Coordinated acquisition of inhibitory and activating receptors and functional properties by developing human natural killer cells

Abstract

The stages of human natural killer (NK) cell differentiation are not well established. Culturing CD34(+) progenitors with interleukin 7 (IL-7), IL-15, stem cell factor (SCF), FLT-3L, and murine fetal liver cell line (EL08.1D2), we identified 2 nonoverlapping subsets of differentiating CD56(+) cells based on CD117 and CD94 (CD117(high)CD94(-) and CD117(low/-)CD94(+) cells). Both populations expressed CD161 and NKp44, but differed with respect to NKp30, NKp46, NKG2A, NKG2C, NKG2D, CD8, CD16, and KIR. Only the CD117(low/-) CD94(+) population displayed cytotoxicity and interferon-gamma production. Both populations arose from a single CD34(+)CD38(-) Lin(-) cell and their percentages changed over time in a reciprocal fashion, with CD117(high)CD94(-) cells predominating early and decreasing due to an increase of the CD117(low/-)CD94(+) population. These 2 subsets represent distinct stages of NKcell differentiation, since purified CD117(high) CD94(-) cells give rise to CD117(low/-)CD94(+) cells. The stromal cell line (EL08.1D2) facilitated the transition from CD117(high)CD94(-) to CD117(low/-)CD94(+) via an intermediate phenotype (CD117(low)CD94(low/-)). EL08.1D2 also maintained the mature phenotype, preventing the reversion of CD117(low/-)CD94(+) cells to the intermediate (CD117(low)CD94(low/-)) phenotype. An analogous population of CD56(+)CD117(high)CD94(-) cells was found in cord blood. The identified stages of NK-cell differentiation provide evidence for coordinated acquisition of HLA-specific inhibitory receptors (ie, CD94/NKG2A) and function in developing human NK cells.

Figure 1.

Phenotype and NKR expression of CD34⁺-derived NK cells. (A) Forward and side scatter appearance of cells at days 14, 21, 28, and 35 of culture (top panels). Early in culture, large cells with high side and forward scatter are seen. Over time these cells decrease and there is an increase in cells within the lymphocyte gate. Concurrent with the increasing percentages of lymphocytes, both CD56⁺ and CD16⁺ cells can be detected (bottom panels, gated on viable cells using forward and side scatter). By day 35 of culture, the vast majority of cells are CD56⁺ with a significant proportion coexpressing CD16. Results were representative of more than 20 healthy UCB donors. (B) Expression of NK-cell receptors on in vitro–generated CD56⁺ cells at day 28. Shown are data from cells falling within the lymphoid gate. Results representative of more than 20 donors. (C) The fraction of CD56⁺ cells expressing an individual NKR as a function of the percentage of CD56⁺ cells in culture. Shown are the percentage of NK cells in culture (x-axis) and the percentage of receptor-expressing cells (y-axis). At all time points studied (days 14, 17, 19, 21, 25, 28), the majority of CD56⁺ cells (> 75%) expressed NKp44 and CD161. There was progressive acquisition of NKp30, NKp46, NKG2A, CD94, and NKG2D as the percentage of NK cells increased in the culture. A third group of receptors (CD16 and KIR) was acquired more gradually. Results were representative of more than 20 donors.

Figure 2.

CD117 and CD94 define 2 separate populations of differentiating NK cells. (A) CD117 defines 2 populations of CD56⁺ cells. FACS staining with CD56-APC versus CD117-PE shows 2 populations: CD56⁺CD117^high and CD56⁺CD117^low/–. (B) Expression of inhibitory receptors on CD56⁺CD117^high and CD56⁺CD117^low/– cell populations. Cells were stained with a monoclonal antibodies (mAbs) directed against CD56, CD117 in combination with CD161, KIR (CD158a and CD158b), NKG2A, and CD94 at day 28 of culture. Shown are histograms generated by gating on either the CD56⁺CD117^high or CD56⁺CD117^low/– populations. Results are representative of more than 20 donors. (C) Expression of activating receptors on CD56⁺CD117^high and CD56⁺CD117^low/– cells. FACS staining for NKp44, NKp46, NKp30, NKG2D, NKG2C, CD16, and CD8 after gating on either CD56⁺CD117^high or CD56⁺CD117^low/– cells. Results were representative of more than 20 donors. (D) CD117 and CD94 define 2 discrete populations of differentiating NK cells. FACS staining for CD117 and CD94 after gating on the CD56⁺ fraction. Results were representative of more than 20 experiments. (E) Adaptor protein expression in CD94^–CD117^high and CD94⁺CD117^low/– populations. Cells were FACS sorted into CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– fractions at days 21 to 28 of culture. RT-PCR was performed using primers specific for DAP12, DAP10, CD3ζ, FcεRIγ, and actin. Results were representative of 3 or more experiments.

Figure 3.

