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HEP_24162_sm_suppinfofig1.tif2119KSupporting Figure 1. Expression of key surface molecules and chemokines/receptors on CD11b+ cells purified from islet allografts. Cells were isolated from BALB/c (H-2d) islet allografts cotransplanted with B6 (H-2b) HSC on POD 7. CD11b+ cells were purified by magnetic beads. Islet alone allografts served for comparison (n=3 in each group). (A) Expression of the indicated key cell surface molecules were analyzed by flow cytometry gated on CD11b+ cells, and demonstrated as histograms (filled areas are isotype controls). The number is percentage of the positive cells. The data are representative of three separated experiments. (B) Expression of CD62L, CCR7, CCR2 and CCR5 mRNA. Total RNA was isolated from the CD11b+ cells, and mRNA expression was assessed by quantitative (q) PCR. The data are expressed as mean relative to 18S ± 1SD.
HEP_24162_sm_suppinfofig2.tif2345KSupporting Figure 2. Impact of HSC on BM cell culture with GM-CSF and IL-4. HSC (B6) were added (HSC:BM cells = 1:80) at the beginning of the culture of B6 BM cells (2 × 106/well) in the presence of GM-CSF (8 ng/ml) and IL-4 (1000 U/ml) for 5 days. The H-MC were harvested and re-suspended in RPMI 1640 medium for flow analysis. (A) Cells were stained with mAbs against CD11b and the indicated surface molecules. Expression of surface molecules was analyzed in CD11b+ cell population, and demonstrated as histograms (clear area is isotype control). The number is percentage of positive cells. The data are representative of two separate experiments. (B) 300 BALB/c islets were mixed with 2, 5 × 106 of H-MC (B6) generated in the presence of GM-CSF and IL-4, and transplanted into diabetic B6 recipients. Islet allografts co-transplanted with H-MC generated in the presence of GM-CSF were used for comparison. Survival of islet allografts were determined by monitoring serum glucose levels as described in Methods.
HEP_24162_sm_suppinfofig3.tif3685KSupporting Figure 3. Identification of the soluble factor (s) produced by HSC that mediates H-MC induction. (A) G-CSF, GM-CSF and VEGF do not mediate induction of H-MC. HSC from WT, G-CSF−/− or GM-CSF−/− mice, or from WT mice whose VEGF expression was silenced by siRNA*, were added at the beginning of the culture of B6 BM cells (2 × 106/well, (HSC:BM cells = 1:80) in the presence of GM-CSF (8 ng/ml) for 5 days. The floating cells were harvested, and analyzed for expression of CD11b and CD11c by flow cytometry. The culture in the absence of HSC (none) served as control. The number is percentage of positive cells. (B) Analysis of HSC supernatant with electrophoresis. Supernatant of the HSC culture in serum free medium for 3 days was fractionized using SDS gel electrophoresis, and stained with Coomassie blue. Compared to medium alone (None), 3 distinct bands were displayed in >100kD. (C) Identification of FH and C3 proteins in HSC supernatant by Western blotting using polyclonal Abs for FH and C3 (ComTech, Tyler, TX), respectively. (D) The effect of C3 and FH in HSC culture supernatant in induction of H-MC. C3 or FH was depleted from the supernatant of HSC culture (serum free medium) by addition of anti-CD3 or -FH specific Ab (5mg/ml). The antigen/antibody complex was precipitated by addition of protein-A agarose and removed by centrifugation. The complete removal of FH or C3 was confirmed by Western blotting assay (data not shown). The supernatant that depleted C3 or FH was added into BM cell culture at 1/2 of total culture medium volume. The harvested floating cells were analyzed for expression of CD11b and CD11c by flow. (E) HSC before cotransplantation with islet allografts were intracellularly stained with goat anti-mouse C3 mAb, analyzed by flow cytometry, and expressed as histograms (dotted area is isotype control). The number is percentage of positive cells. (F) 300 BALB/c islets were mixed with 5 × 105 HSC (B6), and transplanted under renal capsule of STZ-induced diabetic B6 recipients. The animals were sacrificed on POD 14. The graft cryostat sections were double stained with anti-α-SMA (red) and -C3 (green) mAbs, and evaluated under a fluorescent microscope. The dotted line outlines area of islets. The data are representative of three separate experiments.
HEP_24162_sm_suppinfofig3E.tif817KSupporting Information Figure 3E
HEP_24162_sm_suppinfofig3F.tif8556KSupporting Information Figure 3F
HEP_24162_sm_suppinfotable1.doc29KSupporting Table 1. Proteins identified*
HEP_24162_sm_suppinfo.doc36KSupporting Information

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