CD40 Ligand-Induced Apoptosis is Fas-Independent in Human Multiple Myeloma Cells

Abstract

We and others previously demonstrated that human multiple myeloma (MM) cells express CD40 and have an active CD40-growth regulatory pathway. This study characterizes the growth outcome of soluble (gp39) or membrane-bound recombinant human CD40-ligand (rCD4()L) and its relationship with Fas-dependent apoptosis. Contrary to the moderate growth-stimulatory effect of the CD40-MAb G28.5, gp39 inhibited ³H-thymidine uptake of the plasma dyscrasia lines ARH-77, U266, and HS-Sultan in a dose-dependent fashion by up to 82%. By comparison, RPMI 8226 cells were resistant to CD40L-growth modulation, which may be attributable to a single base substitution (TCA→TTA, serine→leucine) at the 3^rd cysteine-rich extramembrane region of CD40. Gp39 similarly reduced myeloma clono-genic colony (MCC) formation in patient primary bone marrow cultures by 50% (40-76%; n=6). Studies using transfectant L cells that constitutively expressed CD40L showed that membrane-bound CD40L inhibited the growth of ARH-77, U266, and HS-Sultan cells (66%, 63%, and 32%. respectively), whereas untransfected L cells did not. Growth inhibition by gp39 or CD40L⁺ L cells was neutralized by coincubation with the CD40L antibodies 5c8 or LL48. CD40L-treatment increased apoptotic activity of MM cells, as defined by oligonucleo-somal DNA fragmentation and an increased binding to annexin V (16-28%). All three untreated CD40-responsive MM lines expressed 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-II. CD95 expression was not affected significantly by gp39 treatment, and growth inhibition by CH-11 was additive to gp39 (from 42% to 64% decrease in H-thmidine uptake). Conversely, the CD95 antagonist antibody ZB4 reversed the Fas-dependent growth inhibitory process but did not significantly alter gp39-mediated growth outcome. Gp39 treatment lowered the expression of TNFR-associated factors TRAF4 and TRAF6 by 38% and 32%, respectively, whereas detectable levels of TRAF1,2,3, and 5 levels remained unchanged. Our observations indicate that the CD40L-binding inhibits human MM cell growth and increases its apoptotic activity. This growth inhibitory effect corresponds to lower levels of cytoplasmic TRAF signaling elements, and appears independent of the Fas-signaling pathway. CD40 receptor mutation may lead to unresponsiveness to CD40 growth modulation in multiple myeloma cells.

Key Words:

• multiple myeloma
• CD40 ligand
• Fas/CD95
• TRAF
• immunotherapy