PGE1-Containing Protocols Generate Mature (Leukemia-Derived) Dendritic Cells Directly from Leukemic Whole Blood

Abstract

Dendritic cells (DCs) and leukemia-derived DC (DC_leu) are potent stimulators of various immunoreactive cells and they play a pivotal role in the (re-) activation of the immune system. As a potential treatment tool for patients with acute myeloid leukemia, we developed and analyzed two new PGE₁-containing protocols (Pici-_PGE1, Kit M) to generate DC/DC_leu ex vivo from leukemic peripheral blood mononuclear cells (PBMCs) or directly from leukemic whole blood (WB) to simulate physiological conditions. Pici-_PGE1 generated significantly higher amounts of DCs from leukemic and healthy PBMCs when compared to control and comparable amounts as the already established protocol Pici-_PGE2. The proportions of sufficient DC-generation were even higher after DC/DC_leu-generation with Pici-_PGE1. With Kits, it was possible to generate DCs and DC_leu directly from leukemic and healthy WB without induction of blast proliferation. The average amounts of generated DCs and DC_leu-subgroups were comparable with all Kits. The PGE₁ containing Kit M generated significantly higher amounts of mature DCs when compared to the PGE₂-containing Kit K and increased the anti-leukemic-activity. In summary PGE₁-containing protocols were suitable for generating DC/DC_leu from PBMCs as well as from WB, which reliably (re-) activated immunoreactive cells, improved the overall ex vivo anti-leukemic activity, and influenced cytokine-release-profiles.

Keywords: AML; PGE1; dendritic cells; immunotherapy; leukemia-derived dendritic cells.

Figure 1

DC/DC_leu-generation from healthy (left side) and leukemic peripheral blood mononuclear cells (PBMCs) (right side). (A) shows the average amounts ± standard deviation of generated dendritic cells (DCs) in the PBMC-fraction and mature DCs in the DC-fraction [CD197⁺DC⁺, (DC_mig/DC⁺)] from healthy PBMCs with Pici-_PGE1, Pici-_PGE2 and control without added cytokines. (B) presents the average amounts ± standard deviation of generated DCs in the PBMC-fraction, DC_leu-subgroups [including DC_leu in the DC-fraction (DC_leu/DC⁺), DC_leu in the blast-fraction (to quantify amounts of leukemic blasts converted to DC_leu) (DC_leu/Bla⁺), DC_leu in the PBMC-fraction (DC_leu/PBMC)] and DC_mig in the DC-fraction (DC_mig/DC⁺) from leukemic PBMCs with Pici-_PGE1, Pici-_PGE2 and control without added cytokines. (C) and (D) show the percentages of sufficient DC-generation from healthy (C) and leukemic (D) PBMCs with Pici-_PGE1, Pici-_PGE2, Pici-_PGE1 or Pici-_PGE2 and control without added response modifiers according to cut-off-values (≥10% DC⁺/PBMC). Each dot (● ▪ ● ▼) represents DC-proportions generated from each individual healthy volunteer or AML-patient. DCs dendritic cells; DC_leu leukemic derived dendritic cells; PBMCs peripheral blood mononuclear cells. The differences were considered as significant*** with p values <0.005.

Figure 2

DC/DC_leu-generation from leukemic whole blood (WB). (A) shows average amounts ± standard deviation of DC- and DC_leu-proportions [including DC_leu-subgroups: DC_leu in the DC-fraction (DC_leu/DC⁺), DC_leu in the blast-fraction (to quantify amounts of leukemic blasts converted to DC_leu) (DC_leu/Bla⁺) and DC_leu in the WB-fraction (DC_leu/WB)] from leukemic WB with Kits (including Kit M, Kit K, Kit I) compared to protocols Picis (including Pici-_PGE1, Pici-_PGE2). (B) shows average amounts ± standard deviation of generated DCs with Kit M, Kit K and Kit I compared to control. (C) presents average amounts ± standard deviation of generated DC_leu subgroups [including DC_leu-subgroups DC_leu in the DC-fraction (DC_leu/DC⁺), DC_leu in the blast-fraction (to quantify amounts of leukemic blasts converted to DC_leu) (DC_leu/Bla⁺) and DC_leu in the WB-fraction (DC_leu/WB)] with Kit M, Kit K and Kit I. (D) shows average amounts ± standard deviation of DC_mig/DC⁺ generated with Kit M, Kit K and Kit I. DC_mig are characterized by the expression of CCR7. DCs dendritic cells; DC_leu leukemic derived dendritic cells; WB whole blood. The differences were considered as significant*, with p values between 0.1 and 0.05 and as significant*** with p values <0.005.

Figure 3

Stimulatory effect of DC/DC_leu generated with Kits from leukemic WB on T cell enriched immunoreactive cells in MLC and the corresponding blast-lysis activity. (A) shows average amounts ± standard deviation of T cell subsets after stimulation of T cell enriched immunoreactive cells with DC/DC_leu generated with Kits from leukemic WB (MLC^{WB-DC Kits}), after stimulation of T cell enriched immunoreactive cells with a WB cell suspension not pretreated with Kits (MLC^WB) and in uncultured cells. Cells were analyzed by flow cytometry and referred to the CD3⁺ cell fraction. (B) shows the improvement of blast-lysis-activity compared to controls after 3 or 24 h of co-culture of target and effector cells (left column) and of cases with improved blast lysis with at least one MLC^{WB-DC Kit} proportions compared to MLC^WB in each individual patient (right column). (C) shows average amounts ± standard deviation of different T cell subsets after MLC^{WB-DC Kit M} and MLC^{WB-DC Kit K}. (D) shows percentage of cases with improved blast-lysis after MLC^{WB-DC Kit M} compared to MLC^{WB-DC Kit K} after 3 h (left column) and after 24 h (right column). MLC mixed lymphocyte culture; WB whole blood. The differences were considered as significant*, with p values between 0.1 and 0.05, as significant** with p values between 0.05 and 0.005 and as significant*** with p values <0.005.

Figure 4

Cytokine-release-profiles after DC/DC_leu-culture from leukemic WB. (A) shows concentrations of MCP-1 (CCL-2) (ng/mL), IL-17A (pg/mL) and IL-10 (pg/mL) in serum and after DC/DC_leu-culture from leukemic WB with Kits (including Kit M, Kit K, and Kit I). (B) shows the comparison of concentrations of MCP-1 (CCL-2), IL-17A, and IL-10 after DC/DC_leu-culture from leukemic WB with Kit M, Kit K and control without added cytokines. All cytokines were measured with ELISA. MCP-1 Monocyte chemotactic protein 1; CCL-2 CC-chemokine ligand 2; IL17-A Interleukin 17A; IL-10 Interleukin 10. The differences were considered as significant** with p values between 0.05 and 0.005 and as significant*** with p values <0.005.

Figure 5

Workflow and FACS analysis of DC/DC_leu generated with Pici-_PGE1 compared to control from leukemic PBMCs in a case of AML. 3–4 × 10⁶ PBMCs were cultured in 12-multiwell-tissue-culure-plates and diluted in 2 mL serum-free X-vivo-15-medium. For the generation of DC/DC_leu with Pici-_PGE1 500 U/mL GM-CSF and 250 U/mL IL-4 were added on day 0. After 6–7 days, 10µg/mL Picibanil a lysis product from Streptococcus pyogenes, which has unspecific immune modulatory effects and 1 µg/mL PGE₁ were added. No response modifiers were added to the control culture. Cells were harvested after 7–10 days of incubation. Half medium exchange was carried out after 3–4 days. x-axis: CD80; y-axis: CD34.