(1) Background: Dendritic cell (DC) vaccination has shown outstanding achievements in cancer treatment, although it still has some adverse side effects. cancer. 0.05). pulsed DCs: A549 tumor cell lysate-pulsed DCs; unpulsed DCs: A549 tumor cell lysate-unpulsed DCs. To analyze the surface phenotype of pulsed and ulpulsed DCs, the cells were characterized by flow cytometry using fluorescently labeled antibodies against human leukocyte antigen D-related (HLA-DR), cluster of differentiation (CD)14, CD11c, CD40, CD56, CD80, CD86, and CD123. Compared to CBMCs at day 0 of cell culture, pulsed DCs and unpulsed DCs at day seven consistently showed an increase expression level of DC markers such as HLA-DR, CD11c, CD40, CD80, and CD86, and low expression of monocyte markers such as CD123, CD144, and CD56 ( 0.05) (Figure 1B). The percentage of cells expressing DC markers was significant higher in pulsed DCs when compared to unpulsed ones ( 0.05). The pulsed DC population expressed DC markers were high on most cell culture such as HLA-DR: 81.0 18.7%, CD11c: 80.9 12.4%, CD80: 80.4 29.1%, CD40: 85.9 9.1%, and CD86: 80.6 16.8% (Figure 1C). This indicated that mature DCs have been generated successfully from cryo CBMCs. 2.2. Typical NBTGR Characteristics of DC-Derived Exosomes To analyze the morphology characteristics of exosomes released by pulsed DCs and unpulsed DCs, exosomes from these cells subjected to negative staining Rabbit polyclonal to ZNF268 and visualized using transmission electron microscopy (TEM). The images showed that the DC-derived exosomes had a cup-shaped morphology and a nanometer-scale size (Figure 2A, arrow). Additionally, protein immunoblot has been applied to detect exosomal marker expression. Data indicated that exosomes released from DCs expressed CD9 and CD63 which are considered as exosome markers (Figure 2B). Besides, CD86 was a DC marker was detected in both DCs and DC-derived exosomes. However, the expression of CD86 was very different that very abundant in DCs but very little in DC-derived exosomes (Figure 2B). This data indicates that DC-derived exosomes carry the DC characteristic. Open in a separate window Figure 2 DCs and their exosomes induced allogeneic T cell proliferation. (A) A cup-shaped morphology was observed for pulsed DC-derived exosomes by TEM. (B) Pulsed DC-derived exosomes expressed CD9, CD63, and CD86. (C) AlloT cells grew as clumps when incubated with pulsed DCs. (D) Carboxyfluorescein succinimidyl ester (CSFE)-stained T cells proliferated during a seven-day incubation with pulsed DCs, unpulsed DCs and exosomes isolated from DCs. Untreated T cells or T cells incubated with exosomes isolated from Exo/unpulsed DCs did not divide. (E) The number of T cells increased highest in the treatment with pulsed DCs, followed by Exo/pulsed DCs, and unpulsed DCs. Exo/unpulsed DCs showed no significant effect on alloT cell proliferation. Data was presented as mean SD in quadruplicate cultures (* 0.05). Exo1: pulsed DC-derived exosome sample 1; Exo2: pulsed DC-derived exosome sample 2; Exo3: pulsed DC-derived exosome sample 3. DCs: dendritic cells; pulsed DCs: A549 tumor cell lysate-pulsed DCs; unpulsed DCs: A549 tumor cell lysate-unpulsed DCs; Exo/unpulsed DCs: NBTGR exosomes isolated from unpulsed DCs; Exo/pulsed DCs: exosomes isolated from pulsed DCs. 2.3. Cryo CBMDCs and their Exosomes Induced the NBTGR Proliferation of Allogeneic NBTGR T Cells T lymphocytes were isolated from healthy donors peripheral blood using a CD3 Microbeads Positive Selection Kit. The isolated T cells were highly pure, as indicated by the calculation that 98.7 1.2% of the cells expressed CD3+. To determine the capacity of DCs and DC-derived NBTGR exosomes to induce allogeneic T cell proliferation, CFSE-stained T cells.