Abstract

Dendritic cells (DCs) are potent of the professional antigen presenting cells (APC) and have the unique ability to stimulate naïve T cells and initiate and regulate immune responses. We have studied human DCs loaded with acute myeloid leukemia (AML) specific lysate and evaluated the in vitro function in the induction of anti-leukemia cytotoxic responses.

Methods. DCs were generated from CD14+ selected monocytes using the MACS system (Miltenyi) and cultured with GM-CSF, and IL-4 (R&D Systems) for 6 days, loaded with AML specific lysate, and matured in the presence of IL1-b, TNF-a, IL-6, and PGE-2 (ITIP). Flow cytometry analysis demonstrated that ITIP-matured DCs showed upregulation of CD80 (80%), CD83 (80 to 90%), and CCR7 (20–40%) molecules.

Characterization of Phagocytosis. DC phagocytic function was assessed by assaying the uptake of FITC-dextran beads or with FITC labeled AML lysate. Day 6 immature DCs were incubated with 0.1 mg/ml FITC-dextran beads or FITC labeled lysate at 37° C for one hour. Cells were analyzed by flow cytometry for the uptake of beads or AML lysate. Approximately 90 % of the immature DCs were positive for the FITC labeled beads or FITC labeled after one hour of incubation.

Allogeneic Mixed Lymphocyte Reaction. To ensure that the loading of antigen onto DCs does not change their immuno-stimulatory function, the control and antigen loaded DCs were tested for their capacity to stimulate the proliferation of allogeneic T cells. In all the DC T cells ratio tested, range from 1:10, 1:25. 1:50, 1:100, there is no difference in the capacity in this allo-MLR assay in comparison to unloaded DCs.

Characterization of Th-1 Response. To generate leukemia specific CTL responses, autologous T lymphocytes were stimulated with antigen (AML lysate) loaded DCs at a DC: lymphocyte ratio of 1:10. Cells were restimulated twice, 9 days apart, in the presence of IL-2 (10 units/ml). At the end of the third stimulation, intracellular IFN-g secretion of CD8+ T cells was assessed by CFC. 5% to 19% of CD8+ T cells were positive for IFN-g, demonstrating that AML lysate loaded DCs can be effective in promoting a Th-1 type immune response.

Killing of Leukemia antigen loaded DC targets by specific CTL We analyzed whether AML specific lysate-loaded DCs were capable of stimulating CTLs from PBMCs that specifically recognize and lyse leukemia specific targets. We have found that T cells primed by both AML lysate loaded and AML mRNA loaded DCs are equally effective in recognizing and lysing AML lysate loaded DC targets. In a standard 5 hour 51Cr release assay, there was 20% killing of target at an E:T ratio of 50:1. CTL killing was significantly lower in a control assay, which utilized unloaded DCs as a target (p<0.006).

Conclusion. We have demonstrated in this study that the CTL killing generated by in vitro cultured and AML lysate loaded DC is antigen specific. Determination of specificity is important prior to the use of AML lysate loaded DCs in a future clinical trial for AML-specific therapy.

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