Journal: The Journal of Clinical Investigation

Article Title: Donor dendritic cell–derived exosomes promote allograft-targeting immune response

doi: 10.1172/JCI84577

Figure Lengend Snippet: (A) FACS sorting of recipient (B6) cDCs bearing donor H2Kd, analyzed 3 days after transplantation of BALB/c hearts. (B) Transmission EM (TEM) image of a recipient cDC, sorted in R1 of A, showing EVs carrying donor H2Dd and IAd. The area in the small rectangle is shown at higher magnification on the right. Recipient cDCs did not express donor (BALB/c) MHC directly on the surface — a section of the plasma membrane is shown at higher magnification below, original magnification, ×2,500–×10,000. One image representative of 60 cells analyzed with gold-labeled EVs attached. Dot plot: size of EVs attached to recipient cDCs. (C) Recipient cDC, FACS-sorted in R1 of A, and labeled with biotin-irrelevant Ab, as a control. Bars: number of 5-nm gold-positive EVs on recipient cDCs, FACS-sorted in R1 of A, and labeled with biotin-H2Dd-IAd or control biotin-irrelevant Abs. (D) Cluster of EVs, attached to a recipient (B6) cDC, FACS-sorted in R1 of A, expressing donor H2Dd and IAd, and CD9 or CD63. One image representative of 50 cells analyzed with gold-labeled EVs attached.

Article Snippet: Day 6 BMDCs were purified with CD11c magnetic beads and transduced (MOI = 100) with the recombinant adenovirus (RAd)-CD63-RFP encoding RFP cDNA (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), or with the control vector RAd-RFP (encoding RFP cDNA alone) or RAd-Empty (no transgene).

Techniques: Transplantation Assay, Transmission Assay, Membrane, Labeling, Control, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Donor dendritic cell–derived exosomes promote allograft-targeting immune response

doi: 10.1172/JCI84577

Figure Lengend Snippet: (A) Passage of IAd from CFSE-labeled BALB/c cDCs to B6 cDCs in vitro (analyzed by FACS). Images of cDCs, FACS-sorted in R1, showing (BALB/c) H2Dd and IAd transferred to (B6) cDCs through EVs expressing CD9 and CD63. R2: BALB/c (donor) cDCs releasing exosomes bearing H2Dd and IAd (rectangle). R3: Recipient cDCs do not express donor MHC on their surface. Results are representative of 3 experiments. Transmission EM, original magnification, ×20,000–×60,000. Images are representative cells from 4 independent experiments. (B) Size of EVs bearing BALB/c H2Dd and IAd, and attached to B6 cDCs. (C) Images of EVs expressing BALB/c H2Dd and IAd, and CD9, which have been internalized by (B6) cDCs. Recipient cDCs were FACS-sorted in R1 (A), then labeled with biotin-H2Kd and -IAd Abs plus CD9 Ab, followed by gold-conjugated secondary reagents, and then maintained at 37°C (30 minutes) to promote internalization of the EVs. Original magnification, ×20,000–×80,000. A representative single cell from 1 of 2 independent experiments is shown.

Article Snippet: Day 6 BMDCs were purified with CD11c magnetic beads and transduced (MOI = 100) with the recombinant adenovirus (RAd)-CD63-RFP encoding RFP cDNA (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), or with the control vector RAd-RFP (encoding RFP cDNA alone) or RAd-Empty (no transgene).

Techniques: Labeling, In Vitro, Expressing, Transmission Assay

Journal: The Journal of Clinical Investigation

Article Title: Donor dendritic cell–derived exosomes promote allograft-targeting immune response

doi: 10.1172/JCI84577

Figure Lengend Snippet: (A) Time-lapse analysis (confocal) of transfer of RFP-tagged exosome clusters from a migrating CD63-RFP BALB/c DC (blue nucleus + RFP-exosomes) to a lymph node cDC (YFP+, in green). Scale bar: 1 μm. (B) Side view of A. The arrow indicates internalized RFP-tagged EVs. (C) Left: 3D panoramic view (confocal) of a lymph node of a CD11c-YFP B6 mouse injected with CD63-RFP BALB/c DCs. Right: RFP-exosome clusters in relationship to host YFP+ cDCs, on the same image analyzed with Imaris X64. Red dots: RFP-exosomes on YFP+ B6 cDCs. Yellow dots: RFP-exosomes internalized by YFP+ B6 cDCs. Blue dots: RFP-exosomes in CD63-RFP BALB/c DCs, free or captured by YFP– cells. (D) 3D view (multiphoton) of the spleen of a CD11c-YFP B6 mouse injected i.v. with CD63-RFP BALB/c DCs. Arrow: interaction between injected CD63-RFP BALB/c DCs and host YFP+ cDCs. Scale bar: 15 μm. (E) Analysis (Imaris X64) of transfer (arrows) of RFP-exosome clusters from a CD63-RFP BALB/c DC to host YFP+ cDCs (in green) in the spleen. In A–E, results are representative of 4 independent experiments.

Article Snippet: Day 6 BMDCs were purified with CD11c magnetic beads and transduced (MOI = 100) with the recombinant adenovirus (RAd)-CD63-RFP encoding RFP cDNA (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), or with the control vector RAd-RFP (encoding RFP cDNA alone) or RAd-Empty (no transgene).

Techniques: Injection

Journal: The Journal of Clinical Investigation

Article Title: Donor dendritic cell–derived exosomes promote allograft-targeting immune response

doi: 10.1172/JCI84577

Figure Lengend Snippet: (A) Recipient MHC class II (B6, IAb) and CD86 expression by cDCs and plasmacytoid DCs (pDCs) from spleens of CD45.1 B6 mice transplanted with CD45.2 BALB/c hearts, analyzed by FACS on successive PODs. Results are representative of 3 mice per variable. (B) Effect of transfer of RFP-tagged exosomes between migrating CD63-RFP BALB/c BMDCs injected i.v. and spleen-resident cDCs of CD11c-YFP B6 mice. Numbers in dot plots indicate percentages of cells in the corresponding quadrants. CD63-RFP BALB/c BMDCs were matured by overnight incubation with IL-1β plus TNF-α. Comparison by FACS analysis of expression of endogenous (B6) MHC class II (IAb), CD40, CD80, CD86, and PD-L1 between YFP+ cDCs without and with RFP+ content, analyzed 16 hours after BMDC injection. Results are from representative experiments with 4 mice per group.

Article Snippet: Day 6 BMDCs were purified with CD11c magnetic beads and transduced (MOI = 100) with the recombinant adenovirus (RAd)-CD63-RFP encoding RFP cDNA (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), or with the control vector RAd-RFP (encoding RFP cDNA alone) or RAd-Empty (no transgene).

Techniques: Expressing, Injection, Incubation, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Donor dendritic cell–derived exosomes promote allograft-targeting immune response

doi: 10.1172/JCI84577

Figure Lengend Snippet: (A) Top dot plots: Detection by FACS of donor (CD45.2+CD45.1–) cells migrated from BALB/c (CD45.2+) fully mismatched skin allografts in B6 (CD45.1+) mice. Donor cells were undetectable by FACS within the “live cell gate” on PODs 1, 3, and 7, in the spleen (A) and in the draining lymph nodes (axillary + inguinal, not shown). Bottom dot plots: Detection by FACS of donor MHC class I (H2Kd + H2Dd) molecules on recipient (CD45.1+CD45.2–) splenic cDCs. Recipient splenic CD8α+ and CD8α– cDCs acquired donor MHC class I molecules. Numbers in dot plots indicate percentages of cells in the corresponding quadrants. Results are representative of 6 mice per time point. (B) Left: FACS analysis of intensity of expression of donor MHC class I (H2Kd + H2Dd) and class II (IAd) molecules on recipient (B6) splenic cDCs after BALB/c skin transplantation. Right: Percentages of recipient (B6) splenic cDCs cross-dressed with donor MHC class I (H2Kd + H2Dd) and class II (IAd) molecules, analyzed by FACS after BALB/c skin transplantation. Results were pooled from 2 experiments, each with 3 mice per time point. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test. (C) Analysis by ImageStream technology of recipient (B6, CD45.1+) splenic cDCs (CD11c+) cross-dressed with donor (BALB/c) MHC class I (H2Kd + H2Dd) and class II (IAd) molecules, both located in spots containing the exosome marker CD63. ImageStream, original magnification, ×60, 5,000 cells analyzed.

Article Snippet: Day 6 BMDCs were purified with CD11c magnetic beads and transduced (MOI = 100) with the recombinant adenovirus (RAd)-CD63-RFP encoding RFP cDNA (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), or with the control vector RAd-RFP (encoding RFP cDNA alone) or RAd-Empty (no transgene).

Techniques: Expressing, Transplantation Assay, Generated, Marker