The functional capacity of thymus subpopulations: limit-dilution analysis of all precursors of cytotoxic lymphocytes and of all T cells capable of proliferation in subpopulations separated by the use of peanut agglutinin.

The functional capability of cortical-type and medullary-type thymocytes, defined by their relative binding of peanut agglutinin (PNA), has been investigated in limitdilution and in bulk cultures stimulated by concanavalin A (Con A) in the presence of T cell growth factors. The PNA+ and PNA− thymocytes were separated by either agglutination-sedimentation or by fluorescence-activated cell sorting, the methods giving 6% and 1% cross-contamination, respectively. Kinetic studies of bulk cultures showed good survival and proliferation from PNA− fractions. However, most cells in PNA+ fractions died rapidly with later proliferation and regrowth from a minor subset; the cells responsible for prolonged growth and function appeared to be mainly PNA− contaminants. Limit-dilution microculture techniques with a cloning efficiency of near 100% for mature T cells were used to measure the frequency of all thymocytes capable of proliferation (PTL-p) and all thymocytes capable of generating a cytolytic response as measured in a lectin-mediated universal readout (CTL-p). In unfractionated thymus the PTL-p frequency was 1 in 9.9, the CTL-p frequency 1 in 28. All this activity (>99%) was concentrated in the PNA− fraction on separation. The low precursor frequency in the separated PNA+ fractions could all be attributed to PNA− contaminants. The lack of activity in the PNA+ cells could not be attributed to suppression. These results oppose the contention that “immature” cortical-type PNA+ thymocytes are able to mature and function in the presence of interleukin II or T cell help. The apparent activity in PNA+ fractions reported by other laboratories probably represents the activity of medullary-type cell contaminants whose proliferation is enhanced by T cell growth factors. The particular difficulties of assessing cortical-type and medullary-type thymocytes by PNA binding are discussed.