( B ) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14 . 5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose , the olfactory bulb ( OB ) and the ventral forebrain ( VFB ) in control and mutant embryos . Scale bar : 50μm . VNO , vomeronasal organ ; OE , olfactory epithelium ; OB , olfactory bulbs ; VFB , ventral forebrain .

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(B) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14.5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose, the olfactory bulb (OB) and the ventral forebrain (VFB) in control and mutant embryos. Scale bar: 50μm. VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulbs; VFB, ventral forebrain.


( C ) Quantitative analysis showing distribution of GnRH neurons in the nose , OB and VFB of mutant and control embryos . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 5 and 6 mouse embryos for mutants and controls , respectively , from at least 3 litters .

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(C) Quantitative analysis showing distribution of GnRH neurons in the nose, OB and VFB of mutant and control embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=5 and 6 mouse embryos for mutants and controls, respectively, from at least 3 litters.


( D ) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs , in a control ( left panel ) and a mutant ( right panel ) postnatal brain . Knock - out animals show an accumulation of GnRH neurons in the accessory olfactory bulb ( circle formed by the GnRH labelling , right panel ) . AOB , accessory olfactory bulb . Scale bar : 300μm .

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(D) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs, in a control (left panel) and a mutant (right panel) postnatal brain. Knock-out animals show an accumulation of GnRH neurons in the accessory olfactory bulb (circle formed by the GnRH labelling, right panel). AOB, accessory olfactory bulb. Scale bar: 300μm.


( E ) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis ( OVLT ) in the median preoptic area , from a control ( left ) and a mutant ( right ) female postnatal brain . Scale bar : 500μm . Oc , optic chiasma .

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(E) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis (OVLT) in the median preoptic area, from a control (left) and a mutant (right) female postnatal brain. Scale bar: 500μm. Oc, optic chiasma.


( F ) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates . OB , olfactory bulb ; DBB , diagonal band of Broca ; 3V , periventricular region of the preoptic area ; Tot , total . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 3 adult mice from 3 litters .

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(F) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates. OB, olfactory bulb; DBB, diagonal band of Broca; 3V, periventricular region of the preoptic area; Tot, total. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 adult mice from 3 litters.


( A ) Schematic representation of the transwell assay used to assess the effect of Sema3A , which was placed in the lower chamber , on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst ( grey ) in the lower part of the transwell membrane after overnight incubation in SFM ( left panel ) , SFM with 100ng / ml Sema3A ( middle panel ) and SFM with 100ng / ml Sema3A + 1μg Nrp1Ab ( right panel ) . Scale bar : 100μm .

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(A) Schematic representation of the transwell assay used to assess the effect of Sema3A, which was placed in the lower chamber, on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst (grey) in the lower part of the transwell membrane after overnight incubation in SFM (left panel), SFM with 100ng/ml Sema3A (middle panel) and SFM with 100ng/ml Sema3A + 1μg Nrp1Ab (right panel). Scale bar: 100μm.


( B ) Quantitative analyses of the mean number of GN11 cells that migrated from the upper chamber ( SFM ) through the perforated membrane in the presence or absence of the indicated doses of Sema3A and Nrp1Ab in the lower chamber . Kruskal - Wallis test , n = 5 to 6 wells in two independent experiments .

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(B) Quantitative analyses of the mean number of GN11 cells that migrated from the upper chamber (SFM) through the perforated membrane in the presence or absence of the indicated doses of Sema3A and Nrp1Ab in the lower chamber. Kruskal-Wallis test, n= 5 to 6 wells in two independent experiments.


( C ) Schematic illustration and representative photomicrograph of an organotypic culture of a nasal explant from E11 . 5 mouse embryos after 8 days in vitro . NE , nasal epithelium ; NMC , nasal midline cartilage . Scale bar 500μm ( 150μm in inset ) .

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(C) Schematic illustration and representative photomicrograph of an organotypic culture of a nasal explant from E11.5 mouse embryos after 8 days in vitro. NE, nasal epithelium; NMC, nasal midline cartilage. Scale bar 500μm (150μm in inset).


( D ) Total number of GnRH neurons that migrated out of nasal explants isolated from control and mutant embryos , quantified after 8 days in culture . Mann - Whitney U test , n = 3 to 5 embryos from at least 3 litters .

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(D) Total number of GnRH neurons that migrated out of nasal explants isolated from control and mutant embryos, quantified after 8 days in culture. Mann-Whitney U test, n=3 to 5 embryos from at least 3 litters.


( E ) Distance travelled by GnRH neurons from the olfactory epithelia ; Cells were quantified within a radius of 1 to 6 mm . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 3 to 5 explants .

(E) Distance travelled by GnRH neurons from the olfactory epithelia; Cells were quantified within a radius of 1 to 6 mm. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 to 5 explants.


( F ) Representative immunofluorescence for GnRH - ( red ) and BrdU - ( green ) positive cells in control and mutant E14 . 5 embryos arising from progenitors born between E9 . 5 and E10 . 5 . BrdU - positive GnRH neurons ( white arrowheads ) were more abundant in mutant than in control embryos . Scale bar : 20μm .

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(F) Representative immunofluorescence for GnRH- (red) and BrdU- (green) positive cells in control and mutant E14.5 embryos arising from progenitors born between E9.5 and E10.5. BrdU-positive GnRH neurons (white arrowheads) were more abundant in mutant than in control embryos. Scale bar: 20μm.


( G ) Number of BrdU - positive GnRH neurons in the nose , olfactory bulb ( OB ) and ventral forebrain ( VFB ) at E14 . 5 and total number of cells in control and mutant embryos . Double - labeled cells were more numerous in the mutant VFB . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 4 embryos from 2 litters .

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(G) Number of BrdU-positive GnRH neurons in the nose, olfactory bulb (OB) and ventral forebrain (VFB) at E14.5 and total number of cells in control and mutant embryos. Double-labeled cells were more numerous in the mutant VFB. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 embryos from 2 litters.


( I ) Number of GnRH neurons in E14 . 5 embryos injected with neutralizing antibodies to Neuropilin - 1 ( Nrp1Ab ) compared to an isotype goat antibody , or the antiapoptotic compound ZVAD compared to its vehicle , together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age . Note that the data are represented as fold change , as raw values are not comparable with transgenic animals that did not undergo surgery . Unpaired t - test , n = 5 to 7 embryos from at least 2 litters .

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(I) Number of GnRH neurons in E14.5 embryos injected with neutralizing antibodies to Neuropilin-1 (Nrp1Ab) compared to an isotype goat antibody, or the antiapoptotic compound ZVAD compared to its vehicle, together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age. Note that the data are represented as fold change, as raw values are not comparable with transgenic animals that did not undergo surgery. Unpaired t-test, n=5 to 7 embryos from at least 2 litters.


( A ) Olfactory sex preference test in P20 ( left panel ) and adult ( right panel ) control and mutant female mice . Half of the mutant females at P20 showed a preference for males , similar to adults of both genotypes , whereas the other half showed an even more pronounced preference for females than their control littermates at P20 . One - way ANOVA , Fisher ' s LSD multiple - comparison test for P20 mice and unpaired t - test for adult mice ; n = 4 to 6 from at least 3 litters .

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(A) Olfactory sex preference test in P20 (left panel) and adult (right panel) control and mutant female mice. Half of the mutant females at P20 showed a preference for males, similar to adults of both genotypes, whereas the other half showed an even more pronounced preference for females than their control littermates at P20. One-way ANOVA, Fisher's LSD multiple-comparison test for P20 mice and unpaired t-test for adult mice; n=4 to 6 from at least 3 litters.


