TLR2 signaling causes long-term impairment of LSC activity. (A) Average ± SEM percent immunophenotypic HSC (left), MPP1 (middle), and MPP2 (right) in CD45.2⁺ BM. Replicates for donor-recipient transplants include: AML-WT (n = 9/group) and AML-Tlr2^−/− (n = 8/group). (B) Kaplan-Meier survival curves of WT mice transplanted with limiting numbers (M million) of CD45⁺Lin⁻cKit⁺ BM cells sorted from transplanted WT mice 24 hours after PBS or Pam₃CSK₄ treatment (n = 4/group). (C) LSC estimation for each treatment group from panel B. (D) Gene set enrichment analysis of significantly differentially expressed genes with Pam₃CSK₄ vs PBS control treatment in HSPC-MPP c5 cells. (E) Volcano plot of transcription factor activity analyses associated with up and downregulated genes in Pam₃CSK₄ vs PBS control treatment for HSPC-MPP c5 cells. (F) Average ± SEM percent MHC class II⁺ cells within the CD45.2⁺Lin⁻cKit⁺Sca1⁺ (LSK) BM from Dnmt3a^+/−Flt3^ITD AML-transplanted CD45.1⁺ WT mice 24 hours after treatment with PBS or Pam₃CSK₄ (n = 5/group). (G) Comparison of the probability of OS in months for patients with AML with high (n = 34) vs low (n = 26) coexpression of TLR2 and MHCII genes. Statistical significance was calculated by unpaired t test (F). ∗∗P < .01 and log rank test (G). Statistical significance by ordinary 1-way ANOVA using Tukey multiple comparison test between cell populations (A), ELDA analysis (C), unpaired t test (F), and log rank test (G). ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05.