Requirement for both extracellular and transmembrane domains for CD47-induced cell death.

(A) Various constructs or/and chimeras of the CD47 molecule (described in “Patients, materials, and methods”) were transfected into U937, Jurkat, or JinB8 (CD47^−/−) cell lines. (B) Expression of the CD47 products (gray histograms) was determined by staining with B6H12 mAb (for hIAP form 1 and 2, IAP/CD7) or antimouse CD8α mAb (for CD8-MC2 and CD8-C2). Expression of endogenous CD47 (Jurkat and U937): cells were cultured overnight on immobilized antimouse CD8-α or CD47 mAb (10 μg/mL). *Jurkat and JinB8 transfectants were pretreated with PMA for 24 hours (2 ng/mL) before killing. CD47-induced cell death was calculated as described in “Patients, materials, and methods.” (C) Light microscopy of PMA-activated Jurkat cells transfected with CD8-C2 construct. Untreated cells (i), cells cultured for 18 hours with soluble (ii), immobilized CD47 mAb (iii) or immobilized antimouse CD8α mAb (iv).