( B ) Time spent sniffing after the introduction of both male and female odors into the cage of control and mutant littermates . One - way ANOVA , Fisher ' s LSD multiple - comparison test for P20 mice and unpaired t - test for adult mice ; n = 4 to 6 from at least 3 litters .

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(B) Time spent sniffing after the introduction of both male and female odors into the cage of control and mutant littermates. One-way ANOVA, Fisher's LSD multiple-comparison test for P20 mice and unpaired t-test for adult mice; n=4 to 6 from at least 3 litters.


( C ) Representative traces of spontaneous firing in GnRH neurons from control ( left , black ) and mutant ( right , red ) animals at week 2 ( upper panels ) and week 4 ( lower panels ) ; lower traces show an enlargement of the upper trace .

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(C) Representative traces of spontaneous firing in GnRH neurons from control (left, black) and mutant (right, red) animals at week 2 (upper panels) and week 4 (lower panels); lower traces show an enlargement of the upper trace.


( D ) Average spontaneous firing frequency in GnRH neurons from control and mutant mice at weeks 2 , 3 and 4 . Individual males and females used for this analysis are represented by blue and grey dots , respectively . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 8 to 14 neurons from at least 3 animals from different litters . Note that individual neurons from male ( n = 3 to 8 ) and female mice ( n = 5 to 8 ) used for this analysis are represented by blue and grey dots , respectively .

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(D) Average spontaneous firing frequency in GnRH neurons from control and mutant mice at weeks 2, 3 and 4. Individual males and females used for this analysis are represented by blue and grey dots, respectively. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=8 to 14 neurons from at least 3 animals from different litters. Note that individual neurons from male (n=3 to 8) and female mice (n=5 to 8) used for this analysis are represented by blue and grey dots, respectively.


( E ) Quantification of evoked firing in GnRH neurons from control and mutant mice in response to current steps . Two - way repeated - measures ANOVA , n = 10 to 11 neurons from at least 3 mice from different litters .

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(E) Quantification of evoked firing in GnRH neurons from control and mutant mice in response to current steps. Two-way repeated-measures ANOVA, n=10 to 11 neurons from at least 3 mice from different litters.


( A ) Evolution of body weight in control and mutant female littermates from 6 to 100 days of age . Mutant females show a significantly higher body weight from P21 . The grey lines mark body weight at puberty onset . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 7 to 17 mice from at least 4 litters .

(A) Evolution of body weight in control and mutant female littermates from 6 to 100 days of age. Mutant females show a significantly higher body weight from P21. The grey lines mark body weight at puberty onset. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=7 to 17 mice from at least 4 litters.


( B ) Fat mass or volume of white adipose tissue ( WAT ) . Adult mutant females showed higher WAT volumes . Unpaired t - test , n = 8 mice .

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(B) Fat mass or volume of white adipose tissue (WAT). Adult mutant females showed higher WAT volumes. Unpaired t-test, n=8 mice.


( C ) Volume of lean tissue . Mann - Whitney U test , n = 8 mice .

(C) Volume of lean tissue. Mann-Whitney U test, n=8 mice.


( D ) Cumulative percentage of the age at vaginal opening , n = 13 mice .

(D) Cumulative percentage of the age at vaginal opening, n=13 mice.


( E ) Cumulative percentage of animals undergoing first ovulation as assessed by the occurrence of first estrus at different ages n = 13 mice .

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(E) Cumulative percentage of animals undergoing first ovulation as assessed by the occurrence of first estrus at different ages n=13 mice.


( F ) Bar graph showing body weight at P45 , which is the mean age at puberty in mutants . Unpaired t - test , n = 9 to 14 mice .

(F) Bar graph showing body weight at P45, which is the mean age at puberty in mutants. Unpaired t-test, n=9 to 14 mice.


( G ) Plasma LH levels at P45 in control and mutant mice . Mann - Whitney U test , n = 5 to 6 mice .

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(G) Plasma LH levels at P45 in control and mutant mice. Mann-Whitney U test, n=5 to 6 mice.


( H ) Mean uterine weight normalized to body weight ( mg / g BW ) . Unpaired t - test , n = 6 to 10 mice .

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(H) Mean uterine weight normalized to body weight (mg/g BW). Unpaired t-test, n=6 to 10 mice.


( A ) Chromatin features mapped here displayed differences between CLL ( patient CLL1 ) and an NBC donor ( H7 ) as shown for the TCF4 locus , which encodes for a transcription factor from the E protein family . Based on the increased H3K4me1 , H3K27ac and ATAC signal , two predicted enhancer loci were marked that became active in CLL . Note that the y - axis for RNA - seq is scaled differently for CLL ( 8000 ) and NBCs ( 100 ) to visualize that the TCF4 gene was not completely silenced but lowly expressed also in NBCs as evident also from the H3K36me3 mark . Light grey depicts active chromatin region and dark grey the confined enhancer locus coinciding with an open chromatin region .

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(A) Chromatin features mapped here displayed differences between CLL (patient CLL1) and an NBC donor (H7) as shown for the TCF4 locus, which encodes for a transcription factor from the E protein family. Based on the increased H3K4me1, H3K27ac and ATAC signal, two predicted enhancer loci were marked that became active in CLL. Note that the y-axis for RNA-seq is scaled differently for CLL (8000) and NBCs (100) to visualize that the TCF4 gene was not completely silenced but lowly expressed also in NBCs as evident also from the H3K36me3 mark. Light grey depicts active chromatin region and dark grey the confined enhancer locus coinciding with an open chromatin region.


( D ) Distribution of ~ 24 , 400 annotated differentially accessible regions ( ATAC - seq ) in NBC and CLL samples according to the chromatin state annotation . In total 7 , 605 annotated regions gained an ATAC - seq signal in CLL , while it was lost at 16 , 790 loci .

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(D) Distribution of ~24,400 annotated differentially accessible regions (ATAC-seq) in NBC and CLL samples according to the chromatin state annotation. In total 7,605 annotated regions gained an ATAC-seq signal in CLL, while it was lost at 16,790 loci.


( A ) Left , example of a large PMD on chromosome 2 derived from a consensus of CLL samples ( n = 11 ) . Right , genome - wide quantification of PMDs across CLL samples ( n = 11 ) and NBCs ( n = 6 ) . Red , methylated DNA ; blue , unmethylated DNA .

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(A) Left, example of a large PMD on chromosome 2 derived from a consensus of CLL samples (n=11). Right, genome-wide quantification of PMDs across CLL samples (n=11) and NBCs (n=6). Red, methylated DNA; blue, unmethylated DNA.


( B ) Expression of genes located inside ( blue ) and outside ( grey ) the PMD regions . ( C ) Fraction of differentially expressed genes inside or outside PMD regions . Up - and downregulated genes are shown in red and green , respectively .

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(B) Expression of genes located inside (blue) and outside (grey) the PMD regions. (C) Fraction of differentially expressed genes inside or outside PMD regions. Up- and downregulated genes are shown in red and green, respectively.


( D ) Upper panel : Average signal of histone modification marks normalized to H3 and standard deviation in 5 - kb windows around the ± 50 kb flanking regions of PMD boundaries . Normalized fold changes were calculated by dividing to the average signal flanking outside the PMD boundaries . Blue box , within PMDs ; thin line , outside PMDs , nor . - normalized . Lower panel : Distribution of bound CTCF sites in CLL cells as determined by ChIP - seq ( blue line ) around the ± 50 kb flanking regions of PMD boundaries in 5 kb windows . The height of the curves gives the sum of the next nearest CTCF peak at the given distance to the PMD boundary .

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(D) Upper panel: Average signal of histone modification marks normalized to H3 and standard deviation in 5-kb windows around the ±50 kb flanking regions of PMD boundaries. Normalized fold changes were calculated by dividing to the average signal flanking outside the PMD boundaries. Blue box, within PMDs; thin line, outside PMDs, nor. - normalized. Lower panel: Distribution of bound CTCF sites in CLL cells as determined by ChIP-seq (blue line) around the ±50 kb flanking regions of PMD boundaries in 5 kb windows. The height of the curves gives the sum of the next nearest CTCF peak at the given distance to the PMD boundary.


( A ) Correlation function of H3K4me3 ChIP - seq reads . A broadening of H3K4me3 domains in CLL by 1 - 2 nucleosomes was detected . Error bars represent the SEM .

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(A) Correlation function of H3K4me3 ChIP-seq reads. A broadening of H3K4me3 domains in CLL by 1-2 nucleosomes was detected. Error bars represent the SEM.


( B ) H3K4me3 peak width distribution at common promoters in CLL and NBC controls .

(B) H3K4me3 peak width distribution at common promoters in CLL and NBC controls.


( C ) Distribution of nucleosome occupancy calculated from histone H3 ChIP - seq averaged over a 1000 bp window within promoters . H3K4me3 regions displayed higher nucleosome density for CLL as compared to NBC samples .

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(C) Distribution of nucleosome occupancy calculated from histone H3 ChIP-seq averaged over a 1000 bp window within promoters. H3K4me3 regions displayed higher nucleosome density for CLL as compared to NBC samples.


( D ) Exemplary region at the TAF13 promoter showing higher H3K4me3 levels upstream of the TSS with lost ATAC signal ( grey bar ) as compared to the NBC control .

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(D) Exemplary region at the TAF13 promoter showing higher H3K4me3 levels upstream of the TSS with lost ATAC signal (grey bar) as compared to the NBC control.


( E ) TF motif analysis of ATAC signal lost at CLL promoters with broadened H3K4me3 regions . An enrichment for the NFY TF motif was detected .

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(E) TF motif analysis of ATAC signal lost at CLL promoters with broadened H3K4me3 regions. An enrichment for the NFY TF motif was detected.


( F ) Heatmap of genome - wide histone modification patterns at promoters within - 2 to 5 kb around the TSS ( x - axis ) for an NBC ( H3 ) and a patient ( CLL1 ) sample . Each line on the y - axis corresponds to one promoter . The clustering revealed one cluster with loss of H3K4me3 at bivalent promoters in CLL .

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(F) Heatmap of genome-wide histone modification patterns at promoters within -2 to 5 kb around the TSS (x-axis) for an NBC (H3) and a patient (CLL1) sample. Each line on the y-axis corresponds to one promoter. The clustering revealed one cluster with loss of H3K4me3 at bivalent promoters in CLL.


( D ) Clustering of samples via expression of bidirectionally regions that are differential between NBCs and CLL and quantified using DESeq2 .

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(D) Clustering of samples via expression of bidirectionally regions that are differential between NBCs and CLL and quantified using DESeq2.


( E ) Volcano plot of differential super - enhancers targeting known leukemia and cancer genes . Examples include SE loss at CDKN1A , PI3KC2B and KMT2B ( MLL2 ) and SE gain at FMOD , CREB3L2 , CTLA4 , TCF4 , LEF1 and BCL2 . Points represent non - differential SEs ( grey ) , and differential SEs ( FDR < 0 . 01 ) with fold change greater than 1 ( orange ) .

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(E) Volcano plot of differential super-enhancers targeting known leukemia and cancer genes. Examples include SE loss at CDKN1A, PI3KC2B and KMT2B (MLL2) and SE gain at FMOD, CREB3L2, CTLA4, TCF4, LEF1 and BCL2. Points represent non-differential SEs (grey), and differential SEs (FDR < 0.01) with fold change greater than 1 (orange).


( F ) RNA expression changes of selected genes associated with differential SEs . Top : genes significantly ( FDR < 0 . 05 ) up - regulated by SEs in CLL . Bottom : Genes significantly down - regulated by SEs in CLL .

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(F) RNA expression changes of selected genes associated with differential SEs. Top: genes significantly (FDR < 0.05) up-regulated by SEs in CLL. Bottom: Genes significantly down-regulated by SEs in CLL.


( G ) Comparison of normalized gene expression of histone deacetylases between CLL and NBCs . Histone deacetylases significantly up - regulated in CLL are shown in bold .

(G) Comparison of normalized gene expression of histone deacetylases between CLL and NBCs. Histone deacetylases significantly up-regulated in CLL are shown in bold.


( H ) HDAC activity and its inhibition by panobinostat in B cells from CLL patients ( red ) in comparison to healthy donors ( grey ) . Error bars indicate standard deviation measured in four biological replicates .

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(H) HDAC activity and its inhibition by panobinostat in B cells from CLL patients (red) in comparison to healthy donors (grey). Error bars indicate standard deviation measured in four biological replicates.


( I ) Genome browser view of H3K27ac tracks ( in grey ) at exemplary genes for NBCs and CLL cells 24 hours after mock and after panobinostat treatment . At genes such as CDKN1A ( cell cycle control ) and KLF13 , reduced H3K27ac signal in CLL was increased upon HDAC inhibition to the level found in NBCs . WNT11 is shown as an example of a de novo gain of an active enhancer due to treatment with panobinostat .

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(I) Genome browser view of H3K27ac tracks (in grey) at exemplary genes for NBCs and CLL cells 24 hours after mock and after panobinostat treatment. At genes such as CDKN1A (cell cycle control) and KLF13, reduced H3K27ac signal in CLL was increased upon HDAC inhibition to the level found in NBCs. WNT11 is shown as an example of a de novo gain of an active enhancer due to treatment with panobinostat.


( J ) Heatmap displaying changes in H3K27ac read occupancy in CLL upon panobinostat treatment for 24 h . A general gain of H3K27ac in enhancers upon panobinostat treatment was observed .

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(J) Heatmap displaying changes in H3K27ac read occupancy in CLL upon panobinostat treatment for 24 h. A general gain of H3K27ac in enhancers upon panobinostat treatment was observed.


( B ) H3K27ac ( left ) and DNA methylation ( right ) at NFAT binding sites . CLL cells showed both an H3K27ac enrichment and DNA - hypomethylation at NFAT target sites , suggesting a higher activity of TFs from the NFAT family in CLL .

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(B) H3K27ac (left) and DNA methylation (right) at NFAT binding sites. CLL cells showed both an H3K27ac enrichment and DNA-hypomethylation at NFAT target sites, suggesting a higher activity of TFs from the NFAT family in CLL.


( D ) ATAC footprints for E2A , EBF and CTCF motifs from the Homer analysis . The E2A motif footprint ( binding site of E protein family TFs like TCF4 ) displayed an increased binding signal in CLL while sites with the EBF and CTCF motif lost the ATAC signal .

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(D) ATAC footprints for E2A, EBF and CTCF motifs from the Homer analysis. The E2A motif footprint (binding site of E protein family TFs like TCF4) displayed an increased binding signal in CLL while sites with the EBF and CTCF motif lost the ATAC signal.


( E ) Expression of the genes nearest to target enhancers with constitutively bound ( " stable " ) CTCF vs . enhancers that lost CTCF in CLL . Loss of CTCF binding correlated with reduced gene expression .

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(E) Expression of the genes nearest to target enhancers with constitutively bound ("stable") CTCF vs. enhancers that lost CTCF in CLL. Loss of CTCF binding correlated with reduced gene expression.


