The rs1024611 Regulatory Region Polymorphism Is Associated with <em>CCL2</em> Allelic Expression Imbalance

<div><p>CC chemokine ligand 2 (CCL2) is the most potent monocyte chemoattractant and inter-individual differences in its expression level have been associated with genetic variants mapping to the cis-regulatory regions of the gene. An A to G polymorphism in the <em>CCL2</em> enhancer region at position –2578 (rs1024611; A>G), was found in most studies to be associated with higher serum CCL2 levels and increased susceptibility to a variety of diseases such as HIV-1 associated neurological disorders, tuberculosis, and atherosclerosis. However, the precise mechanism by which rs1024611influences CCL2 expression is not known. To address this knowledge gap, we tested the hypothesis that rs1024611G polymorphism is associated with allelic expression imbalance (AEI) of <em>CCL2</em>. We used haplotype analysis and identified a transcribed SNP in the 3′UTR (rs13900; C>T) can serve as a proxy for the rs1024611 and demonstrated that the rs1024611G allele displayed a perfect linkage disequilibrium with rs13900T allele. Allele-specific transcript quantification in lipopolysaccharide treated PBMCs obtained from heterozygous donors showed that rs13900T allele were expressed at higher levels when compared to rs13900C allele in all the donors examined suggesting that <em>CCL2</em> is subjected to AEI and that that the allele containing rs1024611G is preferentially transcribed. We also found that AEI of <em>CCL2</em> is a stable trait and could be detected in newly synthesized RNA. In contrast to these in vivo findings, in vitro assays with haplotype-specific reporter constructs indicated that the haplotype bearing rs1024611G had a lower or similar transcriptional activity when compared to the haplotype containing rs1024611A. This discordance between the in vivo and in vitro expression studies suggests that the <em>CCL2</em> regulatory region polymorphisms may be functioning in a complex and context-dependent manner. In summary, our studies provide strong functional evidence and a rational explanation for the phenotypic effects of the <em>CCL2</em> rs1024611G allele.</p> </div

AEI of CCL2 in PBMC, cell lines and brain.

<p>A. Allelic ratios for cDNA and gDNA were determined by pyrosequencing in eight independent donors who were heterozygous for rs13900 and rs1024611 polymorphisms. RNA was extracted from PBMCs treated with LPS for 3 h. and cDNA was synthesized. Pyrosequencing was performed as described under Methods. The ratios of expression (C <i>vs.</i> T) are log₂-transformed and are shown on the y-axis. Statistical significance was determined using a two-tailed Wilcoxon rank sum test (p = 0.0009). B. Stability of allele-specific differences in the CCL2 expression in LPS treated PBMC from heterozygous individuals. AEI was assessed at three different times in 4 independent donors over a period of 4–6 months with a gap of at least 2 weeks between experiments in a single donor. The y-axis indicates the percent level of expression of the C or the T allele. C. AEI in nascent RNA. LPS treated PBMC were cultured in presence of ethylene uridine (EU). The EU RNA was subjected to a click reaction that adds a biotin handle which is then captured by streptavidin beads. cDNA was synthesized from the captured nascent RNA and PCR amplified and subjected to pyrosequencing. Data shown are from two independent biological replicates from a single donor. D. rs13900C allele is expressed at higher levels in heterozygous cell lines. Differential expression of CCL2 alleles in heterozygous cell lines (HeLa- cervical cancer cell line; HL60-myeloid leukemia cell line). cDNA was synthesized using RNA extracted from LPS treated cell lines and PCR and pyrosequencing were performed. E. AEI in brain tissue. RNA was extracted from post-mortem brain tissues obtained from HIV-1 infected and normal donors and the extent of AEI in CCL2 was assessed in heterozygous donors by pyrosequencing. F. Clinical features and pathology associated with the HIV-1 positive donors. HIVE- HIV encephalitis; PML-Progressive Multifocal Leukoencephalopathy; Donors B & D did not exhibit any neuropathology. Statistical significance for differences in the levels of expression between the alleles was calculated using a paired t-test (*, p<0.05, **, p<0.001, #, p<0.0001).</p

Linkage disequilibrium plots for the shared polymorphisms in the <i>CCL2</i> locus.

<p>In all, 9 different polymorphisms that are located within a 25 kb region that spans the rs1024611 polymorphism are shown. The heatmaps display pairwise r² (above diagonal) or D’ (below diagonal) for the each pair of polymorphisms. The relative physical distance between the markers is shown in the bottom of the plots. Populations shown are Yoruba in Ibadan, Nigeria (YRI), Japanese in Tokyo, Japan (JPT), Han Chinese in Beijing, China (CHB) and CEPH (Utah residents with ancestry from northern and western Europe, CEU) populations (HapMap data release #28 August 2010 on Build 36). Only unrelated individuals were used for the analysis. LD maps were constructed using the JLIN program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049498#pone.0049498-Carter1" target="_blank">[61]</a>.</p

Relative minor allele frequencies of <i>CCL2</i> HapMap SNPs in different populations.

<p>The minor allele frequency as well as the Hardy-Weinberg equilibrium p-values (in parenthesis) for the different SNPs in each population are indicated.</p><p>CCL2, CC chemokine ligand 2; SNP, Single nucleotide polymorphism; CEU, Utah residents with Northern and Western European ancestry from the CEPH collection (CEPH, Centre du Etude Polymorphisme Humain); CHB, Han Chinese from Beijing, China; JPT, Japanese from Tokyo, Japan; YRI, Yoruban from Ibadan, Nigeria; the numbers in parenthesis next to the each population group show the genotyped individuals included in the analysis.</p

Inferred haplotypes in the <i>CCL2</i> locus and their frequency.

