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We studied optimal conditions for ex vivo elimination of mature T cells from human bone marrow by T101 immunotoxin (T101-IT) with criteria applicable to graft-versus-host disease (GVHD) prophylaxis prior to allogeneic marrow transplantation. T101-IT consisted of T101 anti-CD5 monoclonal antibody conjugated to purified ricin A-chain toxin. Marrow mononuclear cells isolated by Ficoll-Hypaque or by fractionation with soybean lectin (SBA-cells) were incubated with T101-IT at 37[degrees]C with or without ammonium chloride and/or verapamil as potential enhancers of immunotoxin potency. As controls, competitive inhibition studies with unconjugated T101 or irrelevant IgG2a antibody were carried out. Residual T cells were quantified by limiting dilution in phytohemagglutinin (PHA)-interleukin 2 (IL-2) feeder-cell-containing microcultures and hematopoietic progenitors by CFU-GM assay. We demonstrated that T101-IT in the range of 1-100 nM does not affect early total cell viability; that its delayed cytotoxicity is T-cell-specific, greatly enhanced by ammonium chloride, and moderately by verapamil-which also is not synergistic with ammonium chloride; and that 10 nM x 3 fractionated doses (i.e., added at 0, 1.5, and 3 hr of incubation) in the presence of 10 mM ammonium chloride for 4 hr at pH 7.8 consistently induces 2 log T cell depletion. In addition, if the same T101-IT treatment is preceded by fractionation with soybean lectin (i.e., T101-IT treatment of SBA-marrow cells), 3 log T cell depletion is accomplished. We conclude that T101-IT is highly effective in eliminating T cells from donor grafts. However, data presented here indicate that T101-IT should be associated with additional methods, such as soybean lectin fractionation, to ensure more effective ex vivo T cell depletion and acute GVHD prevention.

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