DC generation from peripheral blood mononuclear cells in patients with chronic myeloid leukemia: Influence of interferons on DC yield and functional properties

Abstract

In Chronic Myeloid Leukemia (CML), standard treatment consists of modern tyrosine-kinase inhibitors (TKI). Nevertheless, there is evidence that immune responses against leukemia-associated antigens (LAA) may play an important role in disease control. Dendritic cell (DC)- based immunotherapy is able to induce T cell responses against LAA and might therefore pose an interesting therapeutic option in CML, especially in the setting of minimal residual disease (MRD). GMP production of DC for clinical vaccination remains a time- and cost- intensive procedure and standardized DC generation is warranted. We asked whether maturation-induction with IFN-γ and IFN-α has an influence on functional properties of DC derived from peripheral blood mononuclear cells (PBMC) in CML patients. Monocyte-derived DC from healthy donors and from patients with CML were analyzed after maturation-induction with our TNF-α-containing standard cytokine cocktail with or without addition of IFN-α and/or IFN-γ. Our results confirm that the addition of IFN-γ leads to enhanced IL-12 secretion in healthy donors. In contrast, in CML patients, IFN-γ was not able to increase IL-12 secretion, possibly due to a higher degree of cell adherence and lower cell yield during the cell culture. Our data suggest, that- in contrast to healthy donors-, additional interferons are not beneficial for maturation induction during large-scale DC production in patients with CML.

Keywords: CML; DC activation; DC generation; dendritic cells; monocyte-derived DC.

Figure 1.

Cell adherence and cell yield of DC from CML patients after maturation induction with IFN-γ. Using our standard cytokine cocktail, DC generation from PBMC of CML patients and healthy individuals gives comparable results (a and b). In contrast, addition of interferons leads to an increase of cell adherence and a lower DC yield (c and d). a: DC culture from a healthy donor, b: DC culture from a patient with CML in chronic phase cultured with our standard cytokine cocktail (GM-CSF, IL-4, TNF-α) without interferons, c: Increased DC adherence in a DC culture containing IFN-γ in a chronic phase CML patient, d: Decreased DC yield (in cells per cm²) in cell cultures from CML patients containing IFN- γ or IFN-γ + IFN-α. *=Difference (as compared to “no IFN”) not statistically significant (p=0.2), possibly due to high variation in a low number of samples (no IFN: n = 3, IFN-γ: n = 3, IFN-γ + IFN-α: n = 3).

Figure 2.

Influence of IFN-γ on the cytokine secretion profile of DC after CD40 ligation in healthy donors. DC from healthy donors were generated under different maturation conditions (standard cytokine cocktail without vs. with IFN-γ). The cytokine profile was measured by CBA (or ELISA for IL-12) after CD40 ligation. a: IL-12 secretion into the culture supernatant without vs. with IFN-γ (p=0.043), b: IL-10 secretion into the culture supernatant without vs. with IFN-γ (p = 0.068),c: IL-12/IL-10 ratio without vs. with IFN-γ.HD = healthy donor **= p<0 .05 as compared to “no IFN;” * = p = not significant as compared to “no IFN.”.

Figure 3.

Influence of IFN-γ on the cytokine secretion profile of DC after CD40 ligation in chronic phase CML patients. a: IL-12 secretion of DC from patients with CML after maturation-induction with our standard cytokine cocktail without vs. with interferons (without IFN vs. IFN-γ: p = 0.055; without IFN vs. IFN-γ plus IFN-α: p = 0.91), b: IL-10 secretion of DC from patients with CML after maturation-induction with our standard cytokine cocktail without vs. with interferons (without IFN vs. IFN-γ: p = 0.28; without IFN vs. IFN-γ plus IFN-α: p = 0.9), c: T cell stimulatory capacity of DC generated with our standard cytokine cocktail without interferons as measured by a ³H-thymidine proliferation assay (mean CPM of cells after maturation-induction without IFN: 27342 + 712; with IFN-γ: 24029 + 727); with IFN-γ plus IFN-α: 24005 +168). IL-12 was determined by ELISA, all other cytokines by CBA. *= not statistically significant.