Rapid cloning of antigen-specific T-cell receptors by leveraging the cis activation of T cells.

It is commonly understood that T cells are activated via trans interactions between antigen-specific T-cell receptors (TCRs) and antigenic peptides presented on major histocompatibility complex (MHC) molecules on antigen-presenting cells. By analysing a large number of T cells at the single-cell level on a microwell array, we show that T-cell activation can occur via cis interactions (where TCRs on the T cell interact with the antigenic peptides presented on MHC class-I molecules on the same cell), and that such cis activation can be used to detect antigen-specific T cells and clone their TCR within 4 d. We used the detection-and-cloning system to clone a tumour-antigen-specific TCR from peripheral blood mononuclear cells of healthy donors. TCR cloning by leveraging the cis activation of T cells may facilitate the development of TCR-engineered T cells for cancer therapy.

Capture of esophageal and breast cancer cells with polymeric microfluidic devices for CTC isolation.

The present study evaluated the capture efficiency of esophageal and breast cancer cells with a modified 'polymeric circulating tumor cells (CTC)-chip' microfluidic device, which was developed for the isolation of circulating tumor cells. Esophageal cancer cell lines KYSE150, KYSE220 and KYSE510, and breast cancer cell lines MCF7, SKBR3 and MDA-MB-231 were used for evaluation. The capture efficiencies of the esophageal cancer cell lines in phosphate-buffered saline (PBS) were ~0.9, irrespective of epithelial cell adhesion molecule (EpCAM) expression, which was represented as the mean fluorescent intensity from 528 to 76. In the breast cancer cell lines, efficient capture was observed for MCF7 and SKBR3 in PBS; however, a low value of ~0.1 was obtained for MDA-MB-231. Fluorescent imaging of immunolabeled cells revealed marginal EpCAM expression in MDA-MB-231. Using whole blood, no clogging occurred in the microstructure-modified CTC-chip and efficiency of capture was successfully evaluated. Capture efficiencies for KYSE220 and MCF7 in whole blood were >0.7, but were of either equal or lesser efficiency in comparison to PBS. Therefore, the modified CTC-chip appears useful for clinical application due to its cost, practicality of use, and efficient cancer cell capture.

Polymeric microfluidic devices exhibiting sufficient capture of cancer cell line for isolation of circulating tumor cells.

Here, we developed polymeric microfluidic devices for the isolation of circulating tumor cells. The devices, with more than 30,000 microposts in the channel, were produced successfully by a UV light-curing process lasting 3 min. The device surface was coated with anti-epithelial cell adhesion molecule antibody by just contacting the antibody solution, and a flow system including the device was established to send a cell suspension through it. We carried out flow tests for evaluation of the device's ability to capture tumor cells using an esophageal cancer cell line, KYSE220, dispersed in phosphate-buffered saline or mononuclear cell separation from whole blood. After the suspension flowed through the chip, many cells were seen to be captured on the microposts coated with the antibody, whereas there were few cells in the device without the antibody. Owing to the transparency of the device, we could observe the intact and the stained cells captured on the microposts by transmitted light microscopy and phase contrast microscopy, in addition to fluorescent microscopy, which required fluorescence labeling. Cell capture efficiencies (i.e., recovery rates of the flowing cancer cells by capture with the microfluidic device) were measured. The resulting values were 0.88 and 0.95 for cell suspension in phosphate-buffered saline, and 0.85 for the suspension in the mononuclear cell separation, suggesting the sufficiency of this device for the isolation of circulating tumor cells. Therefore, our device may be useful for research and treatments that rely on investigation of circulating tumor cells in the blood of cancer patients.

Screening of antigen-specific antibody-secreting cells.

Screening of antigen-specific antibody-producing cells is a key step for obtaining antigen-specific monoclonal antibodies. In murine system, hybridoma between B-lymphocytes and myeloma cells is used to screen and produce antigen-specific monoclonal antibodies. In human system, good hybridoma-producing system is not available. Instead, transformation of B-lymphocytes with Epstein-Barr viruses is used to obtain antibody-secreting cell lines. Furthermore, phage-display system using molecular biology is recently used to obtain antigen-specific human monoclonal antibodies. Here, we describe the new method for screening antigen-specific antibody-secreting cells at single-cell levels using microwell-array chips. The system can be applied to screen antigen-specific antibody-secreting cells from any animal species.

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.

Post-translational modification of TRAIL receptor type 1 on various tumor cells and the susceptibility of tumors to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and TRAIL-R2) are promising targets for tumor therapy. However, their clinical use is limited because some tumors show resistance to TRAIL-treatment. Here, we analyzed epitopes of nine TRAIL-R1-specific human monoclonal antibodies and demonstrated at least five tentative epitopes on human TRAIL-R1. We found that some of the five were post-translationally modified on some tumor cell lines. Interestingly, one of them, an epitope of TR1-272 antibody (TR1-272-epitope) disappeared on the tumor cells that are more susceptible to TRAIL-induced apoptosis compared to TR1-272-epitope positive cells. Treatment of TR1-272-epitope negative cells with TRAIL induced large cluster formation of TRAIL-R1, while treatment of TR1-272-epiope positive cells with TRAIL did not. These results suggest that TR1-272-antibody might distinguish the TRAIL-R1 conformation that could deliver stronger death signals. Further analysis of epitope-appearance and sensitivity to TRAIL should clarify the mechanisms of TRAIL-induced apoptosis of tumor cells and would provide useful information about tumor therapy using TRAIL and TRAIL-R signaling.

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.