TLR Stimulation of Bone Marrow Lymphoid Precursors from Childhood Acute Leukemia Modifies Their Differentiation Potentials

Figure 7

Intracellular TLRs lead to production of mature NK cell in both lymphoid and myeloid acute leukemia. ProB-ALL, C-ALL and AML CD34⁺ cells were purified and stimulated with Pam3CSK4 (TLR2 ligand), Flagellin (TLR5 ligand), MALP2 (TLR2/6 ligand), PolyU (TLR8 ligand) and CpG-ODN (TLR9 ligand) agonists for 48 h. After 30 day-culture, newly produced CD56⁺CD11c⁻ and CD56⁺CD11c⁺ developing and mature NK cells, respectively, were identified by multiparametric flow cytometry (a). CFSE⁺ K562 cells were used as target of cultured NK cells from Mock-ALL progenitors. Cytotoxicity was evaluated by 7AAD incorporation (b), and the expression of the activating receptor NKG2D analyzed by flow cytometry (b). The indicated quadrants in (a) were used to determine cell frequencies for each leukemia subtype (c). Total numbers of mature CD56⁺ recovered cells from the various treatment conditions were calculated and expressed as yields per input progenitor (d). The expression of PAX-5, EBPα and Id-2 transcription factors was analyzed by RT-PCR upon 48 h-CpG stimulation (e). ProB-ALL: B-cell precursor ALL with prevalence of CD34⁺CD10⁺CD19⁺ cells; PreB-ALL: B-cell precursor ALL with prevalence of CD3⁻CD10^+/−CD19⁺ cells; C-ALL: congenital ALL; M-ALL: mixed ALL; AML: acute myeloid leukemia. Mo: mock; Pam: Pam3CSK4; Fla: flagellin; Mal: MALP2; PU: PolyU.