An antigen (K562-P26, P27) was used to stimulate iDCs for 24 hours, and then cytokines (TNF-α + IL-1β) were used for another 24 hours to stimulate iDCs to mature into mDCs. Flow cytometry measured the DC phenotype which showed activated markers of antigen-presenting cells, including HLA-DR, CD86, CD80, CD83, CD40, and CD14. mDCs showed increased expression of these markers, except for CD14 that showed a decrease, on the 7th day compared to marker expression in iDCs on the 5th day, indicating that antigen (K562-P26, P27) combined with cytokines (TNF-α + IL-1β) can stimulate DC maturation (Fig. 2).
We compared the differences in marker expression, cytotoxicity to K562 cells, and the amount of CIK-secreted cytokines between antigen-stimulated DC- CIK and the control. On the 9th day of the 1: 20 DC: CIK coculture, the expression of cell markers CD56, CD3-/CD56, CD4, CD8, and CD28 was not different between Ag + cytokine-stimulated DC-CIK and the control. However, the K562 cell-killing rate of CIK cells increased in Ag + cytokine-stimulated DC- CIK, irrespective of whether the CIK: K562 cell ratio was 25: 1, 5: 1, or 1: 1. The most significant increase in the rate, 21.27%, occurred at the CIK: K562 cell ratio of 25: 1. On the 14th day, the expression of CIK markers, including CD4 and CD28, showed a higher increase in Ag + cytokine-stimulated DC- CIK than in the control. Cytotoxicity to K562 cells also increased, regardless of whether the CIK: K562 cell ratio was 25: 1, 5: 1, or 1: 1. At the CIK: K562 cell ratio of 25: 1, the rate of cytotoxicity showed the most significant change of an increased by up to 37.77%.
The DC: CIK cell ratio was adjusted to 1: 100. The expression of CIK markers, CD56, CD3-/CD56, CD4, CD8, and CD28, was similar in Ag + cytokine-stimulated DC-CIK and control on day 9. Only CD4 and CD28 increased on day 14 in Ag + cytokine-stimulated DC- CIK compared to the control. The K562 cell-killing rate of Ag + cytokine-stimulated was similar to that of the control on the 9th day, irrespective of whether the CIK: K562 cell ratio was 25: 1, 5: 1, or 1: 1. On the 14th day, the K562 cell-killing rate of Ag + cytokine-stimulated DC- CIK increased compared to that of the control at the CIK: K562 cell ratio of 25: 1 and 5: 1, but was similar at the 1: 1 ratio. At the DC: CIK cell ratio of 1: 100 and CIK: K562 cell ratio of 25: 1, the rate of cytotoxicity increased up to 35.97%, which was close to 37.77% at the DC: CIK cell ratio of 1: 20 together with the CIK: K562 cell ratio of 25: 1. However, the more cells are used, the higher the cost. We found that on the 14th day, when the DC: CIK cell ratio was 1: 20 and the CIK: K562 cell ratio was 25: 1, the K562 cell-killing rate of CIK cells was up to 37.77%, which was the most significant and economical rate (Fig. 4).
Regardless of whether the DC: CIK cell coculture ratio was 1: 20 or 1: 100, on the 9th day, the amount of cytokines IFN-γ, TNF-α, IL-10, IL-6, and IL-4 secreted by Ag + cytokine-stimulated DC- CIK increased compared to the control. At the DC: CIK cell ratio of 1: 20, the amount of IFN-γ, TNF-a, IL-10, IL-6, and IL-4 secreted by Ag + cytokine-stimulated DC- CIK increased on the 14th day compared to the control. At the DC- CIK cell ratio of 1: 100, the amount of INF-γ, IL-10, and IL-6 secreted by Ag + cytokine-stimulated DC- CIK was higher but the amount of TNF-a, IL-4, and IL-2 secreted were lower compared to the control. We also found that on the 14th day, the number of cytokines secreted by Ag + cytokine-stimulated DC-CIK cell ratio of 1: 20 than at the ratio of 1: 100.
Cell viability on the 14th day was lower than that on the 9th day, regardless of whether the DC: CIK cell ratio was 1: 20 or 1: 100, indicating that cell viability declined over time. On the 9th day, the number of CIK cells in Ag + cytokine-stimulated DC- CIK was similar to that in the control regardless of whether the DC: CIK cell ratio was 1: 20 or 1: 100. On the 14th day, at the DC: CIK cell ratio of 1: 20, the number of CIK cells in Ag + cytokine-stimulated DC- CIK was lower than that in the control. At the DC: CIK cell ratio of 1: 100, the number of CIK cells in Ag + cytokine-stimulated DC- CIK was slightly higher than that in the control. However, cytokine secretion by CIK cells was at its peak at the DC: CIK cell ratio of 1: 20 on the 14th day, indicating the highest ratio of effecter T cells (Fig. 5).