Identification of the ancestral killer immunoglobulin-like receptor gene in primates
- Jennifer G Sambrook†1,
- Arman Bashirova†2, 5,
- Hanne Andersen3, 6,
- Mike Piatak3,
- George S Vernikos1,
- Penny Coggill1,
- Jeff D Lifson3,
- Mary Carrington4 and
- Stephan Beck1Email author
© Sambrook et al; licensee BioMed Central Ltd. 2006
Received: 12 May 2006
Accepted: 15 August 2006
Published: 15 August 2006
Killer Immunoglobulin-like Receptors (KIR) are essential immuno-surveillance molecules. They are expressed on natural killer and T cells, and interact with human leukocyte antigens. KIR genes are highly polymorphic and contribute vital variability to our immune system. Numerous KIR genes, belonging to five distinct lineages, have been identified in all primates examined thus far and shown to be rapidly evolving. Since few KIR remain orthologous between species, with only one of them, KIR2DL4, shown to be common to human, apes and monkeys, the evolution of the KIR gene family in primates remains unclear.
Using comparative analyses, we have identified the ancestral KIR lineage (provisionally named KIR3DL0) in primates. We show KIR3DL0 to be highly conserved with the identification of orthologues in human (Homo sapiens), common chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), rhesus monkey (Macaca mulatta) and common marmoset (Callithrix jacchus). We predict KIR3DL0 to encode a functional molecule in all primates by demonstrating expression in human, chimpanzee and rhesus monkey. Using the rhesus monkey as a model, we further show the expression profile to be typical of KIR by quantitative measurement of KIR3DL0 from an enriched population of natural killer cells.
One reason why KIR3DL0 may have escaped discovery for so long is that, in human, it maps in between two related leukocyte immunoglobulin-like receptor clusters outside the known KIR gene cluster on Chromosome 19. Based on genomic, cDNA, expression and phylogenetic data, we report a novel lineage of immunoglobulin receptors belonging to the KIR family, which is highly conserved throughout 50 million years of primate evolution.
The Killer Immunoglobulin-like Receptor (KIR) gene family encodes Major Histocompatibility Complex (MHC) class I specific receptors that are expressed on Natural Killer (NK) and T cells [1, 2]. In humans, these are encoded within the Leukocyte Receptor Complex (LRC)  on Chromosome 19q13.4, which like the MHC on Chromosome 6p21.3, is a region characteristic of immune loci: highly plastic, polygenic, polymorphic, rapidly evolving, and associated with disease . As a result, KIR diversity contributes vital variability to our immune system with direct implications for health and disease [5, 6]. KIR working in concert with its Human Leukocyte Antigen (HLA) ligands has been shown to influence directly the resolution of viral infections such as Hepatitis C Virus .
Numerous KIR genes, both in their activating and inhibitory forms, have been identified in all primates examined thus far and shown to be rapidly evolving [8–11]. Inhibitory KIR have longer cytoplasmic tails in comparison to activating KIR, and typically contain two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) which are responsible for repressing the immunoreactivity of NK cells. The emergence of primate activating KIRs can be accounted by two processes: the alteration in the length and sequence of the cytoplasmic tail in an ancestral long-tailed KIR to eliminate the ITIMs, accompanied by nucleotide changes in the transmembrane (TM) domain to introduce a charged residue . Another distinguishing feature of KIR molecules is the number of extracellular immunoglobulin (Ig) domains, numbered D0, D1 and D2. Although KIR2DL4 is conserved in all primates studied to date , it is unlikely to represent the ancestral KIR gene. Structurally, KIR2DL4 has a D0+D2 organisation and has arisen by exon loss from a three Ig-containing progenitor. The ITIMs present in the cytoplasmic tail are also not conserved between all primates: monkeys have two, apes have only one, and in some cases, the motif has diverged from the consensus (gorilla) . KIR2DL4 has a charged amino acid in the TM region, and in this respect, could act as an activating KIR . It binds the non-polymorphic HLA-G molecule, and this may explain why this gene has remained relatively unchanged since the last common ancestor. Here we report a novel lineage (provisionally named KIR3DL0) and show it to be divergent to the previously identified lineages and conserved throughout 50 million years of primate evolution. Characteristics that would be expected to be present in the common ancestral primate KIR, such as three Ig domains, a long cytoplasmic tail, and two ITIMs providing an inhibitory function, are all present in the KIR3DL0 lineage. For the purpose of this report, we define 'ancestral' as the phylogenetically most diverged gene. In support of this discovery, we present genomic, cDNA, expression and phylogenetic data.
Results and discussion
Identification of the KIR3DL0 locus
The relatedness of KIR3DL0 to the KIR gene family is further confirmed by gene structure analyses. Using FINEX, a method that identifies gene families based on the conservation of gene structure (intron/exon phases) rather than sequence similarity , the closest relative of KIR3DL0 is the prototypical human KIR gene KIR3DL1 with a significant z-score of +10.88 (data not shown). Furthermore, using VISTA , a global genomic comparison tool, significant conservation in both coding and non-coding sequences is present between all primate KIR3DL0 sequences and KIR3DL1 [see Additional file 1].
Conservation of KIR3DL0 in primates
We show KIR3DL0 to be highly conserved in primates with the identification of orthologues in the common chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), rhesus monkey (Macaca mulatta) and common marmoset (Callithrix jacchus). The two copies in marmoset are arranged in a head-to-head orientation and are likely to be the result of a species-specific duplication event. These non-human primates diverged from humans approximately 5, 7, 25 and 50 million years ago, respectively. In all cases, KIR3DL0 maps to the syntenic region between LAIR2 and LILRA2. Details of the genomic LRC organization in these primates will be described elsewhere.
The primate KIR3DL0 sequences share the same exon-intron configuration and are predicted to encode functional proteins (Figure 2). In addition to the human cDNA, full length cDNA sequences were isolated from chimpanzee and rhesus monkey. Variations between the genomic and cDNA sequences may indicate KIR3DL0 to be polymorphic. Although only two sequences could be compared per species, we identified five non-synonymous substitutions in chimpanzee and one synonymous substitution in human. The conservation of two immunoreceptor tyrosine-based inhibitory motifs, present in the cytoplasmic tail of the predicted KIR3DL0 protein sequences from all species studied, indicates an inhibitory function for these receptors. These motifs are present in only one copy in marmoset, which has the complete nine-exon gene structure. The short form is missing the last three exons encoding the TM and cytoplasmic domains, resulting in a potentially secreted form, which is similar, but not identical, to the KIR3DL0 cDNA in human (Figure 2). While three N-linked glycosylation sites (NXS/T) are conserved in the majority of the sequences analyzed, one additional site is present in the IgD2 of the two KIR3DL0 copies in marmoset. For the short marmoset form in particular, the extra glycosylation site increases the potential level of glycosylation. High levels of glycosylation have been shown to be essential for secretion of some immunoglobulins .
Based on comparative sequence analysis, we have identified a new lineage of immunoglobulin receptors and show them to be part of the KIR receptor family, supported by four independent lines of evidence. (I) Sequence similarity: Using the BLAST algorithm, the similarity on both the DNA and protein level is highest to KIR. (II) Gene structure similarity: The nine exon gene structure and the phases of the splice site boundaries are identical or more similar to KIR than to any other gene family. (III) Phylogenetics: Full length and domain-by-domain phylogenetic analysis cluster the new sequences with high confidence at the base of the KIR clade. (IV) Expression: cDNA cloning and quantitative RT-PCR analysis demonstrate an expression profile typical of KIR. While the data presented here suggest 3DL0 to be the ancestral KIR gene in primates, we cannot rule out the possibility of selection to have caused the observed divergence. The situation in non-primates remains unclear. Based on our own and previously reported phylogenetic evidence , the primordial non-primate KIR gene would be predicted to be a three-Ig inhibitory receptor, most similar to KIR3DL0. Yet, we were unable to find any significant matches to KIR3DL0 in non-primates, except to previously reported sequences [19–23] that have higher similarity to more recent KIRs. Recently, a cluster of CHIR genes has been identified in chicken, but these resemble the organisation of two-domain KIRs . A possible explanation for these observations is that KIR3DL0 has been lost from non-primate genomes. Polyphyletic loss of multiple human genes from non-primate vertebrate genomes has been observed before and has led to the suggestion that such events are more frequent than previously thought . Both rapid and convergent evolution have probably contributed to or even driven this process. In rodents, for instance, multiple Ly49 genes, which encode C-type lectins, carry out the KIR analogous function. The two murine Kirl1 and Kirl2 genes  lack the characteristic activating/inhibitory motifs of KIRs and map to the X chromosome outside the otherwise conserved LRC. Likewise, the single KIR-like sequence in rat (Kir3dl1)  lacks the stem region and has an elongated TM region. The sequence has, however, the same overall (3DL) structure as KIR3DL0 and maps to the syntenic region in the rat LRC and, in this respect, represents the closest non-primate relative to KIR3DL0.
