Tatsuhiko Ozawa

A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood.

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.

Single lymphocyte analysis with a microwell array chip.

Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for analyzing large populations of cells on a single‐cell basis with multiple parameters in a quantitative manner. We have developed a highly integrated live‐cell microarray system for analyzing the cellular responses of individual cells using a microwell array chip that has 234,000 microwells each of which is just large enough to fit a single cell. Compared with flow cytometry and microscope‐based methods, our system can analyze the history of the cellular responses of a large number of cells. We have successfully applied the system to analyze human antigen‐specific B‐cells and produced human monoclonal antibodies (MoAb) against hepatitis B virus surface antigen. We have also constructed a mouse system to assess hepatitis B virus‐neutralization activity and have demonstrated the neutralization activity of our antibodies. Our technology should expand the horizons of cell analysis as well as enable generation of human MoAb for antibody‐based therapeutics and diagnosis for infectious diseases such as hepatitis viruses.

Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity.

The authors previously developed a cell‐microarray system that effectively detects antigen‐specific B‐cells by monitoring intracellular Ca2+ at single cell levels. Here they present a novel method to detect antigen‐specific B‐cells using cell‐microarray system. To detect antigen‐specific B‐cells, they arrayed live lymphocytes on a chip, stained cells with fluorescence‐labeled nonspecific proteins, and analyzed them with a fluorescence scanner to detect nonspecific protein binding to B‐cells. They then stained cells with fluorescence‐labeled antigen and analyzed them with the scanner. Cells stained with specific antigen, but not with nonspecific proteins, were determined as antigen‐specific B‐cells and harvested. Antibody cDNA was amplified from retrieved B‐cells by single‐cell RT‐PCR, inserted into expression vectors, and was examined for its specificity by ELISA. They could detect antigen‐specific B‐cells at a frequency of 0.01% in a model system using transgenic mice that express antibody to hen‐egg lysozyme on the surface of B‐cells. They applied this system to directly detect hepatitis B virus surface‐antigen (HBs‐Ag)‐specific B‐cells from peripheral blood in HBs‐Ag‐vaccinated volunteers and succeeded in producing HBs‐Ag‐specific monoclonal antibody. This novel system allows us to identify human antigen‐specific B‐cells of very low frequency and is a powerful tool to explore the candidates of antibody therapeutics.

A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days.

Antigen-specific T cell therapy, or T cell receptor (TCR) gene therapy, is a promising immunotherapy for infectious diseases and cancers. However, a suitable rapid and direct screening system for antigen-specific TCRs is not available. Here, we report an efficient cloning and functional evaluation system to determine the antigen specificity of TCR cDNAs derived from single antigen-specific human T cells within 10 d. Using this system, we cloned and analyzed 380 Epstein-Barr virus–specific TCRs from ten healthy donors with latent Epstein-Barr virus infection and assessed the activity of cytotoxic T lymphocytes (CTLs) carrying these TCRs against antigenic peptide–bearing target cells. We also used this system to clone tumor antigen–specific TCRs from peptide-vaccinated patients with cancer. We obtained 210 tumor-associated antigen–specific TCRs and demonstrated the cytotoxic activity of CTLs carrying these TCRs against peptide-bearing cells. This system may provide a fast and powerful approach for TCR gene therapy for infectious diseases and cancers.

Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay

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.

Rapid isolation of viral integration site reveals frequent integration of HTLV-1 into expressed loci

AbstractAlthough there is tight association of the human T-cell leukemia virus type-1 (HTLV-1) with adult T-cell leukemia/lymphoma (ATLL), it has remained unresolved whether the HTLV-1 integration into the host genome has any role in the development of this disease. We isolated a total of 58 HTLV-1 integration sites using newly developed, adaptor-ligated PCR from 33 ATLL patients and five ATLL cell lines. We compared our data as well as the previously reported ones with the complete human genomic sequence for the location of its placement, structure, and expression of genes nearby the integration site. The chromosomal target for integration was selected at random, but the integration favorably occurred within the transcription units; more than 59.5% of total integration was observed within the transcriptional unit. All inserted genes by HTLV-1 integration were expressed in normal T cells. Upregulation of genes due to viral integration was found in two out of nine ATLL cases; about 4.4- and 102-fold elevated ankyrin-1 (ANK-1) and gephyrin (GPHN) gene expressions were observed, respectively. These data suggest that the preferential integration of HTLV-1 into an expressed locus occasionally causes deregulation of corresponding gene, which may lead to leukemogenesis of a fraction of ATLL.

Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array

Here we report a new method for isolating antigen-specific antibody-secreting cells (ASCs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ASCs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. This protocol can be completed in less than 7 h, including 3 h of cell culture. The method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (ELISPOT) but it also overcomes the limitations of ELISPOT in recovering ASCs that can be used to produce antigen-specific human monoclonal antibodies. This method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.

Analysis of the epitope and neutralizing capacity of human monoclonal antibodies induced by hepatitis B vaccine.

Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issue. However, the mechanism by which vaccination-induced antibodies prevent HBV infection remains unclear. To investigate the mechanism by which antibodies induced by hepatitis B surface Ag (HBsAg)-vaccination prevent HBV infection in humans, we prepared human monoclonal antibodies (mAbs) against HBsAg using a novel cell-microarray system from peripheral blood B-lymphocytes from vaccinated individuals. We then characterized the IgG subclass, L-chain subtype, and V-gene repertoire of the H/L-chain, as well as affinities of each of these mAbs. We also determined the epitopes of the individual mAbs using synthesized peptides, and the HBV-neutralizing activities of mAbs using the hepatocyte cell line HepaRG. Consequently, IgG1 and kappa chain was mainly used as the mAbs for HBsAg. Seventy percent of the mAbs bound to the loop domain of the small-HBsAg and showed greater neutralizing activities. There were no relationships between their affinities and neutralization activities. A combination of mAbs recognizing the first loop domain showed a synergistic effect on HBV-neutralizing activity that surpassed conventional hepatitis B-Ig (HBIG) in the HepaRG cell line assay. These results may contribute to the development of effective mAb treatment against HBV infection replacing conventional HBIG administration.

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy.

Unbiased Analysis of TCRα/β Chains at the Single-Cell Level in Human CD8+ T-Cell Subsets

T-cell receptor (TCR) α/β chains are expressed on the surface of CD8+ T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5′-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8+ subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8+ subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8+ T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains.