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) Migrating donor (BALB/c) DCs (IAd+) in T cell areas of the recipient (B6, H2b) spleen (arrows, insets). Confocal microscopy, original magnification, ×200. BALB/c cDCs (CD45.2+CD45.1–CD11c+) homed in the recipient (B6) spleen were undetectable by flow cytometry. Numbers indicate percentages of cells in the corresponding quadrant. Dot plots are representative of 1 untreated or recipient mouse of 3 per time point. (B) Quantities of donor cells mobilized from BALB/c heart grafts to the recipient (B6) spleens estimated by genomic PCR. Mean ± SD, 3 mice per variable. ND, not detected. (C) A donor DC (IAd hi, arrow) in the recipient spleen next to DCs expressing IAd dim or the IAb (B6)–IEα52–68 (BALB/c) complex (arrowheads, detected with the Yae Ab) that likely corresponded to recipient DCs that acquired donor IAd or donor IEα52–68 peptide, respectively. Confocal microscopy, original magnification, ×200. (D) FACS analysis of numbers of recipient (B6) splenic APCs with donor H2Kd or IAd molecules at successive PODs after transplantation of BALB/c hearts. Mean ± SD, 3 mice per variable. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test.

Article Snippet: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

Techniques: Confocal Microscopy, Flow Cytometry, Expressing, Transplantation Assay, Generated

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: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

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) Recipient cDCs carrying donor MHC (FACS-sorted in R1 of Figure 2A) triggered proliferation of 2C CD8 T cells against BALB/c H2Ld. (B and C) Expression of T cell activation/effector markers (B) and cytokine secretion (C) of 2C T cells following stimulation by recipient (B6) splenic cDCs carrying donor MHC (FACS-sorted in R1 of Figure 2A), or by control cDCs. Numbers in dot plots indicate percentages of cells in the corresponding quadrants. IL-5 and IL-17A were undetectable by ELISA. In A–C, 1 representative of 4 experiments is shown. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test.

Article Snippet: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

Techniques: Expressing, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Generated

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: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

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) Transfer efficiency of H2Kd and IAd from BALB/c to B6 cDCs (analyzed by FACS). Mean ± SD. One representative experiment of 2. (B) Percentages of B6 cDCs that acquired H2Kd and IAd from BALB/c cDCs following culture with BALB/c cDCs together, or separated by 0.4-μm-pore transwells (analyzed by FACS). Results are representative of 4 experiments. Numbers in dot plots indicate percentages of cells in the corresponding quadrant. (C) Effect of imipramine or DEVD added to the culture medium, or pretreatment of donor BALB/c DCs with Rab27a siRNA, on transfer of IAd from BALB/c DCs to B6 cDCs in vitro (analyzed by FACS). Results from 1 representative of 4 independent experiments are shown. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test.

Article Snippet: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

Techniques: In Vitro, Generated

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: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

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: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

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) Survival of BALB/c cardiac grafts in CD11c-DTR-B6 BM chimeras depleted of recipient cDCs. Recipient numbers are in parentheses. (B) Quantification by immunofluorescence microscopy of donor (BALB/c) cDCs (CD11c+IAd+) on tissue sections of spleens of B6 (H2b) recipients, on successive PODs. Results represent the analysis of 10 panoramic sections of each spleen per POD and animal group. Results were analyzed with 1-way ANOVA followed by Tukey-Kramer multiple comparisons test. Cells were counted with MetaMorph Offline 7.7.50 software. NS, not significant; ND, not detected. (C) Top: Donor (BALB/c) cDCs detected on tissue sections of spleens from DT-treated WT B6 BM chimeras (control) and DT-injected CD11c-DTR BM chimeras were identified by expression of IAd hi (green) and CD11c (red). Bottom: Homing of donor (BALB/c, IAd+) cDCs (green) to splenic T cell areas (red) in DT-treated WT B6 BM chimeras (control) and DT-injected CD11c-DTR BM chimeras. Arrows indicate the donor DCs shown in detail in the insets. Nuclei were stained blue with DAPI. Immunofluorescence microscopy, original magnification, ×400. Sections are representative of 3 animals per variable. (D) Enzyme-linked ImmunoSpot (ELISPOT) analysis of the recipient T cell response against donor MHC molecules (direct pathway) or donor-derived peptides presented by recipient MHC molecules (indirect pathway) in the spleen on POD 7. Results were pooled from 3–4 mice per group. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test. (E) Anti-donor (BALB/c) Ab titers in serum on POD 7. Recipient numbers are in parentheses.

Article Snippet: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

Techniques: Immunofluorescence, Microscopy, Software, Control, Injection, Expressing, Staining, Enzyme-linked Immunospot, Derivative Assay, Generated

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: Right: RFP-exosome clusters in relationship to host YFP + cDCs, on the same image analyzed with Imaris X64.

Techniques: Expressing, Transplantation Assay, Generated, Marker

Journal: Microorganisms

Article Title: An Analysis of the Gut Microbiota and Related Metabolites following PCSK9 Inhibition in Statin-Treated Patients with Elevated Levels of Lipoprotein(a)

doi: 10.3390/microorganisms12010170

Figure Lengend Snippet: ( A – C ) Boxplots representing the concentration (log10 transformed) of total bile acids ( A ), and primary ( B ) and secondary ( C ) bile acids in fecal samples collected at baseline and on treatment. Samples from the same patient are connected using straight lines. The following primary bile acids were detected in feces: cholic acid, taurocholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid and glycochenodeoxycholic acid. The following secondary bile acids were detected in feces: deoxycholic acid, lithocholic acid, ursodeoxycholic acid, taurolithocholic acid, tauroursodeoxycholic acid, taurodeoxycholic acid and glycodeoxycholic acid. For statistical inference, we used a paired t -test, and the corresponding p -values are shown in each graph. ( D ) Linear relationship between the reduction in plasma LDL-c and the concentration of fecal bile acids (log10 transformed) after alirocumab therapy. Line of best fit for the linear model is plotted with 95% confidence interval (grey shade area). Individual samples are represented with black dots. The regression coefficient, coefficient of determination and the result of the F-test for the linear model are shown on the top of the plot.

Article Snippet: For identification of the different classes of BAs, we used the following pure standards to create a mass spectral–retention time library; taurocholic acid (Santa Cruz, sc220189, Dallas, TX, USA), taurolithocholic acid (Cayman Chemicals, 17275, Ann Arbor, MI, USA), taurochenodeoxycholic acid (Steraloids, C1162-000, Newport, RI, USA), tauroursodeoxycholic acid (Steraloids, C1052-000), glycolithocholic acid (Steraloids, C1435-000), glycochenodeoxycholic acid (Steraloids, C0962-000), glycoursodeoxycholic acid (Steraloids, C1025-000), glycocholic acid (Steraloids, C1927-000), glycodeoxycholic acid (Steraloids, C1087-000), ursodeoxycholic acid (Steraloids, C1020-000), cholic acid (Sigma-Aldrich, C1129, St. Louis, MO, USA), deoxycholic acid (Sigma-Aldrich, D2510), lithocholic acid (Sigma-Aldrich, L6250), chenodeoxycholic acid (Sigma-Aldrich, C1050000).

Techniques: Concentration Assay, Transformation Assay, Clinical Proteomics