?CD56dimNKG2C+NKp46+ NK cells show higher degranulation capacity in non-relapsing CML patients from AST discontinuation trial

SÁNCHEZ, MARÍA BELÉN; VASCONCELOS CORDOBA, BIANCA; PAVLOVSKY, CAROLINA; LEVY, ESTRELLA MARIEL; BIANCHINI, MICHELE

Conferencia; 2023-25th Annual John Goldman Conference on Chronic Myeloid Leukemia: Biology and Therapy.; 2023

Treatment-free remission (TFR) is considered a therapeutic goal in chronic myeloid leukemia (CML) patients in deep molecular response (DMR). Although there is a lack of specific prognostic factors that could determine which patients could discontinue therapy, there is increasing evidence suggesting that NK cells are important in the control of leukemic growth. Accordingly, as previously reported, we set up an immunological sub-study from the 46 patients enrolled in the AST trial (molecular relapse-free survival of 65% at 36 months) and we determined the presence of a specific NK subpopulation (CD56dimCD57+NKG2C+ NKp30+NKp46+) that was present in higher proportion in non-relapsing patients and was also associated with a longer molecular recurrence-free survival time. Therefore, the aim of this work was to analyze the functionality of this subset.Methods: 0,3x106 peripheral blood mononuclear cells from 17 patients from the AST protocol (6 relapsed vs. 11 non-relapsed) were thawed and cultured with K562 cell line (CCL-243, ATCC), at 1:5 NK:K562 cell ratio. The number of target cells was calculated according to the percentage of NK cells, and incubated for 6 h at 37°C with the addition of PE anti-CD107a from the beginning of the assay and Protein Transport Inhibitor after the first hour. Then, cells were stained with BV421 anti-CD56, APC-H7 anti-CD3, AF647 anti-NKG2C, PE-Cy7 anti-NKp46, and PE anti-IFN-γ to identify the CD56dimNKG2C+NKp46+ subset, assuming that it is similar to the one described previously. Phenotypic and functional analyses were performed by flow cytometry (BD FACS Canto?II) and data were analyzed using FlowJo software. For statistical analysis, GraphPad Prism was used. Non-parametric Mann?Whitney test was used for comparing differences between molecular relapsed vs. non-relapsed patients. Molecular recurrence-free survival was estimated by the Kaplan?Meier method and compared within groups by the log-rank test. Differences were considered statistically significant when p