The inhibition of oncogenic BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has significantly improved the prognosis of CML, but the majority of patients need life-long therapy. Before the TKI era, CML patients were treated with interferon-α (IFN-α), and a minor proportion of patients (10–20%) achieved prolonged complete cytogenetic remissions (CCyR). Interestingly, many of the patients in prolonged CCyR were able to discontinue the treatment without imminent disease relapse. As the ultimate goal of current CML therapy is cure, the deeper understanding of the cellular mechanisms behind successful IFN-α discontinuation are of outmost importance. In this study, we analyzed the function of immune effector cells (NK- and T-cells) derived from CML patients in prolonged molecular remission after IFN-α monotherapy in order to understand the putative anti-leukemic effects.
The study cohort included 13 CML patients treated with IFN-α who were in complete molecular remission (CMR) at the time of blood sampling. Five patients were still on IFN-α therapy (IFN-ON, median duration of the treatment 13.7 years) and 8 had stopped treatment successfully (IFN-OFF) and were off from any treatment (median time without treatment 6.3 years). None of the patients had used any TKIs. Samples from 10 healthy volunteers were used as controls. The cytotoxicity of NK-cells in blood was studied by measuring the direct killing of K562 cells and by degranulation assay (CD107). The cytokine secretion (TNF-α and IFN-g) of NK-cells was measured with flow cytometry after stimulation with K562. The function of T-cells was studied by antibody (OKT3) stimulation and measuring TNF-α and IFN-g production by flow cytometry. In addition, NK-cells were phenotyped by multi-color flow cytometry.
IFN-OFF patients had a larger proportion of NK-cells from lymphocytes than IFN-ON patients or healthy controls (IFN-OFF median 23.5 %, IFN-ON 7.0 %, healthy 13.2 %; p=0.0136). Based on the subset analysis, expanded NK-cells in IFN-OFF patients had mature phenotype CD56dimCD62LlowCD27lowCD57high. On the contrary, IFN-ON patients had a larger proportion of immunoregulatory CD56bright NK-cells (20.2% vs. 6.5% in healthy and 3.0% in IFN-OFF, p=0.0035).
When the direct killing of K562 cells was studied, NK-cells from healthy controls killed better than NK-cells from either of the patients groups (in healthy 55 % of K562 cells were alive compared to 79% in IFN-ON and 91% in IFN-OFF, p=0.0042). Similar trend was also observed in degranulation assay (median degranulation in healthy controls 8.3 % compared to 4.3 % in IFN-OFF and 3.6 % in IFN-ON group, p= 0.39). The direct killing capability tended to correlate negatively r=-0.60, p=0.07) with the NK-cell proportion (ie. patients with high NK-cell counts had worse killing).
Instead of cytotoxicity, NK-cells from IFN-OFF patients seemed to secrete more efficiently cytokines (IFN-γ/TNF-α) than NK-cells from healthy controls, but due to low number of patients no firm conclusions can be made (IFN-OFF median 6.9 %, healthy 0.7 %; p= 0.25).
The proportion of potentially cytotoxic CD4+GrB+ T-cells did not significantly differ between the groups, but wide variation was observed between the individual patients (IFN-ON median 9.0 %, IFN-OFF 2.6 %, and healthy controls 3.5 %; p=0.4142). However, the secretion of TNF-α and IFN-γ by CD4+ cells seemed to be increased in IFN-OFF patients when compared to healthy controls (IFN-OFF mean 5.9 %, IFN-ON 3.7 %, healthy 2.5 %; p=0.1055).
CML patients who have been able to discontinue IFN-α therapy (IFN-OFF group) had an expansion of mature CD56dimCD62LlowCD27lowCD57high NK-cells in peripheral blood. Although CD56dim NK-cells have typically been suggested to act as cytotoxic cells, the killing in response to K562 cells was lower in IFN-OFF patients than in controls. It is possible that the expanded NK-cells in IFN-OFF patients represent exhausted terminal stage cells (CD57+) or memory cells that do not react properly against third party target cells (K562). Moreover, they may have an immunoregulatory function instead of cytotoxicity. Further follow-up studies with other patient cohorts (TKI monotherapy and TKI+IFN-α combination therapy treated patients) are ongoing to clarify the issue further.
Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.
Asterisk with author names denotes non-ASH members.