Figure 4.
Platelet counts in St3gal1MK−/− mice are normalized by suppression of CD4+ and Jak3-dependent immune cells. ( A) Platelet count during pan–anti-inflammatory treatment using dexamethasone of St3gal1MK−/− control (blue) and St3gal1MK−/− (red) mice (n = 4). Injection tempo is indicated as a light blue line. (B) Platelet count in Jak3−/− (purple) and DKO mice (orange) (n = 6-11. Average control mice platelet count (dashed blue line, blue circle) and St3gal1MK−/− platelet count (dashed red line, red circle) shown. (C) Binding of PNA to Jak3−/− and DKO platelets as evaluated by flow cytometry. Dashed line indicates fold-change over controls (n = 4). (D) Percentage of TF antigen–positive BM MKs as determined by immunofluorescence of whole femur longitudinal sections using anti-GPIbα and anti–TF antigen antibodies (n = 3). Platelet count during antibody-mediated cell depletion treatments of control (white) and St3gal1MK−/− mice (red). Mice were treated with depleting anti-CD4 antibody (E) (n = 8-13), depleting anti-CD8 antibody (F) (n = 3), and depleting anti-CD20 antibody (G) (n = 6-8). Isotype control injections are shown in gray in panels E, F, and G; the antibody injection tempo is indicated as a light blue line. (H) Confocal microscopy of BM sections from control (bottom right) and ST3Gal1MK−/− (2 examples shown) mice. Tissues were stained for PF4 (MKs in green) and CD4 (CD4 immune cells in red). Overlap between PF4 and CD4 is colored in yellow. Scale bars, 10 μm. (I) Immunofluorescence staining of longitudinal whole femur sections from control (left) and ST3Gal1MK−/− (right) mice, and their respective selected enlargement. Tissues were stained for PF4 (MKs in green) and CD4 (CD4 immune cells in red). Overlap between PF4 and CD4 was colored in yellow. Scale bars, 100 μm. (J) Quantification of colocalization, expressed as percentage of PF4+ cells (n = 5-7). (K) A heat map of pDC-related genes expressed by sel-CD4+ spleen (left) and BM (right) cells from controls (black) and St3gal1MK−/− (red) mice. Significantly differentially expressed genes in the BM are indicated in bold. For multiple comparisons (B-D), 1-way analysis of variance with Tukey’s multiple comparison test was performed. Result displayed is comparison with wild-type control, or as indicated by horizontal lines. For comparison of 2 groups over a treatment period (A,E-G), a multiple Student t test with Holm-Šídák method correction was performed. Comparison of control vs St3gal1MK−/− in black (A,E-G). For comparison of 2 groups (panel J), a 2-tailed, unpaired Student t test was performed. *P < .05, **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; MFI, mean fluorescence intensity; ns, not significant.

Platelet counts in St3gal1MK−/− mice are normalized by suppression of CD4+ and Jak3-dependent immune cells. ( A) Platelet count during pan–anti-inflammatory treatment using dexamethasone of St3gal1MK−/− control (blue) and St3gal1MK−/− (red) mice (n = 4). Injection tempo is indicated as a light blue line. (B) Platelet count in Jak3−/− (purple) and DKO mice (orange) (n = 6-11. Average control mice platelet count (dashed blue line, blue circle) and St3gal1MK−/− platelet count (dashed red line, red circle) shown. (C) Binding of PNA to Jak3−/− and DKO platelets as evaluated by flow cytometry. Dashed line indicates fold-change over controls (n = 4). (D) Percentage of TF antigen–positive BM MKs as determined by immunofluorescence of whole femur longitudinal sections using anti-GPIbα and anti–TF antigen antibodies (n = 3). Platelet count during antibody-mediated cell depletion treatments of control (white) and St3gal1MK−/− mice (red). Mice were treated with depleting anti-CD4 antibody (E) (n = 8-13), depleting anti-CD8 antibody (F) (n = 3), and depleting anti-CD20 antibody (G) (n = 6-8). Isotype control injections are shown in gray in panels E, F, and G; the antibody injection tempo is indicated as a light blue line. (H) Confocal microscopy of BM sections from control (bottom right) and ST3Gal1MK−/− (2 examples shown) mice. Tissues were stained for PF4 (MKs in green) and CD4 (CD4 immune cells in red). Overlap between PF4 and CD4 is colored in yellow. Scale bars, 10 μm. (I) Immunofluorescence staining of longitudinal whole femur sections from control (left) and ST3Gal1MK−/− (right) mice, and their respective selected enlargement. Tissues were stained for PF4 (MKs in green) and CD4 (CD4 immune cells in red). Overlap between PF4 and CD4 was colored in yellow. Scale bars, 100 μm. (J) Quantification of colocalization, expressed as percentage of PF4+ cells (n = 5-7). (K) A heat map of pDC-related genes expressed by sel-CD4+ spleen (left) and BM (right) cells from controls (black) and St3gal1MK−/− (red) mice. Significantly differentially expressed genes in the BM are indicated in bold. For multiple comparisons (B-D), 1-way analysis of variance with Tukey’s multiple comparison test was performed. Result displayed is comparison with wild-type control, or as indicated by horizontal lines. For comparison of 2 groups over a treatment period (A,E-G), a multiple Student t test with Holm-Šídák method correction was performed. Comparison of control vs St3gal1MK−/− in black (A,E-G). For comparison of 2 groups (panel J), a 2-tailed, unpaired Student t test was performed. *P < .05, **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; MFI, mean fluorescence intensity; ns, not significant.

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