( G ) Enhancer - promoter rewiring at the NFKB2 locus . A switch of interactions between the NFKB2 promoter and two different enhancers in CLL ( red line ) vs . NBCs ( grey line ) was observed . Based on the CTCF ChIP - seq analysis both intronic enhancers at the NFKB2 and the FBXL15 gene show ( nearby ) constitutively bound CTCF , even though the targets of the two enhancers are switched .

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(G) Enhancer-promoter rewiring at the NFKB2 locus. A switch of interactions between the NFKB2 promoter and two different enhancers in CLL (red line) vs. NBCs (grey line) was observed. Based on the CTCF ChIP-seq analysis both intronic enhancers at the NFKB2 and the FBXL15 gene show (nearby) constitutively bound CTCF, even though the targets of the two enhancers are switched.


( E ) Analysis of regulation of SNX22 by the intronic H464 enhancer shown in panel B . This enhancer lost its ATAC signal at the predicted EBF1 binding site . EBF1 ChIP - seq analysis validated that EBF1 is indeed lost at this site . Although H3K27ac at the locus was only slightly reduced , transcription of SNX22 was largely reduced . These findings are consistent with a mechanism where EBF1 binding drives gene expression of SNX22 by binding to H464 .

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(E) Analysis of regulation of SNX22 by the intronic H464 enhancer shown in panel B. This enhancer lost its ATAC signal at the predicted EBF1 binding site. EBF1 ChIP-seq analysis validated that EBF1 is indeed lost at this site. Although H3K27ac at the locus was only slightly reduced, transcription of SNX22 was largely reduced. These findings are consistent with a mechanism where EBF1 binding drives gene expression of SNX22 by binding to H464.


Primary human NK cells ( n = 19 different donors per condition ) were co - incubated with either no target cells ( NK ( - ) ) , autologous CD4 T cells ( Mock ) or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) at an effector : target cell ratio of 1 : 2 . NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry . ( A ) Representative histograms ( overlay ) displaying CD107a expression on NK cells after culture with the described culture conditions ( NK ( - ) , Mock , HIV ) . For the HIV - 1 co - culture condition , subsequent analyses of antigen expression were conducted by gating non - responsive ( CD107a - ) and responsive ( CD107a + ) NK cell subsets . ( B ) Relative frequency of CD107a + NK cells ( y - axis ) after culture in the described conditions ( x - axis ) ( n = 19 different donors ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry. (A) Representative histograms (overlay) displaying CD107a expression on NK cells after culture with the described culture conditions (NK(-), Mock, HIV). For the HIV-1 co-culture condition, subsequent analyses of antigen expression were conducted by gating non-responsive (CD107a-) and responsive (CD107a+) NK cell subsets. (B) Relative frequency of CD107a+ NK cells (y-axis) after culture in the described conditions (x-axis) (n = 19 different donors). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Primary human NK cells ( n = 19 different donors per condition ) were co - incubated with either no target cells ( NK ( - ) ) , autologous CD4 T cells ( Mock ) or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) at an effector : target cell ratio of 1 : 2 . NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry . ( C ) HIV - specific response of bulk NK cells displayed as percentage points ( p . p . ) CD107a + NK cells after normalization to mock CD4 T cells ( y - axis ) ( n = 19 different donors ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry. (C) HIV-specific response of bulk NK cells displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 19 different donors). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Primary human NK cells ( n = 19 different donors per condition ) were co - incubated with HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) ( E ) Representative histograms ( overlay ) showing TRAIL surface expression as fluorescence intensity on NK cells after co - incubation with HIV - 1 - infected CD4 T cells . Histograms show TRAIL expression for CD107a + and CD107a - NK cell subsets ( solid lines ) as well as their corresponding FMO controls ( dotted lines ) . Gate defining TRAIL + cells is indicated as well . ( F ) Left panel : Relative frequency of TRAIL + cells ( y - axis ) of CD107a + and CD107a - NK cell subsets ( x - axis ) after co - incubation with HIV - 1 - infected CD4 T cells . Right panel : Difference in relative frequency of TRAIL + cells ( y - axis ) between CD107a + and CD107a - cells displayed as p . p . ( n = 19 different donors ) . Data information : Wilcoxon signed - rank test . Adjustment for multiplicity : Benjamini and Hochberg false discovery rate ( FDR ) F , left panel : ) ; Bonferroni ( F , right panel Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points . Lines connecting CD107a - and CD107a + cells refer to measured values of the same donor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with HIV-1-infected (NL4-3) CD4 T cells (HIV) (E) Representative histograms (overlay) showing TRAIL surface expression as fluorescence intensity on NK cells after co-incubation with HIV-1-infected CD4 T cells. Histograms show TRAIL expression for CD107a+ and CD107a- NK cell subsets (solid lines) as well as their corresponding FMO controls (dotted lines). Gate defining TRAIL+ cells is indicated as well. (F) Left panel: Relative frequency of TRAIL+ cells (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells. Right panel: Difference in relative frequency of TRAIL+ cells (y-axis) between CD107a+ and CD107a- cells displayed as p.p. (n = 19 different donors). Data information: Wilcoxon signed-rank test. Adjustment for multiplicity: Benjamini and Hochberg false discovery rate (FDR) F, left panel:); Bonferroni (F, right panel Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


Primary human NK cells ( n = 19 different donors per condition ) were co - incubated with HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) ( G ) Left panel : TRAIL surface expression ( y - axis ) of CD107a + and CD107a - NK cell subsets ( x - axis ) after co - incubation with HIV - 1 - infected CD4 T cells displayed as median fluorescence intensity . Right panel : Differences in TRAIL surface expression ( y - axis ) between CD107a + and CD107a - cells displayed as fold - difference ( n = 19 different donors ) . Data information : Wilcoxon signed - rank test . Adjustment for multiplicity : ; Bonferroni G ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points . Lines connecting CD107a - and CD107a + cells refer to measured values of the same donor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with HIV-1-infected (NL4-3) CD4 T cells (HIV) (G) Left panel: TRAIL surface expression (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells displayed as median fluorescence intensity. Right panel: Differences in TRAIL surface expression (y-axis) between CD107a+ and CD107a- cells displayed as fold-difference (n = 19 different donors). Data information: Wilcoxon signed-rank test. Adjustment for multiplicity: ; Bonferroni G). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


