IDENTIFICATION OF THYMOCYTE PROGENITORS " 1 • I I oc RELATIVE LIGHT > SCATTER

The thymus of irradiated animals can be repopulated by the progeny of precursors from hemopoietic tissues (1-5). 1 Al though the existence of these functional prothymocytes has long been recognized, only modest progress has been made in identifying and isolating them. In the mouse, functional thymocyte progenitors are part of the "null" (non-T, non-B) cell compar tment of bone marrow and spleen, and enrichments of 10to 30-fold, respectively, have been obtained through depletion procedures in conjunction with density gradient centrifugation (6). Partial separation of prothymocytes and pluripotent hemopoietic stem cells (CFU-S) 2 has been observed using density gradient centrifugation or velocity sedimentation (7, 8). The mouse bone marrow cell fractions that are enriched in pro thymocyte activity have been found to be comparably enriched in terminal deoxynucleotidyl transferase (TdT) activity (6). Moreover, TdT-posi t ive cells have been shown to express T cell surface antigens after brief in vitro induction with thymosin (9) and thymopoiet in (10). Hence, they have been postulated to be the most likely candidates for prothymocytes in bone marrow. Prothymocyte activity has also been found in adult spleen, which lacks T d T positive cells (5); 1 and some TdT-negat ive cells in bone marrow and spleen can be induced to express T cell differentiation antigens (9). This has p rompted the speculation that at least two subsets of prothymocytes exist in the mouse. In the rat, unlike the mouse, the Thy-1.1 antigen is readily detectable on a variety of immature lymphohemopoiet ic cells in bone marrow, spleen, and fetal liver (11-14). By using multiple parameter analysis of relative cell size (low-angle light scatter), relative fluorescence intensity for Thy-1 antigen, and relative sensitivity to cortisone to resolve subsets of rat bone marrow cells on the fluorescence-activated cell sorter (FACS), we have developed protocols for separating and markedly enriching pluri-