Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7- lympho-myeloid thymic progenitors

Abstract

The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34(+)lin(-) thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7(-). CD34(+)lin(-) thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-lymphoid, and myeloid-erythroid conditions. Progressive restriction in lineage potential correlated with CD7 expression, that is, the CD7(hi) fraction produced T and NK cells but lacked B and myelo-erythroid potential, the CD7(int) (CD10(+)) fraction produced B, T, and NK cells, but lacked myelo-erythroid potential. The CD7(-) fraction produced all lymphoid and myelo-erythroid lineages and expressed HSC-associated genes. However, CD34(+)lin(-)CD7(-) thymocytes also expressed early T lymphoid genes Tdt, pTalpha, and IL-7Ralpha and lacked engraftment capacity, suggesting the signals that direct lymphoid commitment and corresponding loss of HSC function are rapidly initiated on arrival of HSC in the human thymus. Thus, differential levels of CD7 identify the progressive stages of lineage commitment in human thymus, initiated from a primitive CD7(-) lympho-myeloid thymic progenitor.

Figure 1

CD34⁺ thymocytes show variable expression levels of CD1a and CD7. (A) Expression of CD1a in CD34⁺ enriched thymocytes. (B) The CD34 enriched, lineage-depleted fraction of thymus may be subfractionated into CD34⁺lin⁻CD7^hi cells, and much rarer CD34+lin⁻CD7^int and CD34⁺lin⁻CD7⁻ subpopulations (shown are percentages of each gated population within CD34⁺lin⁻ cells). (C) Most of the CD34⁺lin⁻CD7^int population expresses CD10 and CD45. Numbers on plots are percentages of total cells.

Figure 2

Expression of stem cell– and lymphoid-associated genes in CD34⁺lin⁻ subsets from thymus and blood. Equal numbers of CD34⁺lin⁻CD7^hi and CD34⁺lin⁻CD7⁻ cells from thymus and CD34⁺lin⁻CD7⁻ cells from normal peripheral blood (PBL) were isolated by FACS and subjected to RT-PCR analysis to define the expression levels of selected stem- and lymphoid-associated genes. All results were confirmed at least twice using independently sorted cells from different thymuses.

Figure 3

Cocultivation on OP9 stroma reveals that lineage potential of CD34⁺lin⁻ thymocytes is related to CD7 expression. Approximately 1500 cells from each population (CD34⁺lin⁻CD7⁻, CD34⁺lin⁻CD7^intCD10⁺, and CD34⁺lin⁻CD7^hi) were isolated by FACS, cocultured on OP9 stroma in FL, TPO, IL-7, and EPO and analyzed at 2 to 4 weeks for expression of erythroid (glycophorin A) and lymphoid (CD19, CD56) markers to evaluate the lineage potential of each subset. Shown is FACS analysis of one representative experiment from a total of 4 independent experiments, using different thymus samples.

Figure 4

Immunophenotypic analysis of OP9-DL1 cocultures generated from CD34⁺lin⁻CD7⁻, CD34⁺lin⁻CD7^intCD10⁺, and CD34⁺lin⁻CD7^hi cells. Approximately 1500 cells from each population were isolated by FACS and cocultured with OP9/DL1 stroma and TPO, IL-7, and FL. Cells in culture were analyzed (A) by FACS at 2 to 4 weeks for expression of T-lineage markers (CD4, CD8) and CD7 and (B) by RT-PCR for pTα expression. Shown are data from one representative experiment of a total of 7 independent experiments for FACS analysis, and 2 independent experiments for RT-PCR, using different thymus samples.

Figure 5

Lineage analysis of clones generated from individual CD34⁺lin⁻CD7⁻ thymocytes. Clones A and B were generated from different single CD34⁺lin⁻CD7⁻ cells isolated by FACS, and cultured on OP9 stroma. (A) FACS of each clone harvested from OP9 stroma analyzing B (CD19) and erythroid (glycophorin; top) and NK (CD56 vs CD7) potential (middle). The same clones were replated into methylcellulose medium and generated CFU, which were analyzed by FACS for myeloid and erythroid potential (CD66b vs glycophorin; bottom). (B) RT-PCR analysis of clones A and B (at a different time point to FACS analysis) reveals the expression of T lymphoid (pTα), erythroid (glycophorin), and/or B lymphoid (Pax 5) genes. In these assays, the lineages produced from each clones fluctuate over time in culture (eg, clone B has CD19⁺ cells at one time but is negative for Pax 5 later in culture). Thus, lineage potential is assigned based on either FACS or RT-PCR marker expression at any time during the life of the clone.