Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow

Using multidimensional flow cytometry we have defined and quantified the human T-cell differentiation pathway, focusing on those events occurring among the most immature thymocytes and putative bone marrow (BM) T-precursors. Early thymocytes were found to express the CD34 antigen and consisted of a mean 1.2% of cells within human pediatric (n = 9) and 2.0% in fetal thymi (n = 4). All CD34⁺ thymocytes were atypical blast by morphology, expressed intracytoplasmatic, but not cell surface, CD3, and were cell surface CD2⁺, CD5⁺, CD7⁺, CD38⁺, CD45⁺, CD45RA⁺, CD49d⁺, and LECAM-1(Leu8)(high). CD34(high) thymocytes lacked surface expression of CD4 and CD8, but as CD34 expression diminished there was a coordinate increase in CD4 levels, followed by the appearance of CD8. The expression of CD1 and CD10 also increased concomitant with the loss of CD34, whereas expression of LECAM-1 diminished with CD34 downregulation. The differential expression of these antigens on early thymocytes (as well as the number of thymocytes displaying these patterns) was highly reproducible among the nine pediatric and four fetal specimens examined, suggesting a precise, stereotyped regulation of early differentiation events. Cell populations with antigen expression patterns suggestive of pluripotent stem cell (CD34(high), CD38^-), or non-T-lineage committed stem cells (CD34⁺, CD33⁺, or CD34⁺, CD19⁺) were not identified in either fetal or pediatric thymi (sensitivity = 1/10⁴). The presence of cells with the antigenic profile of the earliest CD34⁺ thymocytes was explored in human BM. Putative BM T-cell precursors with the appropriate phenotype (CD34⁺, CD7⁺, CD5⁺, CD2⁺, LECAM-1(high)) were readily identified in fetal specimens (constituting ±2% of the CD34⁺ population), but could not be reliably detected in adults. In contrast with thymi, only 13% of these cells expressed cytoplasmatic CD3, suggesting the presence of the immediate precursor of the putative prothymocyte population. This was further supported by the detection of CD34(bright), CD7⁺, CD2^-, CD5^-, LECAM-1(moderate) cells in fetal specimens. Our results document the flow of cell surface differentiation during T-lymphopoiesis and suggest that T-lineage features are first acquired in the BM. The ability to reproducibly identify and isolate T-cell precursor populations of precisely defined maturational stage in marrow and thymus by multiparameter flow cytometry will facilitate characterization of the molecular events controlling T-lineage differentiation.