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
Wei-Feng, C.; Scollay, R.; Shortman, K.
Journal of Immunology 129(1): 18-24
1982
ISSN/ISBN: 0022-1767 PMID: 6211480 Document Number: 183779
The functional capability of cortical-type and medullary-type mouse thymocytes, defined by their relative binding of peanut agglutinin (PNA), was investigated in limit-dilution 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. 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 2 or T cell help. The apparent activity in PNA+ fractions 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.