PRDM1/Blimp-1 Controls Effector Cytokine Production in Human NK Cells Matthew A. Smith, Michelle Maurin, Hyun Il Cho, Brian Becknell, Aharon G. Freud, Jianhua Yu, Sheng Wei, Julie This information is current as Djeu, Esteban Celis, Michael A. Caligiuri and Kenneth L. of September 27, 2021. Wright J Immunol published online 13 October 2010 http://www.jimmunol.org/content/early/2010/10/13/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 13, 2010, doi:10.4049/jimmunol.1001682 The Journal of Immunology

PRDM1/Blimp-1 Controls Effector Cytokine Production in Human NK Cells

Matthew A. Smith,*,† Michelle Maurin,* Hyun Il Cho,* Brian Becknell,‡ Aharon G. Freud,‡ Jianhua Yu,‡ Sheng Wei,* Julie Djeu,* Esteban Celis,* Michael A. Caligiuri,‡ and Kenneth L. Wright*,†

NK cells are major effectors of the innate immune response through cytolysis and bridge to the adaptive immune response through cytokine release. The mediators of activation are well studied; however, little is known about the mechanisms that re- strain activation. In this report, we demonstrate that the transcriptional repressor PRDM1 (also known as Blimp-1 or PRDI-BF1) is a critical negative regulator of NK function. Three distinct PRDM1 isoforms are selectively induced in the CD56dim NK population in response to activation. PRDM1 coordinately suppresses the release of IFN-g, TNF-a, and TNF-b through direct binding to multiple conserved regulatory regions. Ablation of PRDM1 expression leads to enhanced production of IFN-g and Downloaded from TNF-a but does not alter cytotoxicity, whereas overexpression blocks cytokine production. PRDM1 response elements are defined at the IFNG and TNF loci. Collectively, these data demonstrate a key role for PRDM1 in the negative regulation of NK activation and position PRDM1 as a common regulator of the adaptive and innate immune response. The Journal of Immunology, 2010, 185: 000–000. http://www.jimmunol.org/ atural killer cells play critical functions in innate and IFN-g production and increased cytotoxicity (9). IL-18 induces nu- adaptive immunity. Although these lymphocytes were clear localization of NF-kB p50/p65 which, cooperatively with N initially identified by their ability to lyse leukemia cells AP-1, increases IFN-g and cytotoxicity (10). Furthermore, NFAT in a non-MHC–restricted manner, subsequent studies highlighted induces transcription of GM-CSF and TNF-a in NK cells (11). their role in cytokine production. In response to activating stimuli, Conversely, relatively few negative regulators of activation-induced NK cells proliferate, increase cytotoxicity, and produce cytokines, transcription have been identified in NK cells. ATF3 was recently such as IFN-g, TNF-a, and GM-CSF (1). IL-2 upregulates the ex- shown to downregulate IFN-g levels, and ATF2/2 mice exhibit in- pression of effector molecules and enhances natural cytotoxicity creased resistance to murine CMV infection (12). The transcription against a variety of targets. Furthermore, IL-2 and IL-15 signal factor H2.0-like homeobox negatively regulates IFN-g production, by guest on September 27, 2021 through the common gcR to control proliferation, with IL-15 be- primarily through degradation of phosphorylated STAT4 not ing uniquely required for survival in vivo (2). IL-12 and IL-18 direct DNA-binding activity (13). signal through distinct heterodimeric receptor complexes to elicit PRDM1 (also known as Blimp-1 or PRDI-BF1) is a transcrip- increases in IFN-g via several mechanisms, including increased tional repressor encoded by the PRDM1 gene on chromosome transcription, message stability, and nuclear retention (3–5). Syn- 6q21. It was originally identified as a postinduction suppressor of ergistic increases in cytotoxicity and IFN-g production are ob- IFNB in virally infected osteosarcoma cells (14). Subsequent served in response to costimulation with IL-12 and IL-18 (6, 7). work revealed a pivotal role in the terminal differentiation of Ab- Cytokine-mediated activation of NK cells proceeds through producing plasma cells (15). We and other investigators previously several well-characterized nuclear transcription factors, many of showed that PRDM1 exerts its repressive functions through re- which are functionally conserved between T and NK lineages (8). cruitment of histone-modifying enzymes (HDAC2, G9a, PRMT5, STAT4 is induced in response to IL-12 and is required for optimal and LSD1) and Groucho corepressors (16–18). Through silencing of direct (cMyc, CIITA, Pax5) and indirect targets, PRDM1 is a master regulator of terminal differentiation of B lymphocytes, me- *Immunology Program, H. Lee Moffitt Cancer Center and Research Institute; †Department of Molecular Medicine, University of South Florida, Tampa, FL diating cell cycle exit, repression of early B cell factors, and in- 33612; and ‡Ohio State Comprehensive Cancer Center, Columbus, OH 43210 duction of Ig secretion (19, 20). Received for publication May 20, 2010. Accepted for publication September 11, More recently, a role for PRDM1 in T lymphocytes has emerged. 2010. PRDM1 is expressed in CD4 and CD8 T cell lineages and is critical This work was supported by the James and Ester King Biomedical Research Program for maintenance of homeostasis. Conditional knockout in T lym- (09KT-03). phocytes leads to increased effector populations, resulting in severe The sequences presented in this article have been submitted to the Gene Expression Omnibus under accession number GE22919. colitis (21, 22). Upon activation, an autoregulatory loop exists, whereby IL-2 induces PRDM1 expression, which, in turn, nega- Address correspondence and reprint requests to Dr. Kenneth L. Wright, H. Lee Moffitt Cancer Center, MRC4E, 12902 Magnolia Drive, Tampa, FL 33612. E-mail tively regulates IL-2 transcription (23, 24). During CD4 polariza- address: Ken.Wright@moffitt.org tion, PRDM1 is preferentially expressed in Th2 cells and reinforces The online version of this article contains supplemental material. commitment to this lineage through repression of Ifng, cfos, and

Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; Ct, threshold tbx21 (24, 25). Within the CD8 lineage, PRDM1 is expressed at cycle; KD, knockdown; NT, nontargeting; PARP, poly(ADP-ribose) polymerase; higher levels in exhausted subsets and promotes acquisition of poly-IC, polyinosinic-polycytidylic acid; siRNA, small interfering RNA. the effector phenotype through suppression of memory potential Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 (26–28). Thus, in addition to well-characterized B cell-specific

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001682 2 PRDM1 REGULATES NK EFFECTOR CYTOKINE PRODUCTION functions, PRDM1 is a critical regulator of T lymphocytes. In this and counts per minute were determined using a PerkinElmer 1470 auto- report, we provide a functional description of PRDM1 in NK cells. matic gamma counter (PerkinElmer, Wellesley, MA). The percentage of cytotoxicity was calculated using the following formula: (experimental 2 spontaneous release)/(total 2 spontaneous release) 3 100. Materials and Methods Chromatin immunoprecipitation Cells and cytokines A total of 20 3 106 purified NK cells were stimulated for 24 h with IL-2 Primary human NK cells were isolated via negative selection using the (100 U/ml), IL-12 (10 ng/ml), and IL-18 (100 ng/ml). Chromatin EZSep kit (StemCell Technologies, Vancouver, BC, Canada), according to was prepared, as previously described (30). A total of 4.5 3 106 cell the manufacturer’s instructions. Purity was verified by flow cytometry, 2 + + equivalents were used for each immunoprecipitation reaction. Primary and cells were routinely found to be 90–95% CD3 CD56 CD16 . Cells Abs were used at 0.5 mgineach900-ml reaction and were incubated over- were maintained in RPMI 1640 (Life Technologies, Carlsbad, CA), sup- night. Abs used included PRDM1 (PRDI-BF1) (Cell Signaling Tech- plemented with 10% FBS and 1% penicillin-streptomycin. For small in- nology) and normal rabbit IgG (Upstate Biotechnology, Lake Placid, NY). terfering RNA (siRNA) experiments, cells were grown in Accell Delivery Immune complexes were captured with protein A/G beads (Santa Cruz Media (Dharmacon, Chicago, IL), supplemented with 2% FBS. For Biotechnology, Santa Cruz, CA) and washed, as described. Eluted DNA stimulations, the following recombinant human cytokines were used: IL-2 was column purified (Qiagen) after reversal of cross-links (4 h at 65˚C) (100 U/ml; PeproTech, Rocky Hill, NJ), IL-12 (10 ng/ml; PeproTech), IL- and RNase treatment and proteinase K treatment. PCR was performed 18 (100 ng/ml; MBL, Woburn, MA), TNF-a (20–100 ng/ml; eBioscience, using 1.5 ml eluted DNA (∼1/40th) in duplicate. Primers designed against San Diego, CA), and IFN-g (10–50 ng/ml; eBioscience), a-IFN (10 or the second exon of myoglobin B (Diagenode, Liege, Belgium) were used 50ng/ml; Sigma-Aldrich, St. Louis, MO). as a negative control locus. The percentage of input was calculated by 2 Mice linearization of DCt (CtIP Ct1%input) for specific and IgG samples. Downloaded from C57BL/6 mice (n = 4) were immunized i.v. with 250 mg polyinosinic- ELISA polycytidylic acid (poly-IC; Oncovir, Washington, DC) or PBS. Mice were ELISAs were performed in 100-ml volumes in triplicate using commercial sacrificed at 48 h postinjection, and single-cell suspensions were prepared kits for IFN-g and TNF-a, according to the manufacturer’s instructions from pooled splenocytes. Murine NK cells were isolated via negative se- (eBioscience). Plates were read at 450 nm on a Spectramax Plus plate lection using the Murine NK Enrichment Kit (StemCell Technologies). reader (Molecular Devices, Sunnyvale, CA). Lysates were prepared from purified NK cells and total splenocytes and analyzed by immunoblot.

Adenoviral constructs and transduction http://www.jimmunol.org/ Microarray hybridization and data analysis Adenoviral constructs were created in the Ad5/F35 vector from previously Two micrograms of total RNA served as the mRNA source for microarray described constructs (16). This replication-deficient adenovirus uses the analysis. The poly(A) RNA was specifically converted to cDNA and then ubiquitously expressed CD46 molecule to mediate high-level transduction amplified and labeled with biotin, as described (29). Hybridization with the efficiency in a variety of hematopoietic cells (31). Briefly, PRDM1a in the biotin-labeled RNA, staining, and scanning of the chips followed the AdTrack vector was recombined with Ad5/F35 to generate a bicistronic prescribed procedure outlined in the Affymetrix technical manual. Scan- viral vector containing PRDM1a and GFP. Purified stocks were obtained ned output files were visually inspected for hybridization artifacts and then by infecting Ad293T cells for 48 h and concentrating via CsCl banding, analyzed using Affymetrix (Santa Clara, CA) GeneChip Operating Soft- according to standard protocols. Viral titers were calculated using the ware. Heatmaps were generated with Heatmap Builder v1.1 using signal- QuickTiter Adenovirus Quantitation Kit (Cell Biolabs, San Diego, CA).

For transduction experiments, Jurkat cells were transduced using a multi- by guest on September 27, 2021 intensity values of transcripts that were differentially expressed $3-fold. 3 6 Transcripts with $3-fold increases/decreases are provided in Supplemental plicity of infection of 500 at a density of 1 10 /ml for 44 h before Table I, and complete data have been deposited at the Gene Expression stimulation with PMA (1 mg/ml) and PHA (10 ng/ml) for 4 h. Omnibus under accession number GE22919 (http://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE22919). DNA constructs and luciferase assay A fragment of the human IFNG promoter (2507–120) was PCR cloned Western blotting from human genomic DNA into pcr2.1. The fragment was then subcloned Whole-cell lysates from 5 3 105 cells were prepared in lysis buffer (50 mM into pGL3-basic using the HindIII and SmaI restrictions sites to generate Tris [pH 7.2], 150 mM NaCl, 1% Nonidet P-40, 1% Na-deoxycholate, 0.1% pGL3-IFNg_WT. pGL3-IFNg_mut was obtained via site-directed muta- 2 SDS, 2 mM EDTA) on ice, sonicated, and separated by 8% SDS-PAGE. genesis of the 254 site, changing residues AAAAGT to TCTAGA, which Gels were transferred to polyvinylidene difluoride, blocked with 5% skim created a novel XbaI site (Mutagenex, Piscataway, NJ). pGL3-IFNg_D was milk, and probed using Abs directed against PRDM1 (PRDI-BF1) (1:1,000; obtained by ligation of an XbaI-XbaI fragment obtained from pGL3- IFNg_mut with the XbaI-NheI fragment of the pGL3-Basic vector. Cell Signaling Technology, Beverly, MA), poly(ADP-ribose) polymerase 7 (PARP)1 (1:1,000; Cell Signaling Technology), or b-actin (1:10,000; Sigma- Transfections were performed using 20 mg total plasmid into 10 cells by electroporation at 250V, 1070 mF in 300 ml RPMI 1640 using a Bio-Rad Aldrich). Secondary Abs conjugated to HRP that were used for detection 6 included anti-rabbit (1:2,000) and anti-mouse (1:10,000; both from GE, Gene Pulser II. Cells were cultured for 36 h at 10 /ml. Cells were lysed in Piscataway, NJ). 500 ml Passive Lysis Buffer and assayed using the Dual Luciferase kit, per the manufacturer’s instructions (Promega, Madison, WI). Real-time PCR Statistical analyses RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA), according , to the manufacturer’s instructions. Eluted RNA was treated with DNase Two-tailed paired t tests were used for statistical analyses,; p 0.05 were and converted to cDNA, and one twentieth of the cDNA reaction was considered significant. All calculations were performed in Microsoft Office analyzed by real-time PCR in duplicate using the Bio-Rad iCycler (Bio- Excel (Microsoft, Redmond, WA). Rad, Hercules, CA) for 40 cycles, with primer-specific annealing tem- peratures between 55˚C and 60˚C. For expression analysis, data were an- Results alyzed by the DD threshold cycle (Ct) method, with normalization to 18S Human NK cells alter expression of multiple effector molecules or GAPDH. Primers were quality checked for single curve on melt curve, and efficiencies were 90–110%. Sequences of primers (PRDM1a, and transcription factors in response to cytokine stimulation PRDM1b, IFNG, TNF, LTA, DAP10, CIITAp3, CIITAp4, PAX5, IFNB1, NK cells are well known for their ability to upregulate the pro- SLAMF7, MBexon2, IFNG I, II, III, IV, TNF I, II, III, IV) and annealing duction of effector cytokines and cytotoxic potential in response to temperature values are provided in Supplemental Table II. IL-2 and other cytokines, such as IL-12, IL-15, and IL-18. How- [51Cr]-release assay ever, the roles of sequence-specific, DNA-binding transcription NK cells isolated by negative selection were cultured for 72 h prior to the factors in the modulation of NK activity are incompletely char- assay. Cells were incubated with 5000 K562 cells at various E:T ratios in acterized. Todirectlyaddressthis, we isolatedNKcells from peripher- triplicate in a total volume of 100 ml for 4 h. Supernatants were harvested, al blood of healthy donors and performed global gene-expression The Journal of Immunology 3 profiling on RNA isolated immediately or after 24 h of stimulation in value of 0.967 and 0.970 between donors in freshly isolated and the presence of IL-2, IL-12, and IL-18 (Fig. 1). Purity of the NK stimulated samples, respectively (Supplemental Fig. 1). In total, 541 population was determined by flow cytometry to be .96%, and this genes were increased $3-fold in both donors, whereas 609 genes was confirmed by the absence of significant signals for transcripts were decreased $3-fold following 24 h of stimulation (Fig. 1A, associated with B cells, T cells, and monocytes in the microarray (Fig. Supplemental Table I). As expected, genes encoding effector cyto- 1B). Biological reproducibility was extremely close, giving an R2 kines (e.g., IFNG, TNF,andCSF1) and numerous TNF family Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Human NK cells activate effector molecules and transcriptional regulators in response to cytokine stimulation. A, Heatmap depicting top 75 genes found to be up- or downregulated $3-fold in response to 24 h of stimulation with IL-2 (100 U/ml), IL-12 (10 ng/ml), and IL-18 (100 ng/ml) relative to time 0 h. Pixel density (highest values in each are black, lowest are white) represents average hybridization signal intensity from two donors after and before stimulation for increased and decreased genes, respectively. Fold changes are shown in parentheses. Presentation of expression data in this manner allows appreciation of the magnitude of expression and increases/decreases in response to stimulation. B, Average signal intensities of transcripts associated with non-NK cell lineages are shown from the 0-h samples to demonstrate the low frequency of contaminating cells. C, Heatmap depicting the top 15 up- and downregulated DNA-binding transcription factors as in A. D, Average signal intensities of selected secreted cytokines and TNF family members. 4 PRDM1 REGULATES NK EFFECTOR CYTOKINE PRODUCTION members were found to be transcriptionally upregulated in re- sponse to stimulation (Fig. 1C). Decreased levels of the TGFB1 transcript, which is constitutively expressed in NK cells, were also observed in response to stimulation, consistent with TGF-b1’s role as a negative regulator of activation/proliferation. Of the top 150 transcripts found to be modulated, nearly 10% were sequence-specific DNA-binding transcription factors (Fig. 1D). Among these factors, the transcriptional repressor PRDM1 was increased in response to stimulation. PRDM1 has not been previously identified in NK cells; however, PRDM1 plays crucial roles in cell fate decisions and regulation of homeostasis within the immune system. The role of PRDM1 in the terminal differ- entiation of mature B cells into Ig-secreting CD138+ plasma cells is well established. More recently, roles for PRDM1 in the main- tenance of homeostasis and effector versus memory lineage commitment in T lymphocytes were reported (26–28). This sug- gests that PRDM1 may also have key functional roles in NK cells.

