Figure 5.

Targeting of IFNα to the alpha granules

(A) Design of third-generation SIN lentiviral vectors expressed from Pf4P. For the targeting of IFNα to αGs, the sorting domain and signal peptide of RANTES were fused to the N terminus of the IFNα1 coding sequence without the IFNα signal peptide, plus an IRES-EGFP sequence (RSD.IFNα vector). As the control, only the full coding sequence of IFNα with its signal peptide was fused to the IRES.eGFP (IFNα vector). (B) Confocal images of RSD.IFNα- and IFNα-transduced CB-derived MKs showing transgenes (green), P-selectin (red), and DAPI (blue). IFNα was visualized with 1° human IFNα-1 Abs and 2° goat-anti-rabbit Alexa Fluor 647 Abs, P-selectin⁺ αGs were visualized with 1° human/mouse Abs and 2° goat-anti-mouse Cy3 Abs, and GFP was detected in the 488 channel without Ab staining. Images were acquired with a Leica Dmi8 Inverted-3 confocal microscope with a 63× oil immersion objective and 4× digital zoom; scale bar, 10 μm. (C) Box-whisker plots showing colocalization of the GFP and IFNα transgenes with P-selectin⁺ αGs in CB-derived MKs. Line at median on boxplots, n = 8 MKs from two independent transductions; ns, not significant; ∗∗p < 0.01, Mann-Whitney test. (D) ELISA results showing secretion of GFP and IFNα protein from NA and A murine platelets. Data represent meadian and range, n = 5 individually transplanted mice, ∗∗p < 0.01, Mann-Whitney test. (E) MFI of P-selectin expression in A and NA GFP⁺ and GFP⁻ platelets. Native platelets shown for comparison. (F) Platelet size measured by the FSC-A of IFNα-positive and native platelets. (E and F) n = 5, platelets from five independent RSD.IFNα and mock mice, Student's t test with Welch's correction, ns, not significant.