Analysis of Fas antigen gene expression in human thymocytes fractionated with discontinuous gradients using bovine serum albumin

Premature thymocytes proliferate and differentiate from immature to mature T lymphocytes through out both positive and negative selections in thymus. The clonal deletion is a major mechanism of cell selection and mainly depends on apoptosis. Recently, Itoh et al. isolated cDNAs encoding Fas antigen which mediate apoptosis and is expressed on mouse thymocytes. Herein, I further attempted to examine the expression of Fas antigen gene in human thymocytes from thymus resected at cardiac operations. Human thymocytes were separated into 5 fractions with discontinuous gradient using bovine serum albumin (BSA). They consisted of fractions I (BSA concentrations: 10-14%), II (14-16%), III (16-18%), IV (18-20%), and V (20-24%). Human thymocytes in each fraction were characterized regarding the rearrangement of T cell receptor (TCR) genes and the expression of human Fas antigen gene. Human thymocytes were divided into three sub populations according to their stages of differentiation and maturation. First population, thymocytes contained in fraction I, expressed interleukin 2 receptor alpha-chain (IL-2R alpha) and proliferated without the presence of recombinant IL-2 (rIL-2). Second, thymocytes contained in fraction II and III, expressed IL-2R alpha but could not proliferate without the presence of rIL-2. Third, thymocytes contained in fraction IV and V, could not express IL-2R alpha nor proliferate under any conditions assayed, and occupied over 90% of total human thymocytes in number. The southern blot analysis of T cell receptor beta-chain gene constant region (C beta) showed that C beta were rearranged in most of all thymocytes except for small population contained in fraction I. The reverse transcription-polymerase chain reaction (RT-PCR) analysis of Fas antigen gene expression revealed that the thymocytes in fraction I expressed Fas antigen gene more than those in any other fractions and that the thymocytes in fraction V expressed no Fas antigen gene. These results suggested that Fas antigen plays a minor role in the clonal deletion of postnatal human thymocytes.