Fig 2: Flow cytometric analysis of mesenchymal stem cell surface marker CD33 (negative) and CD105 (positive).

Fig 3: Loss of YTHDF2 from Established LSCs and Human AML Cells Compromises Their Ability to Propagate AML(A) Ythdf2fl/fl (Ythdf2CTL) and Ythdf2fl/fl;Mx1-Cre (Ythdf2iCKO) FL c-Kit+ cells were co-transduced with Meis1 and Hoxa9 retroviruses, serially replated, and transplanted into primary recipients. GFP+c-Kit+CD45.2+ cells sorted from leukemic primary recipients were re-transplanted into secondary recipients (n = 14–16).(B) Percentage of GFP-expressing cells as a measure of YTHDF2 expression in Ythdf2CTL and Ythdf2iCKO leukemic cells prior to secondary transplantation.(C) Percentage of CD45.2+ leukemic cells in the PB of the secondary recipient mice 3 weeks after transplantation (n = 14–16 recipients).(D) Survival curve of mice transplanted with Ythdf2CTL and Ythdf2iCKO leukemic cells (n = 14–16 mice).(E) Percentage of GFP-expressing cells in PB CD45.2+ cell compartment of secondary recipient mice.(F) Left: relative levels of YTHDF2 mRNA (normalized to HPRT1) in human AML THP-1 cells transduced with lentiviruses expressing scrambled short hairpin RNA (shRNA) (CTL) and two independent shRNAs targeting YTHDF2 (KD1 and KD2); n = 3. Right: western blot of YTHDF2 in THP-1 cells shown on the left. a-Histone 3 (H3) was used as a loading control.(G) Proliferation assays with THP-1 cells with CTL, KD1, and KD2 shRNAs.(H) Percentage of Annexin V+DAPI- cells.(I) Percentage of CD11b-CD14-, CD11b+CD14-, CD11b+CD14+, and CD11b-CD14+ cells in cultures shown in (G) and (H).(J) NSG mice were injected with THP-1 cells transduced with CTL (n = 4) or KD (n = 4) lentiviruses and analyzed 1 month later. Percentage of human CD45+CD33+ cells in the BM, liver, spleen, and PB of the recipient mice is shown.(K) Survival curve of mice transplanted with 10,000 (n = 6) and 1,000 (n = 3) THP-1 cells.(L) Three independent human primary AML samples (AML1–AML3; detailed in STAR Methods) were transduced with CTL, KD1, and KD2 lentiviruses. The graph shows AML-CFC frequencies after 7 days of culture (n = 3 technical replicates per sample).(M) Representative colony images from (L).Data represent mean ± SEM in (A)–(K) or mean ± SD in (L)–(M); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 4: ILC2-derived IL-13 expands and activates suppressive M-MDSCs in APL. a Representative example of flow cytometry analysis of cell surface expression of the IL-13Ra1 on CD3+, CD14-, CD14+ cells in peripheral blood of an APL patient. b Frequencies of IL-13Ra1 expressing cells in the different compartments (CD3+, CD14-, CD14 + n = 11). c Representative example of flow cytometry phenotypic analysis of CD14+ cells in healthy donors (HD) and APL patients (APL). Evaluated are the expression of HLA-DR and CD33, to define phenotypic M-MDSC. d Expression of arginase-1 and e iNOS in purified CD14+ cells from HD and APL, as assessed by qPCR (n = 11). f Frequency of M-MDSCs in peripheral blood of HD and APL (n = 22). g, h Expression of arginase-1 (g) and iNOS (h) as assessed by qPCR, in purified CD14+ cells cultured in medium supplemented or not with recombinant IL-13 (rIL-13), or cultured in supernatants derived from ILC2 cell lines, activated by PDG2 and the NB4 APL cell line (SN ILC2). A blocking anti-IL-13 antibody (aIL-13) or an Ig control (Ig Ctrl) were added where indicated (n = 6, three independent experiments). i Representative example of CFSE-dilutions of CD3+ T cells co-cultured with CD14+ monocytes, or with in vitro-induced M-MDSCs (CD14+SN ILC2). j Frequencies of proliferating CD3+ T cells upon co-culture with CD14+ cells or with in vitro induced M-MDSCs (CD14+SN ILC2) (n = 6, three independent experiments). Error bars are s.e.m. Statistical analysis was performed using ANOVA test (b), t test (d–f), Kruskal-Wallis test (g, h) and Mann-Whitney test (j)