Dasatinib enhances the expansion of CD56⁺CD3⁻ NK cells from cord blood

To the editor:

Dasatinib can inhibit T-cell activation through inhibition of the Scr family of tyrosine kinases such as p56 (Lck).¹  It has been reported that some chronic myeloid leukemia (CML) patients who were treated with dasatinib developed chronic large-granular lymphocytosis (LGL) with natural killer (NK) or NK T-cell lineage, and that these patients achieved optimal molecular response.²  In addition, Mustjoki et al reported clonal expansion of NK T cells during dasatinib therapy.³  Kreutzman et al reported that mono/oligoclonal T and NK cells were present in CML patients at diagnosis and expanded during dasatinib therapy and that LGL expansion is linked to cytomegalovirus infection.^4,5  Therefore, dasatinib may have a favorable effect on NK-cell proliferation. In this study, we analyzed the effects of dasatinib on the expansion of NK cells from cord blood and transcriptional factors during expansion.

Umbilical cord blood cells (1 × 10⁶/mL; Hokkaido Cord Blood Bank) were cultured with IL-15 (10 ng/mL; PeproTech), IL-2 (5 ng/mL; R&D Systems), and anti-CD3 mAb (OKT3, 10 ng/mL; Janssen Pharmaceutical); with or without dasatinib (10nM; a kind gift from Bristol-Myers Squibb) in culture medium stem cell growth medium (CeeGenix) with 5% human AB serum in 24-well plates, as we reported previously.⁶  After a 7-day culture of umbilical cord blood cells (1 × 10⁶/mL), the absolute number of CD56⁺CD3⁻ NK cells had significantly increased in the culture with dasatinib compared with the culture with cytokines only (before culture 5.3 ± 1.4 × 10⁴ in 10⁶ cord blood cells, after culture with IL-2 + IL-15 26.0 ± 17.8 × 10⁴, and after culture with IL-2 + IL-15 and dasatinib 66.6 ± 29.1 × 10⁴; P < .05, means ± SDs, n = 6; Figure 1A). In addition, the proportion of CD56⁺CD3⁻ cells, CD56⁺NKG2D⁺ cells, and CD56⁺granzyme⁺ cells significantly increased after culture with dasatinib (Figure 1B).

We analyzed the transcriptional factors Eomesodermin (Eomes) and T-bet using an Applied Biosystems 7300 Real-Time PCR System and GAPDH as an endogenous control. Before stimulation, cord blood of CD56⁺ cells showed increased expression of Eomes and T-bet compared with the expression in unfractionated whole cord blood cells and CD3⁺ cells. After 24 hours, Eomes expression was significantly increased in cord blood cells cultured with dasatinib compared with cells cultured with cytokines only (5.96 ± 3.95 vs 0.81 ± 0.62, P < .05; Figure 1C).

At present, there are only a few transcription factors that are known to play an essential role in NK-cell development, especially in humans. T-box proteins, T-bet, and Eomes are involved in NK-cell development.^7-9  T-bet and Eomes are both later required for the differentiation in DX5⁺(CD49b) CD11b⁺ NK cells. In addition, Eomes is highly expressed in fully differentiated NK cells. In this study, we showed NK-cell expansion after culture with dasatinb and increased expression of Eomes after 24 hours. Therefore, dasatinib has some role in NK-cell expansion from cord blood under the condition of IL-2 and IL-15 stimulation through increased expression of transcription factors such as Eomes. This observation may have potentially important implication for the treatment of other diseases with dasatinib.¹⁰