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. 2017 May 15;8:535.
doi: 10.3389/fimmu.2017.00535. eCollection 2017.

The Transcription Factor ZNF683/HOBIT Regulates Human NK-Cell Development

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Free PMC article

The Transcription Factor ZNF683/HOBIT Regulates Human NK-Cell Development

Mirte Post et al. Front Immunol. .
Free PMC article

Abstract

We identified ZNF683/HOBIT as the most highly upregulated transcription factor gene during ex vivo differentiation of human CD34+ cord blood progenitor cells to CD56+ natural killer (NK) cells. ZNF683/HOBIT mRNA was preferentially expressed in NK cells compared to other human peripheral blood lymphocytes and monocytes. During ex vivo differentiation, ZNF683/HOBIT mRNA started to increase shortly after addition of IL-15 and further accumulated in parallel to the generation of CD56+ NK cells. shRNA-mediated knockdown of ZNF683/HOBIT resulted in a substantial reduction of CD56-CD14- NK-cell progenitors and the following generation of CD56+ NK cells was largely abrogated. The few CD56+ NK cells, which escaped the developmental inhibition in the ZNF683/HOBIT knockdown cultures, displayed normal levels of NKG2A and KIR receptors. Functional analyses of these cells showed no differences in degranulation capacity from control cultures. However, the proportion of IFN-γ-producing cells appeared to be increased upon ZNF683/HOBIT knockdown. These results indicate a key role of ZNF683/HOBIT for the differentiation of the human NK-cell lineage and further suggest a potential negative control on IFN-γ production in more mature human NK cells.

Keywords: CD56; NK-cell development; ZNF683/HOBIT; ex vivo differentiation; natural killer cells.

