T-cell development

T cells develop from progenitors that migrate from the bone marrow into the thymus. Thymocytes are subdivided roughly as being double negative (DN), double positive (DP), or single positive (SP), based on the expression of the CD4 and CD8 coreceptors. The DN stage is heterogeneous and can be subdivided into four distinct subsets in mice based on the expression of CD44 and CD25. In human, three distinct DN stages can be recognized: a CD34+CD38−CD1a− stage that represents the most immature thymic subset and the consecutive CD34+CD38+CD1a− and CD34+CD38+CD1a+ stages. Human DN thymocytes mature via an immature single positive (ISP CD4+) and a DP stage into CD4+ or CD8+ SP T cells that express functional T cell receptors (TCR) and that exit the thymus. In this study, gene expression was measured in each of these nine stages. CD34+ lineage negative “stem cell-like” cells were obtained from umbilical cord blood (UCB) and CD34+CD38− CD1a−, CD34+CD38+CD1a−, CD34+CD38+CD1a+, ISP CD4+, DP CD3−, DP CD3+, SP CD4+, and SP CD8+ subpopulations were obtained from thymi which represented consecutive stages of T cell development. Thymi were obtained as surgical tissue discards from children aged 7 wk to 3 yr (median of 6 mo) undergoing cardiac surgery at the Erasmus MC Rotterdam, with informed consent from the parents. The children did not have immunological abnormalities. Thymocytes were isolated by cutting the thymic lobes into small pieces and squeezing them through a metal mesh, and stored at +-80C until further analyses. Mononuclear cells (MNCs) were isolated by Ficoll-Paque (Amersham Biosciences) density centrifugation from human umbilical cord blood (UCB) obtained from full-term normal deliveries and from peripheral blood of healthy volunteers. All samples were obtained according to the guidelines of the Medical Ethical Committee of the Erasmus MC that also approved the human studies. For the isolation of CD34+ lineage (lin)- UCB cells and thymocyte subsets, total MNCs or thymocytes from five donors were pooled to reduce intrasample variation. After thawing, pooling, and Ficoll density separation, thymocytes were labeled with fluorochromeconjugated monoclonal antibodies. To isolate the CD34+ lin- UCB cells, CD34+CD38-CD1a-, CD34+CD38+CD1a-, and CD34+CD38+CD1a+ thymocytes, magnetic beads were used to enrich for CD34+ cells. For isolation of the ISP population, thymocytes were depleted of CD3-expressing cells using magnetic beads. All magnetic beads were used according to the manufacturer’s protocol. After MACS sorting, the enriched cells were labeled with fluorochrome-conjugated monoclonal antibodies for further purification by high speed cell sorting. All cell sorting was performed on a FACS DiVa cell sorter (BD Biosciences). Monoclonal antibodies used, with the clone in brackets: CD4-FITC (SK3), CD38-FITC (HB7), TCRGD- FITC (WT31), CD1a-RDI (T6), CD3-PE (SK7), CD16-PE (B73.1), CD19-PE (4G7), CD56-PE (M431), TCRGD-PE (11F2), CD3-PerCP (SK7), CD8- PerCP (SK1), CD19-PerCP (SJ25C1), CD3-APC (SK7), CD8-APC (SK1) and CD34-APC (8G12) (all obtained from BD Biosciences), CD13- RDI (MY7), and CD33-RDI (906) (obtained from Beckman Coulter). Purity of sorted population was determined on the FACS Calibur (BD Biosciences) and shown to be >95% for all populations. All populations were sorted twice using different donors for each sort.