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Fig. 3 | Journal for ImmunoTherapy of Cancer

Fig. 3

From: Tumor-derived exosomes induce CD8+ T cell suppressors

Fig. 3

Tu167 tumor-derived exosomes are responsible for the induction of suppressor CD3+ T cells. Conditioned media from Tu167 cells had dead cells and debris removed by pelleting during centrifugation and aspirated supernatant was then incubated overnight at 4 °C with ExoQuick-TC (System Biosciences) to purify exsosomes. This produced an exosome fraction (pellet) and a non-exosome membrane vesicle fraction (supernatant). To get Tu167 non-membrane vesicle CGM fraction, Tu167 CGM was ultracentrifuged at 100,000 × g for 16 h at 4 °C. This produced an insoluble pellet (i.e., membrane vesicles) and soluble fraction (Tu167 No-membrane vesicle CGM fraction). After 6 h co-incubation with fractions, freshly purified CD8+ T cells were washed and grown in complete RPMI for 7 days. On day 7, a portion of CD8+ T cells were removed and prepared for flow cytometry. All dot plots are gated off of CD3+ lymphocytes. a. CD28 vs CD27 phenotype distribution for CD3+ T cells incubated under the indicated conditions. On day 6, a portion of CD8+ T cells were removed and used in in vitro suppression assays in anti-CD3- coated plates at a 2:1 responder: suppressor ratio. BrdU incorporation ELISA (b) and 3H-thymidine (c) suppression assay was done for 72 h with BrdU or 3H-thymidine being added 22 h before the end of the experiment. T cell CGM was collected from T cells purified from PBMCs and cultured in cRPMI for 3 days under anti-CD3/CD28 conditions. d. A suppression assay was also examined using KI67 and the antitumor cytokine IFN-γ at the above 2:1 responder: suppressor ratio with treatment of CD3+ T cells with exosomes. Data represents the mean +/− SD of 3. To compare means, a one-way analysis of variance was used with a Bonferroni multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 for the indicated groups vs. responder T cells (Tresps) alone

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