title: T cell receptor-mediated selection of functional rat CD8 T cells from defined immature thymocyte precursors in short-term suspension culture.
abstract: Recent results have indicated that positive and negative repertoire selection act on the major population of CD4,8 double-positive (DP) thymocytes that express 5-10-fold less T cell receptor (TCR) than mature T cells (i.e., they are TCRlow). Since DP cells obtained ex vivo are heterogeneous with regard to their stage within thymic selection, a homogeneous population of virgin DP cells suitable for selection studies was generated in vitro from their immediate precursors, the CD8 single-positive (SP) immature blast cells. To mimic TCR-mediated selection signals, these virgin DP cells were then cultured for another 2 d in the presence of immobilized anti-TCR monoclonal antibodies with or without interleukin 2 (IL-2). Daily monitoring of recovery and phenotype showed that without TCR stimulation, the cells remained DP and became small, TCRlow cells that were lost with a half-life of 1 d, regardless of the presence of IL-2. TCR stimulation resulted in rapid downregulation of CD4 and CD8, maintenance of a larger cell size, and induction of the CD53 antigen that marks mature and CD4,8 double-negative rat thymocytes. In the absence of IL-2, viability decreased as rapidly as without TCR stimulation. Addition of IL-2 rescued TCR-stimulated virgin DP cells and prevented CD8 downregulation, so that 50-80% of input DP cells were recovered after 2 d as CD4-8+53+ cells. After release from modulation, these in vitro generated CD8 SP cells quantitatively upregulated the TCR to the TCRhigh phenotype and were readily induced to proliferate and exhibit cytotoxic T lymphocyte (CTL) activity in a polyclonal readout. Evidence is presented implicating an IL-2 receptor (IL-2R) not containing the p55 chain (i.e., most likely the p70 intermediate affinity IL-2R) in the TCR plus IL-2-driven in vitro differentiation of virgin DP cells towards the mature CD8 SP phenotype.
authors.affiliation: Institute for Virology and Immunobiology, University of Würzburg, Federal Republic of Germany.
authors.forename: T
authors.initials: T
authors.lastname: Hünig
authors.forename: R
authors.initials: R
authors.lastname: Mitnacht
mesh_terms: D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D016827:CD8 Antigens; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D004261:DNA Replication; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008213:Lymphocyte Activation; D008297:Male; D010641:Phenotype; D051381:Rats; D011917:Rats, Inbred Lew; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D013936:Thymidine / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology / Q000276:immunology*
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D016827:CD8 Antigens; D011948:Receptors, Antigen, T-Cell; D013936:Thymidine