Dendritic Cells Pulsed with Leukemia Cell-Derived Exosomes More Efficiently Induce Antileukemic Immunities

<div><p>Dendritic cells (DCs) and tumor cell-derived exosomes have been used to develop antitumor vaccines. However, the biological properties and antileukemic effects of leukemia cell-derived exosomes (LEXs) are not well described. In this study, the biological properties and induction of antileukemic immunity of LEXs were investigated using transmission electron microscopy, western blot analysis, cytotoxicity assays, and animal studies. Similar to other tumor cells, leukemia cells release exosomes. Exosomes derived from K562 leukemia cells (LEX_K562) are membrane-bound vesicles with diameters of approximately 50–100 μm and harbor adhesion molecules (<i>e.g.</i>, intercellular adhesion molecule-1) and immunologically associated molecules (<i>e.g.</i>, heat shock protein 70). In cytotoxicity assays and animal studies, LEXs-pulsed DCs induced an antileukemic cytotoxic T-lymphocyte immune response and antileukemic immunity more effectively than did LEXs and non-pulsed DCs (<i>P</i><0.05). Therefore, LEXs may harbor antigens and immunological molecules associated with leukemia cells. As such, LEX-based vaccines may be a promising strategy for prolonging disease-free survival in patients with leukemia after chemotherapy or hematopoietic stem cell transplantation.</p></div

Therapeutic effect of LEX_L1210-pulsed DCs on established tumors.

<p>To examine the therapeutic effect on established tumors, DBA/2 mice (n = 8 per group) were subcutaneously (s.c.) inoculated with L1210 cells (0.5×10⁶ cells/mouse). After 5 d, when tumors became palpable (∼5 mm in diameter), mice were s.c. immunized with L1210-derived exosomes (LEX_L1210) and different doses of dendritic cells (DCs) pulsed with LEX_L1210 (DC/LEX_L1210) (1.0–4.0×10⁶ cells/mouse). Animal mortality and tumor growth or regression were monitored daily for up to 10 weeks. For ethical treatment of the animals, mice were euthanized when the tumor diameter reached 1.5 cm. Experiments were performed in triplicate. One representative experiment is shown.</p

Exosome-uptaking by dendritic cells (a) Carboxyfluorescein succinimidyl ester (CFSE)-labeled exosomes were co-cultured <i>in vitro</i> with dendritic cells (DCs), and CFSE-positive DCs were detected using flow cytometry at different times during the culture.

<p>Confocal microscopy was used concurrently with flow cytometry to visualize the cultured DCs. (b) Phase changes of CFSE expression in DCs at different time points during the culture. (c) To investigate the rate of decay of exosomes (EXOs) in DCs, DCs were incubated with CFSE-labeled EXOs for 4 h, washed twice with phosphate-buffered saline, cultured in culture medium, and examined at different time points for up to 72 h.</p

LEX_L1210 and LEX_L1210-pulsed DCs induce anti-leukemia protective immunity against L1210 leukemia cells.

<p>Survival of mice prophylactically immunized with different vaccines. DBA/2 mice (n = 8 per group) were immunized with phosphate-buffered saline (PBS), L1210-derived exosomes (LEX_L1210), non-pulsed dendritic cells (DCs), and different doses of LEX_L1210-pulsed DCs (DC/LEX_L1210). On days 7–10 after immunization, all mice were challenged with L1210 leukemia cells. Vaccination with non-pulsed DCs (median survival time [MST], 20 d) did not significantly prolong the survival of mice as compared to the non-vaccinated PBS group (MST, 15 d), whereas LEX_L1210 vaccination (MST, 30 d) improved survival as compared to the control group of non-vaccinated mice (<i>P</i><0.05). In contrast, mice receiving different doses of DC/LEX_L1210 (particularly 2×10⁶ and 4×10⁶) had significantly improved survival as compared to non-vaccinated mice (<i>P</i><0.0001). Results were combined from 2 separate experiments.</p

Vaccination with LEXO and LEXO-targeted DC protects against tumor growth.

<p>To examine the antitumor immunity conferred by LEX_L1210 and DC/LEX_L1210, DBA/2 mice were randomly divided into 4 groups (n = 8) and immunized s.c. with the following vaccines on the inner side of their thighs: PBS (control), LEX_L1210 (30 μg), unpulsed DCs (1×10⁶ cells), and LEX_L1210-pulsed DCs (DC/LEX_L1210) (1×10⁶ cells). On day 7 after immunization, all mice were challenged with L1210 leukemia cells on the outer side of the same thighs (0.5×10⁶ cells/mice). To determine immune specificity, a group of tumor-free mice after immunization, were challenged s.c. with p388 cells (5×10⁵ cells/mouse). Tumor growth was monitored daily for up to 4 weeks using a caliper. For ethical treatment of the animals, all mice were euthanized when the tumor diameter reached 1.5 cm. Three total experiments were performed. One representative experiment is shown.</p

Morophology and expression of heat shock protein 70 and ABL in LEX_K562.

<p>(a) Transmission electron micrograph of K562 cell-secreted exosomes (×100K). (b and c) Electron micrograph of heat shock protein 70 (HSP70)- and ABL-labeled exosomes. (d) Western blot analysis demonstrating the presence of HSP70 and ABL molecules in K562 cells and K562-derived exosomes (LEX_K562).</p

Cytotoxicity assay.

<p>Cytotoxic responses were evaluated by the lactate dehydrogenase (LDH)-releasing method. Splenic T-cells from mice immunized intravenously with L1210-derived exosomes (LEX_L1210) and dendritic cells (DCs) pulsed with LEX_L1210 or phosphate-buffered saline (PBS) as a control were harvested and co-cultured with irradiated L1210 cells. At the end of the culture period, viable T-cells were separated using Ficoll-Paque centrifugation and were thereafter referred to as effector cells. The LDH assay is an enzymatic method used to colorimetrically quantify LDH released from lysed target cells, including L1210 or P388 cells, which served as the control and were mixed at different ratios with effector cells after incubation for 4 h at 37°C. The spontaneous/maximal release ratio was <20% in all experiments. Specific lysis (%) was calculated as follows: (experimental LDH release – effector cell spontaneous LDH release – target spontaneous LDH release)/(target maximum LDH release)×100. * <i>P</i><0.05 compared with the PBS, DC, and P388 groups; ** <i>P</i><0.01 compared with the PBS control group; △ <i>P</i><0.05 compared with the LEX_L1210 group; △△ <i>P</i><0.01 compared with the LEX_L1210 group. Experiments were performed in triplicate. One representative experiment is shown.</p