Figure 4: TLR9 contributes to in vitro production of B cells from mixed acute leukemia, but not from acute lymphoblastic leukemia-derived precursor cells. BM CD34+ cells were purified from ProB-ALL, PreB-ALL, C-ALL, M-ALL, and AML and stimulated with Poly(I:C) (TLR3 ligand), PolyU (TLR8 ligand), and CpG-ODN (TLR9 ligand) agonists for 48 h, followed by a 30 day-stromal cell coculture. Newly produced CD34+ and CD19+ cell fractions were further identified and enumerated by multiparametric flow cytometry (a). The indicated gates were used to discriminate cell populations. Cell frequencies within the culture for both CD34+cells (b) and CD19+ B-lymphoid cells (c) are shown. Total numbers of CD19+ recovered cells from each treatment condition were calculated and expressed as yields per input progenitor (d). ProB-ALL: B-cell precursor ALL with prevalence of CD34+CD10+CD19+ cells; PreB-ALL: B-cell precursor ALL with prevalence of CD3CD10+/−CD19+ cells; C-ALL: congenital ALL; M-ALL: mixed ALL; AML: acute myeloid leukemia.