Cytokine stimulation induces multiple PRDM1 isoforms in Downloaded from human NK cells To directly establish PRDM1 activation in human NK cells, cells were isolated by negative selection and stimulated with multiple combinations of IL-2, IL-12, and IL-18. Immunoblot analysis revealed that freshly isolated and unstimulated NK cells have barely

detectable levels of PRDM1 protein (Fig. 2A). Stimulation with IL-2 http://www.jimmunol.org/ or a combination of IL-12 and IL-18 resulted in increased levels of PRDM1 protein, which is markedly enhanced by costimulation with all three cytokines. Cell lines derived from malignant NK tumors constitutively express PRDM1 and exhibit distinct isoform- expression patterns (Fig. 2B). YT and NK92 cell lines exclusively express the larger a isoform, whereas the NKL cell line most closely resembles the expression pattern observed in primary human NKs. Primary NK cells express three distinct molecular mass. forms of FIGURE 2. Cytokine stimulation induces PRDM1 isoforms in human PRDM1. The largest molecular mass band corresponds to the full- NK cells. A, Western blot analysis of purified human NK cells stimulated by guest on September 27, 2021 length PRDM1a isoform; however, this protein is significantly for 24 h with IL-2 (100 U/ml), IL-12 (10 ng/ml), and IL-18 (100 ng/ml) or underrepresented compared with the smaller PRDM1 proteins. The combination. Blot is representative of numerous experiments. B, Immu- predominant and smallest form corresponds to the PRDM1b origi- noblot analysis of lysates prepared from human NK cell lines demonstrates nally characterized in multiple myeloma tumor cells and shown to differential expression of PRDM1 isoforms. C, Expression values were have partially reduced repressive ability in reporter assays (32). In calculated using the DDCt method, with 18S as the control gene. Upon addition, immunoblot analysis detected an intermediate-sized iso- linearization of Ct values, average time 0 PRDM1a was arbitrarily set to “1” to account for basal differences among donors and the consistently form in primary NK cells. This protein migrating at ∼85 kD likely higher expression levels of PRDM1b. Error bars represent SD from at least represents the human homolog of a previously described murine three biologically independent samples isolated from different donors at splice variant prdm1Dexon7 (33). RT-PCR analysis using primers different times. Paired two-tailed t tests were conducted. pp , 0.05. The p spanning the analogous exon in humans (exon 6) confirmed the values were 0.016 and 0.023 for PRDM1a and PRDM1b, respectively. D, presence this splice variant (Supplemental Fig. 2). Consistent with CD56dim and CD56bright subsets were obtained from IL-12– and IL-18– our immunoblotting experiments, analysis of the mRNA levels stimulated human NK cells by FACS sorting, as previously described. RT- specific for the a and b isoforms indicate that cytokine stimulation PCR analysis was conducted on cDNAs prepared from two donors, as dim upregulates both isoforms and that PRDM1b mRNA is present at above. E, Immunoblot analysis of lysates prepared from CD56 and bright ~20-fold higher levels than is PRDM1a (Fig. 2C). CD56 subsets as in D. Human NK cells can be divided into two subsets based on the surface density of CD56 and the presence of CD16. The CD56bright CD56dim subset in response to stimulation (Fig. 2D,2E). Further- 2 CD16dim/ subset represents ∼10% of the human peripheral NK more, PRDM1b is the predominant isoform present. cell compartment and was suggested to play an immunomodu- latory role based on its increased ability to produce cytokines; PRDM1 associates with NK cell activation the CD56dimCD16+ subset represents ∼90% of human periph- Given that PRDM1 was maximally induced in response to co- eral NK cells and is considered the primary cytotoxic subset. stimulation with IL-2, IL-12, and IL-18, we hypothesized that We questioned whether PRDM1 is restricted to specific subsets levels of PRDM1 correlated with the degree of activation. We or whether it is broadly expressed in NK cells. Human NK cells profiled mRNA levels of the effector cytokines IFN-g and TNF-a in were isolated from healthy donors via negative selection, fol- purified human NK cells after 24 h of stimulation with various lowed by flow-cytometric sorting into CD56bright and CD56dim combinations of cytokines. Quantitative RT-PCR analysis indicates subsets. The cells were then stimulated with IL-12 and IL-18 that IFN-g and TNF-a mRNA are synergistically increased upon for 24 h before analysis. Immunoblot and quantitative RT-PCR stimulation with IL-2, IL-12, and IL-18 (Fig. 3A). Consistent with analysis revealed that PRDM1 is preferentially expressed in the this, IL-2, IL-12, or IL-18 alone each minimally altered PRDM1 The Journal of Immunology 5

of naive and poly-IC–treated mice. PRDM1 is robustly induced specifically in NK cells upon 48 h of treatment (Fig. 4). PRDM1 expression was also detected in total splenocytes, and the level did not change with poly-IC treatment. This is likely due to the pre- sence of PRDM1 in T cells and differentiating plasma cells within the spleen. Interestingly, only the PRDM1a homologous isoform was detectable in murine NK cells. Although an inability of the Ab to cross-react with other murine isoforms cannot be excluded, it is possible that the expression and/or function of PRDM1 iso- forms in murine cells is differentially regulated. PRDM1 is not involved in perforin-mediated cytotoxicity The ability to lyse target cells in an Ag-independent manner is a hallmark of NK cells. Cytotoxicity against heterologous tar- get cells proceeds through the release of perforin and granzymes and is markedly increased upon treatment with IL-2, IL-12 or IL-18 (1, 34). To assess whether PRDM1 regulates cytotoxicity, NK cells were isolated from healthy donors and stimulated with

IL-2 and IL-12 or the combination of IL-2, IL-12, and IL-18 in the Downloaded from presence of a nontargeting (NT) control or a PRDM1-specific siRNA for 72 h. Cytotoxicity against the K562 leukemia cell linewasassessedina4-h[51Cr]-release assay. As expected, cytokine-activated NK cells exhibit significant cytotoxicity against K562 targets (Fig. 5A). Knockdown of PRDM1 expression did not

alter cytolytic activity across several E:T ratios. Distinct from our http://www.jimmunol.org/ experiments that revealed increases in IFNG and PRDM1 mRNA levels with the addition of IL-18 to IL-2 and IL-12, we observed no additive effect of IL-18 in cytotoxicity assays, further demonstrat- ing that cytotoxicity and PRDM1 levels are not directly linked. Knockdown of PRDM1 protein was confirmed by immunoblotting to be highly efficient, and it had no effect on viability, as assessed by trypan blue staining (Fig. 5B, data not shown). Furthermore, no activation-induced cell death was detected in stimulated NK cells,

as measured by PARP cleavage (Fig. 5C). Collectively, these results by guest on September 27, 2021 show that PRDM1 does not have a significant role in regulating perforin-mediated cytotoxicity in NK cells. PRDM1 binds promoters in multiple target genes Because PRDM1 is well-documented as a DNA-binding transcri- FIGURE 3. PRDM1 expression associates with NK activation. A,RT- ptional repressor, we sought to characterize its function in NK cells PCR analysis of cDNA synthesized from freshly isolated or NK cells by identifying DNA elements to which PRDM1 specifically binds. stimulated for 24 h. Expression values were calculated using the DDCt To accomplish this, we performed chromatin immunoprecipitation method with 18S as the control gene. Upon linearization of C values, av- t (ChIP) experiments in primary NK cells isolated from healthy erage time 0 for each donor was arbitrarily set to “1”. Error bars represent SD from at least three biologically independent samples isolated from donors. Cells were stimulated for 24 h with IL-2, IL-12, and IL-18 different donors at different times. B, Immunoblot analysis of purified hu- to induce PRDM1 expression prior to isolation of chromatin and man NK cells stimulated for 24 h with IL-2 (100 U/ml), IL-12 (10 ng/ml), immunoprecipitation with anti-IgG or anti-PRDM1 Abs. Initially, and IL-18 (100 ng/ml) alone or in combination. C, Immunoblot analysis of we assayed promoter regions of genes known to be regulated by lysates prepared from purified NK cells treated with recombinant human PRDM1 (Fig. 6A). A well-documented target is CIITA, the master TNF-a (20 or 100 ng/ml), IFN-g (10 or 50 ng/ml), a-IFN (10 or 50 ng/ml) regulator of MHC class II expression that is transcriptionally si- or IL-2 (100 U/ml), IL-12 (10 ng/ml), and IL-18 (100 ng/ml) for 24 h. lenced as mature B cells differentiate into Ab-producing plasma expression (Fig. 3B). Thus, PRDM1 levels correlate with effector cytokine transcription. We next assessed whether effector cytokines induced PRDM1 through autocrine or paracrine feedback mechanisms. Treatment with rIFN-g or TNF-a failed to induce PRDM1 (Fig. 3C). Simi- larly, stimulation with a-IFN is insufficient to induce PRDM1, suggesting that induction results primarily from cytokine receptor- mediated signaling and likely requires multiple signaling events. To determine whether PRDM1 is induced in vivo, we activated murine NK cells by poly-IC injections into C57/BL6 mice. Poly-IC FIGURE 4. PRDM1 is induced in vivo. C57/BL6 mice were treated with is known to activate NK cells indirectly through TLR3-mediated 100 mg poly-IC via tail vein injection for 48 h. NK cells purified via release of cytokines from dendritic cells and other accessory negative selection (left panel) or total splenocytes (right panel) were an- cells. NK cells were purified by negative selection from the spleens alyzed by immunoblot analysis. 6 PRDM1 REGULATES NK EFFECTOR CYTOKINE PRODUCTION Downloaded from

FIGURE 5. Perforin-mediated cytotoxicity is not affected by PRDM1 knockdown (KD). A, Four-hour [51Cr]-release assays were performed in triplicate from two donors. Purified NK cells were incubated for 72 h in the presence of an NT or PRDM1-specific (knockdown) siRNA and stimulated with http://www.jimmunol.org/ combinations of IL-2 (100 U/ml) and IL-12 (10 ng/ml), with or without IL-18 (100 ng/ml). The percentage of [51Cr] release was calculated as described in Materials and Methods. Error bars represent SD of triplicate wells. B, Western blot analysis to confirm KD in cells used in cytotoxicity assay. IL-2–only sample was not assayed in cytotoxicity assay and is only included to avoid cropping of gel. C, Purified NK cells were incubated for 48 h (upper panel)or72 h(lower panel) in the presence of an NT or PRDM1-specific (KD) siRNA. Cells were stimulated with combinations of IL-2 (100 U/ml), IL-12 (10 ng/ml), and IL-18 (100 ng/ml) and analyzed by Western blot for PRDM1 induction and PARP cleavage. cells. We observed PRDM1 binding at the IFN-g–inducible CII- robust PRDM1 binding was detected in this proximal promoter TApIV promoter but not the lymphoid-specific CIITApIII pro- region, which has not been identified in T cells. We did not detect moter. Although human NK cells can increase surface MHC class binding at CNS 26 or 18–20, neither of which bears potential by guest on September 27, 2021 II expression in response to activation (35), they express low to PRDM1-binding sites. Collectively, these data demonstrate that undetectable levels of CIITA. Binding of PRDM1 to the CIITApIV PRDM1 binds proximally and distally to multiple sites across the promoter may reinforce this repressed state and prevent IFN-g– IFNG locus in human NK cells. mediated autocrine induction. We did not detect binding at the TNF is coordinately induced with IFNG upon cytokine stimu- promoters of PAX5 and IFNB, both of which were shown to be lation of NK cells. Thus, we sought to determine whether this directly regulated via binding of PRDM1 to promoters in B cells locus was also bound by PRDM1. To this end, we assayed four and osteosarcoma cell lines, respectively. Furthermore, we did not distinct locations across the ∼12-kb TNF locus by ChIP. The TNF detect binding at the promoter of SLAMF7, which encodes the locus contains three genes, each of which contains four exons NK-activating receptor CD2-like receptor activating cytotoxic (Fig. 6C). LTA and TNF are separated by ∼1 kb and are tran- cell and contains a potential PRDM1-binding motif within its scribed from the same strand, whereas LTB is transcribed from the proximal promoter. This indicates that PRDM1 binds target-gene opposite strand and is separated from TNF by ∼3 kb. We measured promoters in a selective and cell type-specific manner. As expec- PRDM1 binding at the proximal promoter regions of LTB and ted, no binding was detected using primers to the second exon of TNF, the intergenic region between TNF and LTB, and a recently myoglobin B, which was used as a negative control. identified regulatory site located ∼3.5 kb upstream of LTA. This In murine T cells, Blimp-1 was recently shown to bind to a distal upstream enhancer element coordinately regulates the LTA and conserved regulatory site within the Ifng locus in in vitro polarized TNF genes but not the opposing LTB gene (37, 38). PRDM1 binding CD4+ Th1 lymphocytes (25). Because NK cells are potent pro- was clearly detected at this upstream regulatory site but not at either ducers of IFN-g during the early innate immune response, and proximal promoter or the intergenic region. Furthermore, bio- induction is correlated with PRDM1 expression, we sought to informatic analysis detected a consensus PRDM1-recognition se- evaluate PRDM1 binding to the human IFNG locus in cytokine- quence in the bound enhancer element. These data indicate that stimulated primary NK cells. We assayed four locations across the PRDM1 specifically associates with defined regulatory sequences IFNG locus, which are highly conserved between rodents and hu- of the TNF locus. mans and were demonstrated to regulate IFNG expression (36). We detected binding at the distal regulatory site, which has been de- Blockade of cytokine-induced PRDM1 expression increases marcated as CNS 222 based on the genomic distances relative to effector cytokine production the transcriptional start site in the mouse (Fig. 6B). This site is Our experiments demonstrated that PRDM1 is coordinately in- analogous to the PRDM1-binding site detected in T cells. Addi- duced with effector cytokines upon stimulation and occupies tionally, through bioinformatic analysis, we identified two potential specific regulatory regions within the IFNG and TNF loci. To in- PRDM1-binding sites within the minimal promoter located 370 vestigate the functional effects of PRDM1 in NK cells, we per- and 254 bp upstream of the IFNG transcriptional start site. Very formed gene-expression knockdown (KD) experiments using pri- The Journal of Immunology 7

mary human NK cells. Cells were stimulated with IL-2 and IL-12 in the presence of an NT control or PRDM1-specific siRNA. mRNA was isolated after 48 h and analyzed by real-time quanti- tative RT-PCR. We found significantly higher mRNA levels of IFNG, TNF, and LTA when PRDM1 induction was abrogated via siRNA, yet no significant differences were found for DAP10 (Fig. 7A). We next sought to determine whether these increases corresponded with detectable changes in protein expression. Se- creted IFN-g and TNF-a were measured by ELISA, and consistent increases in secreted protein were detectable in multiple donors in response to PRDM1 expression KD (Fig. 7B). Consistent with this silencing, PRDM1 expression remained elevated over a 4-d post- stimulation time course, whereas IFNG and TNF levels declined (Supplemental Fig. 3). Together, these data indicate that PRDM1 negatively regulates production of these cytokines in response to NK cell activation. Overexpression of PRDM1 mediates repression of activation-induced expression of IFNG and TNF Downloaded from Because we observed occupancy of PRDM1 at effector cytokine loci and increased production in the context of PRDM1 KD, we wanted to assess whether PRDM1 was capable of blocking acti- vation-induced transcription of IFNG and TNF. The Jurkat T cell line was transduced with adenovirus expressing GFP alone or GFP

and PRDM1a prior to stimulation. After PMA/PHA stimulation, http://www.jimmunol.org/ quantitative RT-PCR analysis was performed to assess induction of IFNG and TNF. GFP-transduced cells showed significant up- regulation of IFNG and TNF upon stimulation, which was nearly by guest on September 27, 2021

