Composition of PBMC Population and Functionality of T- and NK-Cells in AML Patients Receiving Cytotoxic Maintenance Therapy,

Abstract 3601

Novel therapeutic venues are following immunotherapeutic strategies by translating the successes of allogeneic stem cell transplantation to immunotherapy in an autologous setting. Several phase I/II clinical trials have been published using AML-associated peptides or RNA-transfected dendritic cells for vaccination of AML patients. The success of immunotherapy is hampered by negative regulatory mechanisms that inhibit anti-leukemic T cell function, leading to evasion from immune attack. A promising approach to overcome negative regulatory immune mechanisms is the combination of immunotherapy with cytotoxic chemotherapy. Within the AML-CG study group, we regularly apply cytarabine in an alternating combination with daunorubicin, 6-thioguanine and cyclophosphamide every 28 days as a maintenance postremission therapy in patients not eligible for stem cell transplantation. We set out to assess the immune status of patients prior to and during this maintenance therapy in order to define a potentially beneficial point of time for active immunotherapies, e.g. dendritic cell vaccination.

We analyzed PBMC subpopulations (CD3⁺CD8⁺ cytotoxic T cells, CD3⁺CD4⁺ T helper cells, CD4⁺CD25^hiCD127^loFoxP3⁺ regulatory T cells, and CD3⁻CD16⁺CD56⁺ natural killer cells) in 8 AML patients at several time-points (days 0, 3, 9, 14, and 21) throughout early maintenance therapy cycles by flow cytometry. Numbers of regulatory T (T_reg) cells were additionally determined by a DNA methylation assay for selected samples. In order to test the proliferative capacity of T helper (Th) cells, CFSE assays with PHA as stimulus were performed in 3 patients. T cell polarization was analyzed by determination of secreted cytokines (IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, IFN-γ) using a cytometric bead array after IL-2 stimulation. Natural killer (NK) cell functionality was assessed in 4 patients using CD69 upregulation after stimulation with IL-2. The resulting data was compared with 15 healthy donors subjected to the same analyses.

The absolute Th cell counts, absolute numbers of T_reg cells and absolute NK cell counts were significantly lower in AML patients prior to the start of maintenance therapy compared to healthy donors (p < 0.05), while the relative T_reg numbers in relation to all lymphocytes were similar. In the course of maintenance therapy, several dynamics were observed: Total leukocyte numbers decreased, with the nadir around 1 G/l on day 21. The proportion of cytotoxic T cells did not vary significantly throughout the course of therapy. However, we observed a temporary increase in Th cells (p < 0.05 for days 9 and 14 compared to day 0). Interestingly, the fraction of T_reg cells more than doubled in the course of the therapy, with a maximum on day 14 (p < 0.05), explaining part of the relative increase in CD4⁺ T cells. In contrast, relative numbers of NK cells declined in the course of maintenance therapy (p < 0.05 for days 9, 14 and 21 compared to day 0).

Functionally, we did not find differences in Th cell proliferation between healthy donors and AML patients before the start of maintenance chemotherapy, while the response was substantially reduced two weeks after the start of the therapy (p < 0.05). When comparing cytokine secretion after stimulation with IL-2, we found that the Th cells isolated from AML patients tended to secrete higher concentrations of IL-4 and IL-17, but less IFN-γ (not statistically significant). This trend did not change in the course of maintenance therapy. Similarly, the activation capacity of NK cells was generally reduced in AML patients relative to healthy donors (p < 0.05), but was not further diminished in the course of the therapy.

In our ongoing study of AML patients, we found that T and NK cell subsets after intense polychemotherapy significantly differed from those of healthy donors. Prior to the start of maintenance chemotherapy, we prominently saw a diminished number and function of NK cells. This is in accordance with previous reports on NK cell dysfunction in AML patients. In the course of maintenance therapy, we observed a significant relative increase in T_reg cells and a decrease in the functional response of Th cells. These results urge to intensively monitor cell populations and functionality during chemotherapy and immunotherapy. Data generated from close immunomonitoring is the basis for creating synergistic combinations of chemotherapy and immunotherapy.