Figure 7.
pDCs inhibit thrombopoiesis primarily through secretion of IFN-1 mediated by Siglec H. Platelet count during treatment with IFNAR blockade (A, n = 12) or anti–Siglec H mAb 440c (B, n = 6-7) in control (blue) and St3gal1MK−/− (red) mice. Injection of isotype controls into St3gal1MK−/− (gray, n = 5). Injection tempo indicated as a light blue dotted line. (C) Quantification of BM pDCs from control and St3gal1MK−/− mice evaluated by using flow cytometry (Lin–, BST2+, CD11c+, Siglech+). (D) BM pDCs were isolated with negative selection magnetic cell sorting kit and evaluated for BST2 reactivity by using flow cytometry. (E) A section of immunofluorescence staining of whole-mount BMs from control and St3gal1MK−/− mice using mAb to BST2. Scale bars, 20 μm. (F) Quantification of BST2+ cells in immunofluorescence stained whole-mount BMs in panel D (n = 3). (G) Colocalization of BST2+ cells with MKs (PF4+) in immunofluorescence-stained whole-mount BMs in panel D (n = 3). (H) Scheme of the pDC and MK coculture experimental setup. (I) IFN-β content in supernatant media of wild-type pDC with control (not detected [ND]) or St3gal1MK−/− (red) cultured MKs after 18 hours of coculture. Addition of anti–Siglec H mAb 440c is indicated . The effect of anti–Siglec H on IFN-β production was not measured (NM) with control MKs. (J) Immunofluorescence staining of wild-type (or control) pDCs cocultured with control (left) or St3gal1MK−/− (right) MKs. MKs and pDCs were identified by using an anti-PF4 (green) and anti-BST2 (red) mAb, respectively. Scale bars, 30 μm. (K) Quantification of percentage of MKs that form proplatelets (proPLTs) as determined by in vitro culture. Control (blue) and St3gal1MK−/−(red) MKs differentiated BM-derived cells cultured in the absence (left panel) or presence (right bars) of control pDCs. For comparison of 2 groups over a treatment period (A-B), multiple Student t tests with Holm-Šídák method correction was performed. Comparison of control vs St3gal1MK−/− in black; St3gal1MK−/− vs St3gal1MK−/− isotype control in salmon. For comparison of 2 groups (D-E,G), a 2-tailed, unpaired Student t test was performed. For multiple comparisons (I), 1-way analysis of variance with Tukey’s multiple comparison test was performed. *P < .05, **P < .01. Only significant changes are annotated. DAPI, 4′,6-diamidino-2-phenylindole; FLT3L, FLT3 ligand; ns, not significant.

pDCs inhibit thrombopoiesis primarily through secretion of IFN-1 mediated by Siglec H. Platelet count during treatment with IFNAR blockade (A, n = 12) or anti–Siglec H mAb 440c (B, n = 6-7) in control (blue) and St3gal1MK−/− (red) mice. Injection of isotype controls into St3gal1MK−/− (gray, n = 5). Injection tempo indicated as a light blue dotted line. (C) Quantification of BM pDCs from control and St3gal1MK−/− mice evaluated by using flow cytometry (Lin–, BST2+, CD11c+, Siglech+). (D) BM pDCs were isolated with negative selection magnetic cell sorting kit and evaluated for BST2 reactivity by using flow cytometry. (E) A section of immunofluorescence staining of whole-mount BMs from control and St3gal1MK−/− mice using mAb to BST2. Scale bars, 20 μm. (F) Quantification of BST2+ cells in immunofluorescence stained whole-mount BMs in panel D (n = 3). (G) Colocalization of BST2+ cells with MKs (PF4+) in immunofluorescence-stained whole-mount BMs in panel D (n = 3). (H) Scheme of the pDC and MK coculture experimental setup. (I) IFN-β content in supernatant media of wild-type pDC with control (not detected [ND]) or St3gal1MK−/− (red) cultured MKs after 18 hours of coculture. Addition of anti–Siglec H mAb 440c is indicated . The effect of anti–Siglec H on IFN-β production was not measured (NM) with control MKs. (J) Immunofluorescence staining of wild-type (or control) pDCs cocultured with control (left) or St3gal1MK−/− (right) MKs. MKs and pDCs were identified by using an anti-PF4 (green) and anti-BST2 (red) mAb, respectively. Scale bars, 30 μm. (K) Quantification of percentage of MKs that form proplatelets (proPLTs) as determined by in vitro culture. Control (blue) and St3gal1MK−/−(red) MKs differentiated BM-derived cells cultured in the absence (left panel) or presence (right bars) of control pDCs. For comparison of 2 groups over a treatment period (A-B), multiple Student t tests with Holm-Šídák method correction was performed. Comparison of control vs St3gal1MK−/− in black; St3gal1MK−/− vs St3gal1MK−/− isotype control in salmon. For comparison of 2 groups (D-E,G), a 2-tailed, unpaired Student t test was performed. For multiple comparisons (I), 1-way analysis of variance with Tukey’s multiple comparison test was performed. *P < .05, **P < .01. Only significant changes are annotated. DAPI, 4′,6-diamidino-2-phenylindole; FLT3L, FLT3 ligand; ns, not significant.

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