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Yang, T. ; Chen, Z.-Z.* ; Kolb, H.-J. ; Buhmann, R.

A novel nonradioactive CFDA assay to monitor the cellular immune response in myeloid leukemia.

Immunobiology 218, 548-553 (2013)
DOI PMC
Open Access Green as soon as Postprint is submitted to ZB.
Background: Donor lymphocyte transfusion (DLT) may induce the graft-versus-leukemia (GVL) effect for patients with AML relapsed after transplant. However, AML is a highly diverse disease and the limited overall efficacy of DLT in clinical practice emphasizes the importance of identifying a specific subgroup of patients who might benefit from this treatment approach. Objective: To monitor the cellular immune response after DLT, we developed an active specific immunization strategy using in vitro generated AML-trained T cells to induce a highly specific antileukemic T-cell response and thus established a novel nonradioactive assay system to assess the antileukemia immunity by flow cytometry, correlated with [3H]-thymidine uptake. Methods: The myeloid blasts derived from five patients with AML relapsed post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) were first labeled with CFDA (5,6-carboxyfluorescein diacetate succinimidyl ester). To analyze the growth inhibitory potential of the donor T cells trained by AML progenitor cells, the myeloid blasts were induced to proliferate by means of a cytokine cocktail (50 ng/mL of SCF; 25 ng/mL of IL-3; 100 ng/mL of GM-CSF; 100 ng/mL of G-CSF; 2 U/mL of EPO; 0.47 g/L of transferrin; and 5 x 10(-5) mmol/L of 2-ME). The T cell mediated growth inhibitory potential was detected after 5 days by flow cytometry and correlated with [3H]-thymidine uptake. The simultaneous use of TO-PRO-dye and calibrate beads allowed not only the cell viability to be known but also allowed quantification of the effector function. Results: Here, we applied a CFDA dye to track the proliferation and expansion of AML blasts in response to the cytokine cocktail in vitro. AML-trained T cells, expressed high levels of the activation markers CD25 and CD69, and were generated to recognize the leukemic progenitor cells and inhibit cytokine-induced leukemic cell proliferation, which is an active specific immunization strategy circumventing the identification of leukemia-associated antigens. The capability of proliferation inhibition of AML-trained T cells evaluated with our nonradioactive, CFDA-based assay provided comparable results with the classic [3H]]-thymidine assay with an even lower ratio of effector to target cells. Conclusion: Taken together, the novel, nonradioactive, CFDA-based assay was a robust tool to monitor the antileukemic immune response after DLT in myeloid leukemias.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords A Nonradioactive Assay ; Cfda Dye ; Flow Cytometry ; Antileukemia Immunity ; Adoptive Immune Response; Diacetate Succinimidyl Ester ; Minor Histocompatibility Antigens ; Bone-marrow-transplantation ; Flow-cytometric Measurement ; T-cells ; Intracellular Cytokines ; Lymphocyte Division ; Affinity Matrix ; Fluorescent Dye ; Dendritic Cells
ISSN (print) / ISBN 0171-2985
e-ISSN 1878-3279
Journal Immunobiology : Experimental and Clinical
Quellenangaben Volume: 218, Issue: 4, Pages: 548-553 Article Number: , Supplement: ,
Publisher Urban & Fischer
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) CCG Hematopoetic Cell Transplants (IMI-KHZ)