Shigeo Koyasu

Cross-linking of T-cell receptors on double-positive thymocytes induces a cytokine-mediated stromal activation process linked to cell death.

To investigate molecular events associated with the intrathymic process of negative selection, we established an in vivo system using an anti‐CD3 epsilon monoclonal antibody to induce synchronous apoptosis in the thymus of AND T‐cell receptor (TCR) transgenic RAG‐2−/− mice in a non‐selecting haplotype. This model eliminates endogenous negative selection as well as gene activation in the mature thymocyte compartment, offering an ideal source of tester (anti‐CD3 epsilon‐treated) and driver (untreated) thymus RNA for representational difference analysis (RDA). Fourteen mRNA sequences that are up‐regulated in the thymuses of such mice 2–6 h after anti‐CD3 epsilon treatment were identified. Surprisingly, the majority of these transcripts were derived from stromal cells rather than the TCR‐cross‐linked CD4+CD8+TCRlow thymocytes including the macrophage products IL‐1, the chemokine Mig and the transcription factor LRG‐21. IFN‐gamma secretion from the CD4+CD8+TCRlow thymocytes regulates macrophage Mig production. Three other cytokines (IL‐4, GM‐CSF and TNF‐alpha), known to activate a variety of stromal cells, are also induced in the same thymocyte population undergoing apoptosis. Expression of a TNF‐alpha‐inducible gene, B94, in stromal cells after TCR ligation further supports the notion of cross‐talk between thymocytes and stroma. Thus, TCR‐triggered immature thymocytes elaborate cytokines which may regulate the delivery of further signals from stromal cells required for apoptosis.

Targeted disruption within the CD3 zeta/eta/phi/Oct-1 locus in mouse.

To elucidate the role of the CD3 eta subunit of the T cell receptor (TCR) in thymic development, a CD3 eta ‐/‐ mouse was generated by gene targeting. Insertion of a neomycin resistance gene into exon 9 of the CD3 zeta/eta/phi locus disrupted expression of CD3 eta and CD3 phi without affecting the expression of CD3 zeta. Little difference was observed between wild type and CD3 eta ‐/‐ mice with regard to cellularity or subset composition in thymus and peripheral lymphoid organs. Furthermore, neither alloproliferative responses nor cytotoxic T lymphocyte generation and effector function was affected by the mutation. The effect of the CD3 eta ‐/‐ mutation on thymic selection was examined by crossing the CD3 eta knockout animals with anti‐HY TCR transgenic animals: the absence of the CD3 eta subunit altered neither positive nor negative selection. Thus, CD3 eta is not required for thymic selection. Of note, the birth rate of the CD3 eta ‐/‐ animals was significantly lower than that of wild type or heterozygous animals (P = 0.041‐0.002). This unexpected result is probably the consequence of an alteration in mRNA expression of the Oct‐1 nuclear transcription factor in CD3 eta ‐/‐ animals. The CD3 zeta/eta/phi locus partially overlaps the gene encoding Oct‐1 whose transcription is dysregulated by the CD3 eta ‐/‐ mutation. Our results clearly underscore the value of characterizing all products of a genetic locus disrupted by gene targeting.

ENERGY OF ADHESION OF HUMAN T CELLS TO ADSORPTION LAYERS OF MONOCLONAL ANTIBODIES MEASURED BY A FILM TRAPPING TECHNIQUE

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.

T-cell receptor isoforms and signal transduction.

Recent cDNA and genomic cloning have identified CD3 eta as an alternatively spliced product of the same gene locus that encodes CD3 zeta. Three distinct T-cell receptor isoforms have now been identified. A current view of the signal transduction function of these isoforms in thymocytes and T cells is discussed.

Interactions Between CD2 and T-Cell Receptor Isoforms in CTL Function

The recognition of specific target cells by cytotoxic T lymphocytes (CTL) involves a set of cell surface proteins. The physical interaction of a T-cell receptor (TCR) with a nominal peptide antigen bound to a specific major histocompatibility complex (MHC) molecule confers specificity on the CTL. The clonally unqiue antigen-specific binding component has been termed Ti and exists as a disulfide-linked Tiα-β heterodimer on the majority of peripheral T cells, although approximately 5% of T cells express Tiγ-δheterodimers (Meuer et al., 1984a; Marrack and Kappler, 1987; Brenner et al., 1988; Davis and Bjorkman, 1988; Raulet, 1989). The immunoglobulin-like Ti structure is noncovalently associated in a molecular complex with the CD3 subunits γ, δ, e, ζ, and η, the latter molecules being involved in signal transduction (Clevers et al., 1988; Ashwell and Klausner, 1990; Koyasu et al., 1991a). The TCR has therefore been referred to as the CD3-Ti complex.