In Chronic Myeloid Leukemia (CML), patients who achieve sustained deep molecular response (DMR) to Tyrosine Kinase Inhibitors (TKIs) are eligible for treatment discontinuation, but only 50% of these patients maintain long-term treatment-free remission (TFR). Biomarkers that predict TFR loss (TFRL) of the molecular response after TKI interruption have not been defined to date.

We hypothesized that antitumoral immunity mediated by Natural Killer (NK) and T cells may contribute to TFR success.

NK and T cells phenotype were correlated with BCR::ABL1 kinetics and TFR success in a cohort of CML Brazilian patients included in the DES-CML study, a multicenter, prospective, open-label, single-arm, non-randomized, phase 2 clinical trial of TKI discontinuation, conducted in Brazilian public healthcare system (SUS). Using flow cytometry, we evaluated frequency, subtypes, maturation, and receptors expression of NK and T cells from diagnosis to TKI discontinuation. Peripheral blood samples from 20 healthy controls and 88 CML patients were analyzed at different time points: 14 diagnosis(DX), 13 Imatinib failure(F), 10 major molecular response(MMR), 20 DMR, 20 TFR and 5 TFR loss(TFRL). Patients in discontinuation had TKI dose reduced to 50% for 6 months before total suspension.

Patients who successfully maintained TFR exhibited more functional and mature immune phenotype compared to those who relapsed. TFR patients showed higher frequencies of cytotoxic NK cells (CD56dim), mature NK (CD57⁺), and activating receptors (NKG2D, DNAM-1, NKp46). They also exhibited increased CD8⁺ effector memory T cells, lower CD8⁺ naïve T cells, and reduced PD-1 expression on NK and CD8⁺ T cells, indicating an activated and immunocompetent profile compared to TFRL patients. In contrast, even during full-dose DMR (DMR100), TFRL patients had reduced total NK cells, CD56dim NKs, CD56⁺CD57⁺ NK, and CD11b⁺CD27⁻ subsets. They also exhibited lower expression of activating receptors (NKG2D, DNAM-1, NKp46). CD8⁺ T cells from TFRL patients showed decreased central memory, effector memory, and terminal effector subsets, indicating early impairment of the cytotoxic compartment. During the 50% dose reduction phase (DMR50), TFRL patients presented elevated frequencies of immature NK subsets (CD11b⁻CD27⁺) and increased expression of inhibitory receptors: TIGIT and PD-1 on NK cells, and PD-1, CTLA-4, TIGIT on CD8⁺ T cells. This phase also showed increased naïve CD8⁺ T cells, reinforcing immune immaturity. Frequencies of mature and cytotoxic NK cells (CD56dim, CD57⁺, NKp80), as well as NKG2D and DNAM-1, were lower in DX, F, and TFRL groups compared to DMR100, TFR, and healthy controls. PD-1 and CTLA-4 expression on CD8⁺ T cells was more pronounced in these groups. No significant differences were found in KIR2DL1 and NKG2A on NK cells or CD3, CD4, CD8 expression on T cells.

These findings suggest that maturation and activation of cytotoxic compartments are crucial for disease control and TFR success. In contrast, immature and exhausted immune profiles may predispose to relapse. Immune dysregulation precedes molecular relapse, reinforcing the role of longitudinal immune monitoring. Progressive loss of mature NK cells and increased checkpoint expression may define an immunological risk profile for TFR failure. Immune assessment during dose reduction could guide clinical decisions on TKI cessation and support individualized strategies in CML based on immunological profiles.