Figure 4.

TCR diversity generated in vitro by a single murine T cell progenitor. Various number of fetal thymocytes (15, 25, or 150) were seeded in individual γ-irradiated lobes. After 2 weeks of FTOC, lobes were examined by flow cytometry for the presence of αβ-TCR positive cells. (A) Representative fluorescence-activated cell sorter profile of cells from an individual reconstituted thymic lobe. The majority of the cells expressed αβ-TCR. No cellularity was observed in nonreconstituted (negative) lobes. For each point, the percentage of negative lobes was calculated. (B) Estimation of T cell precursor frequency. According to the Poisson probability distribution, the inoculum that produces 37% negative recipients is expected to contain a single T cell progenitor. This frequency was estimated to be 1 in 26 cells. Therefore, lobes seeded with 15 cells, when reconstituted, contain the progeny of a single T cell precursor. T cell repertoire analyses were performed on these individual lobes. (C) Analysis of the T cell repertoire in lobes reconstituted with one or five T cell precursors. cDNAs prepared from individual thymic lobes were submitted to PCR by using BV10- (or BV8.2-) and BC-specific primers. Representative CDR3β size distribution profiles corresponding to an unreconstituted lobe or a lobe reconstituted with one or five T cell progenitors are shown. (D) Analysis of BV10-BJ rearrangements in lobes reconstituted with a single T cell precursor.