Kinetics of Cytotoxic Lymphocytes Reconstitution after Induction Chemotherapy in Elderly AML Patients Reveals Progressive Recovery of Normal Phenotypic and Functional Features in NK Cells

Abstract

NK cells are defective in acute myeloid leukemia (AML) at diagnosis. Here, we studied the kinetic of expression of the major activating and inhibitory receptors of NK, CD8 T, and γδ T cells in patients undergoing chemotherapy (CT) for the treatment of AML (n = 29). We showed that NK cells are the main affected population at diagnosis and that expression of activating receptors is partially restored within a few weeks after CT. CD8 T cells and γδ T cells are only weakly affected at diagnosis. Killer cell immunoglobulin-like receptor expression by NK cells, but not NKG2A and CD85j, was downregulated. Interestingly, the development of NK cells appeared altered as the most immature CD56^bright NK cells were seriously underrepresented. Finally, we showed that NK cell functions were only partially restored 6 weeks after CT as degranulation capabilities of NK cells recovered, whereas cytokine production remained low. Our data point out NK cells as antitumor effectors peculiarly hampered by leukemic cells. This study may indicate a timeline when NK-mediated therapies or other immunotherapies could be performed, particularly for patients excluded of hematopoietic stem cell transplantation.

Keywords: NK cells; NK functions; activating receptors; acute myeloid leukemia; chemotherapy.

Figure 1

Kinetics of cytotoxic lymphocyte reconstitution. Kinetics of NK cells (round dots), CD8⁺ T cells (squares) and γδ T cells (triangles) reconstitution at the indicated time points. Cell counts (mean ± SEM) were measured at diagnosis of the disease, before the second consolidation (week 0, W0), at week 2, 4, 6, and 8 (W2, W4, W6, W8). (A) Absolute lymphocyte count. Dotted line corresponds to the mean of lymphocyte counts from 15 healthy donors (HD). (B) NK, γδ T, and CD8⁺ αβ T cell absolute counts. Horizontal lines correspond to the mean of NK, γδ T, and CD8⁺ αβ T cell counts from HD. When indicated a Kruskal–Wallis non-parametric test with Dunn’s posttest was performed to compute the P value for the comparisons (*P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 2

Kinetics of activating and inhibitory receptor expression. (A). Global view of expression of activating receptors was measured by flow cytometry on NK, γδ T, and CD8⁺ αβ T cells at the indicated time points. MFI (unimodal expression) and percentage of positive cells (bimodal expression) were normalized and hierarchically clusterized with Tmev software. Comparison with healthy donors (HDs) was performed, and data are represented with colors reflecting higher (red) or lower (green) expression compared to HDs. Gray squares correspond to non-determined values. (B). Expression of CD158a/h, CD158b1/b2/j, NKG2A, and CD85j by NK cells from HD and patients at the indicated time points. Bars represent the median expression and when indicated a Kruskal–Wallis non-parametric test with Dunn’s posttest was performed to compute the P value for the comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Kinetics of immature NK cells reconstitution. (A) Example of reconstitution of CD56^bright NK cells in a patient following treatment. The figure displays the reapparition of CD56^bright NK cells expressing or not CD16 (top) or NKG2A (bottom). (B) Frequencies of total CD56^bright NK cells (left panel) or CD56^bright NK cells lacking CD16 expression (right panel). (C) Summary figure of reconstitution of the immature CD56^bright NK cell subset after induction and consolidation therapies. Data represent Tukey whisker boxes, with outliers represented as round dots. When indicated a Kruskal–Wallis non-parametric test with Dunn’s posttest was performed to compute the P value for the comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Kinetics of immature NK cells reconstitution. (A) Example of reconstitution of CD56^bright NK cells in a patient following treatment. The figure displays the reapparition of CD56^bright NK cells expressing or not CD16 (top) or NKG2A (bottom). (B) Frequencies of total CD56^bright NK cells (left panel) or CD56^bright NK cells lacking CD16 expression (right panel). (C) Summary figure of reconstitution of the immature CD56^bright NK cell subset after induction and consolidation therapies. Data represent Tukey whisker boxes, with outliers represented as round dots. When indicated a Kruskal–Wallis non-parametric test with Dunn’s posttest was performed to compute the P value for the comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

Effector functions of NK cells following chemotherapy. (A) Specific lysis of target cells (black bars, left axis) and intracellular production of IFN-γ (gray bars, right axis) after interaction between NK from healthy donors (HDs) or patients at the indicated time points and K562 cells. Histogram represents mean and bars represent SEM. (B) Effect of cytokine priming on degranulation and production of IFN-γ and TNF-α upon interaction with K562 cells. NK cells from HDs (n = 3) and patients (n = 5) at the indicated time points were treated overnight with medium only (white bars), IL-2 + IL-15 (black bars) or IL-12 + IL-18 (gray bars). Cells were then washed and incubated for 4 h with K562 cells. Effector responses were analyzed as indicated in the Section “Materials and Methods.” The figure depicts CD56^dim (left) and CD56^bright (right) identified with the flow cytometry analysis software.

Figure 5

Proliferation capacities of NK cells before and during consolidation. Proliferation capacities of NK cells during chemotherapy (CT). NK cells from healthy donors or patients before (W0) and after the first cycle of consolidation CT were isolated from peripheral blood, stained with CellTrace Violet, and cultured for 6 days in IL-2-containing medium. The figure displays CellTrace Violet dilution reflecting cell proliferation for CD56^dim NK cells (top) and CD56^bright NK cells (bottom). The histograms indicate the frequency of non-proliferating cells (generation 0) and proliferating cells (generations 1–6). Data represent mean ± SEM. Insets show a representative donor or patient (n = 3 and n = 5, respectively).