Functional differences between CD94^–CD117^high and CD94⁺CD117^low/– NK cells. (A) CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– populations were FACS sorted at days 21 to 28 of culture, rested for 24 hours, and then used in a ⁵¹Cr release assay with K562 cells as targets. Error bars depict standard deviation. Results are representative of 3 or more experiments. (B) Expression of cytotoxic molecules by RT-PCR in developing NK cells. mRNA was isolated from the FACS-sorted cell populations and was used for RT-PCR with primers specific for perforin, granzyme B, Fas ligand, TRAIL, and actin. Results were representative of 3 or more experiments. (C) Differential expression of perforin, granzyme B, and FasL in CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– cell populations. At days 21 to 28, cultures were analyzed for the expression of perforin and granzyme B using intercellular staining. FasL was assayed using surface staining. High levels of perforin, granzyme B, and FasL could be detected in the CD56⁺CD94^–CD117^low/– population, whereas these proteins were either absent or present at minimal amounts in the CD56⁺CD94^–CD117^high population. Results were representative of 3 or more experiments. (D) Interferon-γ secretion. HPC-derived NK cells were challenged with IL-12 (10 μg/mL) and IL-18 (100 μg/mL) for 18 hours, and cultures were assayed for intracellular interferon-γ. In the gated CD56⁺ population, intracellular interferon-γ was detected only in CD94⁺ cells representing the CD56⁺CD94⁺CD117^low/– population. Results were representative of 3 or more experiments.

Figure 4.

CD56⁺CD94^–CD117^high cells are a precursor population to CD56⁺CD94^–CD117^low/– cells. (A) The percentage of CD94^–CD117^high cells (top) and CD94⁺CD117^low/– cells (bottom) within the CD56⁺ population as a function of the percentage of CD56⁺ cells in culture. CD34⁺Lin^– cells were isolated by FACS and cultured as described in “Materials and methods.” The plots demonstrate that at early time points when the fraction of CD56⁺ cells is low the majority of CD56⁺ cells are CD94^–CD117^high. As the percentage of CD56⁺ cell increases, the fraction of CD56⁺CD94^–CD117^high cells decreases and the fraction of CD56⁺CD94⁺CD117^low/– cells increases (bottom). Results of 2 individual donors tested at days 14, 17, 19, 21, 25, and 28 are shown. (B) Proliferation rate of CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– cells by Ki67 staining. To determine whether there was a differential rate of proliferation in developing NK-cell subsets, cells at days 21 to 28 of culture were permeabilized (as described in “Materials and methods”) and stained for the nuclear antigen Ki67. The results were representative of 3 experiments. (C) CD56⁺CD94^–CD117^high cells are precursors to CD56⁺CD94⁺CD117^low/– cells. At day +21 (±3) of culture, CD56⁺CD94^–CD117^high cells were FACS sorted and cultured with cytokines and the feeder cell line (EL08.1D2). Cells were reanalyzed 1 week later for the presence of CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– populations. Shown are FACS plots after gating on the viable fraction. Results were representative of more than 3 individual experiments.

Figure 5.

The influence of the feeder cell line EL08.1D2 on the transition from CD56⁺CD94^–CD117^high to CD56⁺CD94⁺CD117^low/–. (A) At day 21 (±3) of culture, CD56⁺CD94^–CD117^high, CD56⁺CD94^low/–CD117^low/–, and CD56⁺CD94⁺CD117^low/– populations were FACS sorted and cultured for 1 week on either the fetal liver cell line (EL08.1D2) and cytokines (IL-7, IL-15, SCF, and FLT3L) or in media containing the cytokines alone. After 1 week in culture, the sorted populations were analyzed by FACS. Shown are the results after gating on the CD56⁺ fraction. Results were representative of more than 3 experiments. (B) The percentage of CD56⁺CD94⁺CD117^low/– cells detected after 1 week of culture either in the presence or absence of EL08.1D2. Shown are the results from 3 consecutive experiments (2 donors/experiment) where cells were sorted as in panel A and then cultured in cytokine containing media either without (white) or with (gray) EL08.1D2. Horizontal bar represents average and P is the result of a paired t test. (C) FACS-sorted CD56⁺CD9^–CD117^high cells (20 000) were cultured in triplicate with cytokines alone (left) or with cytokines and the stromal cell line EL08.1D2. Contact with the stromal cell line was either prevented with a transwell (middle) or allowed (right). Values represent the average number (+SD) of CD56⁺CD94⁺CD117^low/– cells detected after 1 week of culture by FACS. Shown are the results from a single donor, representative of 6 individual donors.

Figure 6.

CD56⁺CD94^–CD117high cells are present in UCB. (A) Small numbers of CD56⁺CD94^–CD117^high cells can be detected in UCB. FACS staining for CD117 and CD94 (after gating on CD56) of UCB-derived NK cells (left). FACS analysis after 1 week of culture of UCBderived CD56⁺ cells with either cytokines alone (IL-7, IL-15, SCF, and FLT3L) or with cytokines and EL08.1D2 (middle and right, respectively). Results are representative of 3 or more experiments. (B) Sorting strategy to isolate CD56⁺CD94^–CD117^high cells from UCB. The shown population was first gated on CD56⁺ cells (not shown). (C) Average fold expansion (±SD) of freshly isolated UCB CD56⁺CD94^–CD117^high cells after culture for 1 week either in the presence or absence of the stromal cell line (EL08.1D2). (D) Phenotype of FACSsorted UCB CD56⁺CD94^–CD117^high cells after 1 week of culture either in the presence (left panel) or absence (right panel) of the stromal cell line (EL08.1D2). (E) Expression of granzyme B and perforin in cultured UCB cells after transition to the CD56⁺CD94⁺CD117^low/– phenotype. CD56⁺CD94^–CD117^high cells from UCB were FACS sorted and cultured on the stromal cell line for 1 week. Expression of granzyme B and perforin in either bulk (left), CD56⁺CD94^–CD117^high cells (middle), or CD56⁺CD94⁺CD117^low/– cells (right) was determined by FACS. Panels B to E are the representative results of 4 separate donors.