FIGURE 6. PRDM1 binds promoters in multiple target genes. A, ChIP FIGURE 7. Blockade of cytokine-induced PRDM1 expression increases experiments were performed with chromatin isolated from purified NK effector cytokine production. A, Purified NK cells were incubated for 48 h cells stimulated for 24 h with IL-2 (100 U/ml), IL-12 (10 ng/ml), and IL-18 in the presence of an NT or PRDM1-specific (KD) siRNA and stimulated (100 ng/ml). Induction of PRDM1 was confirmed by Western blot. Percent with IL-2 (100 U/ml) and IL-12 (10 ng/ml). RT-PCR was conducted for input was calculated as described in Materials and Methods. Error bars several genes on cDNA synthesized from RNA isolated from three donors represent SD from three biologically independent experiments performed at different times using 18S as control gene. For each donor, NT was at different times with chromatin preparations from different donors. ChIP normalized to “1.” Data are presented as mean, with error bars representing was performed across the IFNG (B)orTNF (C) loci. Locations of primers SE from three biologically independent experiments. B, ELISA was con- and genes in each locus are shown in diagrams below each graph. Note that ducted on supernatants harvested at 48 h from samples above. Data rep- scales are different for each locus. pp = 0.035; ppp = 0.0009; two-tailed t resent mean of three independent wells. For each analyte, two-tailed paired tests between IgG- and PRDM1-immunoprecipitated samples. t tests demonstrated p , 0.05 among all three donors for NT versus KD. 8 PRDM1 REGULATES NK EFFECTOR CYTOKINE PRODUCTION abolished in PRDM1a-transduced cells (Fig. 8A). Thus, intro- This deletion eliminated ability of PRDM1 to repress transcription duction of PRDM1 prior to stimulation was sufficient to block from the construct. This repression was similarly eliminated by stimulation-induced transcription of IFNG and TNF, providing point mutations at the 2254 site (mutIFNG-pGL3), demonstrating further support that PRDM1 is a negative regulator of effector that this location is critical for PRDM1-mediated repression. Col- cytokine production. lectively, these data demonstrate PRDM1-mediated repression of IFNG through binding to this previously uncharacterized motif. PRDM1 mediates repression of IFNG via elements in the proximal promoter Discussion To directly assess the functionality of PRDM1 binding to the newly In this report, we provide the functional description of PRDM1 in identified elements within the proximal promoter of the IFNG NK cells. We showed that PRDM1 accumulates upon cytokine- gene, we cloned the region containing 2507 to +121 of the human mediated activation and acts as a negative regulator of activation, IFNG gene and inserted it upstream of a luciferase reporter. This attenuating inflammatory cytokine production. Such a negative promoter region contains the two potential PRDM1 binding sites feedback loop has important implications in the context of inflam- located at 2370 and –254. Cotransfection of PRDM1 was suffi- mation and immune homeostasis. cient to repress luciferase activity driven by the wild-type IFNG Using global gene-expression profiling, we identified PRDM1 promoter by ~50% (Fig. 8B). To assess the relative contributions as a highly expressed transcription factor in stimulated NK cells. of the two potential PRDM1-binding motifs, a deletion of ~300 Our data complement previous studies (39–42), providing a global bp encompassing both of these sites (DIFNG-pGL3) was created. gene-expression profile of NK cells using a physiologically rele-

vant combination of cytokines to achieve activation. During the Downloaded from early phases of the innate-immune response, IL-12 and IL-18 are produced by monocytes and epithelial cells, whereas IL-2 is pri- marily produced by activated T cells, typically a later event. An- other early monocyte-derived cytokine, IL-15, signals through the shared IL-2R and can substitute for IL-2 to induce PRDM1 in

the presence of IL-12 and IL-18 (M.A. Smith and K.L. Wright, http://www.jimmunol.org/ unpublished observations). Consistent with this, maximal induction of murine NK cells in response to Salmonella-infected macro- phages was recently shown to require IL-2 and/or IL-15, macro- phage-derived IL-12 and IL-18, and direct NK cell–macrophage contact (6). Thus, the exposure of freshly isolated NK cells to these cytokines ex vivo can mimic the milieu present in vivo during the early innate-immune response and can provide insight into the physiological gene expression changes that are occurring. The ability of NK cells to produce a variety of cytokines in by guest on September 27, 2021 response to stimulation provides a crucial mechanism of cross- regulation between the innate and adaptive arms of the immune system. To maintain homeostasis, regulatory control must be exertedupontheactivationphase,aswellasduringtherecoveryphase to restrain the degree of activation. Although activation events have been thoroughly investigated, molecular events regulating damp- ening or recovery remain poorly characterized. This study now shows that PRDM1 is an important mediator of this phase. PRDM1 attenuates production of multiple effector cytokines in a coordinate manner via direct binding to specific DNA sequences in known regulatory regions. At the TNF locus, PRDM1 binds specifically to a highly conserved regulatory element ∼3.5 kb upstream of the transcriptional start site of LTA. This region exhibits DNaseI hy- persensitivity and contains binding sites for inflammatory activators, such as NF-kB and NFAT (37). Furthermore, this site was shown to be crucial for activation-induced looping (38). This three- dimensional looped chromatin structure mediates interaction be- tween distally and proximally bound NFAT at the TNF promoter, forming an enhanceosome resulting in transcriptional activation of TNF and, to a lesser extent, LTA in response to stimulation. Con- sistent with the notion that this region imparts specific localized FIGURE 8. Ectopic expression of PRDM1 diminishes expression of control at the TNF locus, KD of PRDM1 results in increases in LTA target genes. A, Jurkat cells were transduced for 40 h and then stimulated and TNF, but not LTB, which is located further downstream and is with PMA (50 ng/ml) and PHA (1 mg/ml) for 4 h prior to RNA isolation. encoded on the opposite DNA strand. After correction using 18S, relative expression for IFNG and TNF were The IFNG locus is also subject to PRDM1-mediated regulation determined by assigning a value of 100% to the stimulated GFP-trans- duced treatment. Data are mean of three independent experiments; error in NK cells. Studies in the T cell compartment identified an up- + bars represent SD. pp = 0.0002; ppp = 0.0005. B, Luciferase assays were stream regulatory region in murine CD4 T cells (CNS-22) with conducted in Jurkat cells 36 h posttransfection. Data are mean 6 SD of enhancer activity that contributed to Tbet-dependent Ifng expres- four independent transfections, with each reporter transfected with empty sion in in vitro polarized Th1 cells (36). This site was later shown vector set to 100%. pp=0.005 for wild-type; two-tailed paired t tests. to be bound by the murine homolog Blimp1 (25). In our study, we The Journal of Immunology 9 confirmed the functionality of this distal site in human NK cells. activity might provide a cytoprotective effect, whereby NK cells In addition to this distal element, we identified a promoter-pro- are protected from the antiproliferative and apoptotic effect of ximal element within the IFNG promoter to which PRDM1 binds. PRDM1 but retain the ability to repress selective target genes. This proximal site is required for silencing of a reporter gene driven Characterization of the contribution of each isoform is an im- by the human IFNG promoter. Freshly isolated resting NK cells portant area of future investigation. differ substantially from T cells, with respect to the kinetics of The PRDM1 allele is encoded on chromosome 6 within a region IFNG mRNA induction. Indeed, NK cells induce IFNG rapidly that is known to be frequently associated with B, T, and NK cell- upon stimulation without the requirement for chromatin mod- derived malignancies. A recent report used high-resolution com- ifications, because the entire locus is poised in a hyperacetylated parative genomic-hybridization arrays to precisely map regions state under basal conditions (43). Thus, PRDM1 binding directly deleted within NK lymphomas and identified a minimal common upstream of the transcriptional start site may facilitate potent si- region spanning ~2 Mb of the 6q21 deletion, which is present in lencing in NK cells, without necessarily requiring long-range ∼40% of cases (49). Within this region, the investigators identified chromatin modifications. three genes that were downregulated in tumor specimens harbor- Human NK cells exist in at least two functionally divergent ing the 6q21 deletion: ATG5, PRDM1, and AIM1. More recently, subsets based on the surface density of CD56 and CD16. Several PRDM1-expression levels were shown to be highly variable and groups demonstrated that CD56dimCD16+ NK cells are the major were found to be independent of the 6q21 deletion in an inde- population in peripheral blood, whereas CD56brightCD162/dim NK pendent study of six primary NK lymphoma patient samples (50). cells represent ,10% of peripheral NK cells and primarily lo- Our data clearly establish a functional role for PRDM1 in nor- calize to lymph nodes (44). The CD56bright population has been mal NK cell function. Thus, although PRDM1 can act as a tumor Downloaded from described as a regulator population. These cells express the high- suppressor in diffuse large B cell lymphoma, its contribution to affinity IL-2R, are highly proliferative in response to stimuli, and the transformed phenotype in NK-derived malignancies remains localize at inflammatory sites. Accumulating evidence suggests controversial. that CD56bright cells may be developmental precursors to the more Our studies add NK cells to the expanding list of immune lineages mature CD56dim effector population (45, 46). Conversely, the in which PRDM1 has a key functional role. PRDM1 has been dim CD56 effector population exhibits increased natural cytotoxicity, described in plasma cells, multiple T cell subsets, dendritic cells, http://www.jimmunol.org/ but it produces lower levels of inflammatory cytokines relative to the and myeloid cells. Although lineage-specific regulation of target CD56bright population. Consistent with its role as a negative regu- genes exists, a significant commonality is that PRDM1 regulates lator of cytokine production, we observed that PRDM1 is prefer- final effector function. For instance, negative regulation of Il2 and entially expressed in the CD56dim population. Thus, PRDM1- tbx21 by Blimp1 promotes Th2 polarization in CD4+ T cell dif- mediated transcriptional repression likely contributes to the func- ferentiation, yet the differential expression of these genes does not tional divergence observed in these populations. play a role in B or NK cell effector function. In contrast, coordinate NK cells exhibit a unique pattern of PRDM1 protein isoform ex- regulation of IFNG and TNF is critical to T and NK cell effector pression. In addition to the full-length PRDM1a, NK cells express function. Characterization of overlapping and nonoverlapping fun- high levels of two smaller molecular mass species. Expression of the ctions of PRDM1 across multiple lineages will be an important by guest on September 27, 2021 PRDM1b isoform was documented only in myeloma cell lines and area of focus in future experiments. For example, Lanier and col- samples from myeloma and T cell lymphoma patients, and it is al- leagues (51) recently provided evidence for a memory phenotype ways present at lower levels than is the full-length protein (32, 47). in murine NK cells using a murine CMV infection model wherein NK cells represent the first nontransformed cell type with PRDM1b they demonstrated that Ag-specific Ly49H+ NK cells underwent expression; furthermore, it is consistently observed at higher levels a prolonged contraction period but were subsequently activated to than is the larger PRDM1a.Theb isoform is transcribed from an higher levels upon restimulation. PRDM1-mediated restriction of alternate promoter and uses a distinct transcriptional start site present memory potential was recently described in CD8+ T cells (26). in the third intron of the full-length protein. The resulting protein has Given that PRDM1 is induced in vivo during NK activation, it will a disruption of the highly conserved PR domain. We previously be interesting to ascertain whether it has such a role in NK memory showed that the b isoform localizes to the nucleus and maintains the commitment. capacity for DNA binding, although its repressive activity is damp- In summary, our data suggest that PRDM1 plays a crucial role in ened relative to PRDM1a. All of the cytokine combinations tested the postactivation phenotype of NK cells by negatively regulating activated transcription of both isoforms in NK cells, although there cytokine transcription in a coordinate manner, without compro- remains the potential that isoform-specific activation signals may mising perforin-mediated cytotoxicity or inducing exit from the exist. We also consistently observed an intermediate-sized isoform cell cycle. Such a mechanism may have important implications in (PRDM1aD6) corresponding to a deletion of the amino acids en- innate immunity and tumor surveillance. coded by exon 6. This isoform is generated via splicing to exclude exon 6, resulting in deletion of the second zinc finger, as well as Acknowledgments disruption of a portion of the first and third zinc fingers. An analogous We thank the staff of the Microarray and Flow Cytometry Core Facilities at splice variant was recently described in naive CD19+ murine B cells, H. Lee Moffitt Cancer Center. We also thank Xiaolong Fang for providing although it was present at very low levels (33). The first two zinc recombinant adenoviral vectors and Jerome Ritz (Dana-Farber Cancer In- fingers of PRDM1 have a role in DNA binding and are required for stitute, Boston, MA) for providing the NKL cell line. recruitment of the histone methyltransferase G9a but are dis- Disclosures pensable for interaction with HDAC2 (16, 48). Interestingly, we The authors have no financial conflicts of interest. did not observe induction of apoptosis or evidence of cell cycle exit concomitant with PRDM1 induction in NK cells. Further- more, IL-2–expanded LAK cells continued to proliferate, despite References the acquisition of high-level PRDM1 expression (M.A. Smith and 1. Cooper, M. A., T. A. Fehniger, and M. A. Caligiuri. 2001. The biology of human natural killer-cell subsets. Trends Immunol. 22: 633–640. K.L. Wright, unpublished observations). Thus, the presence of 2. Carson, W. 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Supplemental Figure 1: GEP consistency between donors. Supplemental Figure 2: PCR verification of PRDM1exon6 mRNA structure.

