Cell culture observations of human postnatal thymic epithelium: an in vitro model for growth and humoral influence on intrathymic T lymphocyte maturation.In Vivo. 1996 Sep-Oct; 10(5):515-26.V
Sixteen postnatal human thymuses were obtained at the time of corrective cardiovascular surgery and maintained in vitro as separate cultures of thymocytes and reticulo-epithelial (RE) cells. The stages of differentiation of the thymocytes were investigated in situ with a library of 10 monoclonal antibodies (MoABs) directed against human lymphocyte differentiation antigens. Employing immunofluorescence staining and flow cytometric (FACS) analysis, in vitro immunophenotype (IP) changes were demonstrated, which appeared after use of a combination of mitogen (PHA), recombinant interleukin-2 (rIL-2) and autologous thymic RE cell culture supernatants. RE cell supernatant participated in increasing the expression of the IL-2 receptor (IL-2R) during combined stimulation with phytohaemagglutinin (PHA) and rIL-2. Thymocyte proliferation was measured in 4 hour tritiated-thymidine (3H-TdR) incorporation (proliferation) assay. We were able to isolate the thymic nurse cells (TNC) with and without enzymatic tissue digestion. TNCs were separated from accompanying thymocytes and cultured. They grew as large, sometimes connected cells, but did not display the epithelial type of tissue organization. After in vitro culturing, the cytoskeleton of TNCs expressed high molecular weight cytokeratin and vimentin and intracytoplasmic tonofilaments, characteristic of epithelium. Whole thymic tissue pieces were cultured with and without previous trypsinization. The initial outgrowth of the cuboidal epithelial tissue layer occurred within 24-48 hours, and the RE cells remained functionally active for at least 15 days. RE cell supernatants were collected daily for two weeks and used in thymocyte differentiation experiments. The results indicated that thymic humoral factors contribute to a select, not fully understood differentiation pathway of thymocytes: a) more mature immunophenotype (IP) characterized by CD3 expression; b) de novo synthesis of interleukin-2 receptor (IL-2R); and; c) differentiation of the CD8+ subpopulation, identifying regulatory cells within the two major CD8+ and CD4+ subsets. Use of mitogenic (PHA) stimulation, after 5 days in vitro, resulted in a T helper (CD4+) oriented differentiation pathway of cortical thymocytes. At the same time, the cultured thymocytes expressed CD11 de novo, an early thymocyte differentiation antigen, and CD7, a marker not present on mature peripheral lymphocyte subsets (the IP changes demonstrated a dedifferentiation). Our overall impression, following the studies with the proliferation assays, was that in our experimental in vitro model, thymic hormones did not contribute to the induction of generalized thymocyte proliferation.