NK cells with decreased expression of multiple activating receptors is a dominant phenotype in pediatric patients with acute lymphoblastic leukemia

Abstract

NK cells have unique attributes to react towards cells undergoing malignant transformation or viral infection. This reactivity is regulated by activating or inhibitory germline encoded receptors. An impaired NK cell function may result from an aberrant expression of such receptors, a condition often seen in patients with hematological cancers. Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer worldwide and NK cells have emerged as crucial targets for developing immunotherapies. However, there are important gaps concerning the phenotype and behavior of NK cells during emergence of ALL. In this study we analyze the phenotype and function of NK cells from peripheral blood in pediatric patients with ALL at diagnosis. Our results showed that NK cells exhibited an altered phenotype highlighted by a significant reduction in the overall expression and percent representation of activating receptors compared to age-matched controls. No significant differences were found for the expression of inhibitory receptors. Moreover, NK cells with a concurrent reduced expression in various activating receptors, was the dominant phenotype among patients. An alteration in the relative frequencies of NK cells expressing NKG2A and CD57 within the mature NK cell pool was also observed. In addition, NK cells from patients displayed a significant reduction in the ability to sustain antibody-dependent cellular cytotoxicity (ADCC). Finally, an aberrant expression of activating receptors is associated with the phenomenon of leukemia during childhood.

Keywords: NK cells; acute lymphoblastic leukemia; cancer; immune system; immunooncology.

Figure 1

Percentages of NK cells expressing distinct activating receptors from ALL patients and healthy controls. (A) The frequencies of CD3^-CD20^-CD14^-CD56⁺ cells expressing distinct activating receptors were analyzed by flow cytometry in 72 ALL patients and 21 healthy controls. Vertical lines indicate standard deviation. The horizonal lines represents the mean value. P values reports significance according to U-Mann Whitney Test one-tail. HC: healthy controls; ALL: Acute lymphoblastic leukemia patients. (B) Representative FACS plots depict the percentage of CD3^-CD20^-CD14^-CD56⁺ cells expressing distinct activating receptors from healthy controls (n = 21) and ALL patients (n = 72). HC: healthy controls; ALL: Acute lymphoblastic leukemia patients.

Figure 2

Relative expression of activating and inhibitory receptors on NK cells. Relative expression, represented as the mean fluorescence intensity (MFI), of activating and inhibitory receptors in NK cells from total healthy controls (n = 21) (A) and ALL patients (n = 62) (B). Histograms cover all the values obtained for each control and patient as indicated. (C) Summary of data for frequencies of NK cell expressing distinct activating and inhibitory receptors from21 healthy controls and 62 ALL patients. The vertical lines indicate standard deviation. The horizonal lines represent the mean value. Results were considered significant at * p< 0.05, ** p < 0.01 and *** p < 0.001.

Figure 3

Percentages of NK cells expressing distinct inhibitory receptors on NK cells form healthy controls and ALL patients. (A) The frequencies of CD3^-CD20^-C14^-CD56⁺ cells expressing distinct inhibitory receptors were analyzed by flow cytometry in 72 ALL patients and 21 healthy controls. Vertical lines indicate standard deviation. The horizonal lines represents the mean value. (B) Representative FACS plots depict the percentage of CD3^-CD20^-CD14^-CD56⁺ expressing distinct inhibitory receptors on NK cells from healthy controls (n= 21) and ALL patients (n= 72). HC: healthy controls; ALL: Acute lymphoblastic leukemia patients. NS, Not Significant.

Figure 4

Analysis of concurrent expression of receptors in NK cells among ALL patients. Venn diagrams showing the number of functions, as defined by Boolean gating, simultaneously exhibited by NK cells in patients below the 50th percentile (top panels) or above the 50th percentile (bottom panel) for SLAM, 2B4 and, NTB-A (A), DNAM-1, TIGIT and, CD96 (B), DNAM-1, 2B4 and, NKG2D (C). The seven possible combinations are represented with a different color and the number of patients having one of these possible combinations are indicated as percentages.

Figure 5

Analysis of in the mature NK-cell pool from ALL patients and healthy controls. (A) Representative FACS plots of CD3^-CD20^-C14^-CD56⁺ cells at different stages of maturation according to the expression of NKG2A and CD57 from healthy controls and ALL patients. (B) Summary of data for frequencies of CD3^-CD20^-C14^-CD56⁺ cells according to the expression NKG2A and CD57. Vertical lines indicate standard deviation. The horizonal lines represents the mean value. P values reports significance according to U-Mann Whitney Test one-tail. HC, healthy controls; ALL, Acute lymphoblastic leukemia patients. (C) Summary of data for relative expression, represented as the mean fluorescence intensity (MFI), of CD3^-CD20^-C14^-CD56⁺ cells at different stages of maturation according to expression of NKG2A and CD57. The vertical lines indicate standard deviation. HC, healthy controls; ALL, Acute lymphoblastic leukemia patients. The horizonal lines represents the mean value. Results were considered significant at *p < 0.05, **p < 0.01 and ***p < 0.001. NS, Not Significant.

Figure 6

Degranulation assays in NK cells from ALL patients and healthy controls. Representative FACS plots depict NK cell response (CD107a degranulation) against P815 supplemented or not with anti-CD16 (clone 4G6). (A) PBMCs (1x106/ml) were incubated with P815 cells (2x106/ml) for 3 hours in a total volume of 200 ul in a 96 well plate at 37°C. Degranulation was represented as the fold increase of CD107a on NK cell surface, which is the difference between the percentage of NK cells expressing CD107a at surface after stimulation with P815 cells supplemented with agonist mAb, and the percentage of NK cells expressing CD107a at NK cell surface after incubation with P815 cells with no agonist mAb. NK cell degranulation assays were performed in 32 ALL patients and 10 age-matched controls. (B) Summary of data and statistic from 32 ALL patients and 10 age-matched controls. (C) Venn diagrams showing the number of functions as defined by Boolean gating. The diagrams show the concurrent expression of various NK cell receptors in those patients that displayed degranulation values under the 50th percentile.