- 2 - Supplemental Figure 3: Time Course of Cytokine mRNA and PRDM1 protein Levels

- 3 - Supplemental Table 1 3-fold or more up-regulated

Fold Change Fold Change Donor 1 Donor 2 GenBank ID Gene Symbol 206975_at 4213.85 1686.35 NM_000595.2 LTA 210229_s_at 3408.78 1649.57 NM_000758.2 CSF2 221111_at 1469.52 366.94 NM_018402.1 IL26 230102_at 599.90 102.30 NM_004454 ETV5 220118_at 584.03 542.01 NM_014383.1 ZBTB32 226731_at 514.15 40.95 NM_181501 ITGA1 223767_at 474.60 398.08 NM_020370.1 GPR84 219424_at 423.52 1281.90 NM_005755.2 EBI3 207536_s_at 342.48 236.24 NM_001561.4 TNFRSF9 210831_s_at 341.59 163.69 NM_000957.2 PTGER3 202912_at 291.09 50.16 NM_001124.1 ADM 206341_at 277.84 521.07 NM_000417.1 IL2RA 225316_at 263.00 333.65 NM_032793.2 MFSD2 227860_at 170.62 115.99 NM_019609.3 CPXM 219911_s_at 155.44 195.27 NM_016354.3 SLCO4A1 205542_at 155.05 267.78 NM_012449.2 STEAP1 210118_s_at 124.38 46.46 NM_000575.3 IL1A 207426_s_at 88.03 66.71 NM_003326.2 TNFSF4 209324_s_at 78.17 47.52 NM_002928.2 RGS16 217475_s_at 75.83 33.30 NM_005569.3 LIMK2 205249_at 73.62 41.59 NM_000399.2 EGR2 224461_s_at 64.64 21.87 NM_032797.4 AMID 210448_s_at 62.17 67.73 NM_002561.2 P2RX5 207113_s_at 58.77 69.20 NM_000594.2 TNF 216841_s_at 51.40 52.60 NM_000636.2 SOD2 204014_at 46.25 15.56 NM_001394.5 DUSP4 202437_s_at 46.17 29.11 NM_000104.2 CYP1B1 223062_s_at 45.92 48.09 NM_058179.2 PSAT1 201631_s_at 43.65 27.35 NM_003897.2 IER3 227458_at 42.63 35.12 NM_014143 PDCD1L1 217997_at 42.11 55.78 NM_007350.3 PHLDA1 204533_at 41.79 49.03 NM_001565.1 CXCL10 205768_s_at 40.86 73.53 NM_003645.2 SLC27A2 210354_at 40.70 54.75 NM_000619.2 IFNG 203716_s_at 39.54 192.36 NM_001935.3 DPP4 204032_at 39.43 26.58 NM_003567.2 BCAR3 223383_at 39.22 367.50 NM_032268.3 ZNRF1 210439_at 37.75 73.53 NM_012092.2 ICOS 224799_at 36.85 38.40 NM_019080.1 NDFIP2 205098_at 35.14 49.46 NM_001295.2 CCR1 218266_s_at 34.85 75.35 NM_014286.2 FREQ 208949_s_at 34.52 95.22 NM_002306.1 LGALS3 202431_s_at 34.44 83.94 NM_002467.3 MYC 206486_at 33.52 115.13 NM_002286.4 LAG3 230372_at 33.46 24.53 NM_005328 HAS2 214329_x_at 31.81 18.73 NM_003810 TNFSF10 218888_s_at 31.27 121.02 NM_018092.3 NETO2 225520_at 30.18 44.72 NM_015440.3 MTHFD1L 223454_at 29.30 21.28 NM_022059.1 CXCL16 235965_at 28.77 12.11 NM_015395 DKFZP434B0335 206181_at 26.97 37.54 NM_003037.1 SLAMF1 202934_at 26.52 308.10 NM_000189.4 HK2 204798_at 25.75 76.39 NM_005375.2 MYB 212292_at 25.37 11.93 NM_003045.3 SLC7A1 202609_at 25.34 23.46 NM_004447.4 EPS8 202637_s_at 25.33 17.83 NM_000201.1 ICAM1 227262_at 24.50 28.82 NM_178232.2 HAPLN3 223834_at 23.81 33.84 NM_014143.2 CD274 200951_s_at 22.92 53.94 NM_001759.2 CCND2 202734_at 22.15 15.37 NM_004240.2 TRIP10 202270_at 21.93 11.57 NM_002053.1 GBP1 206991_s_at 21.92 103.31 NM_000579.1 CCR5 219270_at 21.68 76.67 NM_024111.2 CHAC1 223961_s_at 21.49 22.20 NM_145071.1 CISH 203821_at 21.49 36.67 NM_001945.1 HBEGF 213256_at 20.26 14.23 NM_178450.2 MAR3 209765_at 20.10 28.50 NM_033274.2 ADAM19 204440_at 19.69 17.28 NM_001040280.1 CD83 202284_s_at 19.47 16.82 NM_078467.1 CDKN1A 214095_at 17.37 15.85 NM_005412.4 SHMT2 204702_s_at 17.29 28.07 NM_004289.5 NFE2L3 201560_at 17.11 37.76 NM_013943.1 CLIC4 208916_at 17.07 19.14 NM_005628.1 SLC1A5 1555167_s_at 17.07 22.37 NM_005746.1 PBEF1 209636_at 16.62 16.11 NM_002502.2 NFKB2 242943_at 16.42 20.24 NM_005668.3 ST8SIA4 205393_s_at 16.23 19.54 NM_001274.2 CHEK1 219632_s_at 15.80 12.81 NM_018727.4 TRPV1 201489_at 15.79 20.03 NM_005729.3 PPIF 203967_at 15.30 45.70 NM_001254.3 CDC6 242504_at 15.21 19.43 NM_024492 APOARGC 210847_x_at 15.07 25.37 NM_003790.2 TNFRSF25 204466_s_at 15.03 16.34 NM_007308.1 SNCA 243000_at 14.92 11.82 NM_001259.5 CDK6 217738_at 14.56 15.29 NM_005746 PBEF 207229_at 13.60 14.35 NM_006611.1 KLRA1 203971_at 13.53 23.32 NM_001859.2 SLC31A1 200628_s_at 13.50 17.73 NM_004184.3 WARS 205965_at 13.13 25.02 NM_006399.2 BATF 223851_s_at 12.99 30.16 NM_004195.2 TNFRSF18 201014_s_at 12.98 20.04 NM_006452.2 PAICS 207651_at 12.97 9.86 NM_013308.2 GPR171 223225_s_at 12.93 12.70 NM_031216.3 SEH1L 212859_x_at 12.83 13.60 NM_175617.3 MT1E 217599_s_at 12.65 12.46 NM_199072.2 MDFIC 210001_s_at 12.63 13.67 NM_003745.1 SOCS1 210190_at 12.62 20.83 NM_003764.2 STX11 223229_at 12.22 16.57 NM_014176.1 UBE2T 209457_at 11.83 12.86 NM_004419.3 DUSP5 205599_at 11.81 12.46 NM_005658.3 TRAF1 51146_at 11.75 7.20 NM_017837.2 PIGV 218826_at 11.72 5.32 NM_017515.3 SLC35F2 205681_at 11.63 18.34 NM_004049.2 BCL2A1 219159_s_at 11.63 10.54 NM_021181.3 SLAMF7 234976_x_at 11.39 16.51 NM_006636 MTHFD2 201195_s_at 11.39 4.96 NM_003486.5 SLC7A5 210458_s_at 11.35 9.73 NM_133484.1 TANK 231779_at 11.25 11.46 NM_001570.3 IRAK2 1568955_at 10.82 5.31 BE877775 FNBP2 210571_s_at 10.71 10.44 XR_000114 CMAH 212365_at 10.64 9.79 NM_012223.2 MYO1B 223217_s_at 10.46 12.69 NM_031419.2 NFKBIZ 235117_at 10.34 9.95 NM_001008708.1 CHAC2 237759_at 10.19 11.91 NM_001778 CD48 213497_at 10.11 7.82 NM_145804.1 ABTB2 202236_s_at 10.04 11.25 NM_003051.2 SLC16A1 219493_at 10.02 7.51 NM_024745.2 SHCBP1 209715_at 9.85 6.44 NM_012117.1 CBX5 215464_s_at 9.79 6.32 NM_014604.2 TAX1BP3 1569003_at 9.78 8.61 BC024020.1 TMEM49 214228_x_at 9.74 6.40 NM_003327.2 TNFRSF4 203612_at 9.70 11.66 NM_004053.3 BYSL 204947_at 9.69 17.76 NM_005225.1 E2F1 206235_at 9.68 12.71 NM_002312.3 LIG4 226354_at 9.65 4.66 NM_032857.2 LACTB 206665_s_at 9.63 4.94 NM_001191.2 BCL2L1 1553723_at 9.61 3.27 NM_170776.3 GPR97 212561_at 9.45 11.14 NM_015213.2 RAB6IP1 210512_s_at 9.37 13.49 NM_001025366.1 VEGF 213113_s_at 9.29 12.38 NM_014096.2 SLC43A3 213146_at 9.16 7.55 XM_043272.10 JMJD3 206039_at 9.14 10.09 NM_004794.2 RAB33A 224917_at 9.10 10.47 BC053563.1 MIRN21 201577_at 9.00 13.37 NM_000269.2 NME1 219753_at 8.94 10.72 NM_012447.2 STAG3 201251_at 8.93 14.98 NM_182471.1 PKM2 206999_at 8.93 13.30 NM_001559.2 IL12RB2 209304_x_at 8.55 6.59 NM_015675.2 GADD45B 207375_s_at 8.54 8.37 NM_002189.2 IL15RA 225775_at 8.53 41.50 NM_178562.2 TSPAN33 223620_at 8.51 10.67 NM_001033514.1 GPR34 201160_s_at 8.50 21.34 NM_003651.3 CSDA 218145_at 8.49 6.03 NM_021158.3 TRIB3 209239_at 8.46 8.40 NM_003998.2 NFKB1 204057_at 8.42 8.99 NM_002163.2 IRF8 203554_x_at 8.37 10.46 NM_004219.2 PTTG1 204269_at 8.37 12.96 NM_006875.2 PIM2 212641_at 8.37 5.55 NM_006734 HIVEP2 230233_at 8.28 3.66 NM_152545 GPIG4 213056_at 8.19 15.90 XM_114303.3 FRMD4B 200759_x_at 8.17 12.19 NM_003204.1 NFE2L1 223984_s_at 8.09 8.53 NM_001008564.1 NUPL1 209318_x_at 8.06 4.94 NM_002656.2 PLAGL1 202071_at 8.05 8.81 NM_002999.2 SDC4 202307_s_at 7.88 6.08 NM_000593.5 TAP1 229236_s_at 7.84 4.60 NM_178867.3 SFXN4 1555638_a_at 7.75 20.39 NM_022136.3 SAMSN1 205443_at 7.73 5.82 NM_003082.2 SNAPC1 204068_at 7.69 15.43 NM_006281 STK3 205013_s_at 7.68 10.65 NM_000675.3 ADORA2A 222532_at 7.67 8.07 NM_021203.2 SRPRB 228868_x_at 7.61 17.38 NM_030928 CDT1 204027_s_at 7.59 7.15 NM_005371.3 METTL1 209610_s_at 7.59 3.09 NM_003038.2 SLC1A4 223490_s_at 7.49 6.64 NM_016042.2 EXOSC3 206461_x_at 7.42 4.69 NM_005951.1 MT1H 225655_at 7.41 17.20 NM_013282.2 UHRF1 201111_at 7.37 8.23 NM_001316.2 CSE1L 1556820_a_at 7.37 5.59 NM_006021 DLEU2 227027_at 7.37 5.86 NM_002056 GFPT1 218854_at 7.32 18.23 NM_013352.1 SART2 212009_s_at 7.30 9.08 NM_006819 STIP1 202464_s_at 7.14 4.36 NM_004566.2 PFKFB3 211333_s_at 7.13 8.82 NM_000639.1 FASLG 214155_s_at 7.07 4.79 NM_052879.3 LARP4 206365_at 7.03 6.27 NM_002995.1 XCL1 219433_at 7.02 8.17 NM_017745.4 BCOR 218254_s_at 7.00 5.98 NM_001033503.1 SAR1B 202375_at 6.92 6.51 NM_014822.1 SEC24D 200799_at 6.87 6.51 NM_005345.4 HSPA1A 224615_x_at 6.82 5.12 NM_030789.2 HM13 204745_x_at 6.82 4.97 NM_005950.1 MT1G 201263_at 6.82 8.06 NM_152295.3 TARS 223103_at 6.80 3.41 NM_006645.1 STARD10 226448_at 6.79 4.66 NM_198552.1 FAM89A 224285_at 6.74 7.61 NM_032553.1 GPR174 228167_at 6.71 6.25 NM_130446 KLHL6 203293_s_at 6.62 7.14 NM_005570.2 LMAN1 218189_s_at 6.58 6.70 NM_018946.2 NANS 222500_at 6.52 7.15 NM_016059.3 PPIL1 202345_s_at 6.51 9.34 NM_001444.1 FABP5 207237_at 6.48 5.87 NM_002232.3 KCNA3 207339_s_at 6.46 4.67 NM_009588.1 LTB 202043_s_at 6.43 7.35 NM_004595.2 SMS 213198_at 6.41 6.05 NM_004302.3 ACVR1B 210279_at 6.39 6.34 NM_005292.2 GPR18 209193_at 6.37 8.94 NM_002648.2 PIM1 204716_at 6.36 6.55 NM_005436.2 CCDC6 1564520_s_at 6.35 7.64 NM_001039619.1 SKB1 218585_s_at 6.34 15.50 NM_016448.1 DTL 240338_at 6.29 3.78 NM_022350 LRAP 203139_at 6.29 4.19 NM_004938.2 DAPK1 224827_at 6.27 3.75 NM_152277 DC-UbP 203562_at 6.26 3.43 NM_005103.3 FEZ1 218392_x_at 6.24 5.18 NM_022754.4 SFXN1 201619_at 6.19 9.31 NM_014098.2 PRDX3 213427_at 6.15 14.27 NM_006638.2 RPP40 205205_at 6.14 3.64 NM_006509.2 RELB 202710_at 6.08 5.51 NM_005868.4 BET1 203010_at 6.06 5.74 NM_003152.2 STAT5A 217834_s_at 6.02 7.91 AB209098.1 SYNCRIP 210538_s_at 6.02 4.04 NM_182962.1 BIRC3 222981_s_at 5.97 8.22 NM_016131.2 RAB10 210117_at 5.91 3.42 NM_003114.3 SPAG1 225439_at 5.90 7.75 NM_032869.2 NUDCD1 1558102_at 5.86 4.88 NM_023003 TM6SF1 209218_at 5.83 8.21 NM_003129.3 SQLE 208361_s_at 5.80 8.83 NM_001722.2 POLR3D 214062_x_at 5.77 3.68 NM_001001716.1 NFKBIB 1554572_a_at 5.77 6.04 NM_024670.3 SUV39H2 208581_x_at 5.75 4.05 NM_005952.2 MT1X 218739_at 5.71 3.98 NM_016006.3 ABHD5 204523_at 5.70 3.73 NM_003440.2 ZNF140 1555628_a_at 5.64 5.93 NM_032782.3 HAVCR2 228092_at 5.62 5.01 NM_001881.2 CREM 215501_s_at 5.56 3.69 NM_007207.3 DUSP10 208967_s_at 5.51 6.87 NM_001625.2 AK2 214211_at 5.51 4.11 NM_002032.2 FTH1 1557905_s_at 5.51 3.59 NM_000610.3 CD44 203344_s_at 5.50 6.08 NM_002894.2 RBBP8 217299_s_at 5.49 3.39 NM_001024688.1 NBN 212141_at 5.46 5.41 NM_005914.2 MCM4 219201_s_at 5.45 13.64 NM_020648.3 TWSG1 212110_at 5.44 7.11 NM_015359.1 SLC39A14 202014_at 5.37 5.37 NM_014330.2 PPP1R15A 214706_at 5.37 3.25 NM_198087.1 ZNF200 218404_at 5.37 5.68 NM_013322.2 SNX10 201548_s_at 5.35 4.99 NM_006618.3 JARID1B 203033_x_at 5.35 7.29 NM_000143.2 FH 201714_at 5.31 6.11 NM_001070.3 TUBG1 227697_at 5.31 4.50 NM_003955.3 SOCS3 219257_s_at 5.30 8.41 NM_182965.1 SPHK1 216952_s_at 5.28 4.89 NM_032737.2 LMNB2 219920_s_at 5.27 12.65 NM_021971.1 GMPPB 226150_at 5.23 5.01 NM_032483 HTPAP 208813_at 5.23 6.28 NM_002079.1 GOT1 222262_s_at 5.21 5.13 NM_018638.4 ETNK1 207275_s_at 5.18 5.14 NM_001995.2 ACSL1 201144_s_at 5.18 5.17 NM_004094.4 EIF2S1 208748_s_at 5.17 8.78 NM_005803.2 FLOT1 209366_x_at 5.09 4.80 NM_001914.2 CYB5 202205_at 5.08 7.37 NM_003370.3 VASP 202069_s_at 5.07 5.40 NM_005530.2 IDH3A 202748_at 5.06 3.22 NM_004120.3 GBP2 209272_at 5.05 5.00 NM_005966.3 NAB1 203957_at 5.04 6.51 NM_198258.1 E2F6 228490_at 5.04 4.99 NM_007011.5 ABHD2 207785_s_at 5.03 3.89 AK056944.1 RBPSUH 227737_at 5.02 6.88 NM_021203 APMCF1 206034_at 5.00 5.52 NM_198833.1 SERPINB8 224953_at 4.99 3.96 NM_030799.6 YIPF5 205691_at 4.98 3.58 NM_004209.4 SYNGR3 201930_at 4.98 5.27 NM_005915.4 MCM6 221649_s_at 4.96 6.81 NM_020230.4 PPAN 216252_x_at 4.96 15.10 NM_152874.1 FAS 202908_at 4.95 