title: Long term expansion of cytomegalovirus-specific T cell lines in the absence of antigen or antigen-presenting cells. Use of monosized polystyrene particles coated with agonistic antibodies.
abstract: Functional and molecular studies of T lymphocytes involved in normal and abnormal immune responses, i.e., cells infiltrating tissues affected by autoimmune processes, require their previous in vitro expansion. Problems such as unavailability of specific antigen(s) and/or the requirement of large amounts of autologous peripheral blood mononuclear cells (PBMNCs) as feeder cells, demand the development of alternative expansion methods. Cytomegalovirus (CMV)-primed PBMNCs from several seropositive subjects were expanded for 5-6 weeks by stimulation with anti-CD3 coated onto polystyrene beads plus interleukin-2 (IL-2) with a similar efficiency than when the MAb was presented by autologous MNCs. Beads coated with anti-CD4, but not with anti-CD8, were also able to maintain the long term growth of CMV-primed populations of T cells. The expanded T cells of one of these polyclonal populations were cloned by limiting dilution using anti-CD3 or CMV, and autologous PBMNCs as stimuli. 16 and 11 clones, respectively, were obtained and grown to several million cells for 1-2 months by weekly stimulations with anti-CD3-coated beads and IL-2. Proliferation assays performed with most of the clones generated with anti-CD3 stimulation showed that all the tested clones had retained the CMV and the class II MHC restriction specificities for at least 3-4 months after the initial CMV stimulation. All the tested clones secreted IL-2 in response to CMV and were CD3+, CD4+ and CD8-. Comparison of the growth of two of these clones by stimulation with: (a) anti-CD3-coated beads and IL-2; (b) anti-CD3, autologous MNCs and IL-2; or (c) CMV, autologous MNCs and IL-2, showed that the first combination was at least as efficient as the other two in expanding these T cell clones. We conclude that polystyrene monosized particles coated with agonistic antibodies can induce the long term growth of antigen-specific T cell lines in the absence of specific antigen and feeder cells, often unavailable specially in the context of human autoimmune studies.
authors.affiliation: Department of Medicine, University of Minnesota, Minneapolis 55455.
authors.forename: P
authors.initials: P
authors.lastname: Santamaria
authors.forename: M K
authors.initials: MK
authors.lastname: Bryan
authors.forename: J
authors.initials: J
authors.lastname: Barbosa
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000938:Antigen-Presenting Cells; D015703:Antigens, CD / Q000276:immunology; D015704:CD4 Antigens; D002455:Cell Division; D003587:Cytomegalovirus / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D011137:Polystyrenes; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015704:CD4 Antigens; D007376:Interleukin-2; D011137:Polystyrenes