SELECTION AND CHARACTERIZATION OF EARLY HEMATOPOIETIC PROGENITORS USING AN ANTI-CD71 SO6 IMMUNOTOXIN/

Most recently reported methods to select early hematopoietic cells bas ically rely on the depletion of committed progenitors. This task is ge nerally accomplished by laborious procedures, which are sometimes diff icult to reproduce. To simplify the selection method, we took advantag e of the expression of the transferrin receptor (CD71) by proliferatin g committed progenitors and the lack of CD71 on noncycling immature pr ogenitors. A monoclonal antibody (MAB) reactive with CD71 has been con jugated to the Saponaria officinalis seed ribosome-inactivating protei n (SO6). The immunotoxin (IT) complex was used at increasing concentra tions on normal non-phagocytizing bone marrow cells. A complete and re producible killing effect on myeloid (colony-forming unit-granulocyte/ macrophage [CFU-GM]) and erythroid (burst-forming unit-erythroid [BFU- E]) progenitors was observed for IT concentrations of 1x10(-7) M. Unco njugated SO6 or anti-CD71 MAB had no effect on cell growth and viabili ty. IT-resistant cells were able to generate CFU-GM after 7, 14, and 2 1 days of suspension culture in the presence of 5637 CM. Maximal CFU-G M values were obtained at day 21 and nearly approached the pretreatmen t values (mean 2587 vs. 3877 CFU-GM/mL). Growth factor enhancement of CFU-GM yield was obtained only by stem cell factor (SCF) at day 7; SCF , as well as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), had an enhancing effect at days 14 and 21. IT toxicity on highly immature progenitors was ruled out by evaluating the growth of long-term culture-initiating cells (LTC-IC) from IT-tre ated cultures. LTC-IC frequency was found to be 1 out of 1506 seeded c ells, which is within the range of normal untreated BM cells. In concl usion, anti-CD71 IT allows a simple and complete depletion of committe d progenitors while sparing immature hematopoietic cells. The high CD7 1 expression by leukemic cells makes the procedure potentially suitabl e for in vitro purging.