Volume 13, Issue 2, Pages 289-300 (February 2006) Exosomes Derived from Genetically Modified DC Expressing FasL Are Anti- inflammatory and Immunosuppressive  Seon Hee Kim, Nicole Bianco, Rajasree Menon, Eric R. Lechman, William J. Shufesky, Adrian E. Morelli, Paul D. Robbins  Molecular Therapy  Volume 13, Issue 2, Pages 289-300 (February 2006) DOI: 10.1016/j.ymthe.2005.09.015 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 (A) Flow-cytometric analysis of mouse bone marrow DC and DC-derived exosomes. The purified exosomes from bone marrow DC transduced with Ad.control and Ad.FasL were incubated with surfactant-free white aldehyde/sulfate latex beads and stained with MHC I and II, CD11c, CD80, and CD86 antibodies for FACS analysis. (B) Western blot analysis of DC and DC-derived exosomes. Bone marrow-derived DC were isolated from C57BL/6 mice. Half of the DC were infected with Ad.FasL at day 5. Two days later, exosomes were isolated from the DC culture by differential centrifugation. The resulting exosomes as well as DC were analyzed for the expression of FasL. (C) Electron microscopic pictures. Transmission electron microscopic observations were made with a CM120 Twin Phillips electron microscope. Molecular Therapy 2006 13, 289-300DOI: (10.1016/j.ymthe.2005.09.015) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Suppression of DTH in mouse footpad treated with exosomes. Exosomes were isolated from bone marrow-derived DC that were infected with either Ad.Ψ5 or Ad.FasL. The purified exosomes as well as DC were injected into the right footpad of mice that had been systemically immunized with KLH. Twelve hours post-injection of DC or exosomes, KLH antigen was injected into both hind footpads and the extent of swelling measured over 24-, 48-, and 72-h periods. *Significance at P < 0.01. Molecular Therapy 2006 13, 289-300DOI: (10.1016/j.ymthe.2005.09.015) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Importance of the Fas/FasL in the suppression of DTH. BM-DC were generated from wild-type and gld (FasL-deficient) mice infected with either Ad.Ψ5 or Ad.FasL, and exosomes were isolated. (A) The adenovirus-infected DC from either wild-type or gld mice were tested in wild-type DTH recipients. Similarly, the DC-derived exosomes were injected into one hind paw of KLH-immunized (B) wild-type or (C) lpr (Fas-deficient) mice prior to KLH injection. Molecular Therapy 2006 13, 289-300DOI: (10.1016/j.ymthe.2005.09.015) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 DTH suppression of exosomes in the syngeneic mouse. Exosomes were separately isolated from syngeneic C57BL/6 and allogeneic Balb/C mouse bone marrow DC that were infected with Ad.Ψ5 or Ad.FasL. The purified exosomes were injected into the right footpad of KLH-immunized mice. Twelve hours post-injection of DC or exosomes, KLH antigen was injected into both hind footpads, and the extent of swelling is shown for the 48-h time point. *Significance at P < 0.01. Molecular Therapy 2006 13, 289-300DOI: (10.1016/j.ymthe.2005.09.015) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 DTH responses of exosomes from MHC-deficient mice. Exosomes were isolated from bone marrow-derived DC from either wild-type C57BL/6 mice or mice deficient in (A) MHC class I or (B) MHC class II that were infected with either Ad.Ψ5 or Ad.FasL. The purified exosomes as well as DC were injected into the right footpad of KLH-immunized mice. Twelve hours post-injection of DC or exosomes, KLH antigen was injected into both hind footpads, and the extent of swelling is shown for the 48-h time point. *Significance at P < 0.01. Molecular Therapy 2006 13, 289-300DOI: (10.1016/j.ymthe.2005.09.015) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Antigen specificity of the immunosuppressive exosome. Exosomes were isolated from DC that were either nonpulsed or pulsed with KLH or OVA as well as infected with Ad.FasL or Ad.eGFP. The purified exosomes (1 μg of total protein) were injected into the right footpad of KLH-immunized mice. Twelve hours post-injection of DC or exosomes, KLH antigen was injected into both hind footpads, and the extent of swelling is shown for the 48-h time point. *Significance at P < 0.01. Molecular Therapy 2006 13, 289-300DOI: (10.1016/j.ymthe.2005.09.015) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Analysis of trafficking of exosomes following injection into the footpad. PKH26-labeled (red) exosomes obtained from (A, C, E, and G) Ad-Ψ5-transduced DC or from (B, D, F, and H) Ad-FasL-infected DC were internalized by CD11c+ (green) dermal DC by 2 days postinjection. Examples of dermal CD11c+ DC with internalized PKH26 exosomes (in yellow due to overlap of red and green) are indicated with arrows and are shown in detail in the insets. The white lines in (A) and (B) indicate the epidermal–dermal junction. CD11c+ DC with PKH26+ inclusions (arrows) were detected in the draining (popliteal) lymph node in the treated side (C and D), but were absent in the contralateral lymph node (E and F). Analysis of apoptotic lymphoid cells (arrows) was performed by TUNEL staining of the draining lymph nodes of mice injected in the footpad with (G) exosomes obtained from Ad-ψ5-tranduced DC or with (H) exosomes generated by Ad-FasL-infected DC. Nuclei were counterstained with DAPI. Original magnification: A–F, ×200; G, H, and insets, ×400. Molecular Therapy 2006 13, 289-300DOI: (10.1016/j.ymthe.2005.09.015) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 8 Analysis of the therapeutic effect of DC and exosomes in murine collagen-induced arthritis model. Exosomes were isolated from DBA/1 mouse bone marrow DC that were infected with either Ad.Ψ5 or Ad.FasL. (A) The purified DC as well as (B) the exosomes were injected intravenously at day 28 into DBA mice that were immunized with bovine type II collagen. Mice were monitored periodically using an established macroscopic scoring system on a 0 to 4 scale: 0, normal; 1, detectable arthritis with erythema; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; 4, maximal swelling and deformity with ankylosis. The macroscopic score (mean ± SD) was expressed as a cumulative value for all paws, with a maximum possible score of 16 (n = 7). *Significance at P < 0.01. Molecular Therapy 2006 13, 289-300DOI: (10.1016/j.ymthe.2005.09.015) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions