T-cell receptors and T-cell subsets were analysed in T-cell receptor transgenic mice expressing alpha and beta T-cell receptor genes isolated from a male-specific, H-2Db-restricted CD4-8+ T-cell clone. The results indicate that the specific interaction of the T-cell receptor on immature thymocytes with thymic major histocompatibility complex antigens determines the differentiation of CD4+8+ thymocytes into either CD4+8- or CD4-8+ mature T cells.

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.

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Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.

The vast majority of mature T cells are either of the CD4+ 8-or the CD4-8+ phenotype. The CD4 cell surface glycoprotein is expressed primarily on the surface of helper T cells, whose TCR recognize antigen on APCs in the context of class II MHC molecules. The CD8 molecule is primarily expressed on T cells, in particular CTL, whose TCR recognize antigen in the context of class I MHC molecules.The TCR on helper T cells and CTL has been identified as a disulfide-linked ado heterodimer with a molecular weight of 80,000-90,000 (reviewed in reference 1). The binding of CD4 and CD8 molecules to nonpolymorphic regions of class II and class I MHC molecules, respectively, enhances the binding of the TCR to its ligand and may also contribute to signals leading to T cell activation (2, 3).We have constructed TCR transgenic mice with the aim to study the selection ofthe T cell repertoire. To this end, u and a transgenes obtained from an HYspecific, H-21)6-restricted cytolytic CD4-8 + T cell clone were injected into fertilized eggs . We have reported that in female ca/(3 transgenic mice a large proportion of T cells is male specific and expresses both ce and (3 transgenes . In male mice we found that cells with an abnormal CD4/CD8 phenotype accumulated in the periphery, with the vast majority of a//3 T cells being CD4 -8 -and a minority being CD4-8+ . The latter expressed, however, low levels of CD8 accessory molecules. Due to the lack of an appropriate antibody we could not directly address the question as to whether or not these cells expressed the transgenic receptor. Here we describe the use ofmAbs that detect cells expressing both a and a transgenes . Using these antibodies we find that in male H-2D b mice the vast majority of peripheral T cells express both a and a transgenes . Our studies indicate that despite the fact that precursor T cells, which express a low density of the transgenic TCR, are largely deleted in the thymus of male transgenic mice (4), the majority of T cells spared by the deletion express a high density of the transgenic TCR but a low density of CD8 molecules.

Background: 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.Methods:Paired samples of peripheral blood and decidua in induced abortion and miscarriage cases were obtained from consenting patients. CD4+CD25+CD127low/−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.Results: 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%), p < 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%), p = 0.003]. When repertoires in previous pregnancy and subsequent pregnancy were compared, some portions of the repertoire were shared.Conclusion: 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.

HS1, an intracellular protein expressed specifically in hematopoietic cells, is rapidly tyrosine phosphorylated after cross‐linking of antigen receptors on B and T lymphocytes, implicating involvement of this molecule in the signal transduction pathways from the antigen receptors as a substrate of membrane‐associated tyrosine kinase(s). The development of lymphoid cells in HS1‐deficient mice, generated through gene targeting, appeared normal. However, antibody production to T‐independent antigen and proliferative responses of splenic B and T cells after cross‐linking of the antigen receptors were impaired in these mutant mice. Furthermore, B cells in the peritoneal cavity of the mutant mice were resistant to multivalent cross‐linking of the antigen receptor, which causes apoptosis of such cells in normal mice. Crossing the HS1‐deficient mice with the mice harboring transgenes encoding alpha and beta chains of T‐cell antigen receptor against a male H‐Y antigen resulted in a progeny that demonstrated a significantly impaired ability of thymic negative selection. These results indicate that HS1 is a novel molecule involved in the antigen‐receptor‐derived signaling pathways and plays important roles not only in clonal expansion, but also in clonal deletion of B and T cells.

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. '