3.12 NM_006005.2 WFS1 214039_s_at 4.95 3.36 NM_018407.4 LAPTM4B 209572_s_at 4.95 4.74 NM_152991.1 EED 242338_at 4.93 4.98 NM_001008495.1 TMEM64 204858_s_at 4.92 7.98 NM_001953.2 ECGF1 218472_s_at 4.90 4.68 NM_015946.4 PELO 227120_at 4.89 3.50 NM_001012427.1 FOXP4 213012_at 4.88 3.39 NM_006154.1 NEDD4 202138_x_at 4.86 4.07 NM_006303 JTV1 207431_s_at 4.86 3.74 NM_003676.2 DEGS1 204012_s_at 4.86 3.89 NM_014793.3 LCMT2 222734_at 4.81 5.67 NM_015836.2 WARS2 209026_x_at 4.81 6.95 NM_178014.2 TUBB 203598_s_at 4.80 3.45 NM_007187.3 WBP4 212378_at 4.78 9.29 NM_000819.3 GART 202557_at 4.77 3.87 NM_006948.4 STCH 227423_at 4.75 3.17 NM_144598.2 LRRC28 226372_at 4.71 3.45 NM_018413 CHST11 229595_at 4.66 4.72 NM_144636.1 CHCHD4 36564_at 4.66 6.31 NM_153341.1 IBRDC3 203943_at 4.64 5.44 NM_004798.2 KIF3B 201118_at 4.64 7.90 NM_002631.2 PGD 214435_x_at 4.64 4.83 NM_005402.2 RALA 241937_s_at 4.63 6.37 NM_033661.3 WDR4 215165_x_at 4.62 3.72 NM_000373.1 UMPS 202246_s_at 4.62 9.03 NM_000075.2 CDK4 213133_s_at 4.61 3.55 NM_004483.3 GCSH 237215_s_at 4.58 7.93 NM_003234.1 TFRC 1553510_s_at 4.57 4.46 NM_005444.1 RQCD1 212978_at 4.56 3.15 NM_015350 TA-LRRP 203233_at 4.53 5.04 NM_000418.2 IL4R 228234_at 4.53 4.38 NM_021649.3 TICAM2 226897_s_at 4.51 3.10 NM_014153.2 ZC3H7A 64883_at 4.49 8.73 NM_152581 MOSPD2 201169_s_at 4.48 3.68 NM_003670.1 BHLHB2 208726_s_at 4.47 5.01 NM_003908.3 EIF2S2 200806_s_at 4.42 5.64 NM_002156.4 HSPD1 204121_at 4.42 7.11 NM_006705.2 GADD45G 222642_s_at 4.42 3.48 NM_018126.1 TMEM33 205419_at 4.42 26.53 NM_004951.3 EBI2 218264_at 4.40 5.86 NM_016567.2 BCCIP 218408_at 4.40 6.29 NM_012456.2 TIMM10 204459_at 4.38 5.34 NM_001325.2 CSTF2 225291_at 4.36 6.30 NM_033109.2 PNPT1 209567_at 4.36 4.67 NM_015169.3 RRS1 213671_s_at 4.35 4.07 NM_004990.2 MARS 203857_s_at 4.34 6.88 NM_006810.1 PDIA5 216212_s_at 4.34 4.23 NM_001363.2 DKC1 208864_s_at 4.33 4.38 NM_003329.2 TXN 212458_at 4.33 5.39 NM_181784.1 SPRED2 221485_at 4.32 3.97 NM_004776.2 B4GALT5 201096_s_at 4.31 5.07 NM_001660.2 ARF4 214649_s_at 4.30 3.08 NM_201278.1 MTMR2 201397_at 4.28 11.69 NM_006623.2 PHGDH 222867_s_at 4.27 3.79 NM_016060.1 MED31 223299_at 4.25 4.19 NM_033280.1 SEC11L3 205909_at 4.25 4.59 NM_002692.2 POLE2 226996_at 4.25 4.60 NM_001002257.1 LYCAT 212685_s_at 4.25 3.93 NM_012453.2 TBL2 217165_x_at 4.22 4.50 NM_005949.1 MT1F 204125_at 4.22 5.72 NM_016013.2 NDUFAF1 208799_at 4.21 5.69 NM_002797.2 PSMB5 224654_at 4.21 4.58 NM_004728 DDX21 201362_at 4.18 5.03 NM_006469.4 IVNS1ABP 225866_at 4.17 5.65 NM_032194.1 BXDC1 203102_s_at 4.17 3.78 NM_002408.3 MGAT2 224685_at 4.16 4.18 NM_001040001.1 MLLT4 1554696_s_at 4.15 9.03 NM_001071.1 TYMS 202671_s_at 4.14 3.51 NM_003681.3 PDXK 222385_x_at 4.14 3.64 NM_013336.3 SEC61A1 202107_s_at 4.14 5.30 NM_004526.2 MCM2 209662_at 4.13 5.11 NM_004365.2 CETN3 204744_s_at 4.13 5.91 NM_002161.3 IARS 212340_at 4.13 4.74 AK026979.1 YIPF6 231832_at 4.12 3.74 NM_003774.3 GALNT4 231291_at 4.12 4.49 NM_000164 GIPR 216602_s_at 4.12 4.78 NM_004461.1 FARSLA 223240_at 4.10 3.63 NM_012180.2 FBXO8 204767_s_at 4.09 3.83 NM_004111.4 FEN1 214483_s_at 4.06 3.19 NM_001025593.1 ARFIP1 1554351_a_at 4.06 4.01 NM_152902.3 TIPRL 205153_s_at 4.06 5.38 NM_001250.4 CD40 236345_at 4.05 3.67 NM_001061 TBXAS1 232160_s_at 4.04 4.78 NM_024309.2 TNIP2 208598_s_at 4.04 5.33 NM_031407.3 HUWE1 202446_s_at 4.04 3.25 NM_021105.1 PLSCR1 201000_at 4.04 6.00 NM_001605 AARS 207812_s_at 4.03 3.88 NM_015530.3 GORASP2 218512_at 4.01 8.30 NM_018256.2 WDR12 201328_at 4.01 6.36 NM_005239.4 ETS2 235463_s_at 3.99 4.50 NM_203463.1 LASS6 201739_at 3.98 10.84 NM_005627.2 SGK 215629_s_at 3.97 4.76 AF254117.1 BCMSUNL 226481_at 3.97 4.22 BC022792.1 HUMAGCGB 203145_at 3.97 5.96 NM_006461.3 SPAG5 202837_at 3.96 5.63 NM_006700.1 TRAFD1 219390_at 3.95 3.81 NM_017946.2 FKBP14 209971_x_at 3.94 3.47 BC002853.1 HRI 207315_at 3.93 6.78 NM_006566.1 CD226 202451_at 3.92 4.76 NM_005316.2 GTF2H1 218889_at 3.92 3.27 NM_022451.9 NOC3L 219869_s_at 3.91 4.78 NM_022154.5 SLC39A8 218890_x_at 3.90 5.52 NM_016622.2 MRPL35 201098_at 3.90 4.74 NM_004766.1 COPB2 218615_s_at 3.89 3.64 NM_018266.1 TMEM39A 222979_s_at 3.87 4.90 NM_033161.2 SURF4 203201_at 3.86 4.84 NM_000303.1 PMM2 218886_at 3.86 4.01 NM_017906.2 PAK1IP1 200924_s_at 3.86 3.70 NM_001012661.1 SLC3A2 203158_s_at 3.84 4.15 NM_014905.2 GLS 211953_s_at 3.83 3.11 NM_002271.4 RANBP5 204970_s_at 3.82 4.73 NM_032711.2 MAFG 201063_at 3.82 4.15 NM_002901.1 RCN1 217848_s_at 3.81 4.06 NM_021129.3 PPA1 240983_s_at 3.81 3.80 NM_139273.2 CARS 235175_at 3.79 3.72 NM_052941 GBP4 201252_at 3.79 5.16 NM_006503.2 PSMC4 208693_s_at 3.79 5.18 NM_002047.1 GARS 222753_s_at 3.79 4.15 NM_021928.1 SPCS3 239544_at 3.78 4.17 NM_015559 SETBP1 223596_at 3.78 4.15 NM_005135 SLC12A6 202223_at 3.76 5.05 NM_152713.2 ITM1 218670_at 3.75 5.93 NM_001002019.1 PUS1 201298_s_at 3.73 4.09 NM_018221.1 MOBK1B 226651_at 3.73 3.16 NM_004272 HOMER1 221253_s_at 3.72 6.86 NM_030810.2 TXNDC5 201657_at 3.72 3.25 NM_001177.3 ARL1 209822_s_at 3.72 8.16 NM_003383.3 VLDLR 208296_x_at 3.72 4.59 NM_014350.1 TNFAIP8 226537_at 3.72 3.64 NM_138571.3 HINT3 202195_s_at 3.71 3.57 NM_016040.3 TMED5 209276_s_at 3.70 6.53 NM_002064.1 GLRX 201583_s_at 3.69 5.39 NM_006363.4 SEC23B 218488_at 3.69 5.22 NM_020365.2 EIF2B3 203956_at 3.69 3.74 NM_014941.1 MORC2 226181_at 3.68 4.17 NM_016262.3 TUBE1 212296_at 3.67 6.10 NM_005805 PSMD14 218027_at 3.66 4.91 NM_014175.2 MRPL15 224634_at 3.66 4.03 NM_015590.2 GPATC4 208985_s_at 3.66 4.32 NM_003758.2 EIF3S1 203746_s_at 3.66 4.76 NM_005333.2 HCCS 200755_s_at 3.66 3.98 NM_001219.2 CALU 203358_s_at 3.65 3.65 NM_004456.3 EZH2 228964_at 3.64 3.34 NM_001198.2 PRDM1 202483_s_at 3.63 6.30 NM_002882.2 RANBP1 219631_at 3.63 5.32 NM_013437.2 LRP12 204133_at 3.63 5.80 NM_004704.3 RNU3IP2 205644_s_at 3.62 3.06 NM_003096.2 SNRPG 210983_s_at 3.62 5.53 NM_005916.3 MCM7 203120_at 3.62 3.56 NM_001031685.1 TP53BP2 213454_at 3.61 5.30 NM_199294.1 APITD1 222411_s_at 3.61 3.57 NM_007107.2 SSR3 225831_at 3.59 3.17 NM_033631.2 LUZP1 205796_at 3.59 4.70 NM_018393.2 TCP11L1 212185_x_at 3.58 3.80 NM_005953.2 MT2A 226760_at 3.58 3.49 AK056736.1 MBTPS2 218866_s_at 3.56 5.40 NM_016310.2 POLR3K 238581_at 3.55 3.88 NM_052942 GBP5 203880_at 3.55 4.22 NM_005694.1 COX17 202613_at 3.53 4.53 NM_001905.1 CTPS 205192_at 3.53 5.39 NM_003954.1 MAP3K14 223222_at 3.52 8.04 NM_021734.3 SLC25A19 212528_at 3.52 4.83 AI348009 D15Wsu75e 209501_at 3.50 3.30 NM_001802.1 CDR2 201079_at 3.50 4.27 NM_004710.3 SYNGR2 218982_s_at 3.50 3.89 NM_015969.2 MRPS17 226384_at 3.50 4.23 BC033025.1 PPAPDC1B 213144_at 3.49 3.93 NM_004287.3 GOSR2 222684_s_at 3.48 4.74 NM_024894.1 NOL10 203388_at 3.48 5.89 NM_004313.3 ARRB2 216392_s_at 3.48 4.95 NM_007190.2 SEC23IP 203471_s_at 3.48 6.47 NM_002664.1 PLEK 201025_at 3.47 3.18 NM_015904.3 EIF5B 202401_s_at 3.47 3.88 NM_003131.2 SRF 219275_at 3.46 5.31 NM_004708.2 PDCD5 202276_at 3.46 3.69 NM_006304.1 SHFM1 218239_s_at 3.46 3.10 NM_012341.2 GTPBP4 200881_s_at 3.45 3.34 NM_001539.2 DNAJA1 201326_at 3.44 5.21 NM_001009186.1 CCT6A 201946_s_at 3.43 3.59 NM_006431.2 CCT2 201512_s_at 3.43 4.02 NM_014820.3 TOMM70A 203225_s_at 3.43 3.05 NM_018339.3 RFK 205282_at 3.42 4.19 NM_017522.3 LRP8 213191_at 3.42 3.77 NM_182919.1 TICAM1 208680_at 3.42 5.17 NM_002574.2 PRDX1 223335_at 3.42 3.16 NM_016486.2 TMEM69 224428_s_at 3.42 8.92 NM_031942.4 CDCA7 204507_s_at 3.41 3.47 NM_000945.3 PPP3R1 217809_at 3.41 6.01 NM_014038.1 BZW2 219716_at 3.41 3.47 NM_030641.3 APOL6 217995_at 3.40 4.92 NM_021199.2 SQRDL 226241_s_at 3.40 4.34 NM_181304.1 MRPL52 226163_at 3.40 3.76 NM_152735.3 ZBTB9 229074_at 3.40 3.75 NM_139265 EHD4 202418_at 3.39 5.70 NM_020470.1 YIF1A 201176_s_at 3.38 3.86 NM_001655.3 ARCN1 226483_at 3.37 3.63 NM_152417.1 TMEM68 217850_at 3.37 4.23 NM_206826.1 GNL3 211804_s_at 3.37 3.60 NM_001798.2 CDK2 204441_s_at 3.36 6.45 NM_002689.2 POLA2 223100_s_at 3.36 4.94 NM_014142.2 NUDT5 204026_s_at 3.35 6.92 NM_001005413.1 ZWINT 207121_s_at 3.35 3.57 NM_002748.2 MAPK6 218590_at 3.34 4.43 NM_021830.3 PEO1 201872_s_at 3.33 3.92 NM_002940 ABCE1 200894_s_at 3.33 5.29 NM_002014.2 FKBP4 204240_s_at 3.32 4.10 NM_006444 SMC2L1 219819_s_at 3.31 4.62 NM_014018.2 MRPS28 236228_at 3.30 7.24 NM_054027 ANKH 202874_s_at 3.28 4.20 NM_001007254.1 ATP6V1C1 201532_at 3.27 3.72 NM_152132.1 PSMA3 1554020_at 3.27 3.07 NM_001714 BICD1 217919_s_at 3.26 3.51 NM_014050.2 MRPL42 203575_at 3.26 4.81 NM_001896.2 CSNK2A2 225351_at 3.26 3.15 NM_207009.2 FAM45A 206337_at 3.26 6.80 NM_001838.2 CCR7 201892_s_at 3.25 4.68 NM_000884.2 IMPDH2 202805_s_at 3.25 3.59 NM_019898.1 ABCC1 220140_s_at 3.25 5.17 NM_013323.2 SNX11 224414_s_at 3.25 3.76 NM_032587.3 CARD6 1554768_a_at 3.24 4.94 NM_002358.2 MAD2L1 200054_at 3.24 3.32 NM_003904.3 ZNF259 218461_at 3.24 5.21 NM_016301.2 ATPBD1C 225712_at 3.23 5.07 NM_015465 GEMIN5 201274_at 3.22 5.26 NM_002790.2 PSMA5 212218_s_at 3.22 4.59 NM_004104.4 FASN 208787_at 3.21 3.96 NM_007208.2 MRPL3 228019_s_at 3.20 4.91 NM_016067.1 MRPS18C 200989_at 3.20 3.58 NM_001530.2 HIF1A 216942_s_at 3.19 3.53 NM_001779.1 CD58 204203_at 3.19 3.01 NM_001806.2 CEBPG 226750_at 3.18 3.46 NM_032239.2 LARP2 240843_at 3.18 3.17 NM_002828 PTPN2 208649_s_at 3.18 3.75 NM_007126.2 VCP 222502_s_at 3.17 4.41 NM_016617.1 UFM1 221923_s_at 3.17 4.24 NM_001037738.1 NPM1 223758_s_at 3.17 3.84 NM_001515.2 GTF2H2 202295_s_at 3.16 4.90 NM_004390.2 CTSH 201199_s_at 3.16 6.08 NM_002807.2 PSMD1 200660_at 3.15 3.77 NM_005620.1 S100A11 239363_at 3.15 3.23 NM_021218 HSPC043 209434_s_at 3.15 3.73 NM_002703.3 PPAT 46167_at 3.15 3.65 NM_004623.2 TTC4 201945_at 3.14 4.43 NM_002569.2 FURIN 210573_s_at 3.13 3.45 NM_006468.5 POLR3C 214359_s_at 3.13 4.19 NM_007355.2 HSPCB 209507_at 3.12 4.22 NM_002947.3 RPA3 202309_at 3.12 4.67 NM_005956.2 MTHFD1 235113_at 3.12 3.80 NM_152329.3 PPIL5 208975_s_at 3.12 3.16 NM_002265.4 KPNB1 231776_at 3.11 4.15 NM_005442.2 EOMES 221804_s_at 3.11 3.62 NM_018472.1 FAM45B 232432_s_at 3.11 3.76 NM_022902.2 SLC30A5 212636_at 3.10 3.33 NM_206853.1 QKI 205677_s_at 3.10 9.06 NM_005887 DLEU1 207622_s_at 3.10 5.48 NM_007189.1 ABCF2 218049_s_at 3.09 3.78 NM_014078.4 MRPL13 210946_at 3.09 3.01 NM_176895.1 PPAP2A 218235_s_at 3.07 3.48 NM_016037.2 UTP11L 203213_at 3.06 7.86 NM_001786 CDC2 218810_at 3.06 4.73 NM_025079.1 ZC3H12A 219690_at 3.05 3.66 NM_024660.1 U2AF1L4 212116_at 3.05 4.00 NM_006510.3 RFP 208863_s_at 3.05 4.42 NM_006924.3 SFRS1 210996_s_at 3.05 3.79 NM_006761.3 YWHAE 203371_s_at 3.05 4.20 NM_002491.1 NDUFB3 203537_at 3.04 3.81 NM_002767.2 PRPSAP2 224334_s_at 3.04 3.53 NM_016497.2 MRPL51 213951_s_at 3.04 3.61 NM_013290.3 PSMC3IP 201277_s_at 3.03 4.57 NM_004499.3 HNRPAB 200825_s_at 3.02 4.15 NM_006389.2 HYOU1 217499_x_at 3.02 3.91 NR_002163.2 OR7E37P 225766_s_at 3.01 5.02 NM_002270 KPNB2 227335_at 3.01 3.07 NM_033081.1 DIDO1 207629_s_at 3.00 3.42 NM_004723.2 ARHGEF2 218193_s_at 3.00 3.29 NM_016072.2 GOLT1B 3-fold or more down-regualted

