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Blood, Vol. 106, Issue 1, 216-223, July 1, 2005
ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming
Blood Segura et al.
106: 216
Supplemental Figures and Table for: Segura et al, Vol 106, Issue 1, 216-223
Files in this Data Supplement:
- Table S1. Proteins identified in immature and mature exosomes (PDF, 369 KB) -
B indicates B cells; HEK, human embryonic kidney 293T cells; IEC, intestinal epithelial cells; Mast, mast cells; M, melanoma; MC, mesothelioma cells; mov, immortalized Schwann cells; P, platelets; Ret, reticulocytes; T, T cells; and U, urine.
- Figure S1: Enhanced T-cell activation by mature exosomes is not due to contaminants (JPG, 34.2 KB)
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[3H]-thymidine incorporation by Marilyn T cells cultured with mature BMDCs and mature exosomes (Mat HY Exo) or immature exosomes (Imm HY Exo) in the presence of increasing doses of LPS (0, 10 or 100 ng/mL). One representative of 3 independent experiments is shown. The amount of residual LPS in mature exosome preparations was quantified, by limulus amoebocyte assay, as 1.5 ng exosomes per microgram in 2 independent preparations. Thus, at most, 1.5 ng LPS would be added together with mature exosomes in the 150-µL volume of antigen presentation assay, amounting to 10 ng/mL LPS. This quantity is 100 times lower than the amount needed to induce BMDC maturation (i.e. above 1 µg/mL).
- Figure S2: Enhanced in vivo activity of mature exosomes is not due to LPS (JPG, 29.4 KB)
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5 µg exosomes was injected subcutaneously into female C57Bl/6 mice containing adoptively transferred, CFSE-labeled CD45.1+ Marilyn lymph-node cells. Draining lymph-node cells were analyzed by FACS 4 days after injection. The percentage of cells that underwent more than 5 divisions cycles (A), and the number of Marilyn T cells in the draining lymph nodes (B), in mice treated with HY-bearing immature exosomes (clear), HY-bearing immature exosomes + 50 ng LPS (grey) or HY-bearing mature exosomes (black), are shown (3 mice per group). Results are taken from one representative of 3 independent experiments.
- Figure S3: specific MHC-peptide complexes are only slightly increased on mature exosomes (JPG, 85.1 KB)
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The amount of MHC II–peptide complexes on immature (imm exo) and mature (mat exo) exosomes was quantified by FACS analysis of exosome-coated beads using Y-Ae antibody, which recognizes I-Ab-IE 56-73 complexes. 5 µg immature or mature exosomes from D1 cells pulsed with the IE peptide were coated on beads and stained with anti-I-Ab (MHC II), Y-Ae, anti-CD86, and anti-ICAM-1 antibodies. Grey histograms indicate isotype control; bold histograms, specific staining; numbers, the mean fluorescence index. I-Ab is increased 2 times and I-Ab-E is increased 1.5 times in mature exosomes compared with immature exosomes. One representative experiment of 3 is shown.
- Figure S4: ICAM-1 is essential to exosome-mediated T cell activation by B cells (JPG, 37.9 KB)
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[3H]-thymidine incorporation by Marilyn T cells cultured with splenic B cells and HY-bearing mature exosomes from WT (Mat WT Exo) or ICAM-1–/– (Mat ICAM–/– Exo) BMDCs. One representative of 2 independent experiments is shown.
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