Specific ex-vivo depletion of human bone marrow T lymphocytes by an anti-pan-T cell (CD5) ricin A-chain immunotoxin.
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.
D000643:Ammonium Chloride / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface / Q000276:immunology; D001854:Bone Marrow Cells; D016026:Bone Marrow Transplantation; D002478:Cells, Cultured; D004357:Drug Synergism; D006086:Graft vs Host Disease / Q000517:prevention & control; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D037102:Lectins / Q000494:pharmacology; D008212:Lymphocyte Depletion; D037121:Plant Lectins; D012276:Ricin / Q000494:pharmacology*; D030262:Soybean Proteins; D014700:Verapamil / Q000494:pharmacology; D010835:Phytohemagglutinins
D000643:Ammonium Chloride; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface; D000922:Immunotoxins; D037102:Lectins; D037121:Plant Lectins; D012276:Ricin; D030262:Soybean Proteins; D014700:Verapamil; C028824:soybean lectin; D010835:Phytohemagglutinins