Impaired cytotoxicity associated with defective natural killer cell differentiation in myelodysplastic syndromes

Abstract

Natural killer cells are well known to mediate anti-leukemic responses in myeloid leukemia but their role in myelodysplastic syndromes is not well understood. Here, in a cohort of newly diagnosed patients (n=75), widespread structural and functional natural killer cell defects were identified. One subgroup of patients (13%) had a selective deficiency of peripheral natural killer cells (count <10/mm(3) blood) with normal frequencies of T and natural killer-like T cells. Natural killer cell-deficient patients were predominantly found in high-risk subgroups and deficiency of these cells was significantly associated with poor prognosis. In the second subgroup, comprising the majority of patients (76%), natural killer cells were present but exhibited poor cytotoxicity. The defect was strongly associated with reduced levels of perforin and granzyme B. Notably, natural killer cell function and arming of cytotoxic granules could be fully reconstituted by in vitro stimulation. Further phenotypic analysis of these patients revealed an immature natural killer cell compartment that was biased towards CD56(bright) cells. The residual CD56(dim) cells exhibited a significant increase of the unlicensed NKG2A(-)KIR(-) subset and a striking reduction in complexity of the repertoire of killer cell immunoglobulin-like receptors. Taken together, these results suggest that the widespread defects in natural killer cell function occurring in patients with myelodysplastic syndromes are mostly due to either unsuccessful or inefficient generation of mature, functionally competent natural killer cells, which might contribute to disease progression through impaired immune surveillance.

Figure 1.

Selective NK cell deficiency in a subset of MDS patients. Frequencies (left panels) and cell counts (right panels) of (A) NK cells, (B) T cells and (C) NK-like T cells were analyzed in MDS patients (n=75) and healthy age-matched donors (n=30). NK cells were defined as CD56⁺CD3⁻ cells within the lymphocyte gate. NK cell numbers/mm³ were calculated as: (absolute number of lymphocytes) × (frequency of NK cells). The dashed horizontal line in right panel (A) demarcates the NK cell-deficient cases. Each dot represents one individual, and horizontal bars represent mean values. (D) Representative flow cytometric dot plots showing expression of CD56 and CD3 cells in PBMC from a NK cell-deficient patient (left), and a patient with normal NK cell frequency (right). (E) Cell counts of NK cells versus T cells in MDS patients (n=75). Statistical significance was determined by a two-tailed t-test (*P<0.05, ***P<0.001).

Figure 2.

NK cell deficiency correlates with poor prognosis. (A) Absolute NK cell counts and (C) NK cell frequency in patients within WHO subclasses refractory anemia/refractory anemia with ring sideroblasts (RA/RARS) (n=8), refractory cytopenias with multilineage dysplasia (RCMD) (n=43), and refractory anemia with excess blasts (RAEB) I/II (n=13). Statistical significance for subclasses was calculated by one-way ANOVA (*P<0.05). (B) NK cell counts and (D) frequency according to the IPSS score, separated into low/intermediate 1 (Int1) (n=59) and intermediate2 (Int2)/high (n=16) risk groups. Statistical significance was determined by a two-tailed t-test (*P<0.05). Each dot represents one individual, and horizontal bars represent mean values. The dashed horizontal lines in (A) and (B) demarcate the NK cell-deficient cases.

Figure 3.

Impaired NK cell cytotoxicity in MDS patients is associated with low levels of granzyme B and perforin. (A) K562-induced degranulation of NK cells following interleukin-2 stimulation was measured in 30 patients and 20 healthy age-matched donors with flow cytometry (CD3-CD56⁺CD107⁺). (B) Specific lysis of K562 target cells at an E/T ratio of 10:1 using interleukin-2 stimulated PBMC from patients (n=43) and healthy donors (n=20). Patients with <20% lysis were considered to have a functional deficiency. (C) Degranulation of NK cells versus specific lysis of K562 target cells in MDS patients (n=30). (D) CD107 expression levels and K562 cell lysis are shown for a representative MDS patient and a healthy age-matched donor. Spontaneous degranulation of NK cells (upper panel) and spontaneous lysis of K562 cells (lower panel) are depicted by filled histograms. (E) Box plots showing intracellular staining of granzyme B and perforin in CD56^dim NK cells from 37 MDS patients (light gray) and 20 healthy donors (dark gray). (F) Correlation between granzyme B and perforin expression in CD56^dim NK cells from 37 MDS patients. Filled dots represent patients with low NK cell function (specific lysis of K562 cells <20%) and open dots patients with normal NK cell function (specific lysis of K562 cells ≥20%). Error bars represent standard deviation. Statistical significance was determined by a two-tailed t-test (**P<0.01, ***P<0.001).

Figure 4.

Reversibility of deficient NK cell function. (A) Interleukin-2-induced expansion of NK cells from MDS patients (n=5, dashed lines) and healthy age-matched individuals (n=3, solid lines) for 10 days. (B) Cumulative frequency of granzyme B/perforin on days 0 and 10 of NK cell expansion (C) Cytotoxicity of NK cells measured on day 10 in comparison with initial cytotoxicity.

Figure 5.

Selective reduction in CD56^dim NK cell numbers leading to increased CD56^bright frequency in MDS patients. Frequency of (A) CD56^bright and (B) CD56^dim NK cells, as well as (C) absolute number of CD56^bright and (D) CD56^dim NK cells were determined in MDS patients with functionally deficient NK cells (n=35, defined as <20% specific lysis of K562 cells), functional NK cells (n=8, defined as ≥20% specific lysis of K562) and healthy age-matched donors (n=30). Statistical significance was determined by a two-tailed t-test (*P<0.05, ***P<0.001). (E) Correlative analysis of CD56^dim NK cell count and specific lysis of K562 in 43 MDS patients (linear regression analysis, P=0.0004). Error bars represent standard deviation.

Figure 6.

Immaturity of CD56^dim NK cells in MDS patients. (A) Frequency of four NK cell subpopulations according to expression of KIR and/or NKG2A. (B) Frequency of clonal combinations of KIR2DL1, KIR2DL2/3, KIR3DL1, and NKG2A receptors for patients and healthy donors, ordered according to number of expressed receptors. (C) Pie charts showing frequency of CD56^dim NK cells expressing a given single KIR (KIR2DL1, KIR2DL2/3, or KIR3DL1) in healthy donors (left) and patients (right). NK cells without KIR or other KIR constellations were not considered here. (D) Frequency of NK cells expressing a given number of KIR. Analyses were performed with MDS patients (n=30) and healthy age-matched donors (n=20). Error bars represent standard deviation. Statistical significance was determined by a two-tailed t-test (*P<0.05, **P<0.01, ***P<0.001).