Involvement of MM cell-derived exosomes in T lymphocytes immune responses

Abstract

Exosomes were reported to mediate cell communication in the tumor microenvironment; however, the effects of multiple myeloma (MM)-derived exosomes on the quantity and function of T cells remain unknown. Exosomes were extracted from MM cell lines (OPM2 and U266B1) by ultracentrifugation using a Total Exosome Isolation kit. Exosomes were co-cultured with CD4+ T, CD8+ T and regulatory T (Treg) cells that were isolated from healthy donors (HDs) and patients with MM using magnetic beads. Flow cytometry was used to detect T cells apoptosis and expression of perforin and granzyme B in CD8+ T cells. Cell viability was detected using Cell Counting kit-8, and interleukin 10 (IL-10) and transforming growth factor β (TGF-β) in cell supernatants were detected by ELISA. The apoptosis of HD-CD4+ T was higher in the OPM2 group, and viability in the U266B1 group was decreased. The apoptosis of HD-CD8+ T decreased in the OPM2 and U266B1 groups, and cell viability increased in the OPM2 and the U266B1 groups. Perforin of HD-CD8+ T in the U266B1 group was lower while perforin of MM-CD8+ T in OPM2 and U266B1 groups was markedly decreased. The apoptosis of HD-Treg was lower in the U266B1 group, but apoptosis of MM-Treg was higher in the U266B1 group. The viability of HD-Treg in U266B1 group increased but the viability of MM-Treg in OPM2 and U266B1 groups decreased. TGF-β from MM-Treg decreased in the OPM2 and U266B1 groups when compared with the control group (P<0.05). MM-derived exosomes promote apoptosis and inhibit proliferation of HD-CD4+ T, inhibit apoptosis and promote proliferation, but inhibit perforin of HD-CD8+ T, inhibit apoptosis and promote proliferation HD-Treg, and inhibit perforin of MM-CD8+ T and TGF-β secretion of MM-Treg.

Keywords: T lymphocyte; exosomes; immunity; multiple myeloma.

Figure 1.

Exosomes observed by transmission electron microscopy. (A) Exosomes isolated by ultracentrifugation had intact continuous membranes and a diameter of 30–150 nm (magnification, 40.0 k). (B) Exosomes isolated by ultracentrifugation in clusters (magnification, 30.0 k). (C) Exosomes isolated using the Total Exosome Isolation reagent kit had intact continuous membranes and a diameter of 30–150 nm (magnification, 40.0 k). (D) Exosomes isolated using the Total Exosome Isolation reagent kit in clusters (magnification, 20.0 k). (E) Western blotting analysis of CD63 and HSP70 protein expression in exosomes from the OPM2 and U226B1 cells lines and cell lysates of OPM2 and U226B1 cells. HSP70, HSP 70, heat shot protein 70; Exo, exosome; Lys, lysate.

Figure 2.

Effect of MM cell-derived exosomes on CD4+ T lymphocytes. (A) Apoptotic rate of HD-CD4+ T cells co-cultured without exosome (control). (B) Apoptotic rate of HD-CD4+ T cells co-cultured with OPM2-isolated exosomes (50 µg/10⁵ cells). (C) Apoptotic rate of HD-CD4+ T cells co-cultured with OPM2-isolated exosomes (100 µg/10⁵ cells). (D) Apoptotic rate of HD-CD4+ T cells co-cultured without exosome (control). (E) Apoptotic rate of HD-CD4+ T cells co-cultured with OPM2-derived exosomes. (F) Apoptotic rate of HD-CD4+ T cells co-cultured with U266B1-derived exosomes. (G) The apoptotic rate of HD-CD4+ T cells was significantly increased in the OPM2-derived exosomes. (H) The viability of HD-CD4+ T cells was decreased in the U266B1-derived exosomes compared with that of the control (n=15). (I) The apoptotic rate of MM-CD4+T cells showed no statistical significance among the three groups (n=15). (J) The viability of MM-CD4+ T cells showed no statistical significance among the three groups (n=15). HD, healthy donor; MM, multiple myeloma; UL, upper left; UR, upper right; LL, lower left; LR, lower right; exo, exosome; FITC, fluorescein isothiocyanate; PI, propidium iodide; PE, phycoerythrin.

