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Regulatory T (Treg) cells are necessary for the maintenance of allogenic pregnancy. However, the repertoire of effector Treg cells at the feto-maternal interface in human pregnancy remains unknown. Our objective was to study T cell receptor (TCR) repertoires of Treg cells during pregnancy compared to normal and complicated pregnancies. Paired samples of peripheral blood and decidua in induced abortion and miscarriage cases were obtained from consenting patients. CD4 CD25 CD127 CD45RA effector Treg cells were single-cell sorted from mononuclear cells. cDNAs of complementarity determining region 3 (CDR3) in TCRβ were amplified from the single cells by RT-PCR and the sequences were analyzed. The TCRβ repertoires were determined by amino acid and nucleotide sequences. Treg cells were classified into clonally expanded and non-expanded populations by CDR3 sequences. We enrolled nine induced abortion cases in the 1st trimester, 12 cases delivered without complications in the 3rd trimester, 11 miscarriages with abnormal chromosomal karyotyped embryo, seven miscarriages with normal chromosomal karyotyped embryo, and seven cases of preeclampsia [median gestational week (interquartile range): 7 (7-9), 39 (38-40), 9 (8-10), 8 (8-10), and 34 (32-37), respectively]. The frequency of clonally expanded populations of effector Treg cells increased in decidua of 3rd trimester cases compared to 1st trimester cases [4.5% (1.4-10.8%) vs. 20.9% (15.4-28.1%), < 0.001]. Clonally expanded Treg cells were rarely seen in peripheral blood. The ratio of clonally expanded populations of decidual effector Treg cells in miscarriages with abnormal and normal embryos was not significantly different compared with that in 1st trimester normal pregnancy. Interestingly, clonally expanded populations of effector Treg cells decreased in preeclampsia compared with that in 3rd trimester normal pregnancy [9.3% (4.4-14.5%) vs. 20.9% (15.4-28.1%), = 0.003]. When repertoires in previous pregnancy and subsequent pregnancy were compared, some portions of the repertoire were shared. TCR repertoires of decidual effector Treg cells are skewed in the 3rd trimester of normal pregnancy. Failure of clonal expansion of populations of decidual effector Treg cells might be related to the development of preeclampsia.

T‐cell receptor (TCR)‐like Abs that specifically recognize antigenic peptides presented on MHC molecules have been developed for next‐generation cancer immunotherapy. Recently, we reported a rapid and efficient method to generate TCR‐like Abs using a rabbit system. We humanized previously generated rabbit‐derived TCR‐like Abs reacting Epstein–Barr virus peptide (BRLF1p, TYPVLEEMF) in the context of HLA‐A24 molecules, produced chimeric antigen receptor (CAR)‐T cells, and evaluated their antitumor effects using in vitro and in vivo tumor models. Humanization of the rabbit‐derived TCR‐like Abs using the complementarity‐determining region grafting technology maintained their specificity and affinity. We prepared a second‐generation CAR using single‐chain variable fragment of the humanized TCR‐like Abs and then transduced them into human T cells. The CAR‐T cells specifically recognized BRLF1p/MHC molecules and lysed the target cells in an antigen‐specific manner in vitro. They also demonstrated antitumor activity in a mouse xenograft model. We report the generation of CAR‐T cells using humanized rabbit‐derived TCR‐like Abs. Together with our established and efficient generation procedure for TCR‐like Abs using rabbits, our platform for the clinical application of humanized rabbit‐derived TCR‐like Abs to CAR‐T cells will help improve next‐generation cancer immunotherapy. The humanized TCR‐like antibody had a similar specificity and affinity for BRLF1/A24 as the original rabbit antibody. The TCR‐like CAR‐T cells generated from the humanized rabbit‐derived TCR‐like antibody demonstrated efficient cytotoxicity in vitro and in vivo.

