Characterization of the Fab fragments of anti-CD28 antibodies. (A) CD28⁺ Jurkat T cells were incubated with the indicated amounts of anti-CD28 mAb or their Fab fragments and stained with antimouse-FITC antibody. MFI was determined by flow cytometry. (B) Inhibition of CD28/CD80 interactions was assessed by incubating calcein-labeled CD28⁺ Jurkat T cells on monolayers of mouse fibroblasts expressing human CD80 in the presence of saturating doses of anti-CD28 Fab fragments or control Fab fragments. After washing, adherent cells were collected and associated fluorescence was measured by fluorometry. (C) PBMCs (10⁵) were mixed with 10⁵ allogeneic irradiated PBMCs in the presence of the indicated amount of anti-CD28 mAb and Fab fragments or with 10 μg/mL control antibody. Proliferation on day 5 was measured by addition of 10^-6 Ci (37 KBq) ³H-thymidine and measurement of incorporation after 16 hours; ⋄ indicates IgG CD28.1; ▴, IgG CD28.2; ▪, IgG CD28.3; ⋄, Fab CD28.1; ▵, Fab CD28.2; □, Fab CD28.3; and ×, isotype control. The results shown are representative of more than 3 independent experiments.