Figure 3.

Effect of MM cell-derived exosomes on CD8+ T lymphocytes. Assessment of apoptosis by flow cytometry of HD-CD8+ T cells co-cultured with (A) Apoptotic rate of HD-CD8+ T cells co-cultured without exosome (control). (B) Apoptotic rate of HD-CD8+ T cells co-cultured with OPM2-derived exosomes. (C) Apoptotic rate of HD-CD8+ T cells co-cultured with U266B1-derived exosomes. (D) Apoptotic rate of MM-CD8+ T cells co-cultured without exosome (control). (E) Apoptotic rate of MM-CD8+ T cells co-cultured with OPM2-derived exosomes. (F) Apoptotic rate of MM-CD8+ T cells co-cultured with U266B1-derived exosomes. (G) The apoptotic rate of HD-CD8+ T cells co-cultured with OPM2- and U266B1-derived exosomes was significantly decreased compared with the control group (n=15). (H) The viability of HD-CD8+ T cells co-cultured with OPM2- and U266B1-derived exosomes was significantly increased compared with the control group (n=15). (I) The apoptotic rate of MM-CD8+T cells showed no statistical significance among the three groups (n=15). (J) The viability of MM-CD8+ T cells showed no statistical significance among the three groups (n=15). HD, healthy donor; MM, multiple myeloma; UL, upper left; UR, upper right; LL, lower left; LR, lower right; exo, exosome; FITC, fluorescein isothiocyanate; PI, propidium iodide; PE, phycoerythrin.

Figure 4.

Effect of MM cell-derived exosomes on the function of CD8 and Treg cells. (A) Perforin levels of HD-CD8+ T cells co-cultured without exosome (control). (B) Perforin levels of HD-CD8+ T cells co-cultured with OPM2-derived exosomes. (C) Perforin levels of HD-CD8+ T cells co-cultured with U266B1-derived exosomes. (D) Granzyme B levels of HD-CD8+ T cells co-cultured without exosome (control). (E) Granzyme B levels of HD-CD8+ T cells co-cultured with OPM2-derived exosomes. (F) Granzyme B levels of HD-CD8+ T cells co-cultured with U266B1-derived exosomes. (G) The levels of perforin secreted by HD-CD8+ T cells was decreased in the U266B1-derived exosome group compared with the control group. (H) The levels of perforin secreted by MM-CD8+T cells was significantly decreased both in the OPM2 and U266B1-derived exosome groups compared with the control group. (I) The levels of TGF-β in the supernatant of HD-Treg cells showed no statistical significance and (J) the levels of TGF-β secreted by MM-Tregs was decreased in the OPM2 and U266B1-derived exosome groups compared with that of the control (n=15). HD, healthy donor; IL, interleukin; MM, multiple myeloma; TGF-β, transforming growth factor β; UL, upper left; UR, upper right; LL, lower left; LR, lower right; exo, exosome; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 5.

Effect of MM cell-derived exosomes on Tregs. (A) Apoptotic rate of HD-Treg cells co-cultured without exosome (control). (B) Apoptotic rate of HD-Treg cells co-cultured with OPM2-derived exosomes. (C) Apoptotic rate of HD-Treg cells co-cultured with U266B1-derived exosomes. (D) Apoptotic rate of MM-Treg cells co-cultured without exosome (control). (E) Apoptotic rate of MM-Treg cells co-cultured with OPM2-derived exosomes. (F) Apoptotic rate of MM-Treg cells co-cultured with U266B1-derived exosomes. (G) The apoptotic rate of HD-Tregs was significantly decreased in the U266B1-derived exosome group compared with that of the control. (H) The viability of HD-Tregs was significantly increased in the U266B1-derived exosome group. (I) The apoptotic rate of MM-Tregs was significantly increased in the U266B1-derived exosome group compared with that of the control. (J) The viability of MM-Tregs was decreased in the OPM2- and U266B1-derived exosome groups (n=15). HD, healthy donor; MM, multiple myeloma; Treg, regulatory T cell; UL, upper left; UR, upper right; LL, lower left; LR, lower right; exo, exosome; FITC, Fluorescein isothiocyanate; PI, Propidium iodide; PE, Phycoerythrin.