Fig. 2
Percent inhibition of the in vitro proliferative rate in human and mouse T-cell lines. Human (A) and mouse (B) T-cell lines were measured by 3H-thymidine uptake in the presence of sFas and its soluble peptides. Major inhibition occurred in Jurkat and CEM cells treated with the full-length soluble receptor and with VEINCTR-N, compared with the control untreated cells. Pept. #2 also moderately inhibited both lines. The human Fas-transfected mouse T-cell lymphoma, WC8, also was suppressed significantly by both sFas and VEINCTR-N; whereas, the proliferation of human (K-562 and Daudi) and mouse (WR19L) Fas-negative cell lines was not influenced. The preincubation of sFas with the ZB4 MoAb anti-Fas neutralized its inhibitory effect on cell proliferation. (C) Cytofluorimetric measurement of both Ki67 and APO2.7-related antigens as markers of proliferation and apoptosis, respectively. Jurkat and CEM cells were incubated overnight with the soluble molecular forms of Fas and, then, evaluated in parallel for their expression of both antigens in relation to control cultures. Although the suppressive effect of VEINCTR-N and sFas on both cell lines induced a variable decrease of the Ki67-positive population (M2), a significant expansion of the apoptotic subset occurred, since the APO2.7-positive M2 extent in both Jurkat and CEM preparations was greatly increased by both inhibitors (p< 0.02 in all instances). Conversely, pept. #1 and #2 only induced a minor variation of Ki67 expression. (D) Measurement of apoptosis in Fas-positive cell lines by detection of annexin-V. The apoptotic population (M2) was expanded in Fas-positive cell lines of either human (Jurkat and CEM) or mouse (WC8) origin, predominantly in cultures supplemented with VEINCTR-N or with sFas, compared with relative untreated control cultures (p < 0.02). The other soluble peptides of Fas resembling the flank regions of VEINCTR-N failed to induce a similar effect. sFas, soluble Fas; pept., peptide.