Figures

Figure 1
Figure 1
High upregulation of ZNF683/HOBIT mRNA during ex vivo differentiation of human natural killer (NK) cells correlates with preferential expression in peripheral blood NK cells. (A) Ten most highly upregulated transcription factor mRNAs during ex vivo NK cell differentiation: CD34+ stem cells from cord blood were expanded and ex vivo differentiated into NK cells. Cells were sampled at day 10, just before the differentiation into NK cells was initiated by the addition of IL-15, and at day 35, after 25 days of differentiation. RNA was isolated and subjected to real-time RT-PCR analysis. β-actin was used as internal control. Fold upregulation of specific mRNAs at day 35 compared to day 10 is shown. Results were calculated from three series of experiments performed in triplicates with cells from different donors and are displayed as mean ± SEM. (B) High expression of ZNF683/HOBIT mRNA in peripheral NK cells: mononuclear cells were isolated from human peripheral blood and one half of the cells used for isolation of NK cells by negative magnetic sorting. The NK cell fraction was further separated by flow cytometry into CD56bright and CD56dim NK cells. The second half of the mononuclear fraction was used to isolate CD3+ T lymphocytes, CD19+ B lymphocytes, and CD14+ monocytes by flow cytometry. RNA was isolated from the different cell samples and subjected to real-time RT-PCR analysis using β-actin as internal control. ZNF683/HOBIT mRNA levels within the different cell types are compared to the levels in the CD56bright NK cells set to 100%. Results were obtained from three independent experiments using three different donors and are displayed as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 2
Figure 2
Upregulation of ZNF683/HOBIT mRNA during natural killer (NK) cell differentiation. (A) Amplification of three different cell populations in the ex vivo NK-cell differentiation cultures. Cord blood CD34+ cells were differentiated into NK cells over a culture period of 40 days. In regular intervals, cells were analyzed by flow cytometry for expression of the monocytic marker CD14 and the NK cell marker CD56. The numbers of CD56CD14, CD56+CD14, and CD56CD14+ cells were plotted. Results are calculated from 10 independent experiments using cells of different donors and are displayed as mean ± SEM. (B,C) Upregulation of ZNF683/HOBIT mRNA levels. Cell samples were taken at the indicated time points and CD14+ cells separated from the CD14 population using magnetic sorting. RNA was isolated and subjected to real-time RT-PCR analysis with β-actin as internal control. Results are calculated from three independent series of experiments performed in triplicates using different donors. Fold upregulation in comparison to the values obtained for day 8 cells is shown as mean ± SEM (B). To display early ZNF683/HOBIT mRNA upregulation the period until day 21 is shown at a larger scale (C).
Figure 3
Figure 3
Effects of transduction with lentiviruses expressing HOBIT shRNA on total cell expansion. (A) HOBIT shRNA strongly reduces HOBIT expression. Lentiviral vectors expressing HOBIT shRNA were co-transfected with a HOBIT expression construct into HEK293T cells. After 96 h, cells were either used for RNA isolation or lysed in Laemmli sample buffer. The RNA was used for realtime RT-PCR analysis (upper part). Results are derived from two experiments performed in quadruplicates and shown as mean ± SD. The proteins in the lysed samples were separated by SDS-PAGE, Western blotted, and probed with anti-HOBIT antibodies (lower part). As internal control hypoxanthine-guanine phosphoribosyltransferase was detected by respective antibodies. Two experiments with comparable results were performed. (B,C) HOBIT shRNA reduces expansion of cells at day 21. Cord blood CD34+ cells were cultured for 5 days, then cells were transduced with lentiviruses expressing either shHOBIT or a scrambled control shRNA (shControl) or were mock-treated. Transduction efficiency was measured 3 days later by flow cytometry scoring GFP-positive cells. Cells were further cultured and differentiated until day 21. Then flow cytometry was performed to evaluate expansion of transduced GFP-expressing cells. Exemplary dot plots for cells transduced with lentiviruses expressing shHOBIT or shControl or mock-transduced controls are shown in (B). The numbers of the transduced GFP+ cells and the non-tranduced GFP cells in individual cultures was calculated from the measured cell number and the respective percentages determined by flow cytometry and are shown in (C). The values were normalized to the number of transduced or non-tranduced cells measured at day 8 to establish the expansion rates. Results are calculated from four experiments performed in triplicates and are displayed as mean ± SEM (*p < 0.05, ***p < 0.001).
Figure 4
Figure 4
HOBIT shRNA strongly reduces the number of CD56CD14 progenitor cells at day 21. Stem cells at day 5 of culture were transduced and further cultured until day 21. Then CD56 and CD14 expression was assessed by flow cytometry within the GFP+ and GFP parts of the culture. (A) Exemplary flow cytometry dot plots displaying staining for CD56 and CD14 at day 21. (B) The relative expansion of CD56CD14, CD56+, and CD14+ cells within the GFP+ and GFP fractions is shown. Expansion rates were calculated from the measured cell numbers at day 21 and the percentages of the different subsets obtained by flow cytometry. Differences in transduction efficiency were corrected for by normalizing the obtained values to the number of GFP+ and GFP cells at day 8, respectively. Results are calculated from four independent experiments performed in triplicates using different donor cells and are displayed as mean ± SEM (**p < 0.01).
Figure 5
Figure 5
HOBIT shRNA nearly abrogates formation of CD56+ natural killer (NK) cells. Stem cell cultures were transduced and cultured until day 35. The generation of CD56+ NK cells was monitored by flow cytometry in samples taken at days 24, 26, 28, 31 and 35. (A) Exemplary flow cytometry dot plots displaying staining for CD56 within the GFP+ fractions at days 24 and 35. (B) The formation of CD56+ cells within the transduced GFP+ and non-transduced GFP fractions is shown for shHOBIT and shControl cultures. Expansion rates are calculated from measured cell numbers at the respective days and the flow cytometry-derived percentages. Differences in transduction efficiency were corrected for by normalizing the obtained values to the number of GFP+ and GFP cells at day 8, respectively. Results are calculated form four independent experiments, two of them performed in triplicates, using different donor cells and are displayed as mean ± SEM (*p < 0.05, ***p < 0.001).
Figure 6
Figure 6
Effects of HOBIT shRNA on phenotype and functions of natural killer (NK) cells at day 35. (A) Expression of NKG2A/CD94 and KIR receptors. Stem cells were transduced and cultured until day 35. Then expression of NKG2A and KIR was measured by flow cytometry. The percentages of cells within the GFP+ and GFP fractions as well as in the mock-treated cultures are displayed. (B) Degranulation capacity: separate aliquots of the day 35 cultures were cocultured with K562 target cells and surface CD107a measured after 6 h by flow cytometry. Percentages of CD107a-positive cells within the CD56 population are displayed. (C) IFN-γ production: the K562 cocultures were further stained for intracellular IFN-γ. Representative flow cytometry dot plots obtained are shown in the upper part. Percentages of IFN-γ positive cells within the CD56 population are displayed in the lower part. All results are derived from four independent experiments, two of them performed in triplicates, using different donor cells and are shown as mean ± SEM (*p < 0.05).
Figure 6
Figure 6
Effects of HOBIT shRNA on phenotype and functions of natural killer (NK) cells at day 35. (A) Expression of NKG2A/CD94 and KIR receptors. Stem cells were transduced and cultured until day 35. Then expression of NKG2A and KIR was measured by flow cytometry. The percentages of cells within the GFP+ and GFP fractions as well as in the mock-treated cultures are displayed. (B) Degranulation capacity: separate aliquots of the day 35 cultures were cocultured with K562 target cells and surface CD107a measured after 6 h by flow cytometry. Percentages of CD107a-positive cells within the CD56 population are displayed. (C) IFN-γ production: the K562 cocultures were further stained for intracellular IFN-γ. Representative flow cytometry dot plots obtained are shown in the upper part. Percentages of IFN-γ positive cells within the CD56 population are displayed in the lower part. All results are derived from four independent experiments, two of them performed in triplicates, using different donor cells and are shown as mean ± SEM (*p < 0.05).

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