Fold Change Fold Change Donor 1 Donor 2 GenBank ID Gene Symbol 201044_x_at -360.56 -141.86 NM_004417.2 DUSP1 206522_at -342.94 -221.08 NM_004668.1 MGAM 217414_x_at -323.54 -402.43 NM_000558.3 HBA1 206481_s_at -281.69 -88.75 NM_001290.2 LDB2 219452_at -107.77 -136.24 NM_022355.1 DPEP2 242020_s_at -99.83 -683.69 NM_030776.1 ZBP1 218638_s_at -98.67 -92.06 NM_012445.1 SPON2 233743_x_at -96.41 -241.17 NM_030760.3 EDG8 209458_x_at -96.28 -116.24 NM_000517.3 HBA2 212235_at -94.11 -108.28 NM_015103.1 PLXND1 214183_s_at -93.06 -37.63 NM_012253.2 TKTL1 1569225_a_at -66.27 -58.13 BC021582 SCML4 221602_s_at -65.71 -56.81 NM_005449.3 FAIM3 214369_s_at -63.82 -45.89 NM_153819.1 RASGRP2 225803_at -62.17 -60.51 NM_058229.2 FBXO32 209201_x_at -53.71 -44.47 NM_003467.2 CXCR4 201341_at -53.66 -70.76 NM_003633.1 ENC1 205239_at -52.17 -156.69 NM_001657.2 AREG 215189_at -51.80 -124.17 XM_939516.1 KRTHB6 207001_x_at -45.43 -85.81 NM_001015881.1 TSC22D3 213854_at -44.74 -11.25 NM_145731.2 SYNGR1 229952_at -42.39 -31.69 NM_000347 SPTB 204293_at -39.09 -7.74 NM_000199.2 SGSH 228284_at -39.03 -108.92 NM_005077 TLE1 228113_at -38.36 -88.51 NM_175738.3 RAB37 204661_at -36.31 -89.75 NM_001803.2 CD52 232686_at -34.42 -46.82 AF161341.1 SIGLECP3 203036_s_at -33.97 -15.27 NM_014751.2 MTSS1 205826_at -32.57 -122.50 NM_003970.1 MYOM2 201531_at -29.29 -35.35 NM_003407.1 ZFP36 219396_s_at -28.96 -22.24 NM_024608.1 NEIL1 244677_at -28.53 -67.18 NM_002616 PER1 243623_at -28.42 -21.07 NM_006671.3 SLC1A7 227803_at -27.91 -280.60 NM_021572.4 ENPP5 204066_s_at -27.51 -11.62 NM_001037131.1 CENTG2 220384_at -27.47 -46.06 NM_016616.2 TXNDC3 223543_at -27.32 -17.83 NM_032512.2 PDZK4 200965_s_at -26.77 -28.97 NM_001003407.1 ABLIM1 205997_at -24.88 -161.08 NM_014265 ADAM28 202364_at -24.46 -15.27 NM_005962.4 MXI1 1560228_at -23.53 -360.83 NM_178310.1 SNAI3 230707_at -23.31 -38.20 NM_003105 SORL1 215933_s_at -23.19 -50.88 NM_002729.3 HHEX 207606_s_at -23.16 -30.84 NM_018287.4 ARHGAP12 226279_at -23.09 -64.73 NM_007173.3 PRSS23 226489_at -21.95 -50.80 NM_020698.1 TMCC3 204867_at -20.72 -24.28 NM_005258.2 GCHFR 208078_s_at -20.64 -17.10 NM_173354.2 SNF1LK 201367_s_at -20.48 -109.68 NM_006887.3 ZFP36L2 204621_s_at -20.26 -60.73 NM_006186.2 NR4A2 213395_at -19.99 -13.60 NM_015166.3 MLC1 219529_at -19.54 -14.02 NM_004669.2 CLIC3 207840_at -19.43 -33.06 NM_007053.2 CD160 212430_at -19.20 -28.89 NM_017495.4 RNPC1 219371_s_at -18.53 -34.45 NM_016270.2 KLF2 203017_s_at -18.33 -20.28 NM_014021.2 SSX2IP 209185_s_at -18.30 -20.79 NM_003749.2 IRS2 215333_x_at -17.86 -6.10 NM_000561.2 GSTM1 233467_s_at -17.76 -57.02 NM_005705.4 TSPAN32 238429_at -17.59 -30.62 NM_144649.1 TMEM71 219889_at -17.19 -17.09 NM_005479.2 FRAT1 229441_at -16.71 -114.77 NM_012193 FZD4 225562_at -16.69 -33.26 NM_007368.2 RASA3 226549_at -16.51 -22.10 NM_001024401.2 SBK1 206582_s_at -15.76 -16.39 NM_201524.1 GPR56 207118_s_at -15.70 -24.81 NM_006983.1 MMP23B 207008_at -15.68 -34.12 NM_001557.2 IL8RB 202524_s_at -15.35 -27.47 NM_014767.1 SPOCK2 218921_at -15.26 -23.58 NM_021805.1 SIGIRR 209447_at -14.99 -15.34 NM_015293.1 SYNE1 224215_s_at -14.75 -27.25 NM_005618.2 DLL1 227020_at -14.60 -11.53 NM_001005404.3 YPEL2 209880_s_at -14.40 -18.25 NM_003006.3 SELPLG 202973_x_at -14.28 -30.93 NM_001015045.1 FAM13A1 210240_s_at -14.18 -8.80 NM_001800.3 CDKN2D 217159_x_at -14.05 -10.11 NM_016543.1 SIGLEC7 212823_s_at -13.71 -9.89 NM_015549.1 PLEKHG3 228788_at -13.58 -27.71 NM_013313.3 YPEL1 202191_s_at -13.41 -39.24 NM_201432.1 GAS7 235723_at -13.35 -10.08 NM_017637.5 BNC2 211663_x_at -13.34 -15.36 NM_000954 PTGDS 1557166_at -13.16 -34.80 NM_014456 PDCD4 228097_at -13.13 -16.66 NM_013262 MIR 217078_s_at -13.04 -19.93 NM_007261.2 CD300A 227353_at -13.03 -14.80 AK090478.1 TMC8 203281_s_at -12.93 -8.84 NM_003335.2 UBE1L 210202_s_at -12.92 -13.18 NM_004305.2 BIN1 1553132_a_at -12.88 -39.06 NM_152332.2 MTAC2D1 204674_at -12.79 -20.37 NM_006152.2 LRMP 1554827_a_at -12.69 -12.09 NM_001114.2 ADCY7 202388_at -12.65 -14.48 NM_002923.1 RGS2 222895_s_at -12.65 -8.15 NM_022898.1 BCL11B 214623_at -12.56 -23.87 AK024483.1 SHFM3P1 220016_at -12.50 -101.91 NM_024060.2 AHNAK 201280_s_at -12.44 -9.73 NM_001343.1 DAB2 222996_s_at -12.37 -15.12 NM_016463.5 CXXC5 219505_at -12.34 -16.64 NM_017424.2 CECR1 213280_at -12.26 -20.60 NM_015085.3 GARNL4 228708_at -12.05 -16.99 NM_004163 RAB27B 227726_at -11.98 -18.67 NM_178841.2 RNF166 205883_at -11.98 -17.95 NM_001018011.1 ZBTB16 210778_s_at -11.95 -21.76 NM_006454.2 MXD4 216836_s_at -11.79 -8.07 NM_001005862.1 ERBB2 214032_at -11.41 -10.91 NM_001079.3 ZAP70 207460_at -11.41 -17.03 NM_005317.2 GZMM 1555688_s_at -11.38 -16.65 NM_032214.2 SLA2 209189_at -11.23 -22.81 NM_005252.2 FOS 218927_s_at -11.11 -15.90 NM_018641 CHST12 208122_x_at -10.82 -22.78 NM_014513.1 KIR2DS5 209871_s_at -10.79 -16.45 NM_005503.2 APBA2 201417_at -10.72 -14.08 NM_003107.2 SOX4 223130_s_at -10.69 -8.57 NM_013262.3 MYLIP 212599_at -10.58 -8.92 NM_015570.1 AUTS2 229031_at -10.42 -14.39 NM_017623 CNNM3 1568665_at -10.32 -10.09 BC022477.1 RNF103 210077_s_at -10.29 -9.49 NM_006925 SFRS5 226148_at -10.13 -8.79 BC040658.1 BTBD15 228258_at -10.12 -12.62 NM_198517.2 TBC1D10C 218885_s_at -10.12 -6.55 NM_024642 GALNT12 228705_at -9.94 -122.93 NM_144691.3 CAPN12 229164_s_at -9.90 -5.64 NM_172027.1 ABTB1 222862_s_at -9.78 -20.63 NM_174858.1 AK5 222557_at -9.64 -17.01 NM_015894.2 STMN3 57082_at -9.62 -9.27 NM_015627.1 LDLRAP1 212414_s_at -9.61 -13.81 NM_015129.4 SEPT6 231124_x_at -9.48 -12.01 NM_001033667.1 LY9 213279_at -9.48 -12.73 NM_138452.1 DHRS1 235206_at -9.47 -6.01 NM_016558 SCAND1 217983_s_at -9.40 -7.43 NM_003730.3 RNASET2 1557261_at -9.38 -17.04 XM_370838.4 WHDC1L1 225483_at -9.34 -6.73 NM_052875.3 VPS26B 1564424_at -9.32 -35.36 BC035983 FAM62B 1557985_s_at -9.28 -5.69 XM_095991.8 CEP78 221478_at -9.26 -12.73 NM_004331.2 BNIP3L 1569136_at -9.23 -13.00 NM_012214 MGAT4A 203420_at -9.06 -9.81 NM_016255.1 FAM8A1 233309_at -9.03 -38.76 NM_013390 TMEM2 226665_at -8.99 -8.46 NM_152392 AHSA2 228667_at -8.98 -17.17 NM_001012733.1 AGPAT4 221986_s_at -8.98 -19.65 NM_017644.3 KLHL24 229686_at -8.97 -6.38 NM_178129.3 P2RY8 219304_s_at -8.97 -16.30 NM_033135.3 PDGFD 211389_x_at -8.93 -18.80 NM_013289.1 KIR3DL1 204794_at -8.91 -10.40 NM_004418.2 DUSP2 202879_s_at -8.88 -14.41 NM_004762 PSCD1 204164_at -8.81 -6.31 NM_006747.2 SIPA1 1552924_a_at -8.79 -9.37 NM_020845.2 PITPNM2 224482_s_at -8.77 -9.27 NM_032932.3 RAB11FIP4 204773_at -8.72 -9.45 NM_004512.3 IL11RA 202723_s_at -8.71 -9.16 NM_002015.2 FOXO1A 204698_at -8.69 -27.19 NM_002201.4 ISG20 213733_at -8.69 -12.81 NM_012335.2 MYO1F 220974_x_at -8.63 -16.97 NM_030971.3 SFXN3 215111_s_at -8.63 -12.98 NM_006022.2 TSC22D1 214049_x_at -8.59 -14.04 NM_006137.6 CD7 229722_at -8.58 -6.08 NM_014162 HSPC072 221188_s_at -8.57 -10.42 NM_014430.2 CIDEB 225262_at -8.51 -14.79 BX647822.1 FOSL2 227732_at -8.48 -13.52 AB033044.1 ATXN7L1 216552_x_at -8.47 -18.41 NM_178228.2 KIR2DS4 212259_s_at -8.42 -13.95 NM_020524.2 PBXIP1 218631_at -8.41 -13.19 NM_021732.1 AVPI1 216676_x_at -8.41 -22.24 NM_153443.2 KIR3DL3 228261_at -8.39 -6.59 NM_080875.1 MIB2 241402_at -8.38 -15.57 AK094466.1 TSEN54 231922_at -8.32 -5.80 NM_152287.2 ZFP276 211397_x_at -8.28 -25.08 NM_014218.1 KIR2DL1 204917_s_at -8.26 -9.88 NM_004529.1 MLLT3 219229_at -8.24 -8.28 NM_013272.2 SLCO3A1 215894_at -8.23 -14.69 NM_000953.2 PTGDR 231182_at -8.14 -7.11 NM_003387 WASPIP 207314_x_at -8.13 -13.18 NM_006737.1 KIR3DL2 209750_at -8.12 -17.42 NM_005126.2 NR1D2 1553176_at -8.12 -14.52 NM_053282.4 SH2D1B 200696_s_at -8.11 -12.83 NM_000177.4 GSN 231484_at -8.06 -15.18 NM_006095 ATP8A1 1554006_a_at -8.05 -10.78 NM_001015002.1 LLGL2 241018_at -7.91 -9.44 AK124967.1 TMEM59 209906_at -7.84 -19.01 NM_004054.2 C3AR1 212543_at -7.81 -23.60 NM_001624.1 AIM1 208442_s_at -7.69 -14.92 NM_000051.3 ATM 202369_s_at -7.69 -3.92 NM_012288.3 TRAM2 1569642_at -7.60 -10.03 NM_001992 F2R 220987_s_at -7.54 -8.94 NM_030952.1 NUAK2 219014_at -7.52 -4.69 NM_016619.1 PLAC8 212849_at -7.48 -5.79 NM_003502.2 AXIN1 219382_at -7.48 -7.64 NM_203344.1 SERTAD3 223464_at -7.46 -10.75 NM_145638.1 OSBPL5 216979_at -7.41 -15.11 NM_173199.1 NR4A3 243366_s_at -7.38 -7.77 NM_000885 ITGA4 208657_s_at -7.35 -8.58 NM_006640.2 SEPT9 204811_s_at -7.33 -7.63 NM_001005505.1 CACNA2D2 210361_s_at -7.31 -6.10 NM_201999.1 ELF2 202326_at -7.30 -3.18 NM_025256.4 EHMT2 219359_at -7.28 -7.29 AK090428.1 ATHL1 204995_at -7.26 -6.77 NM_003885.2 CDK5R1 227946_at -7.26 -5.21 NM_017731.4 OSBPL7 219132_at -7.24 -11.43 NM_021255.2 PELI2 226843_s_at -7.23 -8.88 NM_001040284.1 PAPD5 236782_at -7.22 -8.89 NM_001017373.1 SAMD3 205005_s_at -7.11 -9.67 NM_004808 NMT2 208179_x_at -7.06 -12.04 NM_015868.2 KIR2DL3 225656_at -7.01 -5.50 NM_018100 EFHC1 219024_at -6.97 -8.76 NM_001001974.1 PLEKHA1 222453_at -6.97 -5.87 NM_024843.2 CYBRD1 201601_x_at -6.94 -10.22 NM_003641.2 IFITM1 223283_s_at -6.90 -6.01 NM_005786.4 SDCCAG33 209889_at -6.90 -4.74 NM_015490.3 SEC31L2 225912_at -6.89 -6.96 NM_033285.2 TP53INP1 212774_at -6.84 -9.77 NM_006352.3 ZNF238 212071_s_at -6.81 -5.56 NM_003128 SPTBN1 205315_s_at -6.79 -12.38 NM_130845.1 SNTB2 202587_s_at -6.78 -5.83 NM_000476.1 AK1 211299_s_at -6.75 -5.79 NM_004475.2 FLOT2 219183_s_at -6.75 -8.54 NM_013385.2 PSCD4 202390_s_at -6.75 -3.44 NM_002111.6 HD 219566_at -6.74 -6.78 NM_024310.2 PLEKHF1 229215_at -6.72 -7.86 NM_005170.2 ASCL2 1568964_x_at -6.71 -21.05 NM_001030288.1 SPN 226605_at -6.70 -4.78 NM_001347.2 DGKQ 46665_at -6.59 -7.60 NM_017789.3 SEMA4C 217838_s_at -6.55 -5.69 NM_016337.2 EVL 202478_at -6.49 -9.21 NM_021643.1 TRIB2 211410_x_at -6.48 -10.32 NM_001018081.1 KIR2DL5B 205786_s_at -6.47 -8.74 NM_000632.3 ITGAM 37117_at -6.47 -8.97 NM_001017526.1 ARHGAP8 209156_s_at -6.46 -13.15 NM_001849.2 COL6A2 234574_at -6.45 -6.10 AK024563 ADAMTS10 201315_x_at -6.43 -10.13 NM_006435.1 IFITM2 208010_s_at -6.42 -13.04 NM_012411.2 PTPN22 208981_at -6.35 -16.33 NM_000442 PECAM1 216218_s_at -6.34 -6.18 NM_015184.2 PLCL2 232693_s_at -6.33 -4.57 NM_018660.2 ZNF395 224882_at -6.31 -6.00 NM_032501.2 ACSS1 227113_at -6.28 -10.41 NM_144650.1 ADHFE1 203085_s_at -6.28 -7.51 NM_000660.3 TGFB1 242644_at -6.26 -13.89 NM_152468 EVER2 220646_s_at -6.25 -8.79 NM_016523.1 KLRF1 201012_at -6.24 -8.59 NM_000700.1 ANXA1 201666_at -6.24 -6.04 NM_003254.2 TIMP1 223179_at -6.22 -9.05 NM_031477.3 YPEL3 226906_s_at -6.20 -7.41 NM_032496.1 ARHGAP9 210288_at -6.19 -8.91 NM_005810.3 KLRG1 1560316_s_at -6.19 -3.96 N32168 GLCCI1 227819_at -6.18 -6.42 NM_021636.2 LGR6 208198_x_at -6.16 -11.21 NM_014512.1 KIR2DS1 229445_at -6.16 -16.25 NM_000101 CYBA 203179_at -6.13 -7.07 NM_000155.2 GALT 213924_at -6.13 -13.32 NM_023075 MPPE1 221221_s_at -6.12 -13.06 NM_017415.1 KLHL3 203236_s_at -6.06 -4.82 NM_002308 LGALS9 212144_at -6.04 -5.59 NM_015374.1 UNC84B 209770_at -6.01 -6.22 NM_007048.4 BTN3A1 208426_x_at -6.01 -11.81 NM_002255.3 KIR2DL4 203206_at -5.99 -3.56 NM_014661.3 FAM53B 227199_at -5.99 -5.12 NM_015151 DIP2 203657_s_at -5.99 -10.29 NM_003793.3 CTSF 230563_at -5.98 -8.18 NM_145313.1 RASGEF1A 241619_at -5.95 -6.61 NM_006888 CALM1 202145_at -5.95 -4.92 NM_002346.1 LY6E 201853_s_at -5.94 -4.96 NM_004358.3 CDC25B 205462_s_at -5.94 -4.21 NM_002149.2 HPCAL1 204122_at -5.92 -7.78 NM_198125.1 TYROBP 213856_at -5.87 -4.53 NM_001777 CD47 219521_at -5.87 -3.69 NM_054025.2 B3GAT1 225763_at -5.85 -10.15 NM_052862.2 RCSD1 236295_s_at -5.84 -7.91 NM_178844 NOD3 205285_s_at -5.80 -7.46 NM_001465.3 FYB 211343_s_at -5.77 -4.73 NM_005203.3 COL13A1 221564_at -5.76 -3.40 NM_001535 HRMT1L1 236172_at -5.75 -5.14 NM_000752 LTB4R 203186_s_at -5.73 -5.79 NM_019554.1 S100A4 230133_at -5.73 -4.94 NM_018835 MNAB 227224_at -5.73 -8.06 NM_018037 FLJ10244 201137_s_at -5.72 -7.56 NM_002121 HLA-DPB1 212203_x_at -5.72 -6.87 NM_021034.1 IFITM3 1556874_a_at -5.71 -5.39 BC032952.2 RKHD2 224973_at -5.71 -5.57 AF350451.1 FAM46A 201752_s_at -5.70 -6.19 NM_001121.2 ADD3 204046_at -5.68 -6.05 NM_004573 PLCB2 226140_s_at -5.67 -8.96 XM_166659.6 OTUD1 224027_at -5.65 -6.81 NM_019846 CCL28 213045_at -5.65 -4.97 XM_038150.5 MAST3 214083_at -5.64 -6.38 NM_002719 PPP2R5C 226602_s_at -5.62 -8.39 NM_004327.3 BCR 205398_s_at -5.61 -6.84 NM_005902.3 SMAD3 1559018_at -5.59 -6.70 NM_006504 PTPRE 239163_at -5.59 -5.11 NM_003337 UBE2B 211005_at -5.57 -6.31 NM_001014987.1 LAT 209864_at -5.57 -4.27 NM_012083.2 FRAT2 205839_s_at -5.54 -10.25 NM_004758.1 BZRAP1 203117_s_at -5.50 -3.95 NM_014871.2 USP52 205718_at -5.43 -4.44 NM_000889.1 ITGB7 225949_at -5.42 -7.58 NM_178564.2 NRBP2 204857_at -5.42 -3.77 NM_001013836.1 MAD1L1 217992_s_at -5.38 -3.80 NM_024329.4 EFHD2 205213_at -5.38 -7.47 NM_014716.2 CENTB1 207361_at -5.34 -7.41 