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.     

Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr–296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr–296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr–296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr–296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr–296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr–296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.     

Contrasting Diversity and Host Association of Ectomycorrhizal Basidiomycetes versus Root-Associated Ascomycetes in a Dipterocarp Rainforest

Root-associated fungi, including ectomycorrhizal and root-endophytic fungi, are among the most diverse and important belowground plant symbionts in dipterocarp rainforests. Our study aimed to reveal the biodiversity, host association, and community structure of ectomycorrhizal Basidiomycota and root-associated Ascomycota (including root-endophytic Ascomycota) in a lowland dipterocarp rainforest in Southeast Asia. The host plant chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) region and fungal internal transcribed spacer 2 (ITS2) region were sequenced using tag-encoded, massively parallel 454 pyrosequencing to identify host plant and root-associated fungal taxa in root samples. In total, 1245 ascomycetous and 127 putative ectomycorrhizal basidiomycetous taxa were detected from 442 root samples. The putative ectomycorrhizal Basidiomycota were likely to be associated with closely related dipterocarp taxa to greater or lesser extents, whereas host association patterns of the root-associated Ascomycota were much less distinct. The community structure of the putative ectomycorrhizal Basidiomycota was possibly more influenced by host genetic distances than was that of the root-associated Ascomycota. This study also indicated that in dipterocarp rainforests, root-associated Ascomycota were characterized by high biodiversity and indistinct host association patterns, whereas ectomycorrhizal Basidiomycota showed less biodiversity and a strong host phylogenetic preference for dipterocarp trees. Our findings lead to the working hypothesis that root-associated Ascomycota, which might be mainly represented by root-endophytic fungi, have biodiversity hotspots in the tropics, whereas biodiversity of ectomycorrhizal Basidiomycota increases with host genetic diversity.     

Medical Decision-Making Incapacity among Newly Diagnosed Older Patients with Hematological Malignancy Receiving First Line Chemotherapy: A Cross-Sectional Study of Patients and Physicians

Background

Decision-making capacity to provide informed consent regarding treatment is essential among cancer patients. The purpose of this study was to identify the frequency of decision-making incapacity among newly diagnosed older patients with hematological malignancy receiving first-line chemotherapy, to examine factors associated with incapacity and assess physicians’ perceptions of patients’ decision-making incapacity.

Methods

Consecutive patients aged 65 years or over with a primary diagnosis of malignant lymphoma or multiple myeloma were recruited. Decision-making capacity was assessed using the Structured Interview for Competency and Incompetency Assessment Testing and Ranking Inventory-Revised (SICIATRI-R). Cognitive impairment, depressive condition and other possible associated factors were also evaluated.

Results

Among 139 eligible patients registered for this study, 114 completed the survey. Of these, 28 (25%, 95% confidence interval [CI]: 17%-32%) were judged as having some extent of decision-making incompetency according to SICIATRI-R. Higher levels of cognitive impairment and increasing age were significantly associated with decision-making incapacity. Physicians experienced difficulty performing competency assessment (Cohen’s kappa -0.54).

Conclusions

Decision-making incapacity was found to be a common and under-recognized problem in older patients with cancer. Age and assessment of cognitive impairment may provide the opportunity to find patients that are at a high risk of showing decision-making incapacity.     

Prolyl Aminopeptidase from Streptomyces thermoluteus subsp. fuscus Strain NBRC14270 and Synthesis of Proline-Containing Peptides by Its S144C Variant