Comparison of CD107a expression levels between various NK cell subsets after co - incubation with either no target cells present ( - ) , autologous CD4 T cells ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) ( n = 13 different donors per condition ) . Predefined NK cell subsets comprise CD56Dim , CD56Bright , KIR - educated cells , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) and negative for NKG2A , NKG2A - educated cells , expressing NKG2A but lacking self - inhibitory KIR , and uneducated cells , lacking self - inhibitory KIR and NKG2A altogether . ( A ) Relative frequency of CD107a + cells ( y - axis ) of predefined NK cell subsets ( x - axis ) . Left panel displays data for bulk NK cells , middle panel for CD56Dim and CD56Bright NK cells , right panel for uneducated , KIR - educated and NKG2A - educated cells ( n = 13 different donors ) . Data information : Wilcoxon signed - rank test adjusted for multiple comparisons ( Bonferroni ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of CD107a expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (A) Relative frequency of CD107a+ cells (y-axis) of predefined NK cell subsets (x-axis). Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of CD107a expression levels between various NK cell subsets after co - incubation with either no target cells present ( - ) , autologous CD4 T cells ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) ( n = 13 different donors per condition ) . Predefined NK cell subsets comprise CD56Dim , CD56Bright , KIR - educated cells , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) and negative for NKG2A , NKG2A - educated cells , expressing NKG2A but lacking self - inhibitory KIR , and uneducated cells , lacking self - inhibitory KIR and NKG2A altogether . ( B ) HIV - specific responses of predefined NK cell sub - populations ( x - axis ) displayed as percentage points ( p . p . ) CD107a + NK cells after normalization to mock CD4 T cells ( y - axis ) ( n = 13 different donors ) . Data information : Wilcoxon signed - rank test adjusted for multiple comparisons ( Bonferroni ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of CD107a expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (B) HIV-specific responses of predefined NK cell sub-populations (x-axis) displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of TRAIL expression levels between various NK cell subsets after co - incubation with either no target cells present ( - ) , autologous CD4 T cells ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) ( n = 13 different donors per condition ) . Predefined NK cell subsets comprise CD56Dim , CD56Bright , KIR - educated cells , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) and negative for NKG2A , NKG2A - educated cells , expressing NKG2A but lacking self - inhibitory KIR , and uneducated cells , lacking self - inhibitory KIR and NKG2A altogether . ( C ) TRAIL expression levels ( y - axis ) of predefined NK cell subsets ( x - axis ) . Left panel displays data for bulk NK cells , middle panel for CD56Dim and CD56Bright NK cells , right panel for uneducated , KIR - educated and NKG2A - educated cells . Expression is displayed as median fluorescence intensity ( MdFI ) ( n = 13 different donors ) . Data information : Wilcoxon signed - rank test adjusted for multiple comparisons ( Bonferroni ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of TRAIL expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (C) TRAIL expression levels (y-axis) of predefined NK cell subsets (x-axis). Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells. Expression is displayed as median fluorescence intensity (MdFI) (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of TRAIL expression levels between various NK cell subsets after co - incubation with either no target cells present ( - ) , autologous CD4 T cells ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) ( n = 13 different donors per condition ) . Predefined NK cell subsets comprise CD56Dim , CD56Bright , KIR - educated cells , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) and negative for NKG2A , NKG2A - educated cells , expressing NKG2A but lacking self - inhibitory KIR , and uneducated cells , lacking self - inhibitory KIR and NKG2A altogether . ( D ) Comparison of TRAIL expression levels ( y - axis ) between CD107a - and CD107a + cells of predefined NK cell subsets . Left panel displays data for bulk NK cells , middle panel for CD56Dim and CD56Bright NK cells , right panel for uneducated , KIR - educated and NKG2A - educated cells . Expression is displayed as MdFI . ( n = 13 different donors ) . ( E ) Fold - differences in TRAIL surface expression ( y - axis ) between CD107a + and CD107a - cells of pre - defined NK cell sub - populations ( x - axis ) . ( n = 13 different donors ) . Data information : Wilcoxon signed - rank test adjusted for multiple comparisons ( Bonferroni ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points . Lines connecting CD107a - and CD107a + cells refer to measured values of the same donor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of TRAIL expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (D) Comparison of TRAIL expression levels (y-axis) between CD107a- and CD107a+ cells of predefined NK cell subsets. Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells. Expression is displayed as MdFI. (n = 13 different donors). (E) Fold-differences in TRAIL surface expression (y-axis) between CD107a+ and CD107a- cells of pre-defined NK cell sub-populations (x-axis). (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


Primary human CD4 T cells from eight different donors ( n = 8 ) were separately infected with lab - adapted and primary HIV - 1 strains . Combined expression of DR4 and DR5 was assessed by flow cytometry four days after infection . Uninfected CD4 T cells were determined as CD4 - positive and HIV - 1 p24 - negative , infected cells as CD4 - negative and p24 - positive . ( A ) Histograms ( overlay ) of one representative donor displaying combined DR4 / 5 surface expression on CD4 T cells previously incubated with either NL4 - 3 , JR - CSF , WITO , CH198 , or CH236 . Expression is displayed as fluorescence intensity ( x - axis ) . ( B ) Comparison of the combined surface expression of DR4 / 5 between mock ( white circles ) , uninfected ( grey circles ) and infected CD4 T cells ( orange circles ) , from left to right : Mock : n = 8 ; NL4 - 3 : n = 8 ; JR - CSF : n = 8 ; WITO : n = 7 ; CH198 : n = 7 ; CH236 : n = 8 ( 7 - 8 different donors per condition ) . Expression is displayed as relative fluorescence intensity ( RFI ) after normalization to the respective secondary - AB - only control ( y - axis ) . Data information Wilcoxon signed - rank test . Values for non - infected and infected cells of the same donor are connected with lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human CD4 T cells from eight different donors (n = 8) were separately infected with lab-adapted and primary HIV-1 strains. Combined expression of DR4 and DR5 was assessed by flow cytometry four days after infection. Uninfected CD4 T cells were determined as CD4-positive and HIV-1 p24-negative, infected cells as CD4-negative and p24-positive. (A) Histograms (overlay) of one representative donor displaying combined DR4/5 surface expression on CD4 T cells previously incubated with either NL4-3, JR-CSF, WITO, CH198, or CH236. Expression is displayed as fluorescence intensity (x-axis). (B) Comparison of the combined surface expression of DR4/5 between mock (white circles), uninfected (grey circles) and infected CD4 T cells (orange circles), from left to right: Mock: n = 8; NL4-3: n = 8; JR-CSF: n = 8; WITO: n = 7; CH198: n = 7; CH236: n = 8 (7-8 different donors per condition). Expression is displayed as relative fluorescence intensity (RFI) after normalization to the respective secondary-AB-only control (y-axis). Data information Wilcoxon signed-rank test. Values for non-infected and infected cells of the same donor are connected with lines.


