PRDM1 decreases sensitivity of human NK cells to IL2-induced cell expansion by directly repressing CD25 (IL2RA)

Abstract

IL2 receptor signaling is crucial for human NK cell activation and gain of effector functions. The molecular mechanisms involved in termination of IL2 activation are largely unknown in human NK cells. PR/SET domain 1 was previously reported to decrease cell growth and increase apoptosis in an IL2-dependent manner in malignant NK cell lines, suggesting the possibility of down-regulation of IL2 signaling pathway gene(s) through direct transcriptional repression. Using ChIP-Seq, we identified a PRDM1 binding site on the first intron of CD25 (IL2RA), which codes for the IL2 receptor subunit regulating sensitivity to IL2 signaling, in primary NK cells activated with engineered K562 cells or IL2. Ectopic expression of PRDM1 down-regulated CD25 expression at transcript and protein levels in two PRDM1 nonexpressing NK cell lines. shRNA-mediated knockdown of CD25 in two malignant NK cell lines led to progressive depletion of NK cells in low IL2 concentrations. By contrast, ectopic CD25 expression in primary human NK cells led to progressive increase in cell number in CD25-transduced cells in low IL2 concentrations. Altogether these results reveal a pivotal role of PRDM1 in inhibition of IL2-induced NK cell expansion through direct repression of CD25 in activated human NK cells. These observations provide additional support for the role of PRDM1 in attenuation of NK cell activation and growth, with implications on neoplastic transformation or NK cell function when it is deregulated.

Keywords: CD25; IL2 signaling; IL2Rα; NK cell expansion; PRDM1.

Figure 1.. Relationship between CD25 expression levels and the expansion capacity of human primary NK cells.

A) The schematic diagram showing the strategy employed to expand the primary human NK cells by coculturing PBLs with the engineered K562 cells. A modified version of a previously reported (Somanchi et. al. JOVE 2011) procedure was employed to expand the human NK-cells. Briefly, equal number of PBLs and 100Gr irradiated K562-Cl9-mb21 cells were seeded together in the presence of the NK-cell expansion medium (NKEM). Seven days after the first induction, equal numbers of cocultured cells were mixed with the 100 Gy irradiated K562-Cl9-mb21 cells and cultured in the presence of the NKEM for 7 more days. The purity of the NK cells was determined with CD56-APC and CD3-PE staining. B) The proliferation assay on cocultured NK cells in the later stages of NK cell activation. NK92 cells were used as positive control. Data are means ± SD of two independent experiments. C) mRNA expression levels of the CD25 (IL2RA) in NK cells activated with engineered K562 cells during the termination of NK cell activation. RPL13A was used to calibrate the mRNA levels of CD25, and the initial time point of the assay was used to normalize the calibrated expression values. Data are shown as means ± SD. Each data point is representative of replicate measurement of CD25 and RPL13A. The p value was calculated with unpaired t test by comparing the values to those in d13. ns, not significant.Western blot analysis of PRDM1 expression in primary NK cells obtained by coculturing for 12, 15, and 18 days (D) or for 10 and 13 days (E). Five-day IL2 activated NK cells (PBNKD5) were used as the positive control and PRDM1-null KAI3 cells were used as the negative control.

Figure 2.. PRDM1 binds to the 1^st intron of CD25 in activated NK cells.

A) The IGV snapshots showing the ChIP-Seq peaks located on the PRDM1 binding site identified downstream of the TSS of CD25 in PBNKD6, NKCOD13, and NKCOD14 samples. Horizontal gray bars indicate the predicted PRDM1 binding sites. The p value was calculated by the MACS program on the basis of the dynamic parameter λ_local as described in the Materials and Methods section. B) Genomic region of CD25 occupied by PRDM1 in three primary NK samples. The nucleotide sequence for the PRDM1 binding site observed in the CD25 ChIP-Seq peak region was displayed with PRDM1 consensus sites bolded and underlined. The p value was calculated by CentriMo as described in the Materials and Methods section. C) ChIP-qPCR cross-validation of PRDM1 occupation on the 1^st intron of CD25 in NKCOD14 sample.

Figure 3.. CD25 expression in normal and neoplastic NK cells.

