CD40L message is detected in RNAs prepared from CLL samples. Fresh CLL B cells (1 × 10⁷) purified from the peripheral blood of 2 patients were exposed overnight in culture to media alone, to SAC and IL-2 (1:60,000 and 40 U/mL), to PMA and ionomycin (10 ng/mL and 1.25 μg/mL), or to IL-4 (10 ng/mL). After 18 hours, RNAs were isolated, cDNAs were prepared by reverse transcriptase, and CD40L sequences were amplified (A). The RNAs analyzed are as follows: lane 1, CLL case no. 15 B cells with media; lane 2, with IL-2 and SAC; lane 3, with PMA and ionomycin; lane 4, with IL-4; lane 5, CLL case no. 16 B cells with media; lane 6, with IL-2 and SAC; lane 7, with PMA and ionomycin; lane 8, with IL-4. Positive and negative controls for detection of CD40L RNA sequences included RNAs from 1 × 10⁷ D1.1 cells (lane 10, Jurkat mutants that constitutively express CD40L) and Ramos Burkitt's lymphoma B cells (lane 9, these do not express CD40L). The cDNA preparations were evaluated by amplification of GAPDH sequences (B).