Materials and Methods

Study Design. Highly purified RA+/RO-peripheral blood lymphocytes were activated in vitro with either II.-2, IL-4, II.-6, IL-7, or with immobilized anti-CD3 mAb. The effects of these different activating agents on the expression of CD45 isoforms, adhesion molecules and proliferation were sequentially evaluated after 4-21 d of culture.

Media and Cytokines. Complete medium (CM) was composed of RPMI 1640 (ICN Biomedicals, Inc., Costa Mesa, CA) supplemented with 10% heat-inactivated filtered human AB serum (Irvine Scientific, Santa Ana, CA), 0.01 M Hepes buffer, and antibiotic-antimycotic mixture (Gibco Laboratories, Grand Island, NY). Recombinant IL-2 (1.8 x 107 U/mg protein) was the generous gift of Cetus Corp.(Berkeley, CA); recombinant IL-4 was obtained from Immunex Corp.(Seattle, WA); recombinant Ib6 was purchased from R & D Systems, Inc.(Minneapolis, MN); and recombinant IL-7 was purchased from Sterling Drug, Inc.(Malvern, PA). CsA in a stock solution of 50 mg/ml was purchased from Sandoz Pharmaceuticals (East Hanover, NJ). mAbs. mAbs used for cell depletion and activation included preservative-free purified anti-CD45RA (Leu-18; Becton Dickinson & Co., Mountain View, CA), anti-CD45RO (UCHL1 clone; Dako Corp., Carpinteria, CA), and anti-CD3 (X35 clone; Amac, Inc., Westbrook, ME). Polyclonal rabbit anti-human IL-2 IgG (Collaborative Biomedical, Bedford, MA) with a neutralizing activity of 9.4 IU//~ g of antibody was used to block the effects of IL-2. FACS| analysis was performed with the following panel of monoclonal antibodies: CD45RA-FITC (Leu-18), CD4-FITC, CD8-PE, CD56-PE, and Leu-8-FITC (LAM-1) from Becton Dickinson & Co.; CD45RO-PE and CD45RO-FITC from Dako Corp.; CD5-FITC, CD11a-FITC, and CDw29-FITC from Amac, Inc.; and CD3-TC (Tri-Color| fluorochrome) and CD2-FITC from Caltag Laboratories (S. San Francisco, CA).