Jin and colleagues introduce a new chip-based system for the rapid identification and isolation of single antigen-specific antibody–secreting cells from human peripheral blood lymphocytes. The approach can be used to detect antibody-secreting cells for multiple antigens on the same chip and should have advantages over current technologies for isolating and producing human monoclonal antibodies of clinical significance. Antigen-specific human monoclonal antibodies (mAbs) are key candidates for therapeutic agents. However, the availability of a suitable screening system for antigen-specific antibody–secreting cells (ASCs) is limited in humans. Here we present a unique method for detecting individual ASCs using microwell array chips, which enables the analysis of live cells on a single-cell basis and offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for identifying and recovering objective ASCs. We applied the system to detect and retrieve ASCs for hepatitis B virus and influenza viruses from human peripheral blood lymphocytes and produced human mAbs with virus-neutralizing activities within a week. Furthermore, we show that the system is useful for detecting ASCs for multiple antigens as well as for selection of ASCs secreting high-affinity antibodies on a chip. Our method can open the way for the generation of therapeutic antibodies for individual patients.

G-protein-coupled receptors (GPCRs) are one of the most important drug targets and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.

To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals. Using an HLA-A*02-restricted cytomegalovirus (CMV) pp65-derived immunogenic peptide, we found that the more dominant pp65-specific TCR clonotypes in the blood of healthy donors have higher binding affinities for the CMV peptide and arise from clonotypes that are highly shared among individuals. Interestingly, these highly shared HLA-A*02-restricted CMV-specific TCRs were detected in a CMV-seronegative individual as well as in HLA-A*02-negative donors albeit at lower frequency. More intriguingly, these shared TCR clonotypes were abundant in the stem memory T-cell subset, and TCR diversity of the stem memory T-cell repertoire was significantly lower than in the central memory and effector memory T-cell repertoires. These results suggest that the stem memory T-cell subset may serve as a reservoir of highly shared and highly functional memory T-cells.

Citrus mosaic virus (CiMV) is one of the causal viruses of citrus mosaic disease in satsuma mandarins (Citrus unshiu). Prompt detection of trees infected with citrus mosaic disease is important for preventing the spread of this disease. Although rabbit monoclonal antibodies (mAbs) exhibit high specificity and affinity, their applicability is limited by technical difficulties associated with the hybridoma-based technology used for raising these mAbs. Here, we demonstrate a feasible CiMV detection system using a specific rabbit mAb against CiMV coat protein. A conserved peptide fragment of the small subunit of CiMV coat protein was designed and used to immunize rabbits. Antigen-specific antibody-producing cells were identified by the immunospot array assay on a chip method. After cloning of variable regions in heavy or light chain by RT-PCR from these cells, a gene set of 33 mAbs was constructed and these mAbs were produced using Expi293F cells. Screening with the AlphaScreen system revealed eight mAbs exhibiting strong interaction with the antigen peptide. From subsequent sequence analysis, they were grouped into three mAbs denoted as No. 4, 9, and 20. Surface plasmon resonance analysis demonstrated that the affinity of these mAbs for the antigen peptide ranged from 8.7 × 10-10 to 5.5 × 10-11 M. In addition to CiMV, mAb No. 9 and 20 could detect CiMV-related viruses in leaf extracts by ELISA. Further, mAb No. 20 showed a high sensitivity to CiMV and CiMV-related viruses, simply by dot blot analysis. The anti-CiMV rabbit mAbs obtained in this study are envisioned to be extremely useful for practical applications of CiMV detection, such as in a virus detection kit.