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270) was obtained using sequence-based screening. From {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.Prolyl aminopeptidase (PAP) (EC 3.4.11.5), belonging to the S33 family, is an exopeptidase that catalyzes the hydrolysis of the N terminus prolyl residue of peptides or proteins. This family has catalytic Ser. To date, few applications of this enzyme for peptide synthesis have been reported. However, from the perspective of biotechnology, PAP might be a good tool for synthesizing proline-containing peptides by catalyzing aminolysis.Recently nutraceutical properties of peptides containing proline have received increasing attention. For example, prolyl hydroxyproline (Pro-Hyp) stimulates the growth of fibroblasts from mouse skin (11). Pro-Arg can protect against oxidative stress/damage and H₂O₂-induced human diploid fibroblast cell death (13). Furthermore, the lactotripeptides Ile-Pro-Pro and Val-Pro-Pro exhibit angiotensin I-converting enzyme-inhibiting activity (9). In addition to these dipeptides and tripeptides, a cyclic dipeptide (namely, diketopiperazine) containing proline shows several physiological functions. Cyclo[Pro-Pro] (cPP) exerts antibacterial activity against Micrococcus luteus and Pseudomonas aeruginosa (8). Caspase-3 activation by cyclo[Pro-Phe] in HT-29 cells has been described (3). However, its synthesis method has not been established. Enzymatic peptide synthesis presents a useful and desirable strategy because it can conduct specific reactions under milder conditions than those of chemical synthesis.Engineered endoserine proteases that have Cys substituted for catalytic Ser have also been applied for peptide synthesis since subtiligase was constructed by Abrahmsén et al. (1). Because of the weakened hydrolytic activity of the parent enzyme, it is considered that Ser/Cys-substituted protease can trap the substrate (acyl donor). Then, a nucleophilic reaction occurs between another substrate (acyl acceptor) and the trapped acyl donor (2). This is a so-called “aminolysis” reaction. Although aminolysis can conduct peptide synthesis in an aqueous solution, the problem of the necessity of using an N-protected amino acid as an acyl donor remains when using endoproteases.These problems would be solved using exoprotease as a catalyst, because N-terminal free amino groups of acyl donors are recognized by enzymes. It is rarely reported that exoprotease was applied for peptide synthesis, except in the report of Oshiro et al., in which Pro-Phe, Pro-Tyr, and Pro-Trp were synthesized (10). Recently our group reported that the Ser/Cys variant of exoprotease, aminolysin-S, has been constructed and has produced l-Phe-l-Phe ethyl ester and their derivatives from non-N-protected phenylalanine and phenylalanine ethyl ester as acyl donors in aqueous solution (12). However, aminolysin-S cannot produce proline-containing dipeptides.In this study, we describe a PAP from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270). Furthermore, synthesis of various proline peptides was attempted through catalysis by its Ser/Cys variant (scPAP14270) from proline ester and several amino acids and their esters in aqueous solution. A basic characterization to determine the effect of pH and the amount of substrate was conducted. Moreover, correlation was found between proline peptide synthesis and the log D value, which is the distribution coefficient between octanol and water, of acyl acceptors in aminolysis mediated by scPAP14270.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

DNA-fiber EPR investigation of the influence of amino-terminal residue stereochemistry on the DNA binding orientation of Cu(II).Gly-Gly-His-derived metallopeptides

DNA fiber EPR was used to investigate the DNA binding stabilities and orientations of Cu(II).Gly-Gly-His-derived metallopeptides containing D- vs. L-amino acid substitutions in the first peptide position. This examination included studies of Cu(II).D-Arg-Gly-His and Cu(II).D-Lys-Gly-His for comparison to metallopeptides containing L-Arg/Lys substitutions, and also the diastereoisomeric pairs Cu(II).D/L-Pro-Gly-His and Cu(II).D/L-Pro-Lys-His. Results indicated that L-Arg/Lys to D-Arg/Lys substitutions considerably randomized the orientation of the metallopeptides on DNA, whereas the replacement of L-Pro by D-Pro in Cu(II).L-Pro-Gly-His caused a decrease in randomness. The difference in the extent of randomness observed between the D- vs. L-Pro-Gly-His complexes was diminished through the substitution of Gly for Lys in the middle peptide position, supporting the notion that the epsilon-amino group of Lys triggered further randomization, likely through hydrogen bonding or electrostatic interactions that disrupt binding of the metallopeptide equatorial plane and the DNA. The relationship between the stereochemistry of amino acid residues and the binding and reaction of M(II).Xaa-Xaa'-His metallopeptides with DNA are also discussed.