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. © 2007 International Society for Analytical Cytology

To identify HLA alleles closely involved in the autoantigen presentation in acquired aplastic anemia (AA), we studied the HLA allelic loss frequencies of 312 AA patients, including 43 patients with loss of heterozygosity of 6p chromosome (6pLOH). An analysis of the HLA alleles contained in the lost haplotype revealed HLA-B*40:02 to be the most frequently lost allele. When we examined 28 AA (12 6pLOH[+] and 16 6pLOH[−]) patients with HLA-B*40:02 for the presence of leukocytes lacking HLA-B4002 (B4002⁻) using a new monoclonal antibody specific to this allele, B4002⁻ granulocytes were detected not only in all 6pLOH(+) patients but also in 9 (56%) of the 16 6pLOH(−) patients. Furthermore, 10 (83%) of the 12 6pLOH(+) patients possessed 1.0% to 78% B4002⁻ granulocytes that retained the HLA-A allele on the same haplotype (B4002⁻A⁺), suggesting the frequent coexistence of granulocytes that underwent mutations restricted to HLA-B*40:02 with 6pLOH(+) (B4002⁻A⁻) granulocytes. Deep sequencing of the HLA-B*40:02 of sorted B4002⁻A⁺ granulocytes revealed various somatic mutations, such as frameshift, nonsense, and splice site mutations, in all 15 patients studied. Surprisingly, missense mutations in the α-3 domain of HLA-B*40:02 that are not involved in the antigen presentation were detected exclusively in the B4002⁺ granulocytes of 3 patients possessing B4002⁻ granulocytes. The markedly high prevalence of leukocytes lacking HLA-B4002 as a result of either 6pLOH or structural gene mutations, or both, suggests that antigen presentation by hematopoietic stem/progenitor cells to cytotoxic T cells via the HLA-B allele plays a critical role in the pathogenesis of AA. Publisher9s Note: There is an Inside Blood Commentary on this article in this issue. Key Points Somatic mutations of HLA-B*40:02 are very frequently detected in granulocyte of patients with acquired aplastic anemia. Antigen presentation via HLA-B4002 may play a critical role in the pathophysiology of acquired aplastic anemia.

The canonical description of transmembrane receptor function is initial binding of ligand, followed by initiation of intracellular signaling and then internalization en route to degradation or recycling to the cell surface. It is known that low concentrations of extracellular ligand leads to a higher proportion of receptor that is recycled, and that non-canonical mechanisms of receptor activation, including phosphorylation by the kinase p38, can induce internalization and recycling. However, no connections have been made between these pathways; i.e., it has yet to be established what happens to unbound receptors following stimulation with ligand. We herein demonstrate that a minimal level of activation of EGFR tyrosine kinase by low levels of ligand is sufficient to fully activate downstream MAPK pathways, with most of the remaining unbound EGFR molecules being efficiently phosphorylated at intracellular serine/threonine residues by activated MAPKs. This non-canonical, p38-mediated phosphorylation of the C-tail of EGFR, near Ser1015, induces the clathrin-mediated endocytosis of the unliganded EGFR monomers, which occurs slightly later than the canonical endocytosis of ligand-bound EGFR dimers via tyrosine autophosphorylation. EGFR endocytosed via the non-canonical pathway is largely recycled back to the plasma membrane as functional receptors, whereas p38-independent populations are mainly sorted for lysosomal degradation. Moreover, ligand concentrations balance these endocytic trafficking pathways. These results demonstrate that ligand-activated EGFR signaling controls unliganded receptors through feedback phosphorylation, identifying a dual-mode regulation of the endocytic trafficking dynamics of EGFR.

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.