Patients with GATA2 deficiency specifically lack CD56bright/CD16- natural killer (NK) cells, while the CD56dim/CD16+ (DP) subset is preserved. CD56bright/CD16- cells have been postulated to be the precursor population for more mature and cytotoxic CD16+NK cells. The mechanism by which an immature population disappears while a putatively more mature population is preserved is not understood. Our group has recently shown that CD16+/CD56dim/- NK cells have a distinct ontogeny, showing little overlap with CD56+/CD16- NK cells, which share clonal derivation with myeloid, T and B lineages (Wu et al, Cell Stem Cell 2014). We hypothesize that the missing CD56bright NK population in GATA2 deficiency is due to a maturation defect in a specific hematopoietic stem/progenitor population, separate from those relatively preserved progenitors producing CD16+ NK cells.

We performed ex vivo expansion of NK cells from 4 healthy volunteers and 5 patients with GATA2 mutations and compared phenotypes at baseline and upon expansion. Sorted peripheral blood total NK (TNK) cells (CD3, CD20, CD14 depleted and expressing CD16 and/or CD56) and double negative (DN) cells (CD3-/CD20-/CD14- and CD16-/CD56-) were cultured in the presence of IL2 and irradiated Epstein-Barr virus-transformed lymphoblastoid cells. After 14 days of culture, each cell culture was characterized and re-sorted into TNK cells and DN cells, and cultured for an additional 14 days with restimulation. Subject 1 had GATA2 c.229+1 G>A mutation and presented with pancytopenia, recurrent warts, classic B/NK lymphopenia/monocytopenia and MDS with trisomy 8. Subjects 2 and 5, and subjects 3 and 4 were from the same families, respectively, and all shared the same intron 5 GATA2 c.1017+572C>T mutation. The proband, subject 2 had low grade MDS, recurrent warts and classic B/NK lymphopenia and monocytopenia. However, subjects 3, 4 and 5 had normal peripheral blood counts and bone marrow morphology, with only mild B or NK lymphopenia.

TNK cells from subject 1 had profound lack of CD56bright cells at baseline, and showed decreased ex vivo expansion efficiency; 18.5-fold increase at day 14 compared to a 39.4-fold increase for control TNK cells. Even after expansion, the CD56bright population was still absent. When DN cells from subject 1 were placed into culture, they did not give rise to a CD56brightpopulation after 14 or 28 days, while control DN cells did give rise to a clear CD56bright population, also expressing additional NK-defining markers such as NKG2D and NKp46. TNK cells from patients with GATA2 intron 5 mutations had a less striking phenotype and were more variable. Subject 2 had greatly decreased CD56bright NK cells at baseline, in contrast to her asymptomatic father, subject 5, and to subjects 3 and 4 from another family. Ex vivo expansion kinetics for the TNK cells from all 4 subjects were similar to controls. By day 14 in culture, their TNK cells showed preserved generation of CD56bright/+ cells and DP cells. DN cells from subject 2 showed no CD56 expression upon expansion at day 14 and only a minor CD56dim population at day 28. Subjects 3, 4 and 5 showed comparable phenotypes at baseline and upon expansion until day 14 (Figure 1). GATA2 DN cells sorted and expanded twice from DN cells at days 1 and 14, at day 28 had much lower CD56bright NK cells (1.1±0.6% vs. 10.1±3.9%; p<0.05) and higher expression of NKG2D, an activating receptor on NK cells, compared to controls (68.23±9.91% vs. 12.3±4.63%; p=0.0036), suggesting a differentiation block in precursor NK cells.

CD107a functional analysis showed impaired NK degranulation capacity upon K562 co-incubation in expanded GATA2 TNK cells and DN cells compared to their controls at day 14 (12.4±0.6 vs. 3.5±0.1 vs. 6.7±0.1 (p<0.001); 8.43±0.38 vs. 4.53±0.09 vs. 1.23±0.03 (p<0.001) fold increase from baseline in control 3, subjects 3 and 4 respectively).

Our findings suggest that NK differentiation defects in GATA2 deficiency may be due to a differentiation block from a precursor present in the DN subset which is impaired in giving rise to CD56bright/CD16- cells upon prolonged culture, and also potentially in vivo. The differences between the GATA2c.229+1 G>A and GATA2 c.1017+572C>T (intron 5) samples in both phenotype and expansion characteristics correlate with the clinical severity of patients, and suggest a mutation-specific, dose-dependent role of GATA2 in NK development, as well as other modifying factors.


Townsley:GSK: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.