The aberrant activation of receptor tyrosine kinases (RTKs) is associated with tumor initiation in various types of human cancer, including non-small cell lung cancers (NSCLCs). Tyrosine kinase-independent non-canonical RTK regulation has also been investigated in tumor malignant alterations, including cellular stress responses. It was recently reported that the phosphorylation of epidermal growth factor receptor (EGFR) at C-terminal Ser-1015 serves a critical role in growth factor and cytokine signaling. In the present study, the role of non-canonical EGFR regulation has been investigated in NSCLC cells treated with cisplatin, a common chemotherapeutic agent. Cisplatin-induced p38 activation triggered the Ser-1015 phosphorylation of EGFR, with similar kinetics to previously reported Ser-1047 phosphorylation, in a tyrosine kinase-independent manner. In addition, phosphorylation around Ser-1015 triggered endocytosis of a dimer deficient mutant of EGFR. The non-canonical endocytosis of EGFR monomers was primarily controlled by the region around Ser-1015 only; however, Ser-1047 on internalized EGFR was equally phosphorylated. The results of the present study provide mechanistic evidence for the cisplatin-induced non-canonical regulation of EGFR.

Tyrosine kinase activity of the asymmetric EGFR homodimer is negatively regulated via ERK-mediated phosphorylation of Thr-669 in the juxtamembrane domain. In the present study, we investigated in human breast cancer cells whether a similar mechanism plays a role in the feedback regulation of the ErbB2/ErbB3 heterodimer, the most potent ErbB receptor dimer. Constitutive tyrosine phosphorylation of ErbB2 and ErbB3 was significantly decreased in phorbol ester- and growth factor-treated BT-474 and MDA-MB-453 cells. In contrast to the decreased tyrosine phosphorylation, Phos-tag Western blot analysis revealed that TPA induced phosphorylation of ErbB2 in an ERK-dependent manner. The target threonine residue corresponding to EGFR Thr-669 and the surrounding residues are highly conserved in ErbB2, but not in ErbB3. Therefore, we demonstrated ERK-mediated phosphorylation of ErbB2 at Thr-677 by generating phospho-specific monoclonal antibodies. Moreover, treatment with trametinib and SCH772984, inhibitors of the MEK-ERK pathway and substitution of Thr-677 to alanine impaired the feedback inhibition of ErbB2 and ErbB3. These results demonstrated that ERK-mediated phosphorylation of the conserved threonine is a common mechanism for the negative feedback control of active ErbB receptor dimers.

Doublecortin-like kinase 1 (DCLK1) is considered a putative tumor stem cell (TSC) marker and a promising therapeutic target, as DCLK1 progeny cells exhibit high expression in tumors. However, the biological function of DCLK1 cells in tumorigenesis and tumor progression remains unclear. We generated rabbit monoclonal antibodies (mAbs) against DCLK1, DCLK1-42, and DCLK1-87 mAbs, using a novel chip-based immunospot array assay on a chip system. First, the specificity of two mAbs to DCLK1 was confirmed by Western blot, which were bound to DCLK1-long in normal colon cells and to DCLK1-short in a cancer cell line as well as colorectal cancer (CRC) cells. Precise localization analysis using immunofluorescence revealed that both mAbs had cytoplasmic signal and exhibited a high degree of overlap with microtubules. Furthermore, bacterial display technology indicated that the antigenic epitope region of DCLK1-87 mAb was consistent with that of a commercial anti-DCLK1 polyclonal antibody. In addition, DCLK1-42 mAb has the common polyclonal antibody characteristic of binding to more than one site on DCLK1. By immunohistochemistry, it was found that DCLK1-87 mAb was more specific for DCLK1 cell labeling than a commercial anti-DCLK1 polyclonal antibody. DCLK1 labeled with DCLK1-87 mAb might be a potential TSC marker because the tissue expression site covers the ALDH1 area in CRC tissues. Finally, we analyzed 100 pairs of cancer tissues and matching paracancerous tissue samples from patients with CRC who received 100 months of follow-up with the DCLK1-87 mAb. The results showed that patients with high DCLK1 expression exhibited a longer survival time than that of patients with low DCLK1 expression ( =0.0029). Our results indicated that we successfully generated an efficient tool for the precise detection of DCLK1 cells in cancer tissues. Moreover, we found that high DCLK1 expression in CRC patients appears to play a protective role against tumor progression.