 for review).Lqh2 is a 64-residue long toxin from L. quinquestriatus hebraeus (Israeli yellow scorpion) (27) that is almost identical in sequence (96% identity) to the most active anti-mammalian toxin, Aah2, whose structure and action are documented (18, 28, 29). By functional expression and mutagenesis we uncovered residues on the Lqh2 exterior that are putatively involved in bioactivity. By construction of these residues on the scaffold of the anti-insect toxin LqhαIT we confirmed their bioactive role and differentiated those that determine toxin potency from those contributing to toxin efficacy. Comparison to other α-toxins was then instrumental for the design of an Lqh2 mutant that exhibits high specificity for the neuronal channels hNa_v1.1, rNa_v1.2a, and rNa_v1.3.     

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8⁺ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8⁺ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8⁺ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8⁺ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8⁺ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.     

Human Adenovirus-Specific γ/δ and CD8+ T Cells Generated by T-Cell Receptor Transfection to Treat Adenovirus Infection after Allogeneic Stem Cell Transplantation

Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected α/β T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into γ/δ T cells, which are not allo-reactive. TCR-transfected γ/δ T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8α-encoding mRNA. In direct comparison with TCR-transfected α/β T cells, TCR-CD8α-co-transfected γ/δ T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected α/β T cells and TCR-CD8α-co-transfected γ/δ T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only α/β T cells but also γ/δ T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.     

Lipid and Carbohydrate Modifications of α-Galactosylceramide Differently Influence Mouse and Human Type I Natural Killer T Cell Activation

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from α-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type I NKT cells in in vivo or in vitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators.     

Effect of Human WEE1 and Stem Cell Factor on Human CD34^＋ Umbilical Cord Blood Cell Damage Induced by Chemotherapeutic Agents

Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE l （WEElHu） and proliferation-stimulating stem cell factor （SCF） was generated. In this study, we selected human umbilical cord blood CD34^＋ cells as the in vitro model in an attempt to investigate whether WEEIHu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay, colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEElHu and SCF in CD34^＋ cells provided the cells with some protection. These findings suggest that the expression of WEElHu and SCF might rescue CD34^＋ cells from chemotherapyinduced myelosuppression.     

Suppression of Tcf1 by Inflammatory Cytokines Facilitates Effector CD8 T Cell Differentiation

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Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting β cells renders them susceptible to the autoimmune attack, and is associated with a diminution in β-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on β cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in β cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing β cells is implicated in β-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated.