LILRB1 Intron 1 Has a Polymorphic Regulatory Region That Enhances Transcription in NK Cells and Recruits YY1

This information is current as Kang Yu, Chelsea E. Davidson and Deborah N. Burshtyn of October 1, 2021. J Immunol published online 22 April 2020 http://www.jimmunol.org/content/early/2020/04/21/jimmun ol.2000164 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/04/21/jimmunol.200016 Material 4.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 22, 2020, doi:10.4049/jimmunol.2000164 The Journal of Immunology

LILRB1 Intron 1 Has a Polymorphic Regulatory Region That Enhances Transcription in NK Cells and Recruits YY1

Kang Yu,* Chelsea E. Davidson,*,1 and Deborah N. Burshtyn*,†,‡

LILRB1 is a highly polymorphic receptor expressed by subsets of innate and adaptive immune cells associated with viral and autoimmune diseases and targeted by pathogens for immune evasion. LILRB1 expression on human NK cells is variegated, and the frequency of LILRB1+ cells differs among people. However, little is known about the processes and factors mediating LILRB1 transcription in NK cells. LILRB1 expression in lymphoid and myeloid cells arises from two distinct promoters that are separated by the first exon and intron. In this study, we identified a polymorphic 3-kb region within LILRB1 intron 1 that is epigenetically marked as an active enhancer in human lymphoid cells and not monocytes. This region possesses multiple YY1 sites, and complexes of the promoter/enhancer combination were isolated using anti-YY1 in chromatin immunoprecipitation–loop. CRISPR-mediated deletion of the 3-kb region lowers LILRB1 expression in human NKL cells. Together, these results indicate the

enhancer in intron 1 binds YY1 and suggest YY1 provides a scaffold function enabling enhancer function in regulating LILRB1 Downloaded from gene transcription in human NK cells. The Journal of Immunology, 2020, 204: 000–000.

he human leukocyte Ig-like receptors (LILRs) family has One of the most highly studied LILR is LILRB1, an inhibitory 11 multifunctional regulatory receptors involved in im- receptor for which genetic variation has been linked to various mune tolerance, inflammation, hematopoietic differenti- autoimmune conditions, including rheumatoid arthritis, systemic

T http://www.jimmunol.org/ ation, and neural functions. LILRs are widely expressed within the lupus erythematosus, and atherosclerosis (6–13). LILRB1 is expressed immune system and interact with diverse ligands ranging from on monocytes, dendritic cells, B cells, and subsets of T and NK cells MHC class I (MHC-I) molecules, CNS-derived molecules, and host (2, 14, 15). LILRB1 regulates cellular responses through binding immune-modulatory to pathogen-derived molecules and classical and nonclassical MHC-I molecules (4, 16) as well as intact pathogens (1). The LILR family contains stimulatory recep- S100A9 (17). LILRB1 is exploited by several bacteria, viruses, tors that transmit signals through associated signaling subunits and and parasites that interact directly with LILRB1, such as dengue inhibitory receptors that repress signaling through ITIMs within virus, malaria, and human CMV (HCMV) (14, 18, 19). For ex- their cytoplasmic tails. The family is characterized by substantial ample, HCMV expresses an MHC-I mimic named UL18 that binds polymorphism and copy number variation, and the genetic diversity tightly to LILRB1 (14, 20). The presumed main function of UL18 by guest on October 1, 2021 has been linked to disease susceptibility (2–4). Each family member is to inhibit NK cell responses. LILRB1-negative NK cells control displays a unique expression pattern on immune cells and can be virus dissemination in vitro better than LILRB1+ NK cells (21– constitutively expressed or induced under particular conditions (5). 23). LILRB1 is the only LILR expressed on NK cells, and its expression is variegated. Moreover, the frequency of LILRB1+ NK cells differs among individuals. HCMV infection is also as- *Department of Medical Microbiology and Immunology, University of Alberta, sociated with the expansion of LILRB1+ NK cells, and LILRB1 Edmonton, Alberta T6G 2S2, Canada; †Alberta Transplant Institute, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; and ‡Li Ka Shing Institute of Virol- expression on other lymphoid cells can fluctuate with time, par- ogy, University of Alberta, Edmonton, Alberta T6G 2H7, Canada ticularly in response to HCMV replication (21, 24–26). We re- 1Current address: College of Veterinary Science, University of Saskatchewan, Sas- cently described that LILRB1 polymorphisms correlated with katoon, Saskatchewan, Canada. LILRB1 expression patterns on NK cells are also correlated with ORCID: 0000-0003-4829-3351 (D.N.B.). the control of HCMV in transplant patients (27). Received for publication February 14, 2020. Accepted for publication March 31, Although the precise mechanism underlying variegated LILRB1 2020. expression in NK cells and the variation in the LILRB1+ NK cell This work was supported by Canadian Institutes of Health Research Grants frequency are not known, we and others have linked particular MOP123257 and PJT162372 (awarded to D.N.B.). K.Y. was funded by studentships + from the China Scholarship Council and a University of Alberta Faculty of Medicine haplotypes with the frequency of LILRB1 NK cells (28, 29), a and Dentistry 75th Anniversary Award. C.E.D. was supported by scholarships from feature reminiscent of the expression patterns of the highly related the Natural Sciences and Engineering Research Council of Canada and Alberta and syntenic killer-cell Ig-like receptors (KIRs). For example, Innovates Health Solutions. KIRs have allele-specific expression patterns (30), and KIR ex- Address correspondence and reprint requests to Dr. Deborah Burshtyn, Department of Medical Microbiology and Immunology, 6-020 Katz Research Centre, University pression is acquired at a late stage of NK differentiation as is of Alberta, Edmonton, AB T6G 2S2, Canada. E-mail address: [email protected] LILRB1 (31). The KIR expression patterns are correlated with The online version of this article contains supplemental material. DNA methylation at the promoter region (32–34), and there is Abbreviations used in this article: BAC, bacterial artificial ; 3C, chro- evidence that DNA methylation is involved in keeping LILR mosome conformation capture; C/EBP, Ccaat-enhancer-binding ; ChIP, chro- quiescent (35). During NK development, a probabilistic matin immunoprecipitation; ChIP-Seq, ChIP sequencing; ddPCR, Droplet Digital PCR; GEO, Omnibus; HCMV, human CMV; KIR, killer-cell Ig- switch controls expression of KIR through a bidirectional pro- like receptor; LILR, leukocyte Ig-like receptor; LINE, long interspersed nuclear moter for which the relative strength of the forward activity over element; MHC-I, MHC class I; qPCR, quantitative PCR; SNP, single-nucleotide the reverse activity correlates with the frequency of a particular of polymorphism; YY1, Yin Yang 1. KIR gene progressing to permanent expression (36, 37). Poly- Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 morphisms within the bidirectional promoter are associated with

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000164 2 LILRB1 INTRON 1 ENHANCER allele-specific frequencies of expression for a given KIR gene (30, cells from each LILRB1+ clone were lysed using the SingleShot Cell Lysis 38). Although multiple KIR genes can be expressed in a single NK Kit (Bio-Rad, Hercules, CA) following the manufacturer’s protocol and cell, typically only one allele for each KIR gene is expressed, and immediately subjected to reverse transcription using iScript Advanced cDNA Synthesis Kit (Bio-Rad). A custom TaqMan genotyping assay (identifier: a piwi-like system arising from the antisense transcript may si- ANGZE69) was used to differentiate the expression of alleles specific for lence the other allele of a KIR gene that has initiated reverse rs1061079 (C/T) that was designed using the online tool (https://www. transcription (39). The LILRB1 gene has 16 exons, and tran- thermofisher.com/order/custom-genomic-products/tools/genotyping/). The scription is initiated at two distinct promoters (15). The first (59) sequences of primers and probes are listed in Supplemental Table I. Each Droplet Digital PCR (ddPCR) reaction was prepared by mixing the cDNA promoter is used by lymphoid cells, and the resulting transcript template derived from ∼1000 cells of every single clone, TaqMan primers has all 16 exons (15). We previously defined the core region of the probes mix, and ddPCR Supermix for Probes (no dUTP; Bio-Rad) and distal promoter with a functional JunD site that is required for then subjected to droplet generation using QX200 Droplet Generator (Bio- expression in NK cells (40). The second promoter is positioned Rad) following the instruction manual. The ddPCRs were amplified in a just upstream of exon 2 and contains functional PU.1 and Sp1 sites thermal cycler according to the kit instructions, and the fluorescence signals were detected and recorded using the QX200 Droplet Reader (Bio-Rad). The in line with its function in myeloid lineage cells (35). copy number per sample of the two alleles for the single-nucleotide poly- In the current study, we investigated the transcriptional regulation morphism (SNP) rs1061079 (C/T) was determined using QuantaSoft Soft- of LILRB1 in NK cells with a view to uncovering how lineage- ware (Bio-Rad). specific patterns of expression are generated and how polymor- DNA methylation analysis phisms selectively control the expression patterns in NK cells. We Genomic DNAwas isolated from ex vivo primary NK cells using the Illustra used the features of KIR transcriptional regulation and publicly tissue and cells genomicPrep Mini Spin Kit (GE Healthcare, Chicago, IL). available epigenomic data as a guide. We show that individual NK Bisulfite conversion was done using the EZ DNA Methylation kit (Zymo Downloaded from cells can express one or both alleles at the same time and that there is Research) following the manufacturer’s protocol. Each conversion reaction a correlation between high cell surface LILRB1 and expressing both was done with 500 ng of genomic DNA, and half of each reaction was used alleles. Although we did not find strong evidence of a bidirectional in the subsequent PCR. Primers were designed to amplify the DNA regions of interest avoiding the inclusion of CpG sites in the primer binding re- promoter, we identified an additional polymorphic enhancer within gions. The regions were amplified using the bisulfite primers listed in the first intron. We provide evidence that the deletion of the region Supplemental Table I. The PCR was done with AccuStart Taq DNA Po- reduces LILRB1 expression in an NK cell line, that the transcription lymerase HiFi (QuantaBio, Beverly, MA). PCR products were run on an factor Yin Yang 1 (YY1) associates with the region as well as the agarose gel and extracted according to the manufacturer’s protocol using http://www.jimmunol.org/ the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). An A-overhang promotors, and that the enhancer and promotors are in proximity, reaction was performed on purified PCR products before performing TA implicating YYI as a scaffold. The identification of this regulatory cloning into the pCRII vector following the TOPO TA Cloning Kit (Life region provides new (to our knowledge) insight into the regulation Technologies, Carlsbad, CA). The cloned PCR products in pCRII were of LILRB1 expression in NK and likely other lymphoid cells. transformed into TOP10 Escherichia coli, as per the manufacturer’s proto- col. Ten to 20 clones for each PCR product were selected and cultured in Luria–Bertani broth with ampicillin for 16 h, and the plasmid DNA was Materials and Methods isolated using the QIAprep Spin Miniprep Kit (Qiagen) and subsequently Cell lines and Abs sent for sequencing at McLab (San Francisco, CA). Each CpG site was analyzed in all clones to determine the percentage of methylation for each The human NK cell line NKL was cultured in Iscove’s medium supple- CpG cytosine. by guest on October 1, 2021 mented with 10% characterized FBS (HyClone), 1 mM L-glutamine, and 200 U/ml recombinant human IL-2 (Tecin; Biological Resources Branch, Putative enhancer fragment knockout by CRISPR-Cas9 National Cancer Institute at Frederick). The human B lymphoma cell line The putative enhancer region was amplified using iProof Hi-Fi DNA Po- 721.221 was maintained in Iscove’s medium supplemented with 10% lymerase (Bio-Rad) with the primers listed in Supplemental Table I. characterized FBS (Life Technologies) and 1 mM L-glutamine. PE-Cy5 CRISPR guide RNAs were designed using the online software (http:// mouse anti-human CD85j (clone: GHI/75) and PE-Cy5 mouse IgG2b k chopchop.cbu.uib.no/). The boundaries of the 3-kb region encompassing isotype control were purchased from BD Biosciences (San Jose, CA). the entire putative enhancer were targeted by two single guide RNAs Allophycocyanin-conjugated mouse anti-human CD85j (clone: HPF1) and (59-AATCAGTACTAAAAATCTTC-39 and 59-TGATGAGCATAGTATT- allophycocyanin-conjugated mouse IgG1k isotype control were purchased GGTG-39) using an Alt-R CRISPR-Cas9 System (Integrated DNA Tech- from eBioscience (Waltham, MA). Chromatin immunoprecipitation (ChIP) nologies, Coralville, IA) combined with a 4D-Nucleofector System (Lonza, grade anti-human YY1 (clone: D5D9Z) was obtained from Cell Signal- Basel, Switzerland). Briefly, the guide RNA and tracrRNA (conjugated with ing Technology, and the normal rabbit IgG control was included in the ATTO 550) were preincubated to form duplex, followed by incubating with SimpleChIP Enzymatic Chromatin IP Kit (no. 9003; Cell Signaling Tech- Cas9 nuclease to form ribonucleoprotein complex. The ribonucleoprotein nology, Danvers, MA). complex was then electroporated into NKL cells within the nucleofector Primary NK cell isolation and culturing system using the reagents from SF Cell Line 4D-NucleofectorTM X Kit S and the CM150 program. Nucleofection efficiency was determined by flow Blood samples were collected from healthy donors with written informed cytometry after 24 h, and the NKL cells were further incubated for 2 d and consent, and the experiments were performed as approved by the Health assessed for LILRB1 surface expression with Ab staining (clone: GHI/75) by Research Ethics Board at the University of Alberta (Edmonton, AB, flow cytometry. The population with decreased LILRB1 expression was Canada). PBMCs were isolated from the whole blood using Lympholyte-H sorted and followed with single-cell sorting to generate clones using a BD Cell Separation Media (Cedarlane, Burlington, ON, Canada). NK cells were FACSAria III sorter (BD Biosciences). The genomic DNA of the clones was then isolated from the PBMCs using the EasySep Human NK Cell Isolation isolated using QuickExtract DNA Extraction Solution (Lucigen, Middleton, kit (STEMCELL Technologies, Vancouver, BC, Canada) following the WI), and the knockout of the putative enhancer was validated using PCR and manufacturer’s protocol. NK cell purity was determined by flow cytom- Sanger sequencing. etry, and at least 90% purity was accepted. Purified NK cells were plated at 3, 1, and 0.3 cells per well with 104 irradiated 721.221 feeder cells per well Quantitative chromosome conformation capture to generate single clones using limiting dilution and cultured in Iscove’s (quantitative PCR) medium with 10% human serum and 1 mM L-glutamine, supplemented The chromosome conformation capture (3C)–quantitative PCR (qPCR) m with 0.5 g/ml PHA-P (Sigma-Aldrich) and 100 U/ml recombinant human assay was performed following the protocol as described (42) with some IL-2, as previously described (41). Clones were derived from plates with , minor modifications. NKL cells were cross-linked by 1% formaldehyde for growth in 30 wells after 30 d. 10 min at room temperature and quenched by 0.125 M glycine. The cells Allele-specific expression assay were then washed twice with ice-cold PBS and lysed with lysis buffer containing 10 mM Tris-HCl (pH = 8), 10 mM NaCl, and 0.2% Nonidet Ex vivo NK cell clones were first stained by anti-human CD85j (clone: HPF1) P-40 with protease inhibitor mixture (Cell Signaling Technology) for 45 min for surface LILRB1 expression using flow cytometry. Approximately 8 3 104 on ice. Nuclei were collected and resuspended in 1.23 restriction enzyme The Journal of Immunology 3 buffer supplemented with 0.3% SDS and then incubated at 37˚C with respectively. In contrast to D183, we detected the rs1061679-T shaking for 1 h. A total of 2% Triton X-100 was added to the nuclei to variant alone in several clones for both D185 and D500. For those sequester the SDS and incubated at 37˚C with shaking for 1 h. Cross- clones in which both alleles were detected, the ratio was variable linked genomic DNA within the nuclei was digested by EcoRI overnight at 37˚C with shaking. A total of 1.6% SDS was then added to the sample, with either allele being dominant (Fig. 1, right panels). and the restriction enzyme was inactivated by incubating at 65˚C for The trend that there is a more frequent expression of the 25 min. The digested DNA was diluted into 1.153 ligation buffer sup- rs1061679-T allele in two out of three donors fits with our earlier plemented with 1% Triton X-100 and incubated for 1 h at 37˚C with gentle observation of a correlation between the frequency of LILRB1+ NK shaking. T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA) was added to ligate the DNA fragments, and the reaction was incubated at 16˚C cells and LILRB1 gene polymorphisms in the regulatory regions for 4 h, followed with 30 min at room temperature. The cross-linking was of the gene (29). The SNP rs1061679 is in the coding domain but reversed by adding proteinase K and incubating at 65˚C overnight. After has strong linkage disequilibrium with the SNPs within the distal treatment with RNase for 45 min at 37˚C, the sample was purified by promoter (r2 = 0.92) and proximal promoter (r2 = 0.91), which we phenol-chloroform extraction and ethanol precipitation. The concentration showed to correlate with the frequency of LILRB1 expression on of the 3C library was determined using Qubit Fluorometric Quantitation System (Thermo Fisher Scientific). Specific ligation products were de- NK cells (27). Therefore, it is fitting that the rs1061679-T allele tected by TaqMan qPCR with the primers and probe listed in Supplemental was detected more frequently and on its own in two donors. Al- Table I. To generate a reference control library, we purchased the bacterial though D183 showed the opposite trend, it also had the fewest artificial chromosome (BAC) clone (WI2-1436-K15), which contains the clones analyzed and perhaps points to additional polymorphic whole LILRB1 gene, from BACPAC Resources (Oakland, CA) and per- formed parallel digestion, ligation, and purification steps as described regulatory regions. above. The values presenting the relative cross-linking frequency were calculated using the formula of 10(Ct 2 b)/a (b = intercept; a = slope) where Methylation analysis of LILRB1 distal promoter the parameters were from the standard curves generated using the BAC As shown earlier, there are a series of SNPs in the distal and Downloaded from reference control library. proximal promoter that are in near-perfect linkage disequilibrium ChIP and ChIP-loop assay with each other (27), forming haplotypes that correlate with the frequency of LILRB1+ NK cells. The two typical haplotypes are ChIP assay was performed using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) following the manufacturer’s protocol. Briefly, denoted as the major GGTG-AGG and minor CGTA-GAA, and the chromatin of NKL cells was cross-linked as described for the 3C assay. there is a very minor G—G-GAA variant, which is illustrated in Nuclei were isolated and incubated with micrococcal nuclease to digest the Fig. 2A. We previously reported there is not a difference in the http://www.jimmunol.org/ chromatin to fragments of between 150 and 900 bp. The nuclei were activity of the allelic variants of the distal promoter using reporter sonicated using the Sonic Dismembrator Model 100 (Thermo Fisher Sci- entific) to break down the nuclear membrane. The digested chromatin was assays (27), which implies that other regulatory mechanisms other collected and then incubated with anti-human YY1 (1:50 dilution) or than differential recruitment of transcription factors to the pro- normal rabbit IgG overnight at 4˚C with rotation. Protein G magnetic moters are involved. Although there are no obvious CpG islands in beads were added to each immunoprecipitated sample and incubated at the region of the distal promoter, methylation of individual CpG 4˚C for 2 h with rotation. The beads were washed, and the chromatin was sites can alter binding to regulate expression eluted at 65˚C for 30 min with vortexing in a thermomixer (Thermo Fisher Scientific). The cross-linking was reversed by incubating with proteinase K (44), and CpG methylation is an epigenetic modification that overnight at 65˚C. Predicted YY1 binding loci were assessed by standard could limit transcription in an allele-specific manner. by guest on October 1, 2021 PCR using the purified ChIP DNA and then analyzed in agarose gel. The To determine if CpG methylation near the promoter was cor- primers used for ChIP and ChIP-loop were designed based on the assay for related with promoter activity in general, we performed bisulfite transposase-accessible chromatin using sequencing results (Supplemental Fig. 2A) and listed in Supplemental Table I. sequencing analysis of the regions around the LILRB1 distal The ChIP-loop assay was performed as described previously (43) promoter from ex vivo purified NK cells, B cells, and monocytes in combined with the SimpleChIP Enzymatic Chromatin IP Kit. The digested four heterozygous donors and one homozygous donor for the AGG and ligated chromatin was generated as described above before proceeding haplotype (Fig. 2B). We found that the extent of methylation was to the immunoprecipitation using the YY1 or normal rabbit IgG Ab. The very low for the CpG site at 214,052 in NK and B cells, the only purified ChIP-loop library was subjected to PCR analysis. CpG in the core distal promoter. The CpG site at 214,052 is quite prominently methylated in monocytes, suggesting its methylation Results might be linked to preventing transcription from the distal pro- NK clones express both LILRB1 alleles to varying degrees moter in monocytes and fit with our observations that THP-1 cells Our previous work showed that both alleles of LILRB1 are can transcribe from the distal promoter when introduced as a expressed in a mixed population of ex vivo NK cells (29). To plasmid (45). In all three cell types, the degree of methylation examine the relative expression of each allele within single NK increases progressively for sites further upstream, and such a cells, we selected three individuals with varying LILRB1+ NK cell pattern is consistent with that location of the core promoter of the profiles as shown in Fig. 1 (left panels) known to be heterozygous LILRB1 gene, as previously reported (40). Curiously, the two at SNP rs1061679. We initially performed a single-cell analysis of CpG sites just upstream of 214,205 have higher methylation in ex vivo NK cells to determine the relative expression of each al- NK cells relative to B cells or monocytes, perhaps indicative of lele for rs1061679, but the sensitivity was not sufficient to draw NK-specific regulation through DNA modification. conclusions (data not shown). Therefore, we grew out clones from To test if there are differences between two alleles in terms of the purified NK cells and analyzed relative rs1061679 expression in at methylation status of the distal promoter, we examined the DNA least eight clones from each donor. The clones had varying surface methylation patterns of the distal promoters in isolated NK cells staining of LILRB1 (Supplemental Fig. 1), and we included a from the four heterozygous donors (Fig. 2C). The bisulfite se- clone without detectable surface expression for each donor in the quencing technique allowed us to segregate methylation status analysis. As expected, detection of LILRB1 transcript was cor- according to the alleles. The CpG site located at 214,290 from the related with surface expression; however, in each donor, we found translational start site is the only site to be substantively differen- a different pattern of expression for the two alleles. For D183, two tially methylated between the two haplotypes (p = 0.02). However, clones expressed only rs1061679-C and three clones expressed the allele with the higher methylation at 214,290 is the haplotype both alleles, whereas three clones had no detectable transcript. For associated with lower expression in NK cells (Fig. 2D). Although it D185 and D500, we detected both alleles in 5 of 12 and 8 of 11 clones, is possible a negative regulator occupies the undermethylated site or 4 LILRB1 INTRON 1 ENHANCER Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. LILRB1 gene allelic expression in ex vivo single NK clones. Primary NK cells were isolated from three donors (D183, D185, and D500), and LILRB1 expression was determined by flow cytometry. The white and black filled peaks indicate the unstained control and the staining using LILRB1 Ab (HP-F1), respectively. The frequency of the LILRB1+ population was shown inside of each plot. The corresponding histogram shows the copy number per sample of the LILRB1 transcript from the rs1061679-T and rs1061679-C allele determined by ddPCR in ex vivo NK cell clones. NK cells of different clones are plotted from low to high of LILRB1 surface expression as measured by flow cytometry (see Supplemental Fig. 1). RT-, negative control without adding reverse transcriptase. a positive regulator requires the methylation, the data do not fit well LILRB1. A reciprocal situation is found at the proximal promoter, with an epigenetic silencing mechanism similar to KIR arising from which is marked as an active promoter only for the CD14 cells. an antisense transcript. In the three lymphoid cell types, there is a 3-kb region within intron 1 with high sensitivity to DNase and three peaks densely Prediction of an enhancer in LILRB1 intron 1 marked by H3K4me1 (red) and H3K27ac (region boxed in Given that enhancers are elements typically controlling the cell Fig. 3A), a pattern of modifications associated with active en- type–specific gene expression that could mediate allele-specific hancers (47). The intensity of the signal is greatest for CD19, effects, we looked for evidence of additional regulatory elements followed by CD56 and then CD3, which corresponds to the ex- in the LILRB1 gene using data from the Roadmap Epigenomics pected proportion of cells that would express LILRB1 for each project (46). The dataset includes patterns of histone modifications lineage, and the region does not exhibit the modifications in CD14 and DNase sensitivity for populations of cells denoted as CD56, cells. Published assay for transposase-accessible chromatin using CD3, CD19, and CD14 markers for NK, T, B, and monocyte cells. sequencing data from decidual NK cells (48) also showed the 3-kb First, we examined the patterns of DNA accessibility and histone region has high accessibility, further supporting a role for this modification at the known promoters in the various cell populations region in regulating transcription (Supplemental Fig. 2A). In addition, (Fig. 3A). Active promoters are marked by DNase hypersensitivity analysis of available ChIP sequencing (ChIP-Seq) data from the En- (green) and H3K4me3 (blue) and H3K27ac (orange). The region cyclopedia of DNA Elements or Gene Expression Omnibus (GEO) upstream of the distal promoter showed both H3K4me3 and H3K27ac datasets (49, 50) also contains histone modifications indicative of an in CD56, CD3, and CD19 cells but not CD14 cells (Fig. 3). active enhancer at the same region in three lymphoblastoid cell lines This pattern of histone modification is consistent with the fact and Jurkat cells, further supporting the 3-kb region as a regulatory that only lymphocytes use the distal promoter to transcribe site in lymphoid cells (Supplemental Fig. 2B, 2C). The region starts The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. DNA methylation analysis on LILRB1 distal promoter. (A) The asterisks indicate the SNPs used to define the low and high haplotypes. The shaded regions indicate the location of the distal and proximal promoter relative to the LILRB1 translational start site. The protein-coding exons are filled with black. (B) Comparison of DNA methylation patterns of the distal regulatory region of LILRB1 in different lineages. DNA methylation percentage at each CpG position was calculated by analyzing 10 or more clones. The x-axis indicates the CpG position relative to the translational start site. The table indicates the p values as determined by Student t test, and those ,0.05 are highlighted in yellow. (C) LILRB1 expression on isolated NK cells from four donors heterozygous for the promotor haplotypes. The isotype control is shown in red and LILRB1 (GHI/75) is shown in cyan. (D) DNA methylation patterns of the distal regulatory region of LILRB1 by haplotype in the NK cells isolated from the four heterozygous donors shown in (B). At least 20 clones

were analyzed for each donor, the haplotype determined by the sequence and grouped into low and high haplotypes. The CpG position relative to the by guest on October 1, 2021 translational start is marked on the x-axis. The table indicates the p values for each CpG site examined as determined by Student t test, and the one site with a p value ,0.05 highlighted. at 55,135,000 and ends at 55,137,700 (human hg19) and, as ex- proteins such as YY1, that facilitate and stabilize the DNA loop- pected for an intron, has a high degree of variability with many ing structure by forming dimers (51–54). YY1 is also a factor that SNPs recorded in the SNP and 1000 genomes databases (Fig. 3B). can promote or prevent transcription, and several YY1 binding sites To characterize the putative enhancer region and the relationship are predicted in the putative enhancer region as well as in the distal to the promoter haplotypes, we amplified the predicted enhancer and proximal promoters. There is also evidence YY1 binds to the region from NKL cells that have both promoter haplotypes as well as putative enhancer region and the two promoters in published ChIP- two donors, D230 and D258, which are homozygous for the pro- Seq data from the Encyclopedia of DNA Elements and GEO moter haplotypes (27). The alignment of a portion of the sequence is datasets (Supplemental Fig. 2B, 2C) (49, 50). These observations shown in Fig. 3C. Despite being heterozygous for the promoter suggest YY1 could mediate physical interaction between the en- haplotypes, only one sequence was derived from NKL for the entire hancer and the two promoters. To test if YY1 also binds to the 3-kb region. Only one sequence was derived from D258, as well; putative enhancer and/or the two promoter elements of the LILRB1 however, it differs from NKL’s in a number of places, four of which gene in NK-type cells, we applied YY1-ChIP in NKL cells. We are shown in Fig. 3C. We obtained two alleles from D230 and al- analyzed 13 YY1 sites within the putative enhancer region and two though the sequence is more similar to that of NKL for the region promoter regions and detected YY1 binding at 10 sites (Fig. 4). Two shown in the left panel of Fig. 3C, it differs in other regions as of the sites were excluded because of high background for the shown in the right panel of Fig. 3C. As will be discussed in more negative control (E1 and D5), and all the remaining 11 sites shown detail below, algorithms to predict transcription factor binding sites except D3 have YY1 association (Fig. 4). The D1 and P3 sites indicate a large number of potential sites for factors known to be consistently provided the most intense signal, suggesting the highest expressed by lymphoid cells, such as STAT4, Pax5, and c-Ets-1 occupancy with YY1 for these sites. The ChIP results indicate YY1 (Table I). The sequence variation in this putative enhancer region is present at the promoter and enhancer regions and support the suggests alleles could differentially recruit transcription factors. In possibility YY1 scaffolds these regulatory elements together. addition, the region has predicted YY1 sites, and YY1 is a factor The putative enhancer and the two promoters form DNA loops that has been implicated in enhancer function. Given genome-wide high throughput 3C by sequencing assay is a YY1 interacts with the enhancer and the LILRB1 promoters powerful tool used to identify chromatin interactions across the The physical interaction of enhancers with promoters can be me- whole genome (55), we first examined the chromatin interacting diated by certain cofactors, including cohesion and DNA-binding pattern at the LILRB1 gene locus using high throughput 3C by 6 LILRB1 INTRON 1 ENHANCER Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Prediction of a putative enhancer region in the intron 1 of the LILRB1 gene. (A) Histone modification markers at LILRB1 gene locus in different types of immune cells. DNase I hypersensitivity sites (DHS) and histone modifications ChIP-Seq data of CD56, CD3, CD19, and CD14 primary cells shown above were achieved from the National Institutes of Health Roadmap Epigenomics Project (http://www.roadmapepigenomics.org/). The GEO accession number (https://www.ncbi.nlm.nih.gov/geo/) for each track is listed as follows: CD56 (DHS-GSM665836; H3K4me3-GSM1027301; H3K4me1- GSM1027297; H3K27ac-GSM1027288), CD3 (DHS-GSM701526; H3K4me3-GSM1058782; H3K4me1-GSM1058778; H3K27ac-GSM1058764), CD19 (DHS-GSM701492; H3K4me3-GSM537632; H3K4me1-GSM1027296; H3K27ac-GSM1027287), and CD14 (DHS-GSM701503; H3K4me3- GSM1102797; H3K4me1-GSM1102793; H3K27ac-GSM1102782). The gray dotted box indicates the location of the lymphoid-specific putative enhancer. The tracks were visualized using the Washington University epigenome browser (80). (B) Polymorphisms of the putative enhancer region. Sequence variation data at the LILRB1 gene locus shown is from the SNP database and 1000 genomes project (phase 3). Signals of H3K4me1 and H3K27ac in CD56 primary cells are also shown to indicate the position of the putative enhancer. The tracks were visualized using the University of California, Santa Clara genome browser (81). (C) A partial region of the alignment of the amplified putative enhancer from NKL, the homozygous high donor (D258) and the homozygous low donor (D230). Several SNPs are marked by white asterisks in the left panel. D230-A1 and A2 indicate the two alleles sequenced from the genomic DNA of donor D230, which can be differentiated by the alignment shown in the right panel. Numbers after each name indicate the size of the putative enhancer sequenced. The alignment was done using the Mafft program in Jalview software (82). The Journal of Immunology 7

Table I. Prediction of transcription factor binding sites in the region of the putative enhancer

No. of Predicted Binding Sites

Transcription Factorsa Expression in NK Cells Transcriptional Activity D258 D230-A1 D230-A2 C/EBPb Yes Activator 74 73 73 C/EBPa No Activator 9 8 8 c-Ets-1 Yes Activator/repressor 11 9 9 EBF1 No Activator/repressor 3 4 4 GCFC2 Yes Repressor 1 0 0 GR-a Yes Activator/repressor 45 45 44 GR-b Yes Dependent on GR-a expression 68 67 67 HIF-1 Yes Activator 0 1 1 NF-kB1 Yes Activator/repressor 0 1 0 NF-Y Yes Activator/repressor 3 3 4 p53 Yes Activator 29 28 28 Pax-5 No Activator 30 31 31 RelA Yes Activator 0 1 1 RXR-a Unknown Activator/repressor 34 33 33 STAT4 Yes Activator 29 27 27 USF1 Unknown Activator 4 3 3 YY1 Yes Activator/repressor 23 23 23 Downloaded from aThe prediction was done using the ALGGEN-PROMO program (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3). sequencing data in the 4D Nucleome Data Portal (56) available for with the anchor in the enhancer than fragments on either side several lymphoblastoid cell lines. We observed signals indicating (Fig. 5A). There were two additional major and minor interactions the interaction between the putative enhancer and both of the observed with 21972 and 21474 in the proximal region, respec-

LILRB1 promoters in most cell lines, but the data were derived tively, where the proximal promoter and the YY1 site denoted as P3 http://www.jimmunol.org/ from the HindIII-digested chromatin, which did not provide the in Fig. 4 are included in the latter fragment (Fig. 5A). The stronger best resolution for the fragments we are interested in. signal for the 21972 fragment suggests the region upstream of the Therefore, we went on to test if the putative enhancer is in direct previously defined “core promoter” is involved. physical contact with the LILRB1 gene promoters in NK cells by To investigate if YY1 is part of the complexes that contain the performing 3C-qPCR on NKL cells. We used EcoRI to generate enhancer and promoter in the 3C assay, we used ChIP-loop, a tech- suitable fragments to resolve the putative enhancer from the nique that combines ChIP and 3C. In brief, the chromatin-capture LILRB1 promoters and specify their interaction (Fig. 5A). The procedure was initiated, and then the YY1-associated complexes probe and the anchoring primer were designed within the en- were immunoprecipitated and subsequently analyzed by PCR for hancer fragment close to the EcoRI site at -7667 from the trans- the ligation produces. As shown in Fig. 5B, specific ligation products by guest on October 1, 2021 lational start codon, and all the test primers for the fragments were with the three fragments denoted as 216,483, 21972, and 21474 designed close to the upstream restriction end (Fig. 5A). Among the were detected in the anti–YYI-immunoprecipitated samples relative three distal fragments examined, the 216,483 fragment encom- to an IgG control and the negative control region at 217,925. passing the distal promoter has a significantly stronger interaction Collectively, the 3C and ChIP-loop data are consistent with the

FIGURE 4. YY1-ChIP analysis on the region of the putative enhancer and the LILRB1 gene distal and proximal promoters in NKL cells. Schematic of partial LILRB1 gene locus and location of the tested predicted YY1 binding sites marked by asterisks. The protein-coding region starts in exon 3 and is filled with black. The primers used for each predicted site shown above are listed in Supplemental Table I. ChIP results are shown as electrophoresis of the PCR products detecting YY1 binding at different sites in NKL cells. Input and IgG worked as a positive and negative control for the ChIP assay, re- spectively, and the YY1-neg was a negative control detecting a non-YY1 binding site for the ChIP Ab targeting human YY1. The results of YY1-D5 and YY1-E1 are excluded because of their high background with IgG control. The results shown are representative of three independent experiments. 8 LILRB1 INTRON 1 ENHANCER Downloaded from http://www.jimmunol.org/ FIGURE 5. Physical contact between the putative enhancer and LILRB1 gene promoters involving YY1 in NK cells. (A) Analysis of physical contact between the putative enhancer and the LILRB1 gene promoters by 3C-qPCR assay in NKL cells. The chromatin was digested by EcoRI, and the EcoRI sites are marked by vertical arrowheads. All the numbers below the EcoRI sites are indicating the distance to the translational start site of the LILRB1 gene. Dark gray shading indicates the anchor fragment, and the light gray shading indicates the fragments tested for the cross-linking frequency with the anchor fragment. The TaqMan probe is indicated by a solid line, and the horizontal arrowheads indicate the positions of primers. Asterisks indicate the 10 sites with YY1 binding shown in Fig. 4. The exact relative cross-linking values listed on the right were calculated referring to the method described previously (42). The results shown are representative of three independent experiments. (B) Involvement of YY1 in the enhancer/promoter physical interaction tested by ChIP-loop assay (ChIP + 3C) shown as electrophoresis of the PCR products using 3C primers. The fragment 217,925 was used as a negative control validated in (A). Library build using BAC DNA encompassing the whole LILRB1 gene was used as a positive control. Target PCR products were sequenced to verify the specificity. The results shown are representative of three independent experiments. by guest on October 1, 2021

YY1-ChIP placing YYI at the site of the enhancer and pro- all significantly decreased compared with the NKL control (Fig. 6D). moters in complex with each other in NKL cells. Importantly, the LILRB1 mRNA levels of those six clones were well matched with the LILRB1 mean fluorescence intensities detected by Deletion of the putative enhancer decreases LILRB1 expression FACS (Fig. 6B, 6D), which suggested the knockout of the putative We tested the ability of the 3.2-kb region to enhance the distal enhancer inhibited the LILRB1 gene transcription. promoter using the pGL3-basic luciferase system. However, the To ensure the lower LILRB1 transcript was not an artifact of region actually repressed transcription, and its size made it difficult subcloning the NKL line, we also isolated 19 NKL clones from to pursue this approach. To more directly test the role of the putative the parental NKL cells. Although these NKL subclones have enhancer in LILRB1 gene expression in the context of chromatin, slightly different LILRB1 surface expression, they were all close we applied CRISPR-Cas9 technology to delete the 3.2-kb region in to the parental level (Fig. 6E). The mRNA was also analyzed for NKL cells as illustrated in Fig. 6A. We sorted single-cell clones six of these clones, showing minimal changes in the transcript with lower LILRB1 expression by FACS and analyzed by PCR to level (Fig. 6F). These results indicate the intronic putative enhancer more readily ascribe the relative LILRB1 expression to the dele- plays a positive role in regulating LILRB1 gene transcription. tion and assess the variability of LILRB1 expression in unmanip- ulated but cloned NKL cells. We selected clones with decreased surface LILRB1 expression compared with the NKL control. In Discussion these six clones, we detected bands corresponding to the intact In the current study, we wanted to characterize the NK-specific locus at 5286 bp from NKL control and the expected deletion at regulation of LILRB1 transcription with a view to understanding around 1500–2000 bp and sequenced the product to ensure the the influence of allelic variation on LILRB1 expression. To this deletion was of the correct locus. Among the six clones, KO-3, end, we investigated the allelic expression of LILRB1 in NK clones KO-4, KO-5, and KO-6 with two alleles knocked out showed from heterozygous individuals. We detected the major allele at a lower surface LILRB1 expression than that of KO-1 and KO-2 higher frequency as it is detectable in more clones from two of the with one allele knocked out (Fig. 6B, 6C). To investigate whether three individuals examined. This correlates well with our previous the decreased surface LILRB1 expression was due to decreased results that associate the major allele with higher overall expression LILRB1 transcription, we isolated total RNA from the six knockout in NK cells (29) and is reminiscent of the allele-specific expression clones and did quantitative real-time PCR to detect the change of patterns of KIRs. However, the expression of LILRB1 alleles LILRB1 transcript. Consistent with the FACS data shown in differs from KIR in important ways. First, a substantial proportion Fig. 6B, the LILRB1 mRNA levels of the six knockout clones were of NK clones express both LILRB1 alleles, whereas, for most The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. CRISPR-based knockout of the putative enhancer in NK cells. (A) Schematic depicts the knockout of the putative enhancer represented by three peaks of H3K4me1 and H3K27ac signals using the CRISPR-Cas9 system in NKL cells. Black arrowheads indicate the location of the guide RNAs. Red arrowheads indicate the positions of primers to validate the fragment knockout. (B) Surface LILRB1 level on parental NKL cells and the knockout NKL clones (KO-1 to KO-6) tested by flow cytometry using the HP-F1 Ab. Different clones stained using the LILRB1 Ab (HP-F1) are indicated by different colors, and the dotted peak in the same color indicates the corresponding clone stained using isotype Ab. Each Geom.MFI value with background subtracted is shown beside the plot. (C) Electrophoresis detecting the knockout fragments in NKL clones shown in (B). Parental NKL cells were used as a negative control. Sanger-sequencing was used to confirm the knockout sequence. (D) Total LILRB1 transcript level was detected in the knockout NKL cells using real-time qPCR (RT-qPCR). Fold change values of the knockout cell lines relative to parental NKL cells were calculated using the 2^2DDCt method. Means of the fold change calculated from three independent experiments were compared using Student t test, and an asterisk (*) indicates a p value ,0.05. (E) Surface LILRB1 expression on six NKL subclones (NKL-1 to NKL-6) ranged from low to high mean fluorescence intensity tested by flow cytometry using the HP-F1 Ab. Different clones stained using the LILRB1 Ab (HP-F1) are indicated by different colors, and the dotted peak in the same color indicates each clone stained using isotype Ab. Each Geom.MFI value with background subtracted is shown beside the plot. (F) Total LILRB1 transcript level was detected in the six NKL subclones using RT-qPCR. Fold change values of the NKL cell clones relative to parental NKL cells were calculated using the 2^2DDCt method.

