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

Abstract

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. CD34high 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 (CD34high, CD38-), or non-T-lineage committed stem cells (CD34+, CD33+ or CD34+, CD19+) were not identified in either fetal or pediatric thymi (sensitivity = 1/10(4)). 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-1high) 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 CD34bright, CD7+, CD2-, CD5-, LECAM-1moderate 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.

CDw108 expression during T‐cell development

“…Specifically, approximately 50% of double‐positive subsets expressed CDw108, and the expression of CDw108 on the other subsets (CD4 bright CD8 dull , CD4 or CD8 single positive, and double negative) was almost negative. Immature CD4 CD8 double‐negative cells initially express CD34, a stem‐cell marker, and the expression of CD34 no longer occurs during T‐cell development in the thymus ( 22–25). CD117 (c‐kit) is also known as a stem‐cell marker, but it is expressed on more mature cells in the thymus ( 26–28).…”

CD7 expression by CD34+ cells in CML patients, of prognostic significance?

“…The T‐cell associated cell surface molecules CD2 and CD7 are expressed on a comparable number of normal CD34 + cells, and CD34 + CD2 + and CD34 + CD7 + cells are considered putativeT‐lymphoid progenitor cells (21, 33). In line with what has previously been reported (26), we found CD2 to be expressed by a small proportion of Ph + CD34 + cells not different from that of controls, whereas the proportion of CD34 + CD7 + cells was significantly increased.…”

“…CD2, CD4, CD5, CD6 and CD7 are all molecules associated with the T‐lymphoid lineage, and CD34 + cells co‐expressing CD2, CD5 and/or CD7 are putative T‐lymphoid progenitors (16, 20, 21). Co‐expression of CD2, CD4, CD5 and CD6 by CD34 + cells was not found to differ between CML patients and controls, but the proportion of CD34 + CD7 + cells was significantly higher in the CML patients than in controls (mean 25.3% vs. 4.9%, P < 0.01; Table 3).…”

Prognostic impact of the CD34+/CD38− cell burden in patients with acute myeloid leukemia receiving allogeneic stem cell transplantation

“…Hematopoietic stem cells (HSCs) are found in the bone marrow (BM) and physiologically give rise to all blood cells . The phenotype of HSCs is not well defined, but HSCs have been suggested to be part of the primitive CD34+/CD38− cell population . A high amount of the CD34+/CD38− cells seem to rest in the G0 cell cycle phase, and are able to initiate retransplantable hematopoiesis in animal models …”