The delta Assay - A Suitable Tool To Detect the Cellular Immune Response Against Acute Myeloid Leukemia Blasts.

Adoptive immunotherapy with donor T-cells has been used successfully for the treatment of both relapsed chronic and acute myeloid leukemia after hematopoietic stem cell transplantation. Here we describe a method for the detection of T cell immunity by the inhibition of cytokine driven growth of blasts. According to the heterogeneous growth pattern of various cases of acute myeloid leukemia (AML) the culture requirements had to be determined individually. The basic cytokine cocktail consisted of SCF (50 ng/mL), GM-CSF (100 ng/mL), IL3 (50 ng/mL), G-CSF (100 ng/mL) and EPO (2 U/mL); this cocktail succeeded to induce blast-proliferation in 15 out of 16 AML samples tested. In a first step we determined optimal growth conditions regarding cell density and the day of linear growth. The peak proliferation was measured by the incorporation of tritium labeled thymidine at various time points. It ranged between days 2 and 5. In a second step, donor cells were stimulated for 10 days with irradiated AML-cell lines (MonoMac6, THP1) or patient derived AML blasts. After harvesting, the donor cells were irradiated (15 Gy) and co-cultured with the AML-cell lines, or the patients cytokine stimulated blast suspensions. Inhibition of growth was measured by the incorporation of tritium labeled thymidine at the previously determined day of linear proliferation of the AML-blasts. A significant inhibition of blast proliferation was observed in the co-culture of normal T cells with both AML-cell lines. In HLA-identical combinations T cells were primed by dendritic cells derived from AML blasts and re-stimulated on days 6 and 10. T cells primed by this method inhibited the growth of AML blasts in higher effector target ratios, in lower ratios no inhibition or even stimulation was observed. In summary, the delta-assay is a suitable tool to monitor specific anti-leukemic immune responses of donor lymphocytes even against very heterogeneous blast populations, if individualized culture conditions are considered.