Apoptotic effect exerted by Fas-L⁺ myeloma cells on erythroblasts.

(A) Cultured erythroblasts were measured in their spontaneous apoptosis (left panels) and after incubation with autologous myeloma cells at a 1:1 ratio (central panels) or with the relative SN (right panels). The cytofluorimetric pattern is related to the erythroblast culture of patient no. 16 and indicates a clear-cut increase of the subdiploid DNA erythroblast population (i) as well as an enlargement of both the TUNEL⁺ subset (ii) and the cell population expressing the mitochondrial APO2.7-reactive antigen, an early marker of apoptosis (iii). A similar, though slightly lower, effect was obtained with the myeloma cell–conditioned SN. The upper section includes the erythroblast populations gated to the cytofluorimetric analysis. (B) Inhibition experiments by PI staining (■) and TUNEL (○) measurements of the apoptogen potential of myeloma cells by preincubation of either erythroblasts or plasma cells with a nonagonist MoAb to Fas (UB2) or with the Fas-Fc construct, respectively. These inhibiting tests of erythroblast apoptosis were completed in 2 patients with advanced MM. Pretreatment with both reagents induced a progressive saturation of the binding sites of Fas on erythroblasts and, alternatively, of Fas-L on plasma cells, leading to exhaustion of the apoptosis induction through Fas/Fas-L pathway. This effect was dose-dependent and promptly induced by blocking Fas on erythroblasts, with amounts of UB2 higher than 1 μg/mL in culture medium. By contrast, saturation of Fas-L on plasma cells required a nearly 10-fold higher concentration of Fas-Fc to produce a similar effect in cultured erythroblasts from both patients.