The cells of origin of lymphocyte clones grown by limiting dilution

A high proportion of the mononuclear cells separated from peripheral blood by Ficoll-Hypaque centrifugation show extensive proliferation when cloned at limiting dilution. The nature of clone-forming cells ( CFCs ) and the cells generated during clone formation were studied by cytotoxicity with antibody and complement and by immunofluorescence. Cytotoxicity prior to cloning with OKT3 plus OKT11 eliminated 99.34% of clones, indicating that virtually all CFCs are T-lymphocytes, and cytotoxicity with OKT4 or OKT8 indicated that helper and suppressor subclasses each contribute approximately half of the CFCs . Immunofluorescence of cells proliferating in clones confirmed that all clones were T-lymphocytes and showed that 47% were OKT4 positive and 47% were OKT8 positive; 6% were negative with both OKT4 and OKT8. The results indicate that in the nearly optimal conditions for proliferation provided by limiting dilution, clones arise entirely from T-lymphocytes and clone formation is a property of a variety of lymphocyte subclasses.