NM_012257 HBP1 214054_at -5.33 -4.95 NM_003974.2 DOK2 1558711_at -5.33 -4.47 AK093650.1 FAM13A1OS 222018_at -5.32 -3.63 NM_005594 NACA 203628_at -5.30 -9.00 NM_000875 IGF1R 206170_at -5.27 -4.93 NM_000024.3 ADRB2 206965_at -5.26 -12.45 NM_016285.2 KLF12 227236_at -5.25 -14.49 NM_005725.3 TSPAN2 212334_at -5.20 -5.40 NM_002076.2 GNS 213902_at -5.20 -3.69 NM_004315 ASAH1 201681_s_at -5.20 -7.97 NM_004747.3 DLG5 212757_s_at -5.19 -5.12 NM_172169.1 CAMK2G 204890_s_at -5.18 -5.87 NM_005356.2 LCK 203402_at -5.14 -4.84 NM_003636 KCNAB2 235139_at -5.11 -3.97 NM_031498.1 GNGT2 205571_at -5.08 -3.97 NM_015929.2 LIPT1 203286_at -5.07 -5.45 NM_014901.4 RNF44 235780_at -5.07 -4.83 NM_207578.1 PRKACB 235695_at -5.07 -4.71 NM_001566 INPP4A 1559975_at -5.06 -7.02 NM_001731 BTG1 207686_s_at -5.06 -12.97 NM_001228.3 CASP8 210006_at -5.05 -3.38 NM_015407.3 ABHD14A 211824_x_at -5.02 -4.46 NM_014922.3 NALP1 203633_at -5.00 -6.09 NM_001876 CPT1A 203908_at -4.97 -6.58 NM_003759.1 SLC4A4 228062_at -4.96 -5.94 NM_153757.1 NAP1L5 235721_at -4.96 -6.60 NM_178502.2 DTX3 231904_at -4.95 -3.31 NM_006758 U2AF1 203029_s_at -4.94 -10.84 NM_002847.2 PTPRN2 212332_at -4.94 -4.71 NM_005611.2 RBL2 204294_at -4.93 -8.25 NM_000481.2 AMT 238025_at -4.93 -5.90 NM_152649.1 MLKL 218807_at -4.93 -7.28 NM_006113.3 VAV3 209398_at -4.91 -6.99 NM_005319.3 HIST1H1C 221769_at -4.91 -4.41 NM_080861.3 SPSB3 211676_s_at -4.91 -7.31 NM_000416.1 IFNGR1 1562368_at -4.90 -5.44 NM_032415 CARD11 226757_at -4.90 -7.21 NM_001547.3 IFIT2 204834_at -4.85 -10.38 NM_006682 FGL2 215275_at -4.85 -9.97 AK126139.1 TRAF3IP3 206296_x_at -4.84 -3.78 NM_007181.3 MAP4K1 211559_s_at -4.84 -6.04 NM_004354.1 CCNG2 227134_at -4.81 -5.12 NM_032872.1 SYTL1 209048_s_at -4.80 -4.45 NM_183047.1 PRKCBP1 1559883_s_at -4.80 -7.87 NM_015474.2 SAMHD1 218168_s_at -4.79 -4.08 NM_020247 CABC1 211395_x_at -4.77 -7.69 NM_001002273.1 FCGR2B 207100_s_at -4.77 -3.84 NM_016830.2 VAMP1 238476_at -4.76 -6.17 NM_153607 LOC153222 202657_s_at -4.75 -6.28 NM_014755.1 SERTAD2 219821_s_at -4.75 -5.06 NM_018988.1 GFOD1 1552623_at -4.74 -7.00 NM_032855.2 HSH2D 207735_at -4.72 -22.32 NM_017831.2 RNF125 244050_at -4.71 -8.12 NM_001010915.1 PTPLAD2 1558002_at -4.71 -4.88 NM_007178 UNRIP 225274_at -4.70 -6.60 NM_016297 PCL1 219854_at -4.70 -3.60 NM_021030.2 ZNF14 1556306_at -4.70 -8.85 NM_018682 MLL5 210647_x_at -4.69 -4.54 NM_001004426.1 PLA2G6 216748_at -4.68 -5.75 AK024890.1 PYHIN1 1554250_s_at -4.68 -8.06 BC033871 TRIM74 205125_at -4.66 -6.80 NM_006225.2 PLCD1 210253_at -4.65 -3.87 NM_006410 HTATIP2 225446_at -4.65 -4.41 NM_001007246.1 BRWD1 210281_s_at -4.65 -11.57 NM_003453.2 ZNF198 224800_at -4.65 -8.55 NM_020830.3 WDFY1 223228_at -4.62 -3.57 NM_032287.2 LDOC1L 203778_at -4.62 -3.59 NM_005908.3 MANBA 204563_at -4.62 -5.70 NM_000655.2 SELL 221666_s_at -4.61 -5.45 NM_145182.1 PYCARD 204425_at -4.60 -3.16 NM_001666.2 ARHGAP4 219657_s_at -4.58 -5.71 NM_016531.3 KLF3 219033_at -4.58 -6.52 NM_024615.2 PARP8 219855_at -4.58 -5.43 NM_018159.2 NUDT11 226694_at -4.57 -13.01 NM_001004065.3 AKAP2 230872_s_at -4.56 -6.68 AK023960.1 TTLL3 223469_at -4.54 -3.23 NM_017712.2 PGPEP1 200661_at -4.54 -3.59 NM_000308.1 PPGB 205898_at -4.52 -3.72 NM_001337.3 CX3CR1 223349_s_at -4.49 -4.61 NM_032515.3 BOK 212510_at -4.48 -3.88 NM_015141.2 GPD1L 226157_at -4.47 -4.33 NM_006286 TFDP2 218487_at -4.47 -5.87 NM_000031.4 ALAD 219228_at -4.46 -18.25 NM_018555.4 ZNF331 230972_at -4.46 -6.00 NM_152326.2 ANKRD9 202704_at -4.45 -7.07 NM_005749.2 TOB1 239122_at -4.44 -9.23 NM_006850 IL24 212914_at -4.40 -3.93 NM_175709.2 CBX7 207556_s_at -4.38 -3.52 NM_003646.2 DGKZ 231271_x_at -4.37 -4.40 NM_020677 HSCARG 228483_s_at -4.36 -8.87 NM_015975 TAF9L 202208_s_at -4.36 -4.16 NM_005737.3 ARL4C 202501_at -4.35 -4.17 NM_014268.1 MAPRE2 228813_at -4.33 -5.69 NM_006037 HDAC4 222912_at -4.33 -4.20 NM_004041.3 ARRB1 202245_at -4.31 -3.78 NM_002340.3 LSS 228370_at -4.30 -3.51 NG_002690.1 SNORD 212689_s_at -4.28 -3.90 NM_018433.3 JMJD1A 202887_s_at -4.27 -4.39 NM_019058.2 DDIT4 1552634_a_at -4.27 -3.79 NM_033204.2 ZNF101 201720_s_at -4.27 -5.36 NM_006762.1 LAPTM5 229036_at -4.26 -4.01 NM_001024843.1 TNRC6B 225954_s_at -4.24 -4.68 NM_177401.4 MIDN 204613_at -4.23 -3.62 NM_002661.1 PLCG2 208498_s_at -4.21 -19.20 NM_020978.3 AMY2B 204499_at -4.20 -6.90 NM_015239 AGTPBP1 229312_s_at -4.18 -4.58 NM_025211.2 GKAP1 213376_at -4.17 -4.24 NM_014950 ZBTB1 243951_at -4.17 -15.34 0 ABCB1 200632_s_at -4.16 -3.36 NM_006096.2 NDRG1 1557257_at -4.15 -6.55 NM_003921 BCL10 213958_at -4.15 -5.60 NM_006725.2 CD6 238633_at -4.15 -4.43 NM_025209 EPC1 204949_at -4.14 -6.25 NM_002162.2 ICAM3 1555476_at -4.12 -6.88 BC017880 IREB2 242829_x_at -4.10 -13.99 NM_012158 FBXL3A 218273_s_at -4.08 -3.65 NM_018444.2 PPM2C 203332_s_at -4.08 -3.23 NM_005541.3 INPP5D 226080_at -4.08 -4.45 NM_033389.2 SSH2 219284_at -4.07 -4.52 NM_024610.3 HSPBAP1 217286_s_at -4.07 -4.34 NM_022477.2 NDRG3 214814_at -4.06 -3.63 NM_133370 YT521 211998_at -4.05 -3.34 NM_005324.3 H3F3B 212001_at -4.05 -3.54 AV738039 SFRS14 202747_s_at -4.05 -3.24 NM_004867.3 ITM2A 204197_s_at -4.05 -3.75 NM_004350.1 RUNX3 205758_at -4.03 -6.17 NM_001768.5 CD8A 1552497_a_at -4.00 -3.53 NM_052931.3 SLAMF6 204079_at -3.99 -5.08 NM_001008566.1 TPST2 224806_at -3.96 -6.58 NM_005082.4 TRIM25 244360_at -3.96 -5.84 BC063316.1 FBXL17 218285_s_at -3.95 -4.22 NM_020139.3 DHRS6 200701_at -3.94 -3.91 NM_006432.3 NPC2 223679_at -3.93 -4.69 NM_001904 CTNNB1 212406_s_at -3.93 -4.92 NM_018257.1 PCMTD2 214714_at -3.93 -4.58 NM_032164.2 ZNF394 231121_at -3.91 -7.27 NM_032383 HPS3 209696_at -3.90 -6.66 NM_000507.2 FBP1 217088_s_at -3.90 -3.61 NM_004829.4 NCR1 232392_at -3.90 -4.54 NM_003017 SFRS3 226218_at -3.89 -6.46 NM_002185 IL7R 230748_at -3.88 -5.78 NM_004694 SLC16A6 228083_at -3.88 -8.19 NM_001005737.1 CACNA2D4 235024_at -3.87 -5.47 NM_024900.2 PHF17 205352_at -3.86 -5.17 NM_005025.2 SERPINI1 201302_at -3.86 -5.93 NM_001153.2 ANXA4 1553117_a_at -3.86 -3.74 NM_007271.2 STK38 1568949_at -3.85 -4.12 NM_012417 PITPNC1 203723_at -3.84 -3.04 NM_002221.2 ITPKB 201087_at -3.84 -3.98 NM_002859.1 PXN 226970_at -3.84 -4.05 NM_203301.1 FBXO33 209348_s_at -3.84 -4.15 NM_001031804.1 MAF 203047_at -3.82 -3.76 NM_005990.2 STK10 212249_at -3.82 -9.22 NM_181504.2 PIK3R1 218178_s_at -3.81 -3.82 NM_020412.3 CHMP1B 216705_s_at -3.80 -5.95 NM_000022.2 ADA 205139_s_at -3.80 -6.59 NM_005715.1 UST 206724_at -3.78 -3.64 NM_003655.2 CBX4 214196_s_at -3.78 -3.24 NM_000391.2 TPP1 243649_at -3.77 -3.74 NM_012179 FBXO7 226352_at -3.77 -6.16 NM_152405 JMY 213620_s_at -3.75 -3.49 NM_000873.2 ICAM2 210140_at -3.75 -6.52 NM_003650.2 CST7 1554037_a_at -3.75 -4.91 NM_014797.1 ZBTB24 236829_at -3.75 -3.92 NM_007114 TMF1 232044_at -3.74 -4.51 AK023612.1 RBBP6 228730_s_at -3.74 -3.02 NM_138355.2 SCRN2 1555962_at -3.74 -5.58 AK000770.1 B3GNT7 214496_x_at -3.73 -5.55 NM_012330 MYST4 208858_s_at -3.73 -3.52 NM_015292.1 FAM62A 200982_s_at -3.73 -5.56 NM_001155.3 ANXA6 205383_s_at -3.72 -5.47 NM_015642.2 ZBTB20 224871_at -3.71 -4.12 NM_182752.3 FAM79A 236778_at -3.71 -6.93 NM_000489 ATRX 223640_at -3.71 -3.87 NM_014266.3 HCST 222279_at -3.69 -4.78 AK092748.1 HLA-F 223746_at -3.69 -8.47 NM_006282 STK4 227308_x_at -3.69 -3.83 NM_021070 LTBP3 218421_at -3.69 -3.09 NM_022766.4 CERK 205255_x_at -3.69 -4.20 NM_201632.1 TCF7 203521_s_at -3.68 -3.19 NM_014345.1 ZNF318 224910_at -3.68 -3.05 NM_014316 CARHSP1 200664_s_at -3.68 -5.12 NM_006145.1 DNAJB1 204346_s_at -3.67 -3.43 NM_170713.1 RASSF1 218456_at -3.67 -4.62 NM_001002259.1 C1QDC1 214963_at -3.67 -4.52 AK026236 NUP160 226344_at -3.66 -3.56 NM_001011656.1 ZMAT1 227811_at -3.66 -3.88 NM_033086.1 FGD3 212727_at -3.65 -3.52 NM_021120 DLG3 226068_at -3.65 -3.54 NM_003177 SYK 201847_at -3.65 -3.22 NM_000235.2 LIPA 201656_at -3.64 -7.54 NM_000210.1 ITGA6 200885_at -3.64 -3.53 NM_175744.3 RHOC 211458_s_at -3.64 -9.43 NM_031412.2 GABARAPL1 228727_at -3.63 -8.82 NM_001157 ANXA11 243631_at -3.63 -6.08 AI630997 HSMPP8 224671_at -3.63 -3.04 NM_145255.2 MRPL10 227616_at -3.62 -6.18 NM_182557.1 BCL9L 217591_at -3.62 -7.34 NM_005414 SKIL 229955_at -3.62 -3.16 NM_012175 FBXO3 219094_at -3.60 -3.58 NM_014154.2 ARMC8 203081_at -3.60 -3.70 NM_020248.2 CTNNBIP1 201813_s_at -3.59 -4.57 NM_014744.1 TBC1D5 201580_s_at -3.59 -3.06 NM_021156.2 TXNDC13 219191_s_at -3.59 -3.48 NM_016293.2 BIN2 1555851_s_at -3.59 -3.20 NM_003009.2 SEPW1 227897_at -3.59 -3.75 NM_002886 RAP2B 237107_at -3.58 -6.87 NM_003690 PRKRA 208454_s_at -3.58 -3.98 NM_016134.2 PGCP 210886_x_at -3.57 -3.21 NM_007233 TP53AP1 203068_at -3.57 -3.15 NM_014851.2 KLHL21 238987_at -3.56 -5.38 NM_001497 B4GALT1 209815_at -3.56 -4.73 AK124593.1 PTCH 1554544_a_at -3.56 -3.84 NM_001025100.1 MBP 209683_at -3.56 -5.45 AK001942.1 FAM49A 235626_at -3.55 -3.12 NM_153498.2 CAMK1D 215392_at -3.55 -10.68 AU148154 USP3 205668_at -3.54 -3.31 NM_002349.1 LY75 214958_s_at -3.54 -4.58 NM_007267.5 TMC6 212242_at -3.53 -4.68 NM_006000.1 TUBA1 218821_at -3.53 -4.99 NM_024663.3 NPEPL1 204862_s_at -3.53 -3.55 NM_002513.2 NME3 225350_s_at -3.53 -3.92 NM_024646.1 ZYG11B 204805_s_at -3.53 -3.14 NM_006026.2 H1FX 208438_s_at -3.52 -4.92 NM_005248.1 FGR 208661_s_at -3.50 -4.18 NM_001001894.1 TTC3 233169_at -3.50 -3.88 NM_021632 ZNF350 38290_at -3.47 -3.85 NM_006480.4 RGS14 204642_at -3.47 -22.76 NM_001400 EDG1 201050_at -3.46 -4.13 NM_001031696.1 PLD3 229141_at -3.46 -4.15 NM_001006623.1 WDR33 218494_s_at -3.46 -5.01 NM_020062.3 SLC2A4RG 213906_at -3.45 -4.71 AW592266 MYBL1 201285_at -3.44 -3.56 NM_013446.2 MKRN1 221645_s_at -3.43 -5.30 NM_018300.2 ZNF83 204912_at -3.42 -3.07 NM_001558.2 IL10RA 221293_s_at -3.42 -4.30 NM_022047.2 DEF6 212843_at -3.41 -4.16 NM_000615 NCAM1 201009_s_at -3.41 -3.06 NM_006472.1 TXNIP 244443_at -3.41 -4.55 NM_001271 CHD2 203020_at -3.40 -4.24 AB007940.1 RABGAP1L 242463_x_at -3.40 -5.14 NM_198457.1 ZNF600 203543_s_at -3.39 -8.49 NM_001206.2 KLF9 202161_at -3.39 -3.14 NM_002741.3 PKN1 200859_x_at -3.39 -4.00 NM_001456.1 FLNA 205771_s_at -3.39 -5.49 NM_138633.1 AKAP7 221953_s_at -3.38 -5.22 NM_006690 MMP24 221727_at -3.38 -3.45 BC071655.1 SUB1 226136_at -3.38 -4.95 NM_007043 HRB2 202439_s_at -3.37 -4.98 NM_000202.2 IDS 217783_s_at -3.36 -3.86 NM_016061.1 YPEL5 202789_at -3.36 -4.15 NM_002660.2 PLCG1 201462_at -3.36 -3.44 NM_014766.2 SCRN1 219095_at -3.35 -4.23 NM_005090.2 PLA2G4B 204206_at -3.34 -3.62 NM_020310.2 MNT 226249_at -3.33 -7.97 XM_941246.1 SNX30 220999_s_at -3.32 -3.23 NM_001037332.1 CYFIP2 230609_at -3.32 -9.82 NM_014666 ENTH 227639_at -3.30 -3.27 NM_005482 PIGK 1562442_a_at -3.30 -4.42 NM_003143 SSBP1 228410_at -3.30 -3.62 NM_080612.1 GAB3 214683_s_at -3.26 -3.64 NM_004071.2 CLK1 219221_at -3.26 -5.79 XM_929657.1 ZBTB38 206279_at -3.26 -3.70 NM_002760 PRKY 203607_at -3.25 -4.25 NM_014937.2 INPP5F 203445_s_at -3.25 -3.15 NM_005730.3 CTDSP2 230529_at -3.24 -3.44 NM_016217 HECA 228940_at -3.24 -3.78 XM_933340.1 NDUFB4 209994_s_at -3.23 -3.96 NM_018850.1 ABCB4 219170_at -3.23 -3.52 NM_024333.1 FSD1 205171_at -3.23 -4.49 NM_002830.2 PTPN4 202595_s_at -3.23 -3.98 NM_015344.1 LEPROTL1 201751_at -3.22 -3.84 NM_014876.3 JOSD1 227223_at -3.22 -7.84 NM_004902 RNPC2 219481_at -3.22 -3.11 NM_024525.2 TTC13 230375_at -3.22 -6.40 NM_032870 DKFZp564B0769 218217_at -3.21 -3.35 NM_021626.1 SCPEP1 214071_at -3.21 -13.01 NM_002071 GNAL 214085_x_at -3.21 -5.58 NM_006851 GLIPR1 208998_at -3.20 -4.18 NM_003355.2 UCP2 215483_at -3.20 -4.43 NM_005751 AKAP9 238719_at -3.20 -4.01 NM_002715 PPP2CA 225557_at -3.19 -5.55 NM_033027.2 AXUD1 213575_at -3.19 -6.86 NM_013293 TRA2A 230141_at -3.18 -4.09 NM_002892 RBBP1 217478_s_at -3.18 -5.11 NM_006120.2 HLA-DMA 210915_x_at -3.16 -4.46 BC030533.1 TRBC1 214482_at -3.15 -4.10 NM_006977.2 ZBTB25 204174_at -3.15 -4.06 NM_001629.2 ALOX5AP 239695_at -3.14 -6.06 BX648044.1 JAK1 209160_at -3.14 -3.64 NM_003739.4 AKR1C3 229586_at -3.14 -7.84 BC027491.2 CHD9 212221_x_at -3.14 -4.29 BX647783.1 AFMID 215731_s_at -3.13 -3.02 NM_022782 MPHOSPH9 202031_s_at -3.11 -3.47 NM_001033518.1 WIPI2 239101_at -3.11 -5.72 NM_031483.3 ITCH 201849_at -3.11 -7.34 NM_004052.2 BNIP3 1558622_a_at -3.09 -3.55 NM_152909.2 ZNF548 238509_at -3.08 -3.10 NM_003592 CUL1 238551_at -3.07 -3.22 NM_173540.1 FUT11 241403_at -3.07 -4.38 BC065732.1 CLK4 212472_at -3.07 -4.18 NM_014632 MICAL2 202878_s_at -3.05 -3.30 NM_012072.3 C1QR1 215605_at -3.04 -5.73 NM_006540 NCOA2 212980_at -3.04 -3.49 AL050376.1 USP34 209604_s_at -3.04 -5.29 NM_001002295.1 GATA3 236662_at -3.03 -5.58 AI139639 CRAMP1L 224856_at -3.02 -3.83 NM_004117.2 FKBP5 221219_s_at -3.01 -4.31 NM_017566.2 KLHDC4 222074_at -3.01 -4.46 NM_000374 UROD 1569030_s_at -3.00 -3.22 NM_016118 NYREN18 Supplemental Table 2: qPCR Primers Used