KIRs, a minority express both alleles (33). Second, assuming there with the surface expression. Curiously, for the majority of clones is concordance between the signal obtained in the ddPCR and the that express both alleles, the amount of transcript for the rare allele total transcript per cell, there is significant variability in the amount is actually the higher one. Previously we showed that the distal of transcript among the NK clones, which correlates imperfectly promoter variants provide similar levels of transcription when tested 10 LILRB1 INTRON 1 ENHANCER ectopically in an NK cell line, indicating polymorphisms in the core alignment of the LILRB1 gene in different species reveals dif- promoter do not directly explain the expression patterns (27). To- ferences between primates and nonprimates not only in sequence gether, the results suggest the mechanism that leads to differential conservation but also in retaining all or partial sequences upstream expression between alleles is not the probability of initiating of the proximal promoter, although some primates such as gorilla transcription-limiting expression to one allele during a tight devel- and rhesus lost exon1 (68) (Supplemental Fig. 3B). opmental window but rather other aspects that vary at a clonal level The evidence indicating the 3-kb region in intron 1 is a positive and determine the set point for transcription of each allele. In ad- regulator of the distal promoter is that the deletion of the region in dition, the inverted frequency of the alleles for D183 suggests the NKL cells reduces LILRB1 transcript and protein expression. element that is involved is distinct from the polymorphisms in the However, we were unable to show that the 3-kb region or 1-kb promoters we previously identified. fragments can enhance the distal promoter in typical luciferase We have previously shown that the pattern of LILRB1 expression reporter assays (data not shown), suggesting the native chromatin on peripheral NK cells remains quite stable over time for healthy structure is required for function. It is tempting to speculate that the individuals (41). LILRB1 is first expressed during later stages of region has silencing activity for the proximal promoter as the 3C NK development but signals that initiate LILRB1 in NK cells are data shows the enhancer in physical complexes with the proximal not known. We were unable to identify any additional promoters promoter as well, but the biological relevance of the interaction for the LILRB1 gene from the epigenomic datasets. However, the with the enhancer and the proximal promoter requires further proximal promoter region does display some elements suggesting investigation. Despite the ability of NKL cells to support tran- a transcript could arise in reverse (TATA box and poly-A site), and scription from the proximal promoter from a plasmid, the use of the using reporter constructs, we previously found modest reverse proximal promoter to transcribe LILRB1 is negligible in ex vivo activity of the proximal promoter in THP-1 cells and very weak NK cells, although it can be increased under cytokine stimulation Downloaded from reverse activity in NKL cells (45). We also could not detect any (15, 40, 41). It is possible the enhancer region identified in this reverse activity from the distal promoter. Therefore, it remains a study may positively regulate the LILRB1 expression through possibility that the proximal promoter or another region has re- recruiting trans-activating transcription factors to the distal pro- verse activity during some point in NK cell development and moter in NK cells and at the same time block the formation of the mechanism of locus activation/repression akin to that of KIRs transcription preinitiation complex at the proximal promoter. might occur. The 3-kb region contains three peaks of enhancer marks with two http://www.jimmunol.org/ The LILRB1 distal promoter region lacks obvious CpG islands; valleys of unmarked DNA in between, suggesting the region may nonetheless, our DNA methylation analysis suggests one CpG site have multiple distinct elements. We used a transcription factor in the distal promoter needs to be unmethylated for expression. A prediction tool (ALGGEN-PROMO program) with the sequence of second CpG site upstream of the distal promoter has differential the putative enhancer and found many binding sites for Ccaat- methylation between the two haplotypes. Although most methyl- enhancer-binding proteins (C/EBPs) and two binding sites (26581 CpG binding proteins are thought to play a repressive role in gene and 26520) of CREB, which could recruit another marker of transcription by recruiting transcriptional corepressor protein, potential enhancers named CREB-binding protein (CBP) (69–72). there are certain methyl-CpG binding proteins that can activate Among the many transcription factors predicted to associate with by guest on October 1, 2021 transcription, which indicates DNA methylation may not always the enhancer element, we focused on YY1 because YYI was be a repressing marker (57–61). Therefore, if this CpG site is shown recently to be a structural regulator mediating enhancer/ playing a role in the difference between the two haplotypes, it is promoter interactions (54). Our results demonstrate YY1 is bound likely by promoting methylation-dependent binding of an activating to the enhancer region as well as both promoters and can pull transcription factor or preventing the association of a negative regu- down the ligated products with each promoter formed by 3C, latory factor. There are several transcription factors predicted to bind suggesting YY1 dimers bridge the elements together. Coinci- to this site, including STAT4 and SPI1, and, as this site is also 83 bp dently, the proximal promoters of KIR genes also bind YY1, and a from one of the YY1 sites, it may warrant further investigation. polymorphic mutation at the YY1 site increases promoter activity In this study, we identified a new regulatory region within intron and the ratio between the reverse and forward transcripts (30, 38, 1 through its enhancer-specific histone modification pattern in 73). In the cell lines and blood donors we analyzed, the YY1 sites CD56+ cells. Of note, we also observed similar profiles in T cells are conserved and therefore unlikely to directly control allele- and B cells but not monocytes, indicating the putative enhancer is specific expression patterns. Nonetheless, polymorphisms do al- lymphoid-specific. The region possesses many sites predicted to ter the prediction of other transcription factor binding sites recruit transcription factors known to be expressed by lymphoid (Table I), which could lead to differential levels of transcription cells, including some that are more T and NK (e.g., STAT5) or B between alleles. It is also worth noting that there are many pre- lineage specific (e.g., Pax5) (Table I). dicted Pax5 sites, and Pax5 is essential for B cell commitment and The presence of the regulatory region in the intron may explain is expressed specifically during B cell development (74). Although why the LILRB1 gene has maintained a 13-kb-long intron 1 when this study has illustrated an additional polymorphic regulatory the leukocyte receptor complex genes tend toward very compact sequence within the LILRB1 gene, additional layers of regulation structures. Analysis with RepeatMasker annotations (62) reveals might also be involved in the steady-state expression of the that the putative enhancer region is flanked by a long interspersed transcript. Along these lines, a very recent report showed that nuclear element (LINE) indicative of its formation by a trans- NF90 associated with the LILRB1 transcript in THP-1 cells and posable element (Supplemental Fig. 3A) (63–66). The distal inhibits LILRB1 protein expression (75). promoter and the enhancer flanking sequences also have LINE, The current and further characterization of the regulatory long terminal repeats, and short interspersed nuclear elements (67) mechanisms of LILRB1 gene expression provides new perspectives (Supplemental Fig. 3A). The presence of the LINE, short inter- to understand the heterogeneity of human NK cells. Beyond the spersed nuclear elements, and long terminal repeats suggests the role of LILRB1 in NK cell control of HCMV and in decidual NK distal promoter and first exon were acquired through insertion cells, there many associations of LILRB1 in other infections, such as events, and this may explain how the locus evolved to differen- dengue and malaria, and in autoimmune diseases, such as systemic tially regulate LILRB1 in myeloid and lymphoid cells. Multiple lupus erythematosus, rheumatoid arthritis, pemphigus foliaceus, and The Journal of Immunology 11 autoimmune thyroiditis (6–13). Mounting evidence also supports 17. Arnold, V., J.-S. Cummings, U. Y. Moreno-Nieves, C. Didier, A. Gilbert, F. Barre´-Sinoussi, and D. Scott-Algara. 2013. S100A9 protein is a novel ligand the immune checkpoint function of LILRB1 in viral chronic for the CD85j receptor and its interaction is implicated in the control of HIV-1 infection and cancer (76–78). 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