TY - JOUR
T1 - A novel nonradioactive CFDA assay to monitor the cellular immune response in myeloid leukemia
AU - Yang, Ting
AU - Chen, Zhi zhe
AU - Kolb, Hans Jochem
AU - Buhmann, Raymund
PY - 2013/4
Y1 - 2013/4
N2 - 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 (50ng/mL of SCF; 25ng/mL of IL-3; 100ng/mL of GM-CSF; 100ng/mL of G-CSF; 2U/mL of EPO; 0.47g/L of transferrin; and 5×10-5mmol/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.
AB - 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 (50ng/mL of SCF; 25ng/mL of IL-3; 100ng/mL of GM-CSF; 100ng/mL of G-CSF; 2U/mL of EPO; 0.47g/L of transferrin; and 5×10-5mmol/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.
KW - A nonradioactive assay
KW - Adoptive immune response
KW - Antileukemia immunity
KW - CFDA dye
KW - Flow cytometry
UR - http://www.scopus.com/inward/record.url?scp=84875256736&partnerID=8YFLogxK
U2 - 10.1016/j.imbio.2012.06.014
DO - 10.1016/j.imbio.2012.06.014
M3 - Article
C2 - 22883564
AN - SCOPUS:84875256736
SN - 0171-2985
VL - 218
SP - 548
EP - 553
JO - Immunobiology
JF - Immunobiology
IS - 4
ER -