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Supplemental Figure 1 Regression analysis was carried out to assess donor-to-donor variability for both resting and 24 hour stimulated samples. Signal intensities obtained from microarray analysis were plotted on an x-y scatter plot and a R2 value was calculated using a linear fit regression line.

Supplemental Figure 2 NKL cells express a PRDM1 transcript variant corresponding to deletion of exon 6. RT- PCR was carried out using primers specific for the 5th and 7th exons of the human PRDM1 gene. Overexpression of cDNA obtained from either U2OS cells expressing a flag-tagged PRDM1 construct or NK-92 cells yielded the expected 311bp product, while cDNA obtained from NCI-H929 or NKL cells yielded both a 311bp and 182bp products.

Supplemental Figure 3 Primary human NK cells were stimulated with IL-2 (100U/mL) and IL-12 (10ng/mL) for 96 hours. A) qRT-PCR analysis for IFNG and TNF mRNA. B) Immunoblot analysis of PRDM1 expression. Data shown are from a representative experiment.

Supplemental Table 1: Excel file containing list of differentially expressed known genes showing 3-fold change in expression in both donors. Unannotated transcripts and genes with signal intensities of <500 after stimulation (for increased list) and <500 before stimulation (for decreased list) were excluded.

Supplemental Table 2: Sequence and annealing temperature of all oligonucleotide primers used in this report.

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