Mapping and sequencing
BAC contigs covering the KIR region were generated and sequenced as described previously . KIR3DL0 genomic sequences were obtained as follows: for human, using ENSEMBL  assembly NCBI-35; for chimpanzee, using ENSEMBL assembly CHIMP-1; for gorilla, by BAC sequencing [EMBL:CR759947 and EMBL:CR759950]; and for marmoset, by BAC sequencing [EMBL:CR925829]. The single sequence gap that covers the transmembrane region in the CHIMP-1 assembly was amplified and sequenced using forward (5'-caccaacattctttggagcaagtt-3') and reverse (5'-aggctgaggtggaagaatggc-3') primers. The frameshift region in the human KIR3DL0 gene was sequenced in 86 healthy individuals (81 Caucasian, 3 African American, 1 Asian, 1 unknown ethnicity) using forward (5'-ttccaggccaacttttctgtgg-3'), and reverse (5'-tctgtgatccagtgggcacca-3') primers. KIR3DL0 cDNA sequences were obtained by reverse transcription of total RNA using Superscript (Invitrogen). Total RNA was isolated using Trizol (Invitrogen). The macaque and chimpanzee KIR3DL0 cDNAs [GenBank:DQ224422 and GenBank:DQ157756] were cloned by two rounds of PCR from peripheral blood lymphocyte cDNA. The first round of PCR was performed with primers KIR3DL0-F2, 5'-ctgtgtcctgcccaatagaag-3', and KIR3DL0-R2, 5'-cctcctaggaatagatccagg-3'. For the second round, we used primers KIR3DL0-F2 and KIR3DL0-R1, 5'-aggaatagatccaggtccttg-3'. The human KIR3DL0 cDNA [GenBank:DQ224421] was cloned by one round of PCR using the KIR3DL0-F2 and KIR3DL0-R1 primers.
Genomic sequences were analyzed using ARTEMIS , ACT , and BLAST . Multiple sequence alignments were carried out in ClustalX , and edited manually to maximize the alignment. Trees were constructed using the Neighbour Joining method  in MEGA version 2.1 , using human KIR3DL0 derived from genomic sequence, with the frameshift in IgD2 manually corrected to bring the sequence back into frame. Trees were rooted at midpoint, using a complete deletion (Figure 1B) or pairwise deletion (Figure 3), the Poisson correction option, and 500 bootstrap replicates. Global genomic comparisons were made using VISTA . FINEX  was used in conjunction with its corresponding gene structure database derived from EMBL version 181 (eukaryotes only).
Isolation and culture of rhesus macaque PBMC and NKp46+ lymphocytes
Peripheral blood mononuclear cells were isolated from rhesus macaque as described previously . To isolate an enriched NK cell population, PBMC were first incubated with a PE-conjugated anti-human NKp46+ mAb (clone BAB281, Beckman Coulter, Miami, FL), washed once, incubated with PE-selection beads (Miltenyi, Auburn, CA) and passed over a positive selection column for enrichment of PBMC expressing the NKp46 surface marker. Successful enrichment was verified by flow cytometric analysis using a FACSCalibur (Becton Dickinson, Immunocytometry Systems, San Jose, CA); selected cells were ≥85% NKp46+. To assess induction of KIR3DL0 expression following in vitro culture with IL-2, NKp46+ cells were cultured in RPMI-10% FBS with 100U/ml recombinant human IL-2 (Peprotech, Rocky Hill, NJ) for 7 days. Cells were counted, transferred into 1.5 ml RNAse-free polypropylene tubes, spun down, and snap-frozen in liquid nitrogen.
Nucleic acid extraction and quantitative PCR/RT-PCR analysis
Cellular nucleic acids for qPCR and qRT-PCR were prepared with modifications to methods typically applied to the isolation of RNA from plasma associated simian immunodeficiency virus . The disruption and suspension of cell pellets in lysis buffer was facilitated by sonication using a Branson Model S-450D sonifier equipped with a high intensity cup horn (Branson Ultrasonics, Corp., Danbury, CT). The final nucleic acid containing pellet was dissolved in 60 μl 1X Turbo DNase Buffer (Ambion, Inc., Austin, TX) and divided into two equal aliquots. To one aliquot, 2 units of Turbo DNase (Ambion, Inc., Austin TX) were added and the aliquot incubated at 37°C. After 30 minutes, 120 μl ~5.7 M GuSCN, 50 mM TrisCl, pH 7.6, 1 mM EDTA was added and the sample mixed thoroughly, followed by 150 μl isopropanol to precipitate and recover nucleic acid. This sample was dissolved in 30 μl nuclease free water supplemented to contain 1 mM DTT and 1 U/ml RNASeOUT (Invitrogen, Inc., Carlsbad, CA) and assayed by quantitative RT-PCR methods following the conditions and protocols described in Cline et al.  with the following modifications to accommodate the increased level of cellular RNA and to specifically amplify the target KIR3DL0 mRNA sequence: in the reverse transcription step, 1.5 μg random primers and 100 Units reverse transcriptase per reaction were employed and the 85°C heat-kill step was increased to 25 minutes; in the PCR step, the primers and probe used were KIR3DLO-FOR02, 5'-tgggagcaaatccctgaagat-3', KIR3DLO -REV02, 5'-agcttgggtgcgctgagat-3', and KIR3DLO -PR02, 5'-FAM-tcgtcacaggcttgtttaccaaaccctc-BlackHole™ Quencher 1 (Biosearch Technologies, Novato, CA). The remaining aliquot of nucleic acids was heated to 100°C for 5 min, quenched on ice, and then assayed to determine the number of cell genome equivalents represented in the original sample by quantifying copies of a gene sequence for the chemokine receptor CCR5 (MP, unpublished data).
Human Leukocyte Antigen
immunoreceptor tyrosine-based inhibitory motifs
Killer Immunoglobulin-like Receptor
Leukocyte Immunoglobulin-like Receptor
Leukocyte Receptor Complex
Major Histocompatibility Complex
peripheral blood mononuclear cells
We thank J. Almeida, H.R. Hotz, K. Howe, S. Palmer, H. Sehra and S. Sims for technical assistance and help with the analysis. JGS, GSV, PC and SB were supported by the Wellcome Trust. The reagent IL-2 was obtained through the NIH AIDS Research and Reference Reagent Program. Part of this work was supported with federal funds from the National Cancer Institute, National Institutes of Health under contract no. NO1-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsements by the U.S. Government.
- Colonna M, Samaridis J: Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science. 1995, 268: 405-408.PubMedView ArticleGoogle Scholar
- Wagtmann N, Biassoni R, Cantoni C, Verdiani S, Malnati MS, Vitale M, Bottino C, Moretta L, Moretta A, Long EO: Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular domains. Immunity. 1995, 2: 439-449. 10.1016/1074-7613(95)90025-X.PubMedView ArticleGoogle Scholar
- Wende H, Colonna M, Ziegler A, Volz A: Organization of the leukocyte receptor cluster (LRC) on human chromosome 19q13.4. Mamm Genome. 1999, 10: 154-160. 10.1007/s003359900961.PubMedView ArticleGoogle Scholar
- Trowsdale J, Parham P: Mini-review: defense strategies and immunity-related genes. Eur J Immunol. 2004, 34: 7-17. 10.1002/eji.200324693.PubMedView ArticleGoogle Scholar
- Carrington M, Norman P: The KIR Gene Cluster. National Center for Biotechnology Information. Edited by: (US) NLM. 2003, Bethesda (MD), Available from http://ncbi.nlm.nih.gov/entrez/query.fcgi?db=BooksGoogle Scholar
- Parham P: MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol. 2005, 5: 201-214. 10.1038/nri1570.PubMedView ArticleGoogle Scholar
- Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, Cheng J, Goedert JJ, Vlahov D, Hilgartner M, Cox S, Little AM, Alexander GJ, Cramp ME, O'Brien SJ, Rosenberg WM, Thomas DL, Carrington M: HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science. 2004, 305: 872-874. 10.1126/science.1097670.PubMedView ArticleGoogle Scholar
- Vilches C, Parham P: KIR: diverse, rapidly evolving receptors of innate and adaptive immunity. Annu Rev Immunol. 2002, 20: 217-251. 10.1146/annurev.immunol.20.092501.134942.PubMedView ArticleGoogle Scholar
- Khakoo SI, Rajalingam R, Shum BP, Weidenbach K, Flodin L, Muir DG, Canavez F, Cooper SL, Valiante NM, Lanier LL, Parham P: Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans. Immunity. 2000, 12: 687-698. 10.1016/S1074-7613(00)80219-8.PubMedView ArticleGoogle Scholar
- Hershberger KL, Shyam R, Miura A, Letvin NL: Diversity of the killer cell Ig-like receptors of rhesus monkeys. J Immunol. 2001, 166: 4380-4390.PubMedView ArticleGoogle Scholar
- Sambrook JG, Bashirova A, Palmer S, Sims S, Trowsdale J, Abi-Rached L, Parham P, Carrington M, Beck S: Single haplotype analysis demonstrates rapid evolution of the killer immunoglobulin-like receptor (KIR) loci in primates. Genome Res. 2005, 15: 25-35. 10.1101/gr.2381205.PubMedPubMed CentralView ArticleGoogle Scholar
- Abi-Rached L, Parham P: Natural selection drives recurrent formation of activating killer cell immunoglobulin-like receptor and Ly49 from inhibitory homologues. J Exp Med. 2005, 201: 1319-1332. 10.1084/jem.20042558.PubMedPubMed CentralView ArticleGoogle Scholar
- Rajalingam R, Parham P, Abi-Rached L: Domain shuffling has been the main mechanism forming new hominoid killer cell Ig-like receptors. J Immunol. 2004, 172: 356-369.PubMedView ArticleGoogle Scholar
- Wilson MJ, Torkar M, Haude A, Milne S, Jones T, Sheer D, Beck S, Trowsdale J: Plasticity in the organization and sequences of human KIR/ILT gene families. Proc Natl Acad Sci U S A. 2000, 97: 4778-4783. 10.1073/pnas.080588597.PubMedPubMed CentralView ArticleGoogle Scholar
- Brown NP, Whittaker AJ, Newell WR, Rawlings CJ, Beck S: Identification and analysis of multigene families by comparison of exon fingerprints. J Mol Biol. 1995, 249: 342-359. 10.1006/jmbi.1995.0301.PubMedView ArticleGoogle Scholar
- Mayor C, Brudno M, Schwartz JR, Poliakov A, Rubin EM, Frazer KA, Pachter LS, Dubchak I: VISTA : visualizing global DNA sequence alignments of arbitrary length. Bioinformatics. 2000, 16: 1046-1047. 10.1093/bioinformatics/16.11.1046.PubMedView ArticleGoogle Scholar
