Fig. 6. Activation of T cells by allogeneic exosomes and cells cross-dressed with allogeneic MHC.
(A) Procedure used to generate allogeneic exosomes and cells cross-dressed with allogeneic MHC molecules. Steps 1 and 2: Exosomes were isolated and quantified (using ExoQuick kits) from the lower compartments of Transwell plates whose upper compartments contained B6 spleen cells stimulated for 4 to 5 days with CD40 and interleukin-4 (IL-4). Step 3: To obtain cross-dressed cells, BALB/c spleen cells (15 × 106 cells/ml) were cultured for 5 days along with B6-derived exosomes (1.9 × 109). Step 4: BALB/c cells cross-dressed with allogeneic H-2b exosomes were isolated using an affinity column composed of beads coated with anti–Kb-PE antibodies. Step 5: The number and purity of cross-dressed cells were evaluated using imaging flow cytometry. (B) In vitro activation of T cells with exosomes or cross-dressed cells. BALB/c T cells (5 × 105 cells per well) were cultured with B6 allogeneic exosomes or BALB/c cells cross-dressed with allogeneic (H-2b) or control syngeneic (H-2d) MHC (5 × 105 cells per well) for 48 hours. The frequencies of activated T cells producing IFN-γ were measured using ELISPOT. (C) BALB/c mice were injected intraperitoneally with allogeneic (B6) or syngeneic (self, BALB/c) exosomes (2 × 108 to 2 × 109 vesicles). Fourteen days later, spleen T cells from these mice as well as control naïve mice were collected and stimulated in vitro with irradiated allogeneic B6 APCs for 72 hours. The frequency of T cells secreting IFN-γ was measured using ELISPOT. BALB/c mice recipient of a B6 skin graft or injected with B6 spleen cells were tested as positive controls. In (B) and (C), the results are expressed as the number of IFN-γ–producing spots per million T cells ± SD obtained from 13 mice tested individually. The P values obtained with unpaired t test comparing T cell responses after syngeneic versus allogeneic exosome stimulation for (B) and (C) were P = 0.0003 and P = 0.0029, respectively.