Both CD4⁺CD25⁺Foxp3⁺ Regulatory T Cells and CD3^-CD56⁺ NK Cells Affect the Response of Newly Diagnosed Chronic Myeloid Leukemia Patients to Treatment with a Tyrosine Kinase Inhibitor

Background: Regulatory T cells (Treg) play a role in the maintenance of chronic myeloid leukemia (CML). In contrast, natural killer (NK) cells have spontaneous cytolytic activity against various tumor cells, including leukemic cells, and play an important role in immune surveillance against tumor cells. In this study, we examined changes in the number of circulating Treg and NK cells and the NK activity of patients with CML before and after treatment with a tyrosine kinase inhibitor (TKI). Our goal was to evaluate the clinical significance of this treatment on these two different cell types.

Patients and Methods: Treg and NK cells were characterized and quantified by flow cytometry in 13 newly diagnosed CML patients, 5 newly diagnosed patients with other myeloproliferative neoplasms (MPN), and 15 healthy controls. We analyzed all CML patients diagnosed with chronic phase (CP) disease and MPN patients from January 2015 and June 2016. Patients with CML, who were initially treated with TKI between January 2015 and June 2016 and were followed up for more than one year, were enrolled in the study. The mRNA level of the BCR-ABL fusion gene was analyzed by real-time PCR analysis at approximately 6-month intervals during the first year and at 12-month intervals thereafter. After obtaining informed consent, blood was drawn from all patients at initial diagnosis and 12 months after the start of treatment. CD4⁺CD25⁺Foxp3⁺ Treg cells, CD3⁺CD4⁺T cells, CD3⁺CD8⁺ T cells, and CD3^-CD56⁺ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard ⁵¹Cr release assay at effector: target (E: T) ratios of 20:1.

Results: The median age for CML patients was 65 years (range: 47-84), and 9 of the patients were male. The median period of observation was 543 days. After 12 months, 8 CML patients (MMR-group) had a major molecular response (MMR), and 5 patients (NR-group) did have not a MMR. Before treatment, the absolute number of Treg cells was significantly lower in the MMR-group than that in the NR-group (27.6 ± 10 vs. 50.0 ± 24.0 /μL, p=0.037). Similarly, the absolute number of Treg cells after treatment was significantly lower in both the MMR-group (27.3±10.3 /μL) and the MPN-group (27.6±11.5 /μL) than that in the NR-group (40.0±9.9 /μL, p=0.05, p<0.01, respectively). Treg% values in peripheral lymphocytes before treatment in the NR (7.4±2.1%) were significantly higher than that in MMR group and in the healthy group (4.7±1.7%, 5.5±1.1%, p=0.024, p=0.016, respectively). The absolute number and percentages of CD3^-CD56⁺ NK cells before treatment as well as after treatment did not differ between each group. However, NK activity before treatment in the NR-group (15±1.4%) was significantly lower than that in MMR-group (47.2± 11.6 vs 41.7±4.1%, p<0.01, p<0.01, respectively). With time, the number of CD8⁺ T cells in the NR group decreased significantly from 940±570 to 348± 167/μL (p=0.040), and the Treg% increased significantly from 0.18± 0.13 to 0.84± 0.29% (p<0.01). The relative percentage of CD3⁺CD4⁺ T cells (4.7±4.4%) in the 15 CML patients increased remarkably after treatment (12.8±4.3%, p<0.01) but the absolute number did not change.

Conclusions: Treg cell numbers were significantly lower in the CML patients with a MMR compared to that in the patients without a MMR. Although the number of NK cells did not change in the MMR group compared to that in the NR group, the NK activity was significantly higher. While the mechanisms for these changes in cell populations are not completely understood, it is also important to note that we have not yet studied the cellular source and production of cytokines which enhance NK activity. The data suggest that some immunological mechanisms are involved in the response to TKI. Moreover, the increased levels of NK activity in patients with MMR suggest that not only Treg cell numbers but also NK activity may have clinical importance in evaluating the effects of TKI therapy.