Fig 2: miR-181a Is Required for Secondary Expansion of Memory CD4 T Cells(A) Equal numbers of congenically marked WT and miR-181a−/−YFP+ SMARTA CD4 T cells were co-transferred into B6 mice infected with LCMV 1 day later. On day 50 after infection, immune mice were rechallenged with Lm-gp61. Representative flow plots show YFP+ SMARTA cells gated on CD4 T cells (top left) and the relative frequencies of WT and miR-181a−/− SMARTA cells (bottom left) in the spleen at indicated time points after infection and SLAM and CXCR5 expression on WT and miR-181a−/− SMARTA CD4 T cells on day 7 after recall (middle). Dot plots on the right show the ratios of miR-181a−/− to WT SMARTA CD4 T cells.(B–F) WT and miR-181a−/− mice were infected with LCMV. On day 80 after infection, immune mice were rechallenged with Lm-gp61 and cells were harvested on day 7 at the peak of the recall response. (B) Representative flow plots of IAb GP66 tetramer+ cells gated on YFP+ CD4 T cells. (C) Fold expansion of IAb GP66 tetramer+ memory CD4 T cells on day 7 after rechallenge compared to before infection. (D) Representative histogram of tetramer binding intensity of IAb GP66 tetramer+ YFP+ CD4 T cells (left) and dot plot of tetramer MFI (right). Filled gray in the histogram indicates naive CD4 T cells. (E) Effective GP61 peptide concentration required to elicit a half-maximal IFNγ production. (F) TRB genes from IAb GP66 tetramer+ YFP+ CD4 T cells in the spleen were sequenced. Dot plots show the total number of unique TRB sequences (left) and the Shannon diversity index (right).Data are pooled from two independent experiments with 5–9 mice per group (A–E) or are from one experiment with 3 mice per group (F). Unless stated otherwise, data are presented as means. Statistical significance by two-tailed unpaired t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; NS, not significant.

Fig 3: T-ALL and B-ALL EVs affect HSPC maintenance in vitro.Murine Lin− cells were treated ex vivo with EVs isolated from NSG control mice or ALL PDX models. A After 24 h, as assessed by flow cytometry on LSK gating cells, treatment with ALL EVs affects the numbers of HSC (SLAM). B This is further corroborated by an attrition of the primitive LSK CD34− cells. C Flow cytometry on LSK gating cells showing a loss of quiescent cells (in G0). On this figure, data are shown as means ± SD; n = 4 mice. P value are measured by one-way Anova with Tukey’s multiple comparison test; **P < 0.01; ***P < 0.001; ns, non-significant.

Fig 4: Administration of β-Glucan Drives Expansion of HSPC SubpopulationsWT mice were injected with β-glucan or PBS, and BM analysis was performed after 24 hr.(A) Representative fluorescence-activated cell sorting (FACS) plots for the identification of hematopoietic progenitor cells. After gating for Lin− cells, LSK cells were characterized as cKit+Sca1+ cells. LSK cells subpopulations were further characterized as MPP (CD48+CD150−LSK), ST-HSC (CD48−CD150−LSK), and LT-HSC (CD48−CD150+LSK).(B and C) Cell numbers of LSKs, MPPs, ST-HSCs, and LT-HSCs (B) and cell percentages of the same populations in total BM cells (C) of mice at 24 hr after the administration of PBS or β-glucan (ns = 11 and 12 mice).(D) Representative FACS plots for the identification of MPP subpopulations. After gating for LSK cells, MPP4 cells are characterized as CD48+Flt3+CD150−LSK, MPP3 cells are characterized as CD48+Flt3−CD150−LSK, and MPP2 cells are characterized as CD48+Flt3−CD150+LSK.(E) Frequency of MPP subpopulations in LSK cells in the BM of mice at 24 hr after the administration of PBS or β-glucan (n = 5 mice per group).(F and G) Representative FACS plots for the identification of CD41+ LT-HSCs (F) and frequency of CD41+ LT-HSCs (in total LT-HSCs) (G) in the BM of mice at 24 hr after the administration of PBS or β-glucan (n = 5 mice per group).Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.See also Figure S1.

Fig 5: Intake of T-ALL and B-ALL EVs by lipid raft-enriched HSPC.A Procedure followed to identify populations of murine Lin− cells that take in fluorescent ALL EVs. Murine Sca1+ cells were purified with magnetic beads and stained with CTB prior to incubation with fluorescent ALL EVs (HSP70-ATTO488). B Flow cytometry on different populations showing that LSK and HSC (SLAM cells) express high levels of lipid rafts (CTB-AF555) as well as intake of ALL EVs (ATTO488 EVs). Sca1+ cells show intermediate levels in expression of lipid rafts and ALL EV intake, while Sca1− cells are not positive for lipid rafts expression or for intake of ALL EVs. Representative of two independent experiments. C Flow cytometry on HSC showing that when Sca1+ cells are pretreated for 2 h with Methyl-β-cyclodextrine (Mβc), this affects the lipid rafts staining (CTB-AF555) as well as the intake of ALL EVs (ATTO488 EVs). D Microscopy on murine Sca1+ cells showing that the population of primitive hematopoietic cells which take in fluorescent EVs (ATTO488 EVs) correspond to cells also displaying lipid raft staining with the cholera toxin subunit B (CTB-AF555). Magnification ×40, scale bar represents 5 µm. Representative of two independent experiments. E Single cell sorting on positive cells showing colocalization score (R) between EV intake and lipid rafts. Data shows box-and-whisker plots (n = 30 cells for each conditions), presented as medians (central line), first and third quartiles (bottom and top of boxes, respectively), and whiskers (extreme values). Fluorescent optical sections of cells, magnification ×63, scale bar represents 5 µm.