Journal: Science translational medicine

Article Title: Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation

doi: 10.1126/scitranslmed.abb0122

Figure Lengend Snippet: (A) Planimetric view of donor Langerhans cells on epidermis of skin allografts and quantification on successive PODs. Images representative of six grafts per variable. Magnification, ×100. (B) Horizontal view of donor DCs (red) in dermal laminas of skin allografts. On POD 1, donor DCs accumulated inside lymphatics (arrows, inset). Images are representative of four grafts per group. Quantification by flow cytometry of donor DCs in dermal cell suspensions of skin allografts. Three to six grafts per group. Magnification, ×200 and ×400. (C) Vertical section of a skin allograft (POD 3) showing a dermal lymphatic vessel (blue) clogged with donor cells (green), with dendritic morphology, some coexpressing CD326 (inset). Dotted line indicates epidermal-dermal junction. Image representative of four allografts. X200, X400. EpCAM, Epithelial Cell Adhesion Molecule. (D) ImageStream of recipient DCs and B cells from LNs draining skin allografts on POD 2 cross-dressed with donor H2Kb and H2Db. Magnification, ×60; 20,000 cells. (E) Quantification by ImageStream in graft-dLNs of recipient DCs and B cells cross-dressed with donor H2Kb and H2Db after transplantation of allogeneic or syngeneic skin set as background (BKGD) staining. Each dot represents a recipient. (F) IEM images of recipient DCs and B cells, FACS-sorted from graft-dLNs on POD 2, cross-dressed with EVs carrying donor-derived H2Kb/IAb and CD63 (inset). N, nucleus. Magnification, ×2,500 to 10,000. Representative images of 60 to 80 immunogold-labeled cells. In (A), (B), and (F), results were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars represent means ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: A targeting vector was designed to contain the mouse CMV, followed by a loxP -flanked stop sequence [three copies of the SV40 polyadenylate poly(A) signal], TagRFP complementary DNA (cDNA) (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), and the phosphoglycerate kinase polyA signal.

Techniques: Flow Cytometry, Transplantation Assay, Staining, Derivative Assay, Labeling

Journal: Science translational medicine

Article Title: Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation

doi: 10.1126/scitranslmed.abb0122

Figure Lengend Snippet: (A) Kaplan-Meier analysis of survival of B6 cardiac allografts in mice treated short or long term via intraperitoneal (i.p.) injection of GW4869 or vehicle (DMSO). Number of recipients and graft survival days are in parenthesis. (B) Representative analysis by ImageStream of recipient splenic APCs cross-dressed with donor H2Kb and H2Db, 2 days after transplantation of B6 CD45.1 cardiac allografts. CD63 colocalized in areas containing H2Kb H2Db. Magnification, X60, 40,000 cells. (C) Assessment by ImageStream of recipient total leukocytes and APCs cross-dressed with donor H2Kb and H2Db in splenocytes of BALB/c mice grafted with B6 CD45.1 hearts and treated with GW4869 or vehicle. Splenocytes of naïve BALB/c mice were included to set background (BKGD) staining (dotted lines). Each dot represents a mouse or graft recipient. No Tx, no transplant. (D) Flow cytometry analysis of division (CFSE dilution) and activation (CD44High) of CFSE-labeled T cell receptor transgenic 4C CD4 T cells, specific for IAd, in response to ex vivo stimulation with recipient (YFPPos) DCs FACS-sorted from spleens of CD11c-YFP B6 mice untreated (negative control) or grafted 3 days prior with BALB/c (H2d) hearts and treated with GW4869 or DMSO. Plots are representative of four transplants per group. (E) Effect of administration GW4869 or DMSO on DSA titers in serum of BALB/c mice grafted with B6 hearts. Each dot represents a mouse. Results in (C) and (E) were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars denote means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: A targeting vector was designed to contain the mouse CMV, followed by a loxP -flanked stop sequence [three copies of the SV40 polyadenylate poly(A) signal], TagRFP complementary DNA (cDNA) (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), and the phosphoglycerate kinase polyA signal.

Techniques: Injection, Transplantation Assay, Staining, Flow Cytometry, Activation Assay, Labeling, Transgenic Assay, Ex Vivo, Negative Control

Journal: Science translational medicine

Article Title: Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation

doi: 10.1126/scitranslmed.abb0122

Figure Lengend Snippet: (A) Detection of donor IAb in SCS macrophages (inset) in graft-dLN after transplantation of skin allografts. Images representative of six recipients. Magnification, ×200. (B) IEM image of SCS of graft-dLN containing EVs carrying donor H2Kb/IAb and CD63 (red arrows, inset). White arrow indicates CD169 expression by SCS macrophages. Diagram shows image interpretation. (C) SCS macrophages (CD169, white arrows) containing EVs bearing donor H2Kb/IAb and CD63 (red arrows, inset) within a system of interconnected vesicles or channels (asterisks). Magnification, x20,000 and x80,000. Size of donor EVs internalized by SCS macrophages. (D) Sequence of allogeneic EVs captured by SCS macrophages in a pLN. Numbers indicate minutes after injection. (E) Two-photon microscopy of allogeneic EVs captured by SCS and medullary macrophages in a pLN. Dotted line indicates cortex-medulla junction. (F) Uptake of footpad-injected EVs by leukocytes in draining pLNs as measured by flow cytometry. Horizontal dotted lines indicate ex vivo uptake of exogenous EVs by bystander CD45.1 leukocytes. (G) STED microscopy of allogeneic EVs crossing a system of interconnected vesicles or tunnels filled with lymph tracer (blue) within pLN SCS macrophages. Line indicates LN capsule. (H) Sequence by STED microscopy in a pLN of transport across an SCS macrophage of allogeneic CM-Dil-EVs (inset). Arrows indicate directional passage of CM-DilPos material. One representative of two pLNs. (A to E) Images representative of graft-dLNs from three to four recipients. In (D) to (H), an equivalent of 55 × 108 allogeneic EVs (87 ± 49 nm in size) in 30 μl of PBS was injected per footpad. In (F), results were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars denote means ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: A targeting vector was designed to contain the mouse CMV, followed by a loxP -flanked stop sequence [three copies of the SV40 polyadenylate poly(A) signal], TagRFP complementary DNA (cDNA) (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), and the phosphoglycerate kinase polyA signal.

Techniques: Transplantation Assay, Expressing, Sequencing, Injection, Microscopy, Flow Cytometry, Ex Vivo