Figure 1.
Characterization of exosomes derived from DCs.
(A) The phenotypic profile of imDex and mDex were analyzed by cytofluorometry. The two kinds of the exosomes were coated on surfactant-free white aldehyde sulfate latex beads, then stained with FITC-coupled monoclonal antibodies against MHC class I, II and CD80, CD86. The isotype monoclonal antibody of IgG was used as control. The results are representative of four independent experiments. (B) Western blotting analysis of imDex and mDex using monoclonal antibodies specific for HSP70, HSP90, ICAM-1, and MFG-E8. The signal intensities in the blot were assessed by densitometry and are represented in the graphs. One representation of three independent experiments is shown. * P<0.01.
Figure 2.
Administration of low-dose donor imDex with a subtherapeutic rapamycin promotes cardiac allograft survival.
Different imDex concentrations were injected via caudal vein 7 d before, the day of, and 7 d after transplantation, and 1 mg/kg/d rapamycin was injected via caudal vein for 11 consecutive days (days –3 to 7) with or without imDex. (A) The cardiac allograft survival for different donor imDex concentrations in the absence of rapamycin. (B) ImDex (10 μg) of the donor (C57BL/6), recipient (BALB/c), or third-party (CBA/H) were injected into recipient mice. (C) The allograft survival following rapamycin treatment alone or with 10 μg donor, recipient, or third-party imDex.
Table 1.
Cardiac allograft survival after treatment with imDex or imDex+rapamycin.
Figure 3.
Donor imDex combined with rapamycin inhibits proliferation and activation of alloreactive T lymphocytes against donor antigen.
At 5 or 10 d post-transplantation, 5×104 total splenocytes (A-B) or purified T lymphocytes (C-D) from untreated, donor imDex-treated, rapamycin-treated or imDex/rapamycin-cotreated recipients (responder cells) were incubated with 2×104 donor or third-party splenocytes (stimulator cells) for 72 h. Label (1 μCi [3H]-thymidine) was added for the last 18 h of culture, and incorporation was determined using a liquid scintillation counter. Upregulation of CD69 on the four groups of responder cells (10 d after transplantation) was measured by FACS 24 h after simulator cell addition (E-F). One representation of four independent experiments is shown. * P<0.01.
Figure 4.
FACS and western blotting analysis of splenic CD4+CD25+Foxp3+ T cells.
Splenic T lymphocytes were isolated from untreated recipients (n = 6), recipients treated with donor imDex (n = 6) or rapamycin (n = 6) alone, and imDex/rapamycin-cotreated (n = 6) recipients at d5 or d10 after transplantation. (A) The percentages of splenic CD4+CD25+ T cells from the four groups of recipient mice were calculated from flow cytometric analysis. The number in the upper-right quadrant of each histogram indicates the percentage of CD4+CD25+ T cells. Results were obtained from three mice per condition, and one representative of four independent experiments is shown. (B) Foxp3 versus β-actin western blotting analysis of splenic T lymphocytes from the groups of recipients upper. Densitometry shows fold induction of Foxp3:β-actin ratio. Bars = mean ± SD (n = 3), * P<0.01, ** P<0.001. One representation of three independent experiments is shown.
Figure 5.
Signs of acute rejection in cardiac allografts.
Cardiac allografts of untreated and imDex/rapamycin-cotreated recipients were harvested at 7 d post-transplantation, and transplantation of a syngenetic heart (BALB/c to BALB/c) was used as control. (A) The left heart is from the syngenetic transplantation recipient, the middle heart is from the imDex/rapamycin-cotreated recipient, and the right heart is from the untreated recipient. Paraffin sections were stained with hematoxylin-eosin and observed by optical microscope. Representative allografts of untreated recipients (B and C), allografts of imDex/rapamycin-cotreated recipients (D and E), and control specimens from syngenetic transplantation recipients (F and G) are shown.
Table 2.
Survival of the second cardiac allograft for tolerant imDex/rapamycin-cotreated recipients.
Table 3.
Cardiac allograft survival for BALB/c recipients injected with splenocytes or serum from tolerant imDex/rapamycin-cotreated recipients.