CD38 and IL-2 signaling upmodulate ZAP-70. (A) Purified CLL cells from 3 representative patients were cultured for 72 hours in the presence of IL-2 (100 IU/mL). Lysates were probed for CD38, ZAP-70, and syk using specific antibodies. Basal refers to freshly purified cells, while “−” refers to cells cultured for 72 hours without IL-2. Vertical lines have been inserted to indicate a repositioned gel lane. (B) PBMCs from 19 patients with CLL were cultured as in panel A, and CD38 and ZAP-70 was assayed by intracellular staining. Specificity for B cells was confirmed by counterstaining with an anti-CD19 mAb, as shown in the dot plot. (C) Effects induced by IL-2 exposure on the number of CD38⁺ and ZAP-70⁺ CLL cells in 7 representative patients. (D) ZAP-70 expression is increased following CD38 signaling in the presence of IL-2. Purified CLL cells from 5 patients were cultured for 5 days in the presence of agonistic anti-CD38 mAb, IL-2, or a combination of the 2 (CD38 + IL-2). The top panel shows the morphologic changes in the population as a dot plot of forward scatter (FSC) and side scatter (SSC). Bottom panels show density plots of ZAP-70 and SSC. A marked increase in ZAP-70 expression is apparent after treatment with anti-CD38 mAb or IL-2 and is boosted by the combination of the 2. Percentages indicate ZAP-70⁺ CLL cells within the red gate, highlighting cells with highest SSC values.