Figure 5.
Induction of annexin V staining and DNA fragmentation in T cells co-incubated with anti-CD44 monoclonal antibody (mAb)-treated dendritic cells (DC). DC or T cells were selectively preincubated for 3 hr at 37° with 10 µg/ml of the indicated mAb, washed and co-incubated with 105 BALB/c T cells at a DC : T-cell ratio of 1 : 10, as described in the legend to Fig. 3. (a) After 24 hr the cells were labelled with phycoerythrin (PE)-conjugated CD3 mAb, stained with fluorescein isothiocyanate (FITC)–annexin V and 7-amino-actinomycin D (7-AAD), and analysed by flow cytometry. Annexin V-positive and 7-AAD-negative cells in the CD3-gated population were counted as apoptotic T cells. The percentage of apoptotic cells from one experiment, representative of three carried out, is shown as a histogram. (b) On day 3 or day 4, T cells were stained with PE–CD3 mAb followed by nicked DNA labelling with FITC-labelled dUTP using a TdT-mediated biotin–dUTP nick-end labelling (TUNEL) assay kit, as described in the Materials and Methods. The percentage of T cells undergoing apoptosis was determined by gating CD3-, dUTP–FITC- and 7-AAD-positive cells by flow cytometry.