Figure 4

ADP-ribosylation inhibits binding of IL-2 and IL-2-dependent proliferation of ARTC2.2-transfected CTLL-2 lymphoma cells.

(a) Untransfected parental and ARTC2.2-transfected CTLL-2 cells were incubated with etheno-NAD⁺ and stained with fluorochrome-conjugated mAbs specific for CD25, ARTC2.2 and etheno-adenosine and analyzed by flow cytometry. (b) CTLL-2^ARTC2.2 cells were incubated with ³²P-NAD⁺ and cell lysates were subjected to immunoprecipitation with anti-CD25 and SDS-PAGE autoradiography as in Fig. 1b. Lanes 1, 2: lysates before and after precipitation of CD25, lane 3: control precipitation with protein G, lane 4: precipitation with anti-CD25. (c) CTLL-2 and CTLL-2^ARTC2.2 cells were pre-incubated in the absence or presence of NAD⁺ or unlabeled IL-2. Cells were then incubated with biotinylated IL-2 before addition of fluorochrome-conjugated streptavidin and analysis by flow cytometry. (d) CTLL-2 and CTLL-2^ARTC2.2 cells were seeded in a 24 well culture plate (3 × 10⁴/well) and incubated for four days in medium containing the indicated concentrations of IL-2. Medium lacking or containing NAD⁺ was added every 12 hours. Cell numbers were assessed by flow cytometry with the aid of Trucount beads (BD). Results are representative of two independent experiments.