Fig 2: Effects of the absence of T cell-intrinsic MyD88, IL-1R or IL-18R signaling on the proliferation and phenotype of CD4+ and CD8+spleen T cells from infected mixed BM chimeras.(A, C, G, E and I) Mean frequencies and (B, D, F, H and J) absolute cell numbers of: (A and B) BrdU+CD4+ and (C and D) CCR5+CD4+ cells gated on WT CD45.1+ or KO CD45.2+ CD4+ T cells, as in Figure 5A. (E and F) IFN-?+CD8+ and (G and H) GzB+CD8+ T cells gated on WT CD45.1+ or KO CD45.2+ CD8+ T cells, as shown in Figure 6—figure supplement 3. (I and J) CD44hiCD4+ T cells, gated on WT or KO CD4+ T cells as in Figure 5A. Bars are the mean of individually analyzed mice from one (C, D, I and J) or two combined independent experiments (A, B, E, F, G and H). (n = 3 to 8); error bars = SEM; ND = not done; ns = non significant; *p=0.05; **p=0.01; ***p=0.001, ****p=0.0001 (two-tailed Student t-test). Gate strategies and representative dot plots are shown in Figure 6—figure supplements 1–5.

Fig 3: Effects of the absence of T cell-intrinsic MyD88, IL-1R or IL-18R signaling on IFN-? production by spleen CD4+ T cells of infected mixed BM chimeras.(A) Representative dot plots showing gating strategy for CD4+ T cells and for CD45.1+ (B6.SJL WT) or CD45.2+ (B6 WT or Myd88-/- or Il1r1-/- or Il18r1-/-) splenocytes from mixed BM chimeras. (B) Representative dot plots of IFN-? expression gated on CD4+CD45.1+ (B6.SJL WT) or CD4+CD45.2+ (B6 WT or Myd88-/- or Il1r1-/- or Il18r1-/-) splenocytes from non-infected control or infected (2 × 103 blood trypomastigotes, ip) mixed BM chimeras, on day 14 pi. (C) Percentages and (D) absolute cell numbers of IFN-?+CD4+ T cells gated as in (A) and (B). Bars are the mean of combined data from 2 independent experiments (n = 3 to 7 individually analyzed chimeric mice); error bars = SEM; ns = non significant; **p=0.01; ***p=0.001, ****p=0.0001 (two-tailed Student t-test). Data are representative of 3 to 4 independent experiments.

Fig 4: DOT1L inactivation in the adult bone marrow leads to rapid depletion of myeloid progenitors, followed by decrease in the numbers of hematopoietic stem cells (A) (left) Bone marrow cellularity in Dot1lf/f Mx-Cre- and Dot1lf/f Mx-Cre+ mice 7 days post poly(I:C) induction (n = 21 mice/group, pooled from 5 independent experiments; mean +/- SEM). (right) Bone marrow cellularity decreases in Dot1lf/f Mx-Cre+ mice 10 days post poly(I:C) induction. (n = 14–16 mice/group, pooled from 3 independent experiments; mean +/- SEM). (B) Western blot shows decreased H3K79me2 in total BM at day 7 (left) and day 10 (right); (C) Flow cytometric analysis quantification shows that Pre MegE progenitors (CD150+CD105-CD16/32-CD41-LK) are significantly decreased as early as day 7 (left) and continue to be depleted by day 10 (right). (D) Quantification of flow cytometric analysis showing that granulocyte-macrophage progenitors (GMP) (CD16/32+CD150-CD41-LK) are significantly decreased as early as day 7 (left) and continue to decrease through day 10 (right). (E) Myeloid colony formation by wild-type vs Dot1l-null BM in CFU-GM assays (n = 8 mice/group with technical triplicates; mean +/- SEM; pooled from 2 experiments) showing a lack of colony formation potential in Dot1l ?/? mice in comparison to Dot1l WT. (F–H) Profound decrease in LT-HSC, CD11b+Gr1+ BM myeloid cells, and B220+ CD19+B cells at day 10 post poly (I:C). (ns p > 0.05, * p = 0.05, ** p = 0.01, *** p = 0.001, **** p = 0.0001).

Fig 5: A20 leukemia cell line is negative for programmed death 1 (PD-1) and programmed death-ligand 2 (PD-L2), but barely expressed CD80, leaving programmed death-ligand 1 (PD-L1) freely available to interact in trans with PD-1 expressed in immune cells. (A) Upper panel: Flow cytometry histograms displaying the staining with the isotype control and anti-Kd (H-2d) and anti-B220 antibodies in A20 PD-L1 wild-type (WT) and A20 PD-L1 knockout (KO) leukemia cells. Middle and lower panels: Flow cytometry histograms showing the pattern of expression of PD-1, CD80, PD-L1, and PD-L2 in A20 PD-L1 WT compared to KO tumor cells. Notice that in A20 PD-L1-deficient cells, PD-L1 protein expression is absent on the cell surface, whereas the level of expression of the other molecules of the pathway is similar to that observed in A20 PD-L1 WT leukemia cells. This experiment was repeated three times with similar results. A representative histogram displaying the mean fluorescence intensity (MFI) value for each biomarker of the PD-L1/PD-L2/CD80/PD-1 pathway is shown along with the expression of MHC class I (Kb) and B220. (B) Overlapped dot plot illustrating simultaneously PD-L1 expression on host B cells (B220-positive cells), host CD4 T cells, and host CD8 T cells (black dots) infiltrating the tumor and on A20 tumor cells (red dots) in metastatic nodules of the liver of F1 recipients collected at day 30 after the adoptive transfer of tumor cells.