- Hickman S, Kornfeld S: Effect of tunicamycin on IgM, IgA, and IgG secretion by mouse plasmacytoma cells. J Immunol. 1978, 121: 990-996.PubMedGoogle Scholar
- Andersen H, Rossio JL, Coalter V, Poore B, Martin MP, Carrington M, Lifson JD: Characterization of rhesus macaque natural killer activity against a rhesus-derived target cell line at the single-cell level. Cell Immunol. 2004, 231: 85-95. 10.1016/j.cellimm.2004.12.004.PubMedView ArticleGoogle Scholar
- Hoelsbrekken SE, Nylenna O, Saether PC, Slettedal IO, Ryan JC, Fossum S, Dissen E: Cutting edge: molecular cloning of a killer cell Ig-like receptor in the mouse and rat. J Immunol. 2003, 170: 2259-2263.PubMedView ArticleGoogle Scholar
- Storset AK, Slettedal IO, Williams JL, Law A, Dissen E: Natural killer cell receptors in cattle: a bovine killer cell immunoglobulin-like receptor multigene family contains members with divergent signaling motifs. Eur J Immunol. 2003, 33: 980-990. 10.1002/eji.200323710.PubMedView ArticleGoogle Scholar
- Welch AY, Kasahara M, Spain LM: Identification of the mouse killer immunoglobulin-like receptor-like (Kirl) gene family mapping to chromosome X. Immunogenetics. 2003, 54: 782-790.PubMedGoogle Scholar
- Takahashi T, Yawata M, Raudsepp T, Lear TL, Chowdhary BP, Antczak DF, Kasahara M: Natural killer cell receptors in the horse: evidence for the existence of multiple transcribed LY49 genes. Eur J Immunol. 2004, 34: 773-784. 10.1002/eji.200324695.PubMedView ArticleGoogle Scholar
- Sambrook JG, Sehra H, Coggill P, Humphray S, Palmer S, Sims S, Takamatsu HH, Wileman T, Archibald AL, Beck S: Identification of a single killer immunoglobulin-like receptor (KIR) gene in the porcine leukocyte receptor complex on chromosome 6q. Immunogenetics. 2006, 58: 481-486. 10.1007/s00251-006-0110-9.PubMedView ArticleGoogle Scholar
- Laun K, Coggill P, Palmer S, Sims S, Ning Z, Ragoussis J, Volpi E, Wilson N, Beck S, Ziegler A, Volz A: The leukocyte receptor complex in chicken is characterized by massive expansion and diversification of immunoglobulin-like Loci. PLoS Genet. 2006, 2: e73-10.1371/journal.pgen.0020073.PubMedPubMed CentralView ArticleGoogle Scholar
- Roelofs J, Van Haastert PJ: Genes lost during evolution. Nature. 2001, 411: 1013-1014. 10.1038/35082627.PubMedView ArticleGoogle Scholar
- ENSEMBL: http://www.ensembl.org/.
- Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualization and annotation. Bioinformatics. 2000, 16: 944-945. 10.1093/bioinformatics/16.10.944.PubMedView ArticleGoogle Scholar
- Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG, Parkhill J: ACT: the Artemis Comparison Tool. Bioinformatics. 2005Google Scholar
- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25: 3389-3402. 10.1093/nar/25.17.3389.PubMedPubMed CentralView ArticleGoogle Scholar
- Higgins DG, Bleasby AJ, Fuchs R: CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci. 1992, 8: 189-191.PubMedGoogle Scholar
- Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987, 4: 406-425.PubMedGoogle Scholar
- Kumar S, Tamura K, Jakobsen IB, Nei M: MEGA2: molecular evolutionary genetics analysis software. Bioinformatics. 2001, 17: 1244-1245. 10.1093/bioinformatics/17.12.1244.PubMedView ArticleGoogle Scholar
- Cline AL, Bess JW, Piatak M, Lifson JD: Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS. Journal of Medical Primatology. 2005, 34: 303-10.1111/j.1600-0684.2005.00128.x.PubMedView ArticleGoogle Scholar
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