CD40 ligand-induced apoptosis is Fas-independent in human multiple myelomacells

We and others previously demonstrated that human multiple myeloma (MM) cell s express CD40 and have an active CD40-growth regulatory pathway. This stud y characterizes the growth outcome of soluble (gp39) or membrane-bound reco mbinant human CD40-ligand (rCD40L) and its relationship with Fas-dependent apoptosis. Contrary to the moderate growth-stimulatory effect of the CD40-M Ab G28.5, gp39 inhibited H-3-thymidine uptake of the plasma dyscrasia lines ARH-77, U266, and MS-Sultan in a dose-dependent fashion by up to 82%. By c omparison, RPMI 8226 cells were resistant to CD40L-growth modulation, which may be attributable to a single base substitution (TCA-->TTA, serine-->leu cine) at the 3(rd) cysteine-rich extramembrane region of CD40. Gp39 similar ly reduced myeloma clonogenic colony (MCC) formation in patient primary bon e marrow cultures by 50% (40-76%; n=6). Studies using transfectant L cells that constitutively expressed CD40L showed that membrane-bound CD40L inhibi ted the growth of ARH-77, U266, and MS-Sultan cells (66%, 63%, and 32%, res pectively), whereas untransfected L cells did not. Growth inhibition by gp3 9 or CD40L(+) L cells was neutralized by coincubation with the CD40L antibo dies 5c8 or LL48. CD40L-treatment increased apoptotic activity of MM cells, as defined by oligonucleosomal DNA fragmentation and an increased binding to annexin V (16-28%). All three untreated CD40-responsive MM lines express ed the Fas/Apo-1/CD95 antigen (65-92% CD95(+)). However, only ARH-77 cells responded to the growth inhibitory effect of the CD95-agonistic antibody CH -11. CD95 expression was not affected significantly by gp39 treatment, and growth inhibition by CH-11 was additive to gp39 (from 42% to 64% decrease i n H-3-thmidine uptake). Conversely, the CD95 antagonist antibody ZB4 revers ed the Fas-dependent growth inhibitory process but did not significantly al ter gp39-mediated growth outcome. Gp39 treatment lowered the expression of TNFR-associated factors TRAF4 and TRAF6 by 38% and 32%, respectively, where as detectable levels of TRAF1,2,3, and 5 levels remained unchanged. Our obs ervations indicate that the CD40L-binding inhibits human MM cell growth and increases its apoptotic activity. This growth inhibitory effect correspond s to lower levels of cytoplasmic TRAF signaling elements, and appears indep endent of the Fas-signaling pathway. CD40 receptor mutation may lead to unr esponsiveness to CD40 growth modulation in multiple myeloma cells.