Degranulation of primary human NK cells after co - culture with various target cells in the presence or absence of αTRAIL ( A ) Comparison of CD107a expression after co - culture with 721 . 221 target cells with either 10 µg / ml αTRAIL or isotype control ( Iso ) using flow cytometry ( n = 10 different donors per condition ) . Each data point represents the mean of 2 technical replicates . Effector : target ratio was 1 : 1 . Left panel : Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor . Right panel : Box plots showing relative frequency of CD107a + NK cells ( y - axis ) . ( B ) Box plots display inhibition of degranulation ( y - axis ) after co - culture with 721 . 221 target cells in presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 10 different donors per condition ) . ( C ) Comparison of CD107a expression after co - culture with autologous HIV - I - infected CD4 T cells with either 10 µg / ml αTRAIL or isotype control ( Iso ) using flow cytometry ( n = 9 different donors per condition ) Each data point represents the mean of 2 technical replicates . Left panel : Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor . Right panel : Box plots showing relative frequency of CD107a + NK cells ( y - axis ) . ( D ) Box plots display inhibition of degranulation ( y - axis ) after co - culture with autologous HIV - I - infected CD4 T cells in the presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank test . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points . Bar graphs represent the mean and the associated whiskers display the SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αTRAIL (A) Comparison of CD107a expression after co-culture with 721.221 target cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry (n = 10 different donors per condition). Each data point represents the mean of 2 technical replicates. Effector:target ratio was 1:1. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis). (B) Box plots display inhibition of degranulation (y-axis) after co-culture with 721.221 target cells in presence of either isotype or αTRAIL as relative reduction compared to no antibody (n = 10 different donors per condition). (C) Comparison of CD107a expression after co-culture with autologous HIV-I-infected CD4 T cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry (n = 9 different donors per condition) Each data point represents the mean of 2 technical replicates. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis). (D) Box plots display inhibition of degranulation (y-axis) after co-culture with autologous HIV-I-infected CD4 T cells in the presence of either isotype or αTRAIL as relative reduction compared to no antibody (n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of primary human NK cells after co - culture with various target cells in the presence or absence of αDR4 / 5 . ( E ) Representative histograms ( overlay , left panel ) and bar graphs ( n = 3 independent experiments , right panel ) showing the individual and combined surface expression of DR4 and DR5 on 721 . 221 cells . Each data point represents the mean of 3 technical replicates . Data information : Wilcoxon signed - rank test . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points . Bar graphs represent the mean and the associated whiskers display the SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αDR4/5. (E) Representative histograms (overlay, left panel) and bar graphs (n = 3 independent experiments, right panel) showing the individual and combined surface expression of DR4 and DR5 on 721.221 cells. Each data point represents the mean of 3 technical replicates. Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of primary human NK cells after co - culture with various target cells in the presence or absence of αDR4 / 5 . ( F ) Comparison of CD107a expression after co - culture with 721 . 221 target cells in the presence of either αDR4 / 5 ( 10 µg / ml each ) , or 20 µg / ml isotype control ( Iso ) using flow cytometry ( n = 9 different donors per condition ) . Effector : target ratio was 1 : 1 . Box plots showing relative frequency of CD107a + NK cells ( y - axis ) . ( G ) Box plots display inhibition of degranulation after co - culture with 721 . 221 target cells in presence of either isotype or αDR4 / 5 as relative reduction compared to no antibody ( n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank test . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points . Bar graphs represent the mean and the associated whiskers display the SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αDR4/5. (F) Comparison of CD107a expression after co-culture with 721.221 target cells in the presence of either αDR4/5 (10 µg/ml each), or 20 µg/ml isotype control (Iso) using flow cytometry (n = 9 different donors per condition). Effector:target ratio was 1:1. Box plots showing relative frequency of CD107a+ NK cells (y-axis). (G) Box plots display inhibition of degranulation after co-culture with 721.221 target cells in presence of either isotype or αDR4/5 as relative reduction compared to no antibody (n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of primary human NK cells after incubation with plate - coated antibodies ( A ) Comparison of CD107a expression after incubation in either uncoated wells ( PBS ) or wells coated with αTRAIL , αNKG2D , αNKp46 , or isotype using flow cytometry ( n = 12 different donors per condition ) . Left panel : Concatenated density plot depicting CD107a expression as fluorescence intensity ( y - axis ) for one representative donor and 10 µg / ml antibody concentration . Right panel : Box plots showing relative frequency of CD107a + NK cells ( y - axis ) after incubation with plate - coated antibodies of different concentrations ( x - axis ) . ( B ) Comparison of CD107a expression after incubation with plate - coated DR4 protein , DR5 protein or human IgG using flow cytometry ( n = 11 different donors per condition ) . Left panel : Concatenated density plot depicting CD107a expression as fluorescence intensity ( y - axis ) for one representative donor and 10 µg / ml protein concentration . Right panel : Box plots showing relative frequency of CD107a + NK cells ( y - axis ) after incubation with plate - coated proteins of different concentrations ( x - axis ) . Data information : Wilcoxon signed - rank test adjusted for multiple comparisons ( Bonferroni ) . Spearman rank analysis . ( A , B , Each data point represents the mean of 2 technical replicates . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated antibodies (A) Comparison of CD107a expression after incubation in either uncoated wells (PBS) or wells coated with αTRAIL, αNKG2D, αNKp46, or isotype using flow cytometry (n = 12 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml antibody concentration. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated antibodies of different concentrations (x-axis). (B) Comparison of CD107a expression after incubation with plate-coated DR4 protein, DR5 protein or human IgG using flow cytometry (n = 11 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml protein concentration. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. (A, B, Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Degranulation of primary human NK cells after incubation with plate - coated antibodies ( C ) Comparison of granzyme B release after incubation with various stimuli ( 10 µg / ml each ) . Box plots showing Granzyme B concentration in the supernatant as determined by ELISA ( left panel : n = 8 different donors per condition , right panel : n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank test adjusted for multiple comparisons ( Bonferroni ) . Spearman rank analysis . C , Each data point represents the mean of 2 technical replicates . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated antibodies (C) Comparison of granzyme B release after incubation with various stimuli (10 µg/ml each). Box plots showing Granzyme B concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. C, Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Degranulation of primary human NK cells after incubation with plate - coated antibodies ( D ) Correlation analysis between relative frequency of CD107a + NK cells and granzyme B concentration ( n = 53 , data points obtained from A , B , and C , 11 different donors ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated antibodies (D) Correlation analysis between relative frequency of CD107a+ NK cells and granzyme B concentration (n = 53, data points obtained from A, B, and C, 11 different donors).


Degranulation of primary human NK cells after incubation with plate - coated whole proteins . ( E ) Comparison of CD107a expression after incubation with plate - coated DcR1 protein , osteoprotegerin ( OPG ) or human IgG using flow cytometry ( n = 9 different donors ) . Box plots showing relative frequency of CD107a + NK cells ( y - axis ) after incubation with plate - coated proteins of different concentrations ( x - axis ) . Data information : Wilcoxon signed - rank test adjusted for multiple comparisons ( Bonferroni ) . Spearman rank analysis . E ) Each data point represents the mean of 2 technical replicates . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated whole proteins. (E) Comparison of CD107a expression after incubation with plate-coated DcR1 protein, osteoprotegerin (OPG) or human IgG using flow cytometry (n = 9 different donors). Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. E) Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( A ) Comparison of IFNγ production ( y - axis ) after incubation with plate - coated antibodies or proteins ( 10 µg / ml each ) ( x - axis ) . Box plots showing IFNγ concentration in the supernatant as determined by ELISA ( left panel : n = 8 different donors per condition , right panel : n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank test . ( A , Samples were acquired in duplicate and the mean was calculated for each donor and condition . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Comparison of IFNγ production (y-axis) after incubation with plate-coated antibodies or proteins (10 µg/ml each) (x-axis). Box plots showing IFNγ concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. (A, Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( B ) Comparison of IFNγ levels in the supernatant after co - culture of NK cells with 721 . 221 target cells in the presence or absence of either 10 µg / ml αTRAIL or isotype control ( Iso ) using ELISA ( n = 8 different donors per condition ) . Effector : target ratio was 1 : 1 . Left panel : Box plots showing IFNγ levels ( y - axis ) for the described culture conditions ( x - axis ) . Right panel : Box plots display inhibition of IFNγ production by NK cells ( y - axis ) in presence of either isotype or αTRAIL as relative reduction compared to no antibody . Data information : Wilcoxon signed - rank test . B , Samples were acquired in duplicate and the mean was calculated for each donor and condition . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Comparison of IFNγ levels in the supernatant after co-culture of NK cells with 721.221 target cells in the presence or absence of either 10 µg/ml αTRAIL or isotype control (Iso) using ELISA (n = 8 different donors per condition). Effector:target ratio was 1:1. Left panel: Box plots showing IFNγ levels (y-axis) for the described culture conditions (x-axis). Right panel: Box plots display inhibition of IFNγ production by NK cells (y-axis) in presence of either isotype or αTRAIL as relative reduction compared to no antibody. Data information: Wilcoxon signed-rank test. B, Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( C ) Representative contour plots showing conjugate formation between NK cells and 721 . 221 target cells as determined by flow cytometry . Plots showing three distinct populations at the beginning of the co - culture ( 0 min ) and after 50 min : single 721 . 221 cells ( CT Orange - positive ) , single NK cells ( CT Violet - positive ) and conjugates ( double - positive ) . Relative frequency of NK cells in conjugates is calculated based on the total number of gated NK cells . ( D ) Comparison of conjugate formation after co - culture between NK cells and 722 . 221 cells after 0 min and after 50 min in the presence or absence of 10 µg / ml αTRAIL or isotype control ( n = 8 different donors ) . Left panel : Box plots showing relative frequency of NK cells in conjugates ( y - axis ) for different time points and conditions ( x - axis ) . Right panel : Box plots display inhibition of conjugate formation ( y - axis ) in the presence of either isotype or αTRAIL as relative reduction compared to no antibody . Data information : Wilcoxon signed - rank test . D ) Samples were acquired in duplicate and the mean was calculated for each donor and condition . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative contour plots showing conjugate formation between NK cells and 721.221 target cells as determined by flow cytometry. Plots showing three distinct populations at the beginning of the co-culture (0 min) and after 50 min: single 721.221 cells (CT Orange-positive), single NK cells (CT Violet-positive) and conjugates (double-positive). Relative frequency of NK cells in conjugates is calculated based on the total number of gated NK cells. (D) Comparison of conjugate formation after co-culture between NK cells and 722.221 cells after 0 min and after 50 min in the presence or absence of 10 µg/ml αTRAIL or isotype control (n = 8 different donors). Left panel: Box plots showing relative frequency of NK cells in conjugates (y-axis) for different time points and conditions (x-axis). Right panel: Box plots display inhibition of conjugate formation (y-axis) in the presence of either isotype or αTRAIL as relative reduction compared to no antibody. Data information: Wilcoxon signed-rank test. D) Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( E ) Expression levels of phosphorylated signaling proteins . Upper panel : Concatenated contour plots of one donor depicting the expression of phosphorylated signaling proteins Syk , p38 MAPK , Akt and PLC - γ2 for the following culture conditions and controls ( x - axis : left to right ) : FMO , PBS , isotype , αTRAIL , αNKp46 , αNKG2D , αCD16 . Lower panel : Bar graphs showing the percentage of p - Syk + , p - p38 MAPK + , p - Akt + and p - PLC - γ2 + NK cells after 30 min of stimulation ( n = 3 different donors ) . Bar graphs represent the median , whiskers display minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Expression levels of phosphorylated signaling proteins. Upper panel: Concatenated contour plots of one donor depicting the expression of phosphorylated signaling proteins Syk, p38 MAPK, Akt and PLC-γ2 for the following culture conditions and controls (x-axis: left to right): FMO, PBS, isotype, αTRAIL, αNKp46, αNKG2D, αCD16. Lower panel: Bar graphs showing the percentage of p-Syk+, p-p38 MAPK+, p-Akt+ and p-PLC-γ2+ NK cells after 30 min of stimulation (n = 3 different donors). Bar graphs represent the median, whiskers display minimum and maximum data points.


Lysis of different target cells in co - culture with NK cells was quantified in various cytotoxicity assays . ( A ) Left panel : Representative contour plots showing depletion of 721 . 221 target cells in the presence of NK cells . Middle panel : Percentage of target cells remaining ( y - axis ) after co - culture with NK cells in the presence of either αTRAIL or isotype control , in reference to target cells kept alone . Right panel : Box plots displaying difference of target cells remaining ( y - axis ) between αTRAIL and isotype conditions displayed as p . p . ( n = 12 different donors ) . Each data point represents the mean of at least 2 technical replicates . Data information : Wilcoxon signed - rank test . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for all donors in each batch . Lines connect each data value of the NK cell condition with their designated " No NK " control . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (A) Left panel: Representative contour plots showing depletion of 721.221 target cells in the presence of NK cells. Middle panel: Percentage of target cells remaining (y-axis) after co-culture with NK cells in the presence of either αTRAIL or isotype control, in reference to target cells kept alone. Right panel: Box plots displaying difference of target cells remaining (y-axis) between αTRAIL and isotype conditions displayed as p.p. (n = 12 different donors). Each data point represents the mean of at least 2 technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Lysis of different target cells in co - culture with NK cells was quantified in various cytotoxicity assays . ( B ) Left panel : Representative contour plots showing the percentage of . 221 - DR4 / 5KO ( control ) and . 221 - Cas9 cells ( target ) in the presence or absence of NK cells . Middle panel : Ratio between . 221 - DR4 / 5KO and . 221 - Cas9 cells ( y - axis ) in the presence or absence of NK cells . Right panel : Specific lysis of . 221 - Cas9 cells displayed as % ( n = 12 different donors ) . Each data point represents the mean of at least 3 technical replicates . Data information : Wilcoxon signed - rank test . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for all donors in each batch . Lines connect each data value of the NK cell condition with their designated " No NK " control . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (B) Left panel: Representative contour plots showing the percentage of .221-DR4/5KO (control) and .221-Cas9 cells (target) in the presence or absence of NK cells. Middle panel: Ratio between .221-DR4/5KO and .221-Cas9 cells (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of .221-Cas9 cells displayed as % (n = 12 different donors). Each data point represents the mean of at least 3 technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Lysis of different target cells in co - culture with NK cells was quantified in various cytotoxicity assays . ( C ) Left panel : Representative contour plots showing the percentage of Raji - pSIP ( control ) and Raji - DR5 + + ( target ) in the presence or absence of NK cells . Middle panel : Ratio between Raji - pSIP and Raji - DR5 + + ( y - axis ) in the presence or absence of NK cells . Right panel : Specific lysis of Raji - DR5 + + cells displayed as % ( n = 12 different donors ) . Each data point represents the mean of at least three technical replicates . Data information : Wilcoxon signed - rank test . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for all donors in each batch . Lines connect each data value of the NK cell condition with their designated " No NK " control . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate minimum and maximum data points .

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Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (C) Left panel: Representative contour plots showing the percentage of Raji-pSIP (control) and Raji-DR5++ (target) in the presence or absence of NK cells. Middle panel: Ratio between Raji-pSIP and Raji-DR5++ (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of Raji-DR5++ cells displayed as % (n = 12 different donors). Each data point represents the mean of at least three technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( A ) Example regions exhibiting the IPOD - HR occupancy where EPOD calls are made . Loose EPODs ( pink ) and Strict EPODs ( teal ) represent spans of high protein occupancy at lower and higher thresholds , respectively . Neighboring areas showing lower robust z scores are not EPODs . Note that following the definitions in [ 12 ] , the presence of an EPOD requires a sustained region of high average occupancy , without any regions in which occupancy drops to background levels . Thus , breaks in EPOD calls represent cases where a rolling average occupancy drops below the calling threshold ( e . g . , the breakpoint in the middle of pinQ in the top panel ) , and regions of apparent occupancy may nevertheless not be called due to being below the minimum length threshold ( as is the case for nohA ) .

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(A) Example regions exhibiting the IPOD-HR occupancy where EPOD calls are made. Loose EPODs (pink) and Strict EPODs (teal) represent spans of high protein occupancy at lower and higher thresholds, respectively. Neighboring areas showing lower robust z scores are not EPODs. Note that following the definitions in [12], the presence of an EPOD requires a sustained region of high average occupancy, without any regions in which occupancy drops to background levels. Thus, breaks in EPOD calls represent cases where a rolling average occupancy drops below the calling threshold (e.g., the breakpoint in the middle of pinQ in the top panel), and regions of apparent occupancy may nevertheless not be called due to being below the minimum length threshold (as is the case for nohA).


( E ) Specific locations across all conditions remain occupied by protein where EPODs across a representative region , the waa operon , were called ( left ) and unoccupied by RNA polymerase ( right ) . The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy .

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(E) Specific locations across all conditions remain occupied by protein where EPODs across a representative region, the waa operon, were called (left) and unoccupied by RNA polymerase (right). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy.


