Effect of CD38 overexpression in T-cell function. (A,B) Jurkat J77 cells were transiently transfected with the pEGFP-C1 or CD38-GFP constructs by nucleofection. At 24 hours, after transfection cells were subjected to Histopaque-1077 centrifugation and then left alone, or mixed with SEE-pulsed or unpulsed Raji B cells. After a brief centrifugation to favor conjugate formation, ectocellular GDP-ribosyl cyclase activity was continuously measured at 37°C using NGD⁺ as a substrate. The cGDPR production in fluorescence units was plotted against time (minutes). ● indicates J77 CD38-GFP⁺ T cells incubated with SEE-pulsed Raji B cells; ○, J77 CD38-GFP⁺ T cells incubated with unpulsed Raji B cells; ■, Raji B cells alone; ♦, J77 CD38-GFP⁺ T cells alone; ▾, J77 pEGFP-C1⁺ T cells incubated with SEE-pulsed Raji B cells; ◇, J77 pEGFP-C1⁺ T cells incubated with unpulsed Raji B cells; and ▴, J77 pEGFP-C1⁺ T cells. Arrows indicate the 20-minute time frame during which individual cells showed negligible enzymatic activity. (C) Ag-induced Ca²⁺ released in J77 CD38-GFP⁺ T cells (● and ○) and J77 pEGFP-C1⁺ T cells (▾ and ▿). Fura-2/AM–loaded J77 CD38-GFP⁺ or J77 pEGFP-C1⁺ T cells were left alone (♦ and ▴, respectively), or mixed with SEE-pulsed (● and ▾, respectively) or unpulsed (○ and ▿, respectively) Raji B cells. [Ca2⁺]_i changes were measured using a fluorescence plate reader, as described in “Methods.” Characteristic tracings are shown (mean of 3 independent experiments). (D) CD38-GFP–transfected or GFP-transfected Jurkat T cells were stimulated with Raji B cells pulsed with different amounts of SEE. Supernatants were collected after overnight incubation and were assayed for IL-2 by enzyme-linked immunosorbent assay (ELISA). Representative experiment out of 3 independent experiments performed in triplicate (mean ± SEM). (E) CD38 expression by fluorescence-activated cell sorter (FACS) in oligo control–transfected or CD38 siRNA-transfected Jurkat T cells 48 hours after nucleofection. Cells were stained with PE-conjugated anti-CD38 (FL2). The number in each quadrant represents the percentage of cells within each region relative to all viable cells. (F) T cells were then collected, loaded with Fura-2/AM, and stimulated with SEE-pulsed Raji B cells. [Ca2⁺]_i changes were measured as in panel C.