A) CD25 expression was determined with qRT-PCR in primary human NK cells activated by IL2 up to 24h. B) The bar graph showing CD25 protein expression levels in resting and IL2 activated primary NK cells, which were determined as mean flourescent intensity (MFI) values with flow cytometry on CD56⁺/CD3⁻ gated cells. Each data point is average of four measurements derived from two independent experiments with biological replicates. The p values were calculated with unpaired t test by comparing the values to those of 0h time point. C) CD25 mRNA expression in 9 malignant NK cell lines were determined with qRT-PCR. RPL13A was used as a reference gene to calibrate CD25 expression. CD25 mRNA expression in resting NK cells were used to normalize the expression level in malignant NK cell lines. The names of NK cell lines are shown below the bar graphs. Data represent means ± SD. D) Representative FACS plots showing CD25 protein expression in malignant NK cell lines. E) CD25 protein expression levels determined with flow cytometry as MFI values in malignant NK cell lines are shown as a bar graph using logarithmic scale. IM-9, a multiple myeloma cell line with no CD25 expression based on Expression Atlas database (https://www.ebi.ac.uk/gxa/experiments/E-MTAB-2770/Results), was used as the negative control sample. Data represent means ± SD of two independent experiments of which each has biological replicates.

Figure 4.. CD25 is transcriptionally downregulated by PRDM1 in PRDM1α-transduced NK cell lines.

A) The retroviral vector used to ectopically express PRDM1α in PRDM1-null NK92 and KHYG1 cell lines. B) Schematic representation of the approach used to determine changes in CD25 expression in PRDM1α-transduced NK cell lines. GFP⁺ cells were sorted from NK92 or KHYG1 cells 48h post-transduction. Demonstration of transcriptional downregulation of CD25 in PRDM1α-transduced KHYG1 and NK92 cell lines by DNA microarray (C), RNA-Seq (D) or qRT-PCR (E). RNA expression values of PRDM1α-transduced NK cell lines were normalized to the values of the EV-transduced cells. FPKM: Fragments Per Kilobase of transcript per Million mapped reads. For Cuffdiff RNA-Seq expression data, FDR (i.e. q-value), which was obtained after Benjamini-Hochberg correction of the p value is shown. For DNA microarray and qRT-PCR data, paired t test was applied on log2 transformed expression values to obtain p values. F) Representative FACS profiles showing the CD25 protein levels of GFP⁺ cells in empty vector or PRDM1α transduced NK92 or KHYG1 cells. Each FACS plot is representative of two independent experiments with two biological replicates in each experiment (n=4).

Figure 5.. Stable knock-down of CD25 results in decreased cell growth in low IL2 concentrations in malignant NK cell lines.

A) Schematic representation of the retroviral construct used to stably express CD25 shRNA(s) in the context of mir30 backbone. B) Representative FACS plots showing the percentage of GFP⁺ KHYG1 cells 3 (day 0) or 14 days (day 11) post-transduction with the empty vector or each of two individual CD25 shRNA(s). Transduced KHYG1 cells were cultured in regular IL2 concentrations for 3 days after transduction. Three days after transduction, transduced cells were switched to the NK medium having low doses (50IU or 25IU) of IL2. Day 0 represents the day in which transduced cells were placed in medium with low IL2 concentrations. Three days post-transduction, transduced cells were switched to the NK medium having 50 IU (C) or 25 IU (D) of IL2. The same experiment was repeated in NK92 cell line starting from Day 0 in which transduced NK92 cell line was switched into culture medium with low IL2 concentrations. The percentage of GFP⁺ cells was quantified before and after empty vector or CD25 1^st or 2^nd shRNA-transduced NK92 cells were placed under culture with 25 IU (E) or 12.5 IU (F) of IL2. Data represent means ± SD of two independent experiments. The percentage of GFP⁺ cells quantified for later time points were normalized to the initial time point of GFP⁺ cell quantification. Paired t test was calculated for values in comparison to those of EV-transduced cells for each time point. .

Figure 6.. Ectopic expression of CD25 in primary NK cells leads to cellular growth advantage under low IL2 concentrations.

A) Schematic representation of the retroviral construct used for ectopic CD25 expression. B) Representative FACS plots of empty vector or CD25-transduced cocultured primary NK cells. The percentage of GFP⁺ cells was quantified with FACS four days post-transduction (day 0), and cells were then switched to NK cell culture medium with 12.5 IU IL2 concentrations. C) The bar graph showing the relative ratio of GFP⁺ cells after normalization to the levels at day 0 for empty vector (EV) or CD25-transduced cocultured NK cells. EV: Empty vector. Data represent means ± SD of two biological replicates. p values were calculated using paired t-test as a comparision to empty vector-transduced sample for each time point.