( A ) Number of EPODs called in different NAP deletions . D . S . denotes genotypes where cells were collected in the deep stationary phase of growth .

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(A) Number of EPODs called in different NAP deletions. D.S. denotes genotypes where cells were collected in the deep stationary phase of growth.


( D ) Distributions of mean protein occupancy at WT EPOD boundaries and background . The blue dots denote the median and the black line displays the interquartile ranges in each condition . The dashed pink line represents the WT median . ( * ) indicate the Wilcoxon Rank Sum test p value comparing the change in pseudomedian vs . WT for each condition that has been adjusted using the Benjamini and Hochberg method ( against a null hypothesis of no difference in pseudomedians ) . The grey line denotes the same comparison between the D . S . conditions . Underlined ( * ) ' s indicate a gain in the pseudomedian compared to baseline conditions , while no underline indicates a loss in the pseudomedian compared to baseline conditions . p value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * * . Three biological replicates were used for the " WT , Rich " case and " WT ( deep stat . ) " case , and two biological replicates for the " WT , Min " and " WT ( 2 ) , Rich " conditions .

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(D) Distributions of mean protein occupancy at WT EPOD boundaries and background. The blue dots denote the median and the black line displays the interquartile ranges in each condition. The dashed pink line represents the WT median. (*) indicate the Wilcoxon Rank Sum test p value comparing the change in pseudomedian vs. WT for each condition that has been adjusted using the Benjamini and Hochberg method (against a null hypothesis of no difference in pseudomedians). The grey line denotes the same comparison between the D.S. conditions. Underlined (*)'s indicate a gain in the pseudomedian compared to baseline conditions, while no underline indicates a loss in the pseudomedian compared to baseline conditions. p value < 0.05 = *, <0.005=**, <0.0005=***. Three biological replicates were used for the "WT, Rich" case and "WT (deep stat.)" case, and two biological replicates for the "WT, Min" and "WT (2), Rich" conditions.


( E ) Protein occupancy over the waa operon . The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . The EPOD over the waa operon is lost in the ∆ stpA ∆ hns condition which results in increased accessibility and RNA polymerase

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(E) Protein occupancy over the waa operon. The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. The EPOD over the waa operon is lost in the ∆stpA∆hns condition which results in increased accessibility and RNA polymerase occupancy


( G ) RNA - seq analysis shows log fold change compared to WT of waa operon expression upon deletion of hns and stpA . ( * ) indicate q value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * *

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(G) RNA-seq analysis shows log fold change compared to WT of waa operon expression upon deletion of hns and stpA. (*) indicate q value < 0.05 = *, < 0.005 = **, < 0.0005 = ***


( B ) Protein occupancy over the idn operon for each condition ( colors are denoted in ( A ) ) . The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . There is a large loss in protein occupancy when cells are shifted to 5KDG , leading to the loss of the call ed EPOD . Protein occupancy is restored once cells are grown in the second glucose condition . The 500bp normalized average of previously published H - NS ChIP - seq [ 20 ] exhibits high H - NS binding on the idnD promoter region .

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(B) Protein occupancy over the idn operon for each condition (colors are denoted in (A)). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. There is a large loss in protein occupancy when cells are shifted to 5KDG, leading to the loss of the call ed EPOD. Protein occupancy is restored once cells are grown in the second glucose condition. The 500bp normalized average of previously published H-NS ChIP-seq [20] exhibits high H-NS binding on the idnD promoter region.


( C ) To examine the expression of the idn operon at each shift , RNA - seq was performed . RNA - seq expression estimates log2 scaled for idn operon genes for WT cells grown in each condition ( as colored in ( A ) ) . Comparisons are denoted with colored dots with significance stars representing the adjusted p - value where ( * * * ) signify q - values < 0 .

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(C) To examine the expression of the idn operon at each shift, RNA-seq was performed. RNA-seq expression estimates log2 scaled for idn operon genes for WT cells grown in each condition (as colored in (A)). Comparisons are denoted with colored dots with significance stars representing the adjusted p-value where (***) signify q-values <0.0005


( B ) Difference in Log2 ratio of fitnesses of the KDG - exposed cells with unexposed lacZ : : cat relative to that observed prior to KDG exposure , given as a function of time since exposure to KDG . Day - 1 corresponds to the day before KDG exposure , Day 0 is cells taken immediately after growth in KDG , and subsequent timepoints reflect different durations of growth in M9 Min Glu prior to competition ( See ( A ) ) . Points show medians across replicates ( 3 biological replicates for 0 . 5 days , 6 biological replicates for all other timepoints ) ; error bars show 95 % confidence intervals for the difference relative to the Day - 1 timepoint , calculated using the R wilcox . test function . Significance was assessed using a Wilcoxon rank sum test comparing the distribution at each timepoint to the - 1 day ( naive ) timepoint : * , p < 0 . 05 ; * * , p < 0 . 005 .

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(B) Difference in Log2 ratio of fitnesses of the KDG-exposed cells with unexposed lacZ::cat relative to that observed prior to KDG exposure, given as a function of time since exposure to KDG. Day -1 corresponds to the day before KDG exposure, Day 0 is cells taken immediately after growth in KDG, and subsequent timepoints reflect different durations of growth in M9 Min Glu prior to competition (See (A)). Points show medians across replicates (3 biological replicates for 0.5 days, 6 biological replicates for all other timepoints); error bars show 95% confidence intervals for the difference relative to the Day -1 timepoint, calculated using the R wilcox.test function. Significance was assessed using a Wilcoxon rank sum test comparing the distribution at each timepoint to the -1 day (naive) timepoint: *, p<0.05; **, p<0.005.


( C ) Mean protein occupancy was calculated across all WT EPOD locations that contain annotated prophages . The change in mean protein occupancy compared to WT was calculated for each condition , where anything negative is a loss in occupancy compared to WT . Hierarchical clustering was performed to examine which genotypes clustered together and were more similar . Permutation tests comparing the change in occupancy over EPODs containing prophages were performed against the rest of the genome . P value < 0 . 05 = * , < 0 . 005 = * * indicate a negative mean change in occupancy compared to WT .

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(C) Mean protein occupancy was calculated across all WT EPOD locations that contain annotated prophages. The change in mean protein occupancy compared to WT was calculated for each condition, where anything negative is a loss in occupancy compared to WT. Hierarchical clustering was performed to examine which genotypes clustered together and were more similar. Permutation tests comparing the change in occupancy over EPODs containing prophages were performed against the rest of the genome. P value <0.05 = *, <0.005=** indicate a negative mean change in occupancy compared to WT.


( A ) Example prophage region that is annotated with the Fis - and Hfq - associated HMM class 5 in our genome - wide HMM classification The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . Prophage genes are highlighted with a red box . The major peak associated with WT IPOD - HR occupancy is represented in a grey dashed line as a reference to compare IPOD - HR occupancy in the other genotypes . Modest loss of protein occupancy was observed at the same prophage - containing EPOD for ∆ fis ( light purple ; see color key in ( B ) ) and ∆ hfq ( dark purple ; see color key in ( B ) ) conditions compared to WT ( gold ) .

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(A) Example prophage region that is annotated with the Fis- and Hfq-associated HMM class 5 in our genome-wide HMM classification The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. Prophage genes are highlighted with a red box. The major peak associated with WT IPOD-HR occupancy is represented in a grey dashed line as a reference to compare IPOD-HR occupancy in the other genotypes. Modest loss of protein occupancy was observed at the same prophage-containing EPOD for ∆fis (light purple; see color key in (B)) and ∆hfq (dark purple; see color key in (B)) conditions compared to WT (gold).