Transfer of endogenous exosomes between BMDCs. (A) FACS analysis of transfer of eGFP between 10⁶ CD45.2⁺ BMDCs transfected with RAd-eGFP-tmFasLΔ (or control RAd-Empty or RAd-eGFP) and 10⁶ acceptor CD45.1⁺ BMDCs. Numbers are percentages of cells. (B) Effect of EDTA and temperature on transfer of exosomes between CD45.2⁺ RAd-eGFP-tmFasLΔ-transfected BMDCs and CD45.1⁺ BMDCs. (C top) Confocal microscopy of CD45.1⁺ BMDCs (in red) incubated with CD45.2⁺ BMDCs transfected with RAd-eGFP-tmFasLΔ. Arrows indicate acceptor (CD45.1⁺) BMDCs with eGFP⁺ content. As control, arrowheads show some acceptor BMDCs without eGFP. (Bottom) eGFP inside an acceptor BMDC surface labeled with CD45.1 (in red; ×1000). (D) Detection by FACS of transfer of eGFP between BMDCs and TCR transgenic (tg) T cells in a 3-cell culture system. One million BMDCs transfected with RAd-eGFP-tmFasLΔ (or control RAd-Empty or RAd-eGFP) and pulsed with IEα_52-68 or OVA_323-339 peptide were cocultured with in vitro–activated (blasts) 1H3.1 (Thy1.1⁺) and OT-II (Thy1.2⁺) CD4 T cells (5 × 10⁶ of each). CD11c⁺ cells (BMDCs) were gated out. Numbers are percentages of cells. One of 3 (A,D) or 2 (B-C) experiments is shown.