Purification of peripheral blood hematopoietic cells using Campath-1M monoclonal antibody.
The purpose of this study was to develop a method for exhaustive depletion of T cells in preparation for allogeneic peripheral blood (PB) hematopoietic stem cell (HSC) transplantation. Our goal was to achieve 4 logs of T-cell reduction while minimizing the loss of colony-forming progenitor cells. Campath-1M, a rat IgM cytolytic to T cells, B cells, and monocytes in the presence of human complement, was chosen for the investigation. To determine the best target population for Campath treatment, three different PB mononuclear cell (MNC) populations were examined: 1) total MNC isolated with Ficoll-sodium diatrizoate; 2) MNC after T-cell depletion by sheep red blood cell rosetting and high capacity Percoll density gradient fractionation (E-MNC); and 3) B/Null cells, a population of E-MNC obtained by lysing monocytes with L-phenylalanine methyl ester (PME). Of the three target MNC populations, Campath-treated B/Null cells had the highest cloning efficiencies (CE) for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFUe) (56.7 and 38.5 colonies per 5 x 10(3) cells, respectively) and the highest enrichments for CFU-GM (108.2-fold, range 18- to 440-fold) and BFUe (178.5-fold, range 78- to 314-fold). Recoveries of progenitor cells were comparable for Campath-treated MNC (49.7% and 84.5%, respectively, for CFU-GM and BFUe) and Campath-treated B/Null (44.8% and 76.1%), and significantly higher than those from Campath-treated E-MNC (19.5% and 34.3%). The T-cell content in the Campath-treated B/Null cells were reduced by 5.0 logs from the starting MNC, as determined by limiting dilution analysis (LDA). We conclude that Campath-1M can be used for T-cell depletion of PB HSC. The best results were obtained when T cells and monocytes were removed prior to Campath treatment. This cell population is highly enriched for hematopoietic cells and may prove useful for in vitro studies as well allogeneic transplantation.
Law
P
P
Transplantation Laboratory, American Red Cross, Jerome H. Holland Laboratory for Biomedical Sciences, Rockville, Maryland 20855.
Chau
W K
WK
Dooley
D C
DC
D000911:Antibodies, Monoclonal; D001773:Blood Cells / Q000166:cytology; D002469:Cell Separation / Q000379:methods*; D003114:Colony-Forming Units Assay; D015672:Erythroid Precursor Cells / Q000166:cytology; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D007958:Leukocyte Count; D008264:Macrophages / Q000166:cytology; D009000:Monocytes; D013234:Stem Cells / Q000166:cytology; D013601:T-Lymphocytes
D000911:Antibodies, Monoclonal