T lymphoid differentiation in human bone marrow

Abstract

The unique role of the thymus in the development of T cells was established >4 decades ago. To elucidate how uncommitted lymphoid progenitor cells are instructed to migrate from bone marrow to the thymus to undergo T lymphoid differentiation, we generated and analyzed a genome-wide gene expression profile of CD7+ CD10+ human bone marrow T cell lineage precursors (TLPs) by using the serial analysis of gene expression technique. Unexpectedly, the serial analysis of gene expression profile identified a high number of (pre-) T cell receptor antigen (TCR)-related transcripts in bone marrow TLPs. To determine the configuration of the TCRbeta locus in these cells at a quantitative level, we sorted and analyzed bone marrow TLPs from five donors by single-cell PCR. Similar proportions of TLPs harbored TCRbeta germ-line alleles, D-J, or V-DJ gene rearrangements. Thus, bone marrow TLPs are heterogenous with respect to TCRbeta rearrangement status, suggesting an active recombination machinery that is consistent with the expression of RAG1, RAG2, and TdT in this population. As a hallmark of ongoing TCRbeta V-DJ rearrangement, we could amplify broken-ended recombination-signal sequence DNA intermediates from bone marrow TLPs, but not from mature T cells by ligation-mediated PCR. Approximately half of the TCRbeta rearrangements were compatible with the expression of a functional pre-TCR, which is in agreement with surface expression of pre-Talpha on bone marrow TLPs as shown by confocal laser microscopy and flow cytometry. At a frequency <0.5% of mononucleated cells in human bone marrow, this population is rare, yet it exemplifies T lymphoid differentiation in the human already before immigration into the thymus.

Fig. 1.

Single-cell PCR strategy for the determination of the configuration of TCRβ loci and strategy for ligation-mediated PCR to detect broken-ended TCR Dβ-recombination signal sequence (RSS) DNA intermediates. (A) The organization of the human TCRβ locus and the relative position of PCR primers are depicted for the amplification of specific fragments for germ-line configuration (Top), D-J gene rearrangement (Middle), and V-DJ gene rearrangement (Bottom). (B) The rearrangement of V segments to preexisting DJ joints is initiated by DNA double-strand breaks at the heptamer sequence immediately flanking the D segment (i). DNA double-strand breaks result in blunt ends (ii), which can be ligated (hence “ligation-mediated”) to a blunt-ended linker molecule (iii). By using oligonucleotides specific for sequences upstream of the RSS and the ligated linker sequence, broken-ended Dβ-RSS DNA intermediates can be amplified (iv).

Fig. 2.

Expression of RAGs and ongoing recombination of TCRβ V to DJ segments in bone marrow TLPs. (A) By using immunomagnetic beads, CD3⁺ T cells from umbilical cord blood, bone marrow, peripheral blood, and tonsillar tissue were isolated and subjected to semiquantitative RT-PCR analysis of mRNA expression of pre-Tα, RAG1, RAG2, and HPRT, which was used for standardization. PCR products after 30 and 36 cycles of amplification are shown. (B) Genomic DNA was isolated from bone marrow TLPs and mature peripheral blood T cells (PBLs). Comparable amounts of DNA (as shown by amplification of a specific fragment of the MDR1 gene) were ligated to a linker molecule and linker-ligated RSS-DNA intermediates carrying double-strand breaks at 5′ heptamers of Dβ1 or Dβ2 gene segments were amplified by ligation-mediated PCR. “Buffer” indicates control without DNA template.

Fig. 3.

Expression of pre-Tα within pre-TCR complexes on human bone marrow T lineage precursors. (A) Mononuclear bone marrow cells were depleted from myeloid progenitor cells and were double-stained by using antibodies against CD3 (FITC) and pre-Tα, together with a PE-labeled secondary antibody. (B) Bone marrow TLPs were FACS sorted based on staining for CD7 (PE) and CD10 (FITC) and were subsequently labeled with an anti-pre-Tα antibody, together with a Cy5-conjugated secondary antibody (false-color images). Single cells were analyzed by laser microscopy and staining for CD7 and CD10, and pre-Tα was colocalized in an overlay image (Upper). As a control, single sorted B cells were double-stained by using an antibody against CD20 and pre-Tα, together with a Cy5-conjugated secondary antibody (Lower).