Soluble CTLA4 production by CLL. (A) mRNA of total, soluble, and membrane-bound CTLA4 was measured by qPCR using isoform-specific primers (n = 14 total CTLA4, 14 soluble, 8 membrane-bound). (B) CLL cells were treated with 1 μM ibrutinib or acalabrutinib for 1 hour before washing. sCTLA4 mRNA levels were measured 4 hours later by qPCR (n = 4 ibrutinib, 6 acalabrutinib). (C) Plasma was obtained from healthy donors, patients with CLL, and patients with AML by centrifugation of whole blood, and sCTLA4 levels were measured by ELISA (n = 4 healthy donors, 16 patients with CLL, 4 patients with AML). Graphs show mean ± standard deviation. *P < .05 by mixed effect modeling. Ct, threshold cycle.