Figure 4

Molecular profile of CD34^-lineage^- cells differentiated with IL-15 plus IL-21. Panel A: Molecular features of cytokine-differentiated CD34^-lineage^- cells. The expression of mRNA specific for the NK-associated transcription factor Id2 and for Bcl-2 and GATA-3 was investigated by quantitative PCR (qPCR) in freshly isolated (day 0) and cytokine-differentiated CD34^-lineage^- cells (week +4), as previously detailed [5]. CD34⁺ cells from the same UCB samples were used as control. Panel B: TCRγ rearrangement status after in vitro exposure to IL-15 plus IL-21. The analysis of TCRγ rearrangement status was performed as detailed in Materials and Methods. Peripheral blood CD3⁺ T cells from healthy donors were used as control for TCRγ rearrangement status. CD34^-lineage^- cells cultured with IL-15 and IL-21 harboured un-rearranged TCRγ genes, similar to freshly isolated CD34^-lineage^- cells (day 0) and to cells maintained with SCF and Flt3-L either alone or supplemented with IL-21. M = marker. JP = rearrangement involving the Jγ and Cγ segments. The forward primer mapped to the JγP cassette, whereas the reverse primer mapped to the constant (C) region. JP1 = rearrangement involving the JγP1 region and the Cγ segment. The forward primer mapped to the JγP1 cassette, whereas the reverse primer mapped to the constant (C) region. JP2 = rearrangement involving the JγP2 region and the Cγ segment. The forward primer mapped to the JγP2 cassette, whereas the reverse primer mapped to the constant (C) region. J1/2 = rearrangement involving the Jγ1/2 region and the Cγ segment. The forward primer mapped to the Jγ1–2 cassette, whereas the reverse primer mapped to the constant (C) region. L = linker region (no rearrangement). Forward and reverse primers mapped to the spacer region between Vγ9 and Vγ10 segments.