Humanized anti-CD52, in the presence of complement, alters moDC stimulation of allogeneic T-cell proliferation in a dose-dependent manner.

(A) Mature CD83⁺ moDCs were combined with allogeneic T cells at a fixed ratio of 1 DC/30 T cells in alloMLRs. Humanized anti-CD52 (alemtuzumab) was added in graded doses from 1 μg/mL to 1 mg/mL. Complete RPMI was supplemented with complement-replete human plasma (not heat inactivated) or an equivalent amount of commercial complement. The proliferation of responder T cells in the continuous presence of humanized anti-CD52/complement, divided by the proliferation in the presence of control humanized anti-CD20/complement, yielded a stimulation index at each dose of MAb; 100% control proliferation ranged from 50 000 to 200 000 cpm³HTdR incorporation in 3 different allogeneic combinations. This inhibitory effect is due to MAb/complement-dependent lysis of both stimulator and responder cells; although the mature moDCs are relatively less sensitive, so the predominant effect is likely T-cell–based. The graph summarizes 3 independent experiments, and the error bars represent the SD of the averaged triplicate means from each experiment. Comparison of these 2 groups based on Wilcoxon rank sum test showed P < .02 for doses between 1 μg/mL and 1000 μg/mL. (B) Immature moDCs were opsonized with an excess of humanized anti-CD52 (alemtuzumab, 1 mg/mL final) or nonreactive control humanized anti-CD20 (rituximab, 1 mg/mL final) for 30 to 40 minutes on ice, thoroughly washed, and then exposed to complement or plasma (50% vol/vol) for 1 hour or overnight at 37°C. Both conditions started with the same number of cells and were handled identically. Yields and cell concentrations for reculture and terminal maturation of the moDCs, after MAb/complement treatment, but before addition as stimulators to an alloMLR, were based solely on the control anti-CD20 condition. Already matured DCs proved too variable in their sensitivity to MAb/complement lysis; so immature moDCs were treated, then matured and used for these experiments. Incorporation of³HTdR was assessed during the last 12 hours of a 4- to 5-day culture. The percent inhibition at each T/DC dose was calculated as follows: 100 − (mean cpm of T cells stimulated by anti-CD52–treated moDCs/mean cpm of T cells stimulated by anti-CD20 treated moDCs) × 100. Shown are the results of 3 independent experiments and the SD of the mean percent inhibition at each T/DC dose.