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. 2012 Nov 1;21(16):2926-38.
doi: 10.1089/scd.2011.0659. Epub 2012 Jul 17.

Ex Vivo Generated Natural Killer Cells Acquire Typical Natural Killer Receptors and Display a Cytotoxic Gene Expression Profile Similar to Peripheral Blood Natural Killer Cells

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Free PMC article

Ex Vivo Generated Natural Killer Cells Acquire Typical Natural Killer Receptors and Display a Cytotoxic Gene Expression Profile Similar to Peripheral Blood Natural Killer Cells

Dorit Lehmann et al. Stem Cells Dev. .
Free PMC article

Abstract

Ex vivo differentiation systems of natural killer (NK) cells from CD34+ hematopoietic stem cells are of potential importance for adjuvant immunotherapy of cancer. Here, we analyzed ex vivo differentiation of NK cells from cord blood-derived CD34+ stem cells by gene expression profiling, real-time RT-PCR, flow cytometry, and functional analysis. Additionally, we compared the identified characteristics to peripheral blood (PB) CD56(bright) and CD56(dim) NK cells. The data show sequential expression of CD56 and the CD94 and NKG2 receptor chains during ex vivo NK cell development, resulting finally in the expression of a range of genes with partial characteristics of CD56(bright) and CD56(dim) NK cells from PB. Expression of characteristic NK cell receptors and cytotoxic genes was mainly found within the predominant ex vivo generated population of NKG2A+ NK cells, indicating the importance of NKG2A expression during NK cell differentiation and maturation. Furthermore, despite distinct phenotypic characteristics, the detailed analysis of cytolytic genes expressed within the ex vivo differentiated NK cells revealed a pattern close to CD56(dim) NK cells. In line with this finding, ex vivo generated NK cells displayed potent cytotoxicity. This supports that the ex vivo differentiation system faithfully reproduces major steps of the differentiation of NK cells from their progenitors, constitutes an excellent model to study NK cell differentiation, and is valuable to generate large-scale NK cells appropriate for immunotherapy.

Figures

FIG. 1.
FIG. 1.
Expansion of progenitors, monocytic cells, and NK cells in the culture system. (A) Scheme of ex vivo NK cell differentiation protocol: CD34+ cells were isolated from fresh umbilical cord blood and expanded for 10 days in the GBGM® medium with the indicated cytokines and growth factors. Generation of CD56+ NK cells was induced by the replacement of TPO by IL-15 at day 10, and of Flt3L by IL-2 at day 14, as indicated in the middle and lower line. Cytokines and growth factors added throughout the whole culture period are displayed in the upper line. Factors included only until or after day 10 or 14 are shown in the middle and lower lines, respectively. (B) Accumulation of CD56CD14, CD56+, and CD14+ cells over culture time: total cell numbers and composition of the cultures regarding CD56CD14, CD56+, and CD14+ cells were established by cell counting and flow cytometry over the culture period as indicated. Values display the accumulated cell numbers±SEM from 4 independent experiments. NK, natural killer; IL, interleukin; SEM, standard error of the mean; TPO, thrombopoetin.
FIG. 2.
FIG. 2.
A wide range of genes characteristic of NK cells is upregulated during ex vivo differentiation. In parts (A–D), 17 relevant genes selected from the 25 most strongly upregulated genes identified by gene profiling (see Table 1) were analyzed by real-time RT-PCR. Part (E) displays a flow cytometry analysis of the consecutive acquisition of CD56 and CD94. (A) depicts the real-time RT-PCR results obtained for genes of a range of receptors characteristic of NK cells, (B) of receptor chains of the C-type lectin-like CD94/NKG2 receptor family, (C) of cytolytic proteins, and (D) of several signaling proteins potentially involved in NK cell differentiation. In addition to genes displayed in Table 1, the expression of the NK cell receptor CD16 was included in this analysis. Total RNA was isolated, and real-time RT-PCR performed as described in the Materials and Methods section. Values were normalized to β-actin mRNA as an internal standard. Mean values of one representative culture of 3 performed with triplicate wells±SD are shown. (E) At day 19 of culture, CD56+CD94 cells were isolated by preparative FACS sorting and separately cultured for additional 6 days. The consecutive acquisition of CD94 by the CD56+CD94 NK cells was analyzed by flow cytometry at day 25. FACS, fluorescence-activated cell sorting; SD, standard deviation.
FIG. 3.
FIG. 3.
Functional markers of mature NK cells are most prominently expressed in ex vivo generated CD56+NKG2A+ NK cells. In parts (A–D), ex vivo generated NK cells were analyzed by real-time RT-PCR and in part (E), by flow cytometry. For real-time R-PCR analyses, cells were FACS-sorted at day 27 of culture into CD14+ cells (open bars), CD56+NKG2A (gray bars), and CD56+NKG2A+ NK cells (black bars). Total RNA was extracted, and mRNA levels within the FACS-sorted subsets were analyzed by real-time RT-PCR (A) for general receptor characteristic of NK cells, (B) the C-type lectin-like receptor chains of the CD94/NKG2 family, (C) cytolytic proteins, including perforin and granzymes, and (D) several signaling molecules. All values were normalized to β-actin as internal standard. To display the results, the sample with the highest relative induction rate of each gene was arbitrarily set to 100%, which usually was the CD56+NKG2A+ subset, except for CD16, NKG2C, and GZMA. In these cases, the sample of the CD14+ cells and the sample of CD56+NKG2A cells were set to 100%, respectively. Results depicted represent relative mRNA levels±SEM calculated from triplicate wells. (E) For flow cytometry, ex vivo generated NK cells from day 27 were analyzed after staining with antibodies for the proteins indicated and setting a gate for CD56-positive cells. Representative flow cytometry dot blots are shown.
FIG. 4.
FIG. 4.
Ex vivo generated NK cells display phenotypic properties of PBNK cells in flow cytometry. Ex vivo generated NK cells in cultures from day 28 and NK cells in PB mononuclear cells were analyzed in parallel by 10-color flow cytometry using a panel of antibodies for characteristic surface markers and NK cell receptors as described in the Materials and Methods section. (A) Displays the analysis of ex vivo differentiated NK cells and (B) of NK cells in PB mononuclear cells. In (C), data are shown separately for CD56bright and in (D) for CD56dim PBNK cells. The left upper panels in parts (A–D) display the staining with CD56 and CD3, indicating the gate further analyzed in the consecutive boxed panels of these figure parts. A representative example of 5 is shown. PB, peripheral blood.
FIG. 5.
FIG. 5.
Comparison of cytolytic profiles of ex vivo generated NK cells and PBNK cells. (A) Comparison of expression of cytolytic proteins: the expression of GNLY, GZMA, GZMB, GZMK, and PRF1 mRNA was compared for day 30 ex vivo differentiated NK cells (n=4, black bars) and FACS-sorted CD56dim (n=3, open bars) and CD56bright PBNK cells (n=3, gray bars). Real-time RT-PCR analysis was performed using total RNA isolated from the different cell types, and values were normalized to β-actin levels as internal standard. Mean values±SEM are shown in relation to PB CD56dim NK cells, which were arbitrarily set to 100%. (B) Comparison of natural cytotoxic activities: europium-release cytotoxicity assays were performed using K562 cells and ex vivo generated NK cells (open circle) and PBNK cells preincubated for 3 days with a differentiation medium containing 1,000 U rhIL-2 (black quadrant) or with a basal medium (black triangle) at various effector-to-target (E:T) ratios. Mean values±SD calculated from triplicates are shown for one representative experiment of 3 performed. GNLY, granulysin; GZMA, granzyme A; GZMB, granzyme B; GZMK, granzyme K; PRF1, perforin 1.
FIG. 6.
FIG. 6.
Ex vivo generated NK cells can produce IFN-γ and display potent ADCC. (A) Comparison of IFN-γ mRNA levels after induction with IL-12 or PMA plus ionomycin: ex vivo generated NK cells or PBNK cells kept in a basal or differentiation GBGM medium for 12 h were induced with IL-12 or a combination of PMA and ionomycin for 4 h or left untreated. Then, total RNA was isolated from the cells and subjected to real-time RT-PCR analysis. Mean values of relative IFN-γ mRNA levels±SD calculated from 3 independent cell samples are shown. The values obtained for noninduced ex vivo differentiated NK cells were arbitrarily set to 1. (B) Comparison of intracellular IFN-γ production after induction with IL-12 or PMA plus ionomycin: ex vivo generated NK cells or PBNK cells preincubated in a basal or differentiation GBGM medium for 24 h were induced with IL-12 or a combination of PMA and ionomycin for 16 or 6 h, respectively, or left untreated. GolgiPlug was added the last 4 h of induction before analysis. Then, cells were permeabilized and stained for intracellular IFN-γ. The percentages of IFN-γ-positive cells within the CD56-positive population are shown as mean values±SD calculated from 3 independent samples obtained from different individuals. (C) Rituximab strongly increases cytotoxicity against Nalm-6 and 721.221 cells: ex vivo generated NK cells from day 28 of culture were purified and subsequently used in Europium-release killing-assays. 721.221 and Nalm-6 cells were used at an effector-to-target ratio of 12:1. Cells were preincubated with Rituximab as indicated. Mean values±SD calculated from triplicate wells are shown for a representative experiment performed. ADCC, antibody-dependent cellular cytotoxicity; IFN, interferon; PMA, phorbol myristic acetate.

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