<p>Only haplotypes with frequency greater than 2% in any population are shown. Data were downloaded from the Hapmap web site (<a href="http://hapmap.ncbi.nlm.nih.gov/" target="_blank">http://hapmap.ncbi.nlm.nih.gov/</a>) and haplotypes inferred using the ARLEQUIN program. Only the reference polymorphisms that were shared by all the population groups examined were used to generate the haplotypes. Population description is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049498#pone-0049498-t003" target="_blank">Table 3</a>. The numbers within the parenthesis indicate the number of individuals (n). The h18, h19, h20, h21, h22 were present exclusively in the YRI each with a frequency of 0.0045. h7 and h8 were present exclusively in CEU each with a frequency of 0.0045. h12 and h13 were present exclusively in CHB each with a frequency of 0.0061. h14 was exclusively present in JPT at a frequency of 0.0059. The haplotype frequency of h10 was 0.005 in CEU, 0.006 in CHB, and 0.009 in YRI. The haplotype frequency of h11 was 0.005 in CEU, 0.012 in CHB, and 0.012 in JPT. The ancestral state of the reference SNPs (Anc.) was obtained from the dBSNP database (<a href="http://www.ncbi.nlm.nih.gov/projects/SNP/" target="_blank">http://www.ncbi.nlm.nih.gov/projects/SNP/</a>).</p

Disease associations of the <i>CCL2</i> rs1024611 polymorphism.

<p>Disease associations of the <i>CCL2</i> rs1024611 polymorphism.</p

Epigenetic features associated with <i>CCL2</i> locus in cell lines and normal human astrocytes.

<p>(A–B) Relative location of the SNPs linked to rs13900 (highlighted by red dashed lines) to Encode DNase 1 hypersensitivity sites in the <i>CCL2</i> locus as depicted in the UCSC genome browser (Panel A) and histone and DNase 1 tracks in the human epigenome browser (Panel B). Nucleotide numbering is according to hg19. The DNase 1 sites in panel A are depicted as boxes. The shade of the box is proportional to the signal strength detected with darker shaded boxes representing increased sensitivity to digestion. The numbers next to the boxes indicate the numbers of cell lines in which the region is hypersensitive. Panel B shows a wiggle plot depicting relative enrichment of the histone activation markers (H3K4me1, H3K4me3, H3K27Ac, indicated in red) and histone repressive marks (H3K27me3, indicated in green) across the <i>CCL2</i> locus. The heatmap track was configured to set the threshold for the peaks at 20 and values higher than the threshold are shown in brown. Also shown are the tracks for DNAse 1 sensitivity (purple tracks). Regions in gray indicate the regions with higher peaks than the set threshold. (C–E) Heatmaps showing localized histone tracks in <i>CCL2</i> 5′-flanking regions that overlap with the linked polymorphisms. A 500 bp region that spans the indicated polymorphism is shown. No separate panel is shown for rs2857654 due to its proximity to rs1024611. Other details are as in Panel B. The source of the data used for the generation of the DNase 1 tracks is from the Geo accession number GSE29692 (DNaseI Hypersensitivity by Digital DNaseI from ENCODE/University of Washington; public release on June 03, 2011) and for the histone tracks is Geo Accession numbers GSM733763 (H3K27Ac), GSM733729 (H3K27Me), GSM733747 (H3K4me3), and GSM733710 (H3K4me1) deposited by the Bernstein Lab at the Broad Institute (Histone Modifications by ChIP-seq from ENCODE/Broad Institute; public release on Jun 2, 2011). The tracks were generated using ENCODE database and UCSC genome browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049498#pone.0049498-Kent1" target="_blank">[63]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049498#pone.0049498-Rosenbloom1" target="_blank">[64]</a> and Human epigenome browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049498#pone.0049498-Zhou2" target="_blank">[65]</a>.</p

Chromosomal positions and genomic location of the <i>CCL2</i> locus HapMap SNPs.

<p>CCL2, CC Chemokine ligand 2; SNP, Single nucleotide polymorphism; cds-coding sequence; hg18 corresponds to NCBI build 36.1 (Release Date Mar. 2006). Ancestral allele shown is from the dBSNP database (<a href="http://www.ncbi.nlm.nih.gov/projects/SNP/" target="_blank">www.ncbi.nlm.nih.gov/projects/SNP/</a><i>).</i></p

Transcriptional effects of the <i>cis</i>-regulatory region SNPs that are in LD with rs1024611.

<p>(A–B). On the left is the schematic of the <i>CCL2</i> haplotype-specific constructs that were examined for differences in transcriptional strength. The following pair of constructs bearing the indicated polymorphisms, <i>CCL2 -6.0TCAG</i> and <i>CCL2 -6.0AAGC</i> (rs1860190T, rs2857654C, rs1024611A, rs2857656G and rs1860190A, rs2857654A, rs1024611G, rs2857656C, respectively), were tested. The constructs were obtained from a single heterozygous donor exhibiting AEI. The constructs were transfected into U87MG astroglioma cells and were tested at basal level (panel A) and following TNF-α treatment (panel B). Luciferase activity was determined as described in the <i>Methods</i>. The relative luciferase units refer to the fold increase in activity obtained from the <i>CCL2 -6.0</i> constructs relative to that obtained with the promoterless pGL4.16 vector. The data shown were obtained from 7 independent experiments and the error bars indicate the standard error of mean and statistical significance was calculated using two-tailed Student’s <i>t</i> test (**, p<0.0001, *, p = 0.02).</p

Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell-derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood-derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell-mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory