Homologs of CD83 from Elasmobranch and Teleost Fish

Yuko Ohta; Eric Landis; Thomas Boulay; Ruth B. Phillips; Bertrand Collet; Chris J. Secombes; Martin F. Flajnik; John D. Hansen

doi:10.4049/jimmunol.173.7.4553

Homologs of CD83 from Elasmobranch and Teleost Fish

Ohta, Yuko;Landis, Eric;Boulay, Thomas;Phillips, Ruth B.;Collet, Bertrand;Secombes, Chris J.;Flajnik, Martin F.;Hansen, John D.; 2004-10-01 00:00:00 Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 RESEARCH ARTICLE | OCTOBER 01 2004 Yuko Ohta; ... et. al J Immunol (2004) 173 (7): 4553–4560. https://doi.org/10.4049/jimmunol.173.7.4553 Related Content B Cells in Teleost Fish Act as Pivotal Initiating APCs in Priming Adaptive Immunity: An Evolutionary Perspective on the Origin of the B-1 Cell Subset and B7 Molecules J Immunol (March,2014) B cells in primitive vertebrate act as pivotal antigen presenting cells in priming adaptive immunity (P5035) J Immunol (May,2013) Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 The Journal of Immunology 1,2 † ‡ § ¶ Yuko Ohta,* Eric Landis, Thomas Boulay, Ruth B. Phillips, Bertrand Collet, ¶ 3 Chris J. Secombes, Martin F. Flajnik,* and John D. Hansen Dendritic cells are one of the most important cell types connecting innate and adaptive immunity, but very little is known about their evolutionary origins. To begin to study dendritic cells from lower vertebrates, we isolated and characterized CD83 from the nurse shark (Ginglymostoma cirratum (Gici)) and rainbow trout (Oncorhynchus mykiss (Onmy)). The open reading frames for Gici-CD83 (194 aa) and Onmy-CD83 (218 aa) display 28 –32% identity to mammalian CD83 with the presence of two conserved N-linked glycosylation sites. Identical with mammalian CD83 genes, Gici-CD83 is composed of ﬁve exons including conservation of phase for the splice sites. Mammalian CD83 genes contain a split Ig superfamily V domain that represents a unique sequence feature for CD83 genes, a feature conserved in both Gici- and Onmy-CD83. Gici-CD83 and Onmy-CD83 are not linked to the MHC, an attribute shared with mouse but not human CD83. Gici-CD83 is expressed rather ubiquitously with highest levels in the epigonal tissue, a primary site for lymphopoiesis in the nurse shark, whereas Onmy-CD83 mRNA expression largely paralleled that of MHC class II but at lower levels. Finally, Onmy-CD83 gene expression is up-regulated in virus-infected trout, and the promoter is responsive to trout IFN regulatory factor-1. These results suggest that the role of CD83, an adhesion molecule for cell-mediated immunity, has been conserved over 450 million years of vertebrate evolution. The Journal of Immunology, 2004, 173: 4553– 4560. ver the last decade, the critical importance of dendritic activation (7–9). The expression of CD83 on DCs is accompanied by cells (DCs) in Ag presentation and regulation of T cells the up-regulation of costimulatory molecules (CD80 and CD86), im- O has been well documented (1). Surface expression of plicating CD83 in the induction of immune responses. CD83 is the standard lineage maker for activated or differentiated The functional role of CD83 was recently examined via trans- DCs. Mammalian CD83, a cell surface membrane glycoprotein fection assays using a CD83-Ig fusion protein. The fusion protein (45 kDa) whose surface expression is largely restricted to DCs was found to bind peripheral blood monocytes and a subset of including Langerhans cells (human), circulating DCs, and inter- CD8 lymphocytes, suggesting that CD83 mediates adhesion to digitating DCs within T cell zones of secondary lymphoid tissues, circulating monocytes and some T cell subsets via interaction of is a member of the Ig superfamily (IgSf) (2– 4). CD83 expression CD83 with a sialated 72-kDa ligand (10). This led to the classiﬁ- has also been found on some germinal center B cells in vivo and cation of CD83 as a sialic acid-binding Ig-like lectin. More on T cells upon Ag or TCR-based activation (2, 5). In addition, recently, though, CD83 appears to be a central component for CD83 expression has been documented in the mammalian brain by lineage development of cell-mediated immunity, because CD83- Northern blotting, but the source of this expression is not known deﬁcient mice display a dramatic decrease (90%) in peripheral (2, 6). CD83 transcription is largely controlled by SP1 and NF-B CD4 populations (11, 12). The deﬁcit in naive single-positive elements within the CD83 promoter, which is in agreement with the CD4 cells is likely due to the loss of CD83 expression by thymic up-regulation of CD83 upon infection or TNF-, IL-1, and mitogen epithelium and DCs, which are involved in the coordination of T cell lineage commitment. Structurally, CD83 is composed of a single extracellular V-set *Department of Microbiology and Immunology, University of Maryland School of IgSf domain, a hydrophobic transmembrane domain, and a cyto- Medicine, and Molecular and Cellular Biology Program, University of Maryland plasmic tail. In humans, the CD83 gene spans roughly 19 kb and Graduate School, Baltimore, MD 21201; Department of Research, University of is composed of ﬁve exons including two exons that encode the Ig Basel, Basel, Switzerland; School of Biological Sciences, Washington State Univer- sity, Vancouver, WA 98686; Scottish Fish Immunology Research Centre, Zoology V-set domain. The gene maps to murine and human chromosomes Building, Aberdeen University, Aberdeen, Scotland, United Kingdom; and Depart- 13 and 6, respectively (3, 6, 13), and the human CD83 gene ﬂanks ment of Pathobiology, University of Washington, Seattle, WA 98195 the human MHC. Our laboratories are interested in the evolution- Received for publication October 17, 2003. Accepted for publication July 22, 2004. ary events that have shaped the adaptive immune response. So far, The costs of publication of this article were defrayed in part by the payment of page only a handful of potential CD markers have been characterized charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. from all ﬁsh including CD3 (14), CD8 (15), CD9 (16), CD18 (17), CD45 (18 –20), and CD81 (20, 21). In this report, we describe the This work was supported in part by National Institutes of Health Grant AI 27877 (to M.F.F.) and U.S. Department of Agriculture-National Research Initiative Competitive isolation and initial characterization of CD83 homologs from ﬁsh. Grants Program 2002-03472 and National Science Foundation 0324069 (to J.D.H.). The sequences presented in this article has been submitted to GenBank under ac- cession numbers AY183667, AY263793-797, AY437982-983, and AY650049-050. 3 Materials and Methods Address correspondence and reprint requests to Dr. John D. Hansen, Department of Pathobiology, University of Washington, Seattle, WA 98195. E-mail address: cDNA cloning of ﬁsh CD83 [email protected] A nurse shark PBL ZAPII cDNA library (Stratagene, La Jolla, CA) was Abbreviations used in this paper: DC, dendritic cell; IgSf, Ig superfamily; UTR, used as the template for a degenerate PCR approach for amplifying unique untranslated region; EST, expressed sequence tag; ORF, open reading frame; Gici, V-set genes. Three different degenerate primers (reverse/complementary Ginglymostoma cirratum; Onmy, Oncorhynchus mykiss; gDNA, genomic DNA; for D/S/N-X-G-X-Y-X-C; the successful primer corresponded to (A/ BAC, bacterial artiﬁcial chromosome; IHNV, infectious hemopoietic necrosis virus; G)CA-III-RTA-III-NCC-III-RTC) corresponding to the F strand of IgSf IRF, IFN regulatory factor; CHO, Chinese hamster ovary; ISRE, IFN-stimulated reg- ulatory element. V-set domains were used in conjunction with an anchored T3 primer Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 4554 HOMOLOGS OF CD83 FROM ELASMOBRANCH AND TELEOST FISH (within the ZAP vector) for the ampliﬁcation of potential V domains. Prim- was hybridized with a radiolabeled cDNA probe corresponding to the ers were used in the following PCR proﬁle for ampliﬁcation: 94°C for 1 Onmy-CD83 V region. Blots were washed at a ﬁnal stringency of 0.4 min followed by 35 cycles of 94°C for 20 s, 43°C for 25 s (0.1°C increase SSC/0.4% SDS at 68°C and then exposed to ﬁlm for 2 days at 80°C. per cycle), and 72°C for 30 s. The PCR samples were then extended for an Linkage analysis for Gici-CD83 additional 10 min at 72°C to facilitate TA-based cloning (pTOPO; BD Clontech, Palo Alto, CA). Aliquots of the ampliﬁed cDNA library were The isolation of gDNA from 39 nurse shark pups (multiple paternity) has diluted in 5 mM Tris (pH 8.0) and denatured at 98°C for 10 min before been previously described (23). Five micrograms of gDNA was digested to usage as a template to release the cDNA templates from the phagemids. completion with EcoRI, which produced a useful restriction fragment poly- Restriction digests were performed, and 60 random inserts in the proper morphism using the Gici-CD83 probe. The PCR-generated probe corre- size range (250 – 400 bp) were sequenced via automated sequencing (Ap- sponded to the entire ORF for Gici-CD83 (primers, exon 2F.21455 and plied Biosystems 310; Applied Biosystems, Foster City, CA). One partic- exon 5R.21701; see above for details) and procedures for Southern blotting ular clone showed strong similarity to V-set domains and was used as the under high-stringency conditions with randomly primed probes have been basis for nested 3 anchored PCR to amplify the full-length gene from the described previously (24). Segregation patterns for Gici-CD83 were then library. Nested PCR was performed using two gene-speciﬁc primers cor- compared with previously identiﬁed patterns for the nurse shark MHC (23). responding to the putative 5-untranslated region (UTR) (forward, 5-CAT GATGCTTTAAACGTG-3) and ATG codon (forward 2, 5-AACGTGT Onmy-CD83 in situ hybridization and karyotyping TCGAAAGGATGATG-3) with the anchored T7 primer corresponding to the 3 region of all inserts within the library. Full-length cDNAs were The V region of trout CD83 was used as a probe to screen high-density ampliﬁed and cloned into pTOPO and sequenced in both directions (Ap- ﬁlters corresponding to 4.5 times coverage of a bacterial artiﬁcial chromo- plied Biosystems 377). some (BAC) genomic library from the OSU-142 all-female clonal line of Rainbow trout CD83 was obtained via a tBlastN search of the trout trout. DNA was isolated from positive BAC clones using the Qiagen (Va- expressed sequence tags (ESTs) within GenBank using sequence informa- lencia, CA) columns. Both PCR and direct sequencing were used to con- tion from the Ginglymostoma cirratum (Gici)-CD83 open reading frame ﬁrm positive BAC clones harboring Onmy-CD83. Peripheral blood was (ORF). The EST clone was completely sequenced and found to lack an cultured from the Donaldson strain of rainbow trout using standard meth- initiation codon. The 5 end of Oncorhynchus mykiss (Onmy)-CD83 was ods (25). This strain has the same chromosome number as the OSU clonal ampliﬁed from a directional PBL cDNA library (UniZap; Stratagene) using line (n 30). Onmy-CD83 BAC DNA was labeled with Spectrum Orange a vector (T3) and an Onmy-CD83 gene-speciﬁc primer (CD83-E3R1; 5- (Vysis, Downers Grove, IL) using nick translation kit (Vysis). Human pla- CCAGGAGACACTTGTACCTT-3) in anchored PCR. Products were cental DNA (0.2 g) and Cot-1 DNA (1 g; prepared from rainbow trout cloned and sequenced to derive a consensus 5 ATG primer. Primers gDNA) were added to the probe mixture for blocking. Hybridizations were (Onmy-CD83.ATG.F, 5-CATTGCTGTAGTTCTACAAATATG-3; and conducted at 37°C overnight and posthybridization washes were as rec- Onmy-CD83-STOP.R, 5-TTCCTCTTTATGTTCAAGTATAC-3) were ommended by the manufacturer (Vysis) with minor modiﬁcations (26). then used to amplify full-length clones of CD83 cDNA from splenic cDNA Abs to Spectrum Orange (Molecular Probes, Eugene, OR) were used to derived from the homozygous strains of trout, OSU-142, HotCreek, and amplify the signal in some cases. Slides were counterstained with 4,6- Arlee (22). Naive spleens from the OSU-142, HotCreek, and Arlee clonal diamidino-2-phenylindole at a concentration of 125 ng of 4,6-diamidino- lines were generously provided by Dr. G. Thorgaard (Washington State 2-phenylindole in 1 ml of antifade solution. Images were captured with a University). Products were cloned (pTOPO) and sequenced. Sensys camera (Photometrics, Tucson, AZ) and analyzed with Cytovision The following accession numbers were used for assembling the ﬂoun- Genus (Applied Imaging, San Jose, CA) software. Chromosomes were der, zebraﬁsh, chicken, rat, and bovine CD83-like sequences: ﬂounder, classiﬁed using a combination of relative size, chromosome arm ratios, and AU091120; zebraﬁsh, BI671547/BM157226; chicken, BI384417/BU457418/ the intensity of 4,6-diamidino-2-phenylindole bands at the centromeres BU452135/BM440129/AI981465; rat, XM_225224; and bovine, BE755445/ (27). It should be mentioned that the Onmy-CD83 BAC contained two BI539602. ESTs were assembled using AssemblyLine, version 1.0.9, within Onmy-CD83 genes as determined by direct sequencing. MacVector (Accelrys, San Diego, CA). The V domains (amino acid se- Northern blot hybridization and RT-PCR quences) from the various ESTs and full-length clones were used for phylo- genetic analysis as previously described (15). The human CD83 pseudogene RNA isolation and Northern blotting protocols have been previously de- was found using the human CD83 protein (NM_004233) in conjunction with scribed (24, 28). Brieﬂy, 15 g of total RNA was electrophoresed under the BLAT server (http://genome.ucsc.edu/cgi-bin/hgBlat). alkaline conditions and transferred to Nytran (Fisher Scientiﬁc, Atlanta, GA) using 20 standard saline citrate phosphate/EDTA. Two different Genomic structure probes were ampliﬁed from the pGici-CD83 plasmid to conﬁrm the ex- Long-range PCR (Elongase; Invitrogen Life Technologies, Carlsbad, CA) pression pattern for Gici-CD83, one corresponding to the V domain exon was used to deduce the exon/intron structure for Gici-CD83. PCR condi- 2F.21455 and exon 3R.21699 (see genomic structure for details) and the tions consisted of the following proﬁle: 2 min at 95°C followed by 35 other to the cytoplasmic domain plus the 3-UTR (exon 5F.21800, 5- cycles of 94°C for 30 s, 57°C for 30 s, and 68°C for either 3, 6, or 9 min GTACAAGCAACCCAATGTTC-3 and 3-UTR-R.21801, 5-GTTTAT depending on the primer pairs. Products were then cloned into pBLUNT TTGAAATCACAAGCT-3). Ampliﬁed fragments were puriﬁed and then (Invitrogen Life Technologies) for verifying the sequence and to determine randomly primed (Invitrogen Life Technologies) with [ P]dCTP (Amer- the exon/intron splice site and phase. Intron size estimates were based upon sham Biosciences, Piscataway, NJ). Nonincorporated nucleotides were re- standard 1% agarose gel electrophoresis of PCR products minus exon con- moved using G-50 spin columns (Invitrogen Life Technologies) before tributions. The following primers were used: exon 1 to exon 2: exon hybridization. The Gici-MHC class IIA and NDPK probes have been pre- 1F.21789, 5-ATGTTTCACCTTAAGAAATGT-3, and exon 2R.21790, viously described. For Onmy-CD83 Northern blotting, 12 g of total RNA 5-TTCTCCACACTTTACTGTAAC-3; exon 2 to exon 3: exon 2F.21455, was electrophoresed, transferred to nylon (Nytran), and hybridized over- 5-TCCGAAGTTACAGTAAAGTGT-3, and exon 3R.21700, 5-TTGA night using an Onmy-CD83 V domain probe (VaVb). The blot was washed ACACTGATACTTTCCAA-3, plus nested exon 3R.21699, 5-ACATTGA at high stringency and exposed for 6 days whereupon the ﬁlter was stripped ACACTGATACTTTC-3; exon 3 to exon 4: exon 3F.21457, 5-GACTT and reprobed sequentially with trout MHC class IIA (exon 2, 28-h expo- TGGAAAGTATCAGTGT-3, and exon 4R.21788, 5-TCAGAACAACT sure) and then with EfTu-1 (12-h exposure). The protocols for acute in- GGTAAGATAG-3; and exon 4 to exon 5, exon 4F.21787, 5-CTAT fectious hemopoietic necrosis virus (IHNV) infection have been described CTTACCAGTTGTTCTGA-3, and exon 5R.21701, 5-GAACATTGGG previously (29). Brieﬂy, trout weighing 200 g were infected by injection TTGCTTGTACT-3. (i.p.) with 1 10 PFU of IHNV, and tissues were harvested on speciﬁed Primers within the A (Onmy-CD83-E2F1.5-GTCAGTTTGTGGAGA days. Controls consisted of saline-injected ﬁsh. GGATTC-3)andF(Onmy-CD83-E3R1.5-CCAGGAGACACTTGTAC Promoter and reporter assays CTT-3) strand of the trout CD83 V domain were used in PCR to determine whether the Onmy-CD83 V domain is encoded by a single or multiple The promoter region for Onmy-CD83 was obtained using the promoter exon(s) using OSU-142 genomic DNA (gDNA) as the template. Products trapping kit (Genome Walking; Bio S&T, Montreal, Quebec, Canada) in were cloned (pTOPO2.1) and sequenced to identify splice site location and conjunction with speciﬁc reverse primers found within exon 1 (exon 1R, phase. 5-CATTGCACGGAGGCAGCTAG-3)of Onmy-CD83. Two different promoters were ampliﬁed from the OSU-142 Onmy-CD83 BAC clone that Southern blotting for Onmy-CD83 was used for physical mapping. Products were cloned into pBlunt (Invitro- For Southern blotting, 15 g of rainbow trout gDNA was digested, elec- gen Life Technologies), sequenced, and assessed for potential transcription trophoresed, and transferred to nylon as previously described (15). The blot factor binding sites using the MatInspector Professional software suite Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 The Journal of Immunology 4555 (www.genomatix.de/index.html). The following settings were used: core An amino acid alignment (Fig. 1A) was generated to display similarity, 0.85; and matrix similarity, 0.90, against the Transfac database features that have been conserved during vertebrate evolution for (version 7.4). Four different versions of the Onmy-CD83 promoter were CD83. The leaders for all sequences are predicted to be 19 –20 ampliﬁed from the longest promoter clone (956 bp) by PCR and then aa via pSort (http://.psort.nibb.ac.jp/). The N-terminal cleavage site cloned into XhoI/BglII sites of pGL3-Basic. All constructs were sequence (TK) for the Gici-CD83 leader was conﬁrmed using N-terminal veriﬁed before transfection. The four different Onmy-CD83-pGL3 (A–D) constructs were cotransfected (0.2 g) with the pRNL (Renilla coreporter) amino sequencing of Gici-CD83-Ig fusion proteins (J. D. Hansen, to normalize transfection efﬁciencies (i.e., pGL3-OmCD83-full plus unpublished data). The Gici-CD83 mature ORF displays conser- pRNL). Deletion constructs were ampliﬁed by PCR from the 956-bp vation of critical residues found within CD83, and overall, the cloned promoter using forward 1/reverse 1 (Om-CD83-full), forward 2/re- sequences display 20%. Both trout and ﬂounder share 37% sim- verse 1 (Om-CD83-2), forward 3/reverse 1 (Om-CD83-3), and forward 1/reverse 2 (Om-CD83-4), digested, and cloned into the XhoI/HindIII-cut ilarity with Gici-CD83 and 63% similarity (45% identity) between pGLL3. Forward 1 (5-GAGAGCTCGAGGAAGCATCGTTACCCATCG themselves for the entire ORF. The cysteine residues in strands B CGCCACAA-3), forward 2 (5-GAGAGCTCGAGACTAATAAACTGC and F that forms a disulﬁde bond within most Ig domains, as well TAAATAAAGTATTGAGT-3), forward 3 (5-GAGAGCTCGAGTTCA as the invariant tryptophan in the C strand, are present in CD83. ATGAGATCACATTAACACCTCAT-3), reverse 1 (5-GAGAGAAGC One unique feature shared by all CD83 sequences is the presence TTTTGTAGAACTACAGCAATGAAGATGAT-3) and reverse 2 (5- GAGAGAAGCTTTGCTTCGTCACAGTGGGTGTAGATGGA-3) XhoI of a cysteine residue (also found in CD90) within the A strand of and HindIII restriction sites are underlined. Plasmids were cotransfected the IgSf V domain. However, Gici-CD83 does not possess the (1.2 g: 0.6 g of pGL3 constructs and 0.6 g of pcDNA3.1 Onmy-IFN conserved cysteine residue between the G strand and the trans- regulatory factor (IRF)-1 and/or IRF-2) with Lipofectin (Invitrogen Life membrane region. The relevance of this additional cysteine residue Technologies) using standard protocols for Chinese hamster ovary (CHO) cell transfection in 24-well format. Cells were passively lysed (Promega, is not known, but mammalian CD83 is monomeric, and thus, the Madison, WI), and luciferase activity (ﬁreﬂy and Renilla) was measured additional cysteine residue does not play a role in dimerization. (MicroLumat; Berthold Technologies, Oak Ridge, TN) using the dual-lu- Immunoprecipitation of human and murine CD83 under both ciferase reporter system (Stop-n-Glow; Promega). Two wells for each reducing and nonreducing conditions demonstrated that CD83 pro- transfection situation were read in triplicate (n 6 per sample) and were teins are heavily glycosylated (broad 45-kDa band), because the consistent between two independent experiments. Values are represented as fold induction over luciferase activity for the complete CD83 promoter predicted molecular masses for the mature proteins are roughly alone (i.e., CD83-full alone set to 1). only 20 kDa (3). The relative locations of two of the three N-linked glycosylation sites (N-X-S/T) are conserved in evolution, suggest- ing that ﬁsh CD83 proteins are also heavily glycosylated. The third Results and Discussion mammalian N-glycosylation site in the G strand is absent in ﬁsh. Cloning and sequence analysis of Gici- and Onmy-CD83 Conserved O-linked glycosylation sites (XPXX, glycosylated if We used a degenerate PCR primer strategy that incorporates fea- X S or T) were not observed within extracellular domain for any tures of the V domain F strand in conjunction with a unidirectional of the CD83 sequences. The transmembrane and cytoplasmic do- cDNA library for the ampliﬁcation of new V domain sequences mains display 17– 41% amino acid identity among the various from the nurse shark Gici. Sixty random products ﬁtting the proper CD83-like sequences and teleost, rat, and avian cytoplasmic do- size range for V domains plus 5-UTR (300 – 450 bp) were se- mains are much longer than those of the other vertebrates. The quenced and subjected to BlastX/N searches of the National Center trout, ﬂounder, and chicken cytoplasmic domains had to be aligned for Biotechnology Information vertebrate database. One fragment separately from the other sequences due to their low sequence (413 bp) showed strong similarity to both mouse and human CD83 identity. A role for the cytoplasmic domain has yet to be reported (BlastX 7e-04/2e-03) and to Ig L chains (1e-02) using the entire for CD83, but it has been shown that the conserved serine and vertebrate database. Because this fragment likely corresponded to threonine residues in mice and humans are not phosphorylated (3). the 5 end of the putative Gici-CD83 cDNA, we used 3 anchored Neither ITIM nor ITAM motifs are found in any of the sequences, PCR to amplify the full-length sequence from the nurse shark although several tyrosine residues are found especially within the cDNA library. Gici-CD83 (accession no. AY183667) contains 86 chicken cytoplasmic domain. Finally, cysteine residues present in bp of 5-UTR followed by single ORF coding for 194 aa and a the ﬁsh CD83 (also in rat and chicken) cytoplasmic domains war- short 3-UTR (137 bp) that includes a consensus polyadenylation sig- rant further investigation. Cysteine residues were also found in the nal and tail. BlastX analysis of the ORF again showed strongest sim- human CD83 pseudogene (6p22:27636584 –27637508)) upon ilarity to both human and mouse CD83 (8e-08/7e-07, respectively) BLAT (http://genome.ucsc.edu/cgi-bin/hgBlat) inspection. followed by V domains for Ig chains (7e-04) and TCR (1e-03). Phylogenetic analysis of the CD83 V domains Gici-CD83 was then used a template for tBlastN analysis of the public domain databases resulting in potential matches for trout, To compare the ﬁsh CD83 V domains with other members of this ﬂounder, zebraﬁsh, bovine, and chicken CD83 homologs. The family present as cell surface proteins on blood cells, we con- trout clone was completely sequenced and used to design primers ducted a phylogenetic analysis of CD83, CD7, CD8, CD28, CD83, for the ampliﬁcation of full-length versions of Onmy-CD83 from and CD152. As shown in the V region neighbor-joining tree (Fig. splenic cDNA from OSU-142 (AY263793), Arlee (AY263794/ 2), all of the CD83 sequences cluster as one group as supported by 795), and Hotcreek (AY263796/797) clonal trout. Two different bootstrap analysis. In addition, the ﬁsh and higher vertebrates form cDNAs were ampliﬁed from Arlee and Hotcreek and one from their own separate clusters within the CD83 branch, with the OSU-142, indicating that Onmy-CD83 is not a single-copy gene, chicken sequence found between the two groups, proving that because these sequences were derived from homozygous ﬁsh. A these sequences represent homologs. second OSU-142 CD83 gene (partial gDNA sequence) was ob- Genomic structure tained by PCR ampliﬁcation using an OSU-142 CD83 BAC clone as the template. The majority of amino acid differences were We used long-range PCR to determine the exon/intron organiza- focused in the leader and within the A, B, and C strands for the V tion for Gici-CD83 and found that Gici-CD83 possesses the same domain (Fig. 1B). Differing from Gici-CD83, the trout ORF is exon/intron pattern as mammalian CD83. The Gici-CD83 gene is larger (218 aa) but still possesses essential features that are shared composed of ﬁve exons and spans roughly 18 kbp from the ATG between the vertebrate CD83 genes. initiation codon through the polyadenylation site (Fig. 3). Exon 1 Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 4556 HOMOLOGS OF CD83 FROM ELASMOBRANCH AND TELEOST FISH FIGURE 1. CD83 protein alignment. Amino acid sequences were aligned using ClustalX and by manual adjustment. A, Alignment of CD83 genes from various ver- tebrate species. Solid vertical lines represent the borders between the leader, extracellular domain, transmembrane region, and the cy- toplasmic domain. Presumed strands (A–G) are indicated with horizontal lines above the amino acid translation. The loca- tions of exon/intron boundaries are denoted by underlining adjacent amino acids (i.e., GL in the nurse shark). Conserved cysteine res- idues involved in the V domain structure are shaded, and the location of conserved N-linked glycosylation sites are boxed. Iden- tical () and similar (:) amino acids are also shown within the alignment. B, Amino acid alignment of Onmy-CD83 from three differ- ent homozygous trout. Two different Onmy- CD83 genes were found in the Arlee and Hotcreek clonal lines. A second partial (2P) OSU-142 CD83 gene was obtained from the OSU-142 CD83 BAC clone (AY437982). encodes the 5-UTR and the ﬁrst 13 aa of the leader; exons 2 and (exons Va and Vb) and used it as a probe for Southern blot analysis 3 encode the IgSf V domain; exon 4 encodes the remainder of the (Fig. 4). Four to seven strong and two to four weak bands can be V domain (last 10 residues) and the transmembrane domain; and seen for each individual (outbred) and digest (EcoRV and HindIII). exon 5 encodes the cytoplasmic domain and 3-UTR. In all cases, Computer-based restriction analysis of sequenced genomic clones splice junctions correlated with consensus 5 and 3 donor and for the V region, indicated the presence of one HindIII and one acceptor splice sites. Overall, the Gici-CD83 genomic structure is EcoRV site within this region for Onmy-CD83. Thus, our Southern nearly identical with that of mammals including conservation of blot analysis is in agreement with Fig. 1B in which two different phase for the four intron splice sites. Finally, we also determined Onmy-CD83 genes were found in the Arlee and Hotcreek homozy- that the Onmy-CD83 V region is composed of two exons, but the gous, clonal rainbow trout. Furthermore, based upon the presence intron between the Onmy-CD83 Va and Vb exons is much smaller of distinct RFLPs among the different individuals, it appears that (150 or 763 bp, phase 0) than that for Gici or murine CD83. polymorphic variants for Onmy-CD83 may exist. Probably the most interesting and conﬁrmatory aspect from pre- MHC linkage analysis vious genomic analysis of CD83 is that the V domain gene struc- ture is composed of two split exons (phase 0 splice site), Va and In mammals, amphibians, and birds, MHC class I, II, and III re- Vb, with the splice location occurring in the C strand of the V gions are closely linked. However, in all teleost ﬁsh, the MHC domain (3, 6). To date, only a few other genes aside from CD83 class I and II regions are not linked and reside on different chro- have this particular genomic architecture including members of the mosomes (31). Recently, the genomic architecture for the elasmo- greater cortical thymocyte marker in Xenopus (CTX) family (30) branch MHC was examined. In two different shark species, class I, (CTX, CTH, A33, and one JAM member), all of which are poten- II, and III genes were shown to be linked, strongly suggesting that tial adhesion and receptor molecules involved in the cellular im- the primordial organization of the MHC was similar to that found mune response. for all other vertebrates, and the lack of linkage for the bony ﬁsh MHC is a derived characteristic (23, 32, 33). Southern blot analysis of Onmy-CD83 The human CD83 gene maps near the human MHC (6p23, te- Based upon the genomic organization of the Onmy-CD83 V do- lomeric of myelin oligodendrocyte glycoprotein) (6, 13). In mice, main, we ampliﬁed a cDNA probe corresponding to the V region this association is not found, because CD83 maps outside of the Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 The Journal of Immunology 4557 FIGURE 3. The genomic structure for human and nurse shark CD83 is evolutionarily conserved. Exons are boxed (exon 1 encodes the leader, exons 2 and 3 code for IgSf V domain, exon 4 encodes the transmembrane domain, and exon 5 codes for the cytoplasmic domain and 3-UTR). In- tronic distances (in kilobase pairs) are found between the exons with the phase of the given intron in parentheses. The partial genomic structure for Onmy-CD83 is also shown (based upon AY437982 and AY437983). highest levels within the brain, epigonal tissue, gills, intestine, PBLs, spleen, and testis (Fig. 7A). A nurse shark MHC class IIA probe was then used as a comparative marker for the expression FIGURE 2. Fish CD83 proteins are homologous to mouse and human analysis, because some DC lineages express high levels of MHC CD83. Phylogenetic tree of IgSf V domains from proteins with similar class II, consistent with their role in Ag presentation. As expected, structures as CD83. An amino acid alignment (ClustalX) of the V domains nurse shark MHC class IIA also displayed strongest expression was used for the generation of an unrooted neighbor-joining tree. Values at within the gills, intestine, PBLs, and spleen. Transient transfection the tree nodes represent bootstrap values from 1000 replicants. of N terminus Flag-tagged Gici-CD83 and subsequent FACS anal- ysis, demonstrated surface expression on CHO cells (data not shown), thus supporting that Gici-CD83 is a type-1 transmembrane MHC on chromosome 13 (6, 13). To determine whether the Gici- protein. We then examined the expression of Onmy-CD83 in se- CD83 gene is linked to the shark MHC, we studied segregation of lected trout tissues. Onmy-CD83 (Fig. 7B) was primarily expressed Gici-CD83 in a nurse shark family of 39 pups. According to RFLP within the spleen and testis by Northern blotting (V domain probe) analysis with a classical class IA probe, pups were sorted into at at much lower levels in comparison to MHC class IIA (DAA). least 13 groups (A–M). These groups represent the combination of maternal and paternal alleles; therefore, a maximum of four groups can be obtained from a single-copy gene. The increased number of groups is due to multiple paternities (at least ﬁve fathers in this family) (23). Because most of the groups contain only a few pups, we used groups of more than three pups (A, D, and H) for the linkage analysis. Furthermore, groups A and D appear to share the same paternal allele p2. In Fig. 5, a 6.6-kb band is absent in the maternal lane; therefore, the 6.6-kb band represents a paternal band. In all cases, only 50% of the pups carry the 6.6-kb band (i.e., 50% recombinant between two paternal chromosomes), strongly suggesting that Gici-CD83 is not linked to the shark MHC. Recently, the rainbow trout chromosomes and linkage groups harboring the trout MHC have been determined (34). Interestingly, a duplication event has led to the development of two class I re- gions in trout on chromosomes 14 and 18, whereas a compact class II region containing both DAA and DAB was found on chromo- some 17. In addition, ABCB2 (TAP1), a transporter gene located in mammalian class II region, was found on chromosome 3. In this study, we determined that Onmy-CD83 is found near the centro- mere on the short arm of chromosome 9 (9p) using in situ hybrid- ization (Fig. 6). Therefore, as in sharks and mice, Onmy-CD83 is not linked to the trout MHC, and thus the linkage of the two human CD83 genes (CD83 and the CD83 pseudogene) to the MHC is most likely a derived feature. Tissue-speciﬁc expression of Gici- and Onmy-CD83 We then examined the tissue-speciﬁc expression of Gici-CD83 by FIGURE 4. Onmy-CD83 is not a single-copy gene as assessed by Southern Northern blotting using two probes, one designed for the V domain blotting. gDNA from four different individual trout (outbred) were digested and the other encompassing the cytoplasmic tail and 3-UTR. Both with EcoRV and HindIII, blotted, and hybridized with a cDNA probe corre- probes showed identical results with ubiquitous expression and sponding to the Onmy-CD83 IgSf V domain (exons Va and Vb). Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 4558 HOMOLOGS OF CD83 FROM ELASMOBRANCH AND TELEOST FISH FIGURE 5. The nurse shark CD83 gene is not linked to the MHC. M, Maternal RFLP pattern. Individual pups (numbered) corresponding to MHC segregating groups H, D, and A are shown. Recombinants are noted by the presence or absence of the 6.6-kbp band. Expression of Onmy-CD83 was also found in the thymus, prone- phros (bone marrow equivalent), and mesonephros. RT-PCR dem- onstrated that all tissues examined were positive for Onmy-CD83, including skin (data not shown). Finally, we determined that two Onmy-CD83 genes are up-regulated during acute IHNV (a ﬁsh rhabdovirus) infection in the spleen, pronephros, and intestine at day 6 postinfection (Fig. 7C). Mammalian CD83 gene expression is largely controlled by SP1 and NF-B(7–9), with no apparent involvement of IRFs. Onmy-CD83 up-regulation occurred as early as 24 h postinfection and remained induced up to 192 h postin- fection (data not shown). Recently, we showed that STAT-1 and members of the class I pathway are also up-regulated during acute IHNV infection (29). In that study, STAT-1, ABCB2 (TAP1), and PSMB9A (LMP2) were clearly induced by 24 h postinfection, im- FIGURE 7. Tissue-speciﬁc mRNA expression for Gici- and Onmy- plicating the involvement of type-I IFNs. CD83. A, Northern blot hybridization of nurse shark tissues (Br, brain; Ep, In mice, CD83 tissue mRNA expression was found in the heart, epigonal; Gi, gills; Hr, heart; Int, intestine; Kd, kidney; Lv, liver; Pa, pan- brain (high), spleen (high), lung, and muscle, and weakly within creas; PBLs; Sp, spleen; Ts, testis; and Thy, thymus) with the Gici-CD83 the kidney, but similar to Gici-CD83 and Onmy-CD83, prolonged probe (V domain) demonstrates that a single transcript for Gici-CD83 exposure or RT-PCR demonstrated expression within all tissues (900 bp) is expressed at highest levels within the brain, epigonal tissue, examined (6). In humans, CD83 mRNA expression was also de- gills, intestine, PBLs, spleen, and testis (6-day exposure). Strong signals for Gici-MHC class IIA transcription were found in gills, intestine, and spleen (3-day exposure). The Gici-NPDK housekeeping probe was used to show relative equivalency in loading (overnight exposure). B, Onmy-CD83 is expressed mainly in trout lymphoid tissues (9-mo-old trout), including the thymus, pronephros (bone marrow equivalent), and spleen (7-day expo- sure) at lower levels in comparison to MHC class IIA (36-h exposure). Thy, Thymus; PN, pronephros; MN, mesonephros; Sp, spleen; Lv, liver; Int, Intestine; Ms, muscle; Hr, heart; Ts, testis. EfTu-1 was used to show relative equivalency in loading (overnight exposure). C, Onmy-CD83 mRNA expression is up-regulated in the spleen, pronephros, and intestine during acute IHNV infection (day 6 postinfection). Two CD83 messages are induced upon infection. CD83 blots were exposed for 5 days, and EfTu-1 served as a control for loading (overnight exposure). tected in the brain, lungs, and mitogen-activated B lymphocytes, and within some T cell populations. Flow cytometric analysis has shown that CD83 expression is limited to cells of hemopoietic lineages, especially to cells of DC lineages (3, 35). Interestingly, CD83 expression has also been associated with a rather unique murine cell type, one that possesses both B cell CD markers and DC morphology and function, because they were found to be po- tent allo-APCs in MLRs (36). Thus, it will be interesting to see whether a similar cell type can be found in ﬁsh. Because DCs have a diverse tissue distribution, the above results (Fig. 6) are consis- tent with CD83 expression by DC lineages in the nurse shark. Recently, the primary and secondary lymphoid tissues of the nurse shark have been examined (37). Based upon expression of key FIGURE 6. Fish CD83 is not linked to the MHC. Onmy-CD83 resides markers including RAG1 and TdT, the epigonal tissue and thymus on the short arm of rainbow trout chromosome 9 as visualized by in situ were found to be the primary lymphoid tissues throughout the life hybridization using CD83 BAC clones as probes. The trout MHC class I and II regions (boxed) are found on chromosomes 3, 14, 17, and 18 (34). of the nurse shark. Interestingly, the nurse shark spleen strongly Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 The Journal of Immunology 4559 resembles the mammalian splenic architecture in possessing dis- tinct/organized T and B cell zones and the presence of MHC class II DC networks. In salmonids, MHC class IIA and -B mRNA and protein expression are mainly limited to lymphoid tissues including the thymus, kidney, spleen, and intestine (28, 38). Additionally, At- lantic salmon class IIB protein expression was primarily localized to cells of the white pulp with a few positive cells (likely melanomac- rophages) in the red pulp. This suggests that salmonid splenic APCs are found in the same location as that for sharks and mammals. Promoter analysis of Onmy-CD83 In this study, we found that Onmy-CD83 is up-regulated during acute rhabdoviral infection similar to that for the trout MHC class I pathway (29). Based on this result, we cloned the putative promoter region for Onmy-CD83 to assess the presence of transcription factor motifs that may be involved in regulating Onmy-CD83. Two different sequences were obtained (AY650049 and AY650050, 98% identity) from the OSU-142 clonal line cor- responding to the 5-ﬂanking region of Onmy-CD83. Upon MatInspector analysis, transcription factor binding motifs known to be implicated in the regulation of immune relevant genes were found, including NF-B, IRF-7, STAT6, PU.1, IFN-stimulated regulatory element (ISRE), in addition to a consensus TATA-box (Fig. 8A). The location of the TATA-box is 28 bp upstream of the initiation site of Onmy-CD83 as judged by 5-RACE analysis, which conforms to consensus location for TATA-boxes. Peak promoter activity for the human CD83 promoter (7) was conﬁned to a 261 fragment relative to the ATG containing four SP1 and a single NF-B element. Putative SP1 sites were not found in the Onmy-CD83 promoter, but interestingly, a consensus NF-B site (100) was found in the same overall location as that for the human CD83 promoter (123) relative to the ATG. To initially characterize the Onmy-CD83 promoter, four different versions (Fig. 8B)ofthe Onmy-CD83 promoter were cloned into the pGL3- Basic luciferase reporter vector to assess their activity in CHO cells upon cotransfection with trout IRF-1 and/or IRF-2 (39) expression constructs. Upon single transfection of the four Onmy-CD83 pGL3 luciferase constructs, only pOm-CD83-4 (minus NF-B/ISRE/TATA) showed slightly diminished activity in comparison to the pOm-CD83.1-3 pGL3 constructs alone, sug- gesting that this region (103 to 3) may be involved in basal transcription. Poly(I:C) treatment of CHO cells transfected with the various pGL3-OmCD83 constructs did not result in enhanced FIGURE 8. Deletion analysis of the Onmy-CD83 promoter. A,5- luciferase activity (data not shown), suggesting that hamster- Flanking region for Onmy-CD83. Numbers on the left and right borders are derived IRFs and NF-B do not physically interact with sites relative to the inferred ATG for Onmy-CD83. Putative transcription factor found within the Omny-CD83 promoter. In direct contrast, binding sites are in bold with the name of site above. Numbers in paren- enhanced luciferase activity (6- to 7-fold induction) was found for theses reﬂect MatInstpector Professional values for the matrix and core, pGL3-OmCD83-1-3 constructs cotransfected with the IRF-1 where 1 is a perfect match. The potential interaction site for Onmy IRF-1 expression construct but not IRF-2 (Fig. 8C). The enhanced is indicated with asterisks underneath. The TATA-box is underlined. B, luciferase activity was not found upon cotransfection of pGL3- Schematic depiction of the four Onmy-CD83 pGL3 reporter constructs. Om-CD83-4 with IRF-1, indicating that trout IRF-1 interacts Om-CD83-4 represents a deletion from 102 to 4, thereby deleting the with a site located between 103 to 3 relative to the Onmy-C second NF-B and IRF potential binding sites as well as the TATA-box. C, D83 ATG. A true consensus IRF-1 site (SAAAGYGAAAC; Luciferase activity of various reporter constructs for the trout CD83 pro- moter transfected alone or cotransfected with Omny-IRF-1 and/or Onmy- www.gene-regulation.com/) is not found in the 103 to 3 IRF-2 expression constructs in CHO cells. Samples indicated as “none” are region, but a TTTCNNTT motif (83) found within the putative single transfectants. All transfections were performed in the presence of ISRE site (85) is identical with the IRF-1 and -2 physical pRL (Renilla luciferase) for the normalization of transfection efﬁciencies. recognition site as determined by crystallography (40, 41). This Samples were lysed and ﬁreﬂy (pGL3) and Renilla (pRL) luciferase ac- implies a direct interaction of trout IRF-1 with the TTTCACTT tivities were using the Stop-n-Glow substrate system. Fireﬂy luciferase motif. Cotransfection of IRF-1 and IRF-2 together with the pGL3- values (reporter constructs) are presented as fold induction: averaged sam- OmCD83 constructs resulted in lower activities in comparison to ple value over the averaged value of Om-CD83-full alone (i.e., Om-CD83- IRF-1 alone, suggesting that IRF-2 likely competes for the IRF-1 full “none”). The data are shown from a representative experiment reported binding site as is found in higher vertebrates (41, 42). as the mean (n 6) SEM. Considering the presence of a consensus NF-B site at position 100, and the interaction of trout IRFs with the Onmy-CD83 Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 4560 HOMOLOGS OF CD83 FROM ELASMOBRANCH AND TELEOST FISH 17. Qian, Y., A. J. Ainsworth, and M. Noya. 1999. Identiﬁcation of a 2 (CD18) promoter, our results imply that Onmy-CD83 mRNA expression molecule in a teleost species, Ictalurus punctatus Raﬁnesque. Dev. Comp. Im- is likely controlled by innate mechanisms including NF-B and munol. 23:571. type-I IFNs. Taken together, the expression patterns for Gici-C 18. Nagata, T., T. Suzuki, Y. Ohta, M. F. Flajnik, and M. Kasahara. 2002. The leukocyte common antigen (CD45) of the Paciﬁc hagﬁsh, Eptatretus stoutii: im- D83 and Onmy-83 are consistent with the expression of CD83 plications for the primordial function of CD45. Immunogenetics 54:286. in mammals. 19. Okumura, M., R. J. Matthews, B. Robb, G. W. Litman, P. Bork, and M. L. Thomas. 1996. Comparison of CD45 extracellular domain sequences from Concluding remarks divergent vertebrate species suggests the conservation of three ﬁbronectin type III domains. J. Immunol. 157:1569. We have a used a degenerate primer strategy for the isolation of a 20. Uinuk-Ool, T., W. E. Mayer, A. Sato, R. Dongak, M. D. Cooper, and J. Klein. primordial gene that, based upon our analysis, corresponds to a 2002. Lamprey lymphocyte-like cells express homologs of genes involved in immunologically relevant activities of mammalian lymphocytes. Proc. Natl. CD83 homolog of the nurse shark. We then performed a broad Acad. Sci. USA 99:14356. comparison of CD83 from a variety of vertebrates that suggest that 21. Yoder, J. A., and G. W. Litman. 2000. The zebraﬁsh fth1, slc3a2, men1, pc, fgf3, all CD83 proteins are heavily glycosylated, consistent with the and cycd1 genes deﬁne two regions of conserved synteny between linkage group 7 and human chromosome 11q13. Gene 261:235. identiﬁcation of CD83 as a sialic acid-binding Ig-like lectin pro- 22. Young, W. P., P. A. Wheeler, R. D. Fields, and G. H. Thorgaard. 1996. DNA tein. Finally, expression analysis indicates that CD83 is largely ﬁngerprinting conﬁrms isogenicity of androgenetically derived rainbow trout expressed within immunologically important tissues in ﬁsh, and lines. J. Hered. 87:77. 23. Ohta, Y., E. C. McKinney, M. F. Criscitiello, and M. F. Flajnik. 2002. Protea- that expression is up-regulated during acute viral infection in trout. some, transporter associated with antigen processing, and class I genes in the This represents the ﬁrst isolation of a putative ﬁsh marker for DC nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages. J. Immunol. 168:771. lineages, thus opening the door for studies examining DC biology 24. Bartl, S., M. A. Baish, M. F. Flajnik, and Y. Ohta. 1997. Identiﬁcation of class in elasmobranchs and teleosts, as well as the evolution of adaptive I genes in cartilaginous ﬁsh, the most ancient group of vertebrates displaying an immunity and Ag presentation. adaptive immune response. J. Immunol. 159:6097. 25. Reed, K. M., and R. B. Phillips. 1995. Molecular cytogenetic analysis of the double CMA3 chromosome in lake trout, Salvelinus namaycush. Cytogenet. Cell Acknowledgments Genet. 70:104. We thank Louis Du Pasquier for his comments in regard to the manuscript 26. Phillips, R. B., and K. M. Reed. 2000. Localization of repetitive DNAs to ze- and David Avila (Roche, Basel, Switzerland) for N-terminal sequencing of braﬁsh (Danio rerio) chromosomes by ﬂuorescence in situ hybridization (FISH). Chromosome Res. 8:27. the Gici-CD83-Ig fusion protein. 27. Reed, K. M., M. O. Dorschner, and R. B. Phillips. 1998. Characteristics of two salmonid repetitive DNA families in rainbow trout (Oncorhynchus mykiss). Cy- References togenetics 79:184. 1. 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DOI: 10.4049/jimmunol.173.7.4553
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Abstract

Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 RESEARCH ARTICLE | OCTOBER 01 2004 Yuko Ohta; ... et. al J Immunol (2004) 173 (7): 4553–4560. https://doi.org/10.4049/jimmunol.173.7.4553 Related Content B Cells in Teleost Fish Act as Pivotal Initiating APCs in Priming Adaptive Immunity: An Evolutionary Perspective on the Origin of the B-1 Cell Subset and B7 Molecules J Immunol (March,2014) B cells in primitive vertebrate act as pivotal antigen presenting cells in priming adaptive immunity (P5035) J Immunol (May,2013) Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 The Journal of Immunology 1,2 † ‡ § ¶ Yuko Ohta,* Eric Landis, Thomas Boulay, Ruth B. Phillips, Bertrand Collet, ¶ 3 Chris J. Secombes, Martin F. Flajnik,* and John D. Hansen Dendritic cells are one of the most important cell types connecting innate and adaptive immunity, but very little is known about their evolutionary origins. To begin to study dendritic cells from lower vertebrates, we isolated and characterized CD83 from the nurse shark (Ginglymostoma cirratum (Gici)) and rainbow trout (Oncorhynchus mykiss (Onmy)). The open reading frames for Gici-CD83 (194 aa) and Onmy-CD83 (218 aa) display 28 –32% identity to mammalian CD83 with the presence of two conserved N-linked glycosylation sites. Identical with mammalian CD83 genes, Gici-CD83 is composed of ﬁve exons including conservation of phase for the splice sites. Mammalian CD83 genes contain a split Ig superfamily V domain that represents a unique sequence feature for CD83 genes, a feature conserved in both Gici- and Onmy-CD83. Gici-CD83 and Onmy-CD83 are not linked to the MHC, an attribute shared with mouse but not human CD83. Gici-CD83 is expressed rather ubiquitously with highest levels in the epigonal tissue, a primary site for lymphopoiesis in the nurse shark, whereas Onmy-CD83 mRNA expression largely paralleled that of MHC class II but at lower levels. Finally, Onmy-CD83 gene expression is up-regulated in virus-infected trout, and the promoter is responsive to trout IFN regulatory factor-1. These results suggest that the role of CD83, an adhesion molecule for cell-mediated immunity, has been conserved over 450 million years of vertebrate evolution. The Journal of Immunology, 2004, 173: 4553– 4560. ver the last decade, the critical importance of dendritic activation (7–9). The expression of CD83 on DCs is accompanied by cells (DCs) in Ag presentation and regulation of T cells the up-regulation of costimulatory molecules (CD80 and CD86), im- O has been well documented (1). Surface expression of plicating CD83 in the induction of immune responses. CD83 is the standard lineage maker for activated or differentiated The functional role of CD83 was recently examined via trans- DCs. Mammalian CD83, a cell surface membrane glycoprotein fection assays using a CD83-Ig fusion protein. The fusion protein (45 kDa) whose surface expression is largely restricted to DCs was found to bind peripheral blood monocytes and a subset of including Langerhans cells (human), circulating DCs, and inter- CD8 lymphocytes, suggesting that CD83 mediates adhesion to digitating DCs within T cell zones of secondary lymphoid tissues, circulating monocytes and some T cell subsets via interaction of is a member of the Ig superfamily (IgSf) (2– 4). CD83 expression CD83 with a sialated 72-kDa ligand (10). This led to the classiﬁ- has also been found on some germinal center B cells in vivo and cation of CD83 as a sialic acid-binding Ig-like lectin. More on T cells upon Ag or TCR-based activation (2, 5). In addition, recently, though, CD83 appears to be a central component for CD83 expression has been documented in the mammalian brain by lineage development of cell-mediated immunity, because CD83- Northern blotting, but the source of this expression is not known deﬁcient mice display a dramatic decrease (90%) in peripheral (2, 6). CD83 transcription is largely controlled by SP1 and NF-B CD4 populations (11, 12). The deﬁcit in naive single-positive elements within the CD83 promoter, which is in agreement with the CD4 cells is likely due to the loss of CD83 expression by thymic up-regulation of CD83 upon infection or TNF-, IL-1, and mitogen epithelium and DCs, which are involved in the coordination of T cell lineage commitment. Structurally, CD83 is composed of a single extracellular V-set *Department of Microbiology and Immunology, University of Maryland School of IgSf domain, a hydrophobic transmembrane domain, and a cyto- Medicine, and Molecular and Cellular Biology Program, University of Maryland plasmic tail. In humans, the CD83 gene spans roughly 19 kb and Graduate School, Baltimore, MD 21201; Department of Research, University of is composed of ﬁve exons including two exons that encode the Ig Basel, Basel, Switzerland; School of Biological Sciences, Washington State Univer- sity, Vancouver, WA 98686; Scottish Fish Immunology Research Centre, Zoology V-set domain. The gene maps to murine and human chromosomes Building, Aberdeen University, Aberdeen, Scotland, United Kingdom; and Depart- 13 and 6, respectively (3, 6, 13), and the human CD83 gene ﬂanks ment of Pathobiology, University of Washington, Seattle, WA 98195 the human MHC. Our laboratories are interested in the evolution- Received for publication October 17, 2003. Accepted for publication July 22, 2004. ary events that have shaped the adaptive immune response. So far, The costs of publication of this article were defrayed in part by the payment of page only a handful of potential CD markers have been characterized charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. from all ﬁsh including CD3 (14), CD8 (15), CD9 (16), CD18 (17), CD45 (18 –20), and CD81 (20, 21). In this report, we describe the This work was supported in part by National Institutes of Health Grant AI 27877 (to M.F.F.) and U.S. Department of Agriculture-National Research Initiative Competitive isolation and initial characterization of CD83 homologs from ﬁsh. Grants Program 2002-03472 and National Science Foundation 0324069 (to J.D.H.). The sequences presented in this article has been submitted to GenBank under ac- cession numbers AY183667, AY263793-797, AY437982-983, and AY650049-050. 3 Materials and Methods Address correspondence and reprint requests to Dr. John D. Hansen, Department of Pathobiology, University of Washington, Seattle, WA 98195. E-mail address: cDNA cloning of ﬁsh CD83 [email protected] A nurse shark PBL ZAPII cDNA library (Stratagene, La Jolla, CA) was Abbreviations used in this paper: DC, dendritic cell; IgSf, Ig superfamily; UTR, used as the template for a degenerate PCR approach for amplifying unique untranslated region; EST, expressed sequence tag; ORF, open reading frame; Gici, V-set genes. Three different degenerate primers (reverse/complementary Ginglymostoma cirratum; Onmy, Oncorhynchus mykiss; gDNA, genomic DNA; for D/S/N-X-G-X-Y-X-C; the successful primer corresponded to (A/ BAC, bacterial artiﬁcial chromosome; IHNV, infectious hemopoietic necrosis virus; G)CA-III-RTA-III-NCC-III-RTC) corresponding to the F strand of IgSf IRF, IFN regulatory factor; CHO, Chinese hamster ovary; ISRE, IFN-stimulated reg- ulatory element. V-set domains were used in conjunction with an anchored T3 primer Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 4554 HOMOLOGS OF CD83 FROM ELASMOBRANCH AND TELEOST FISH (within the ZAP vector) for the ampliﬁcation of potential V domains. Prim- was hybridized with a radiolabeled cDNA probe corresponding to the ers were used in the following PCR proﬁle for ampliﬁcation: 94°C for 1 Onmy-CD83 V region. Blots were washed at a ﬁnal stringency of 0.4 min followed by 35 cycles of 94°C for 20 s, 43°C for 25 s (0.1°C increase SSC/0.4% SDS at 68°C and then exposed to ﬁlm for 2 days at 80°C. per cycle), and 72°C for 30 s. The PCR samples were then extended for an Linkage analysis for Gici-CD83 additional 10 min at 72°C to facilitate TA-based cloning (pTOPO; BD Clontech, Palo Alto, CA). Aliquots of the ampliﬁed cDNA library were The isolation of gDNA from 39 nurse shark pups (multiple paternity) has diluted in 5 mM Tris (pH 8.0) and denatured at 98°C for 10 min before been previously described (23). Five micrograms of gDNA was digested to usage as a template to release the cDNA templates from the phagemids. completion with EcoRI, which produced a useful restriction fragment poly- Restriction digests were performed, and 60 random inserts in the proper morphism using the Gici-CD83 probe. The PCR-generated probe corre- size range (250 – 400 bp) were sequenced via automated sequencing (Ap- sponded to the entire ORF for Gici-CD83 (primers, exon 2F.21455 and plied Biosystems 310; Applied Biosystems, Foster City, CA). One partic- exon 5R.21701; see above for details) and procedures for Southern blotting ular clone showed strong similarity to V-set domains and was used as the under high-stringency conditions with randomly primed probes have been basis for nested 3 anchored PCR to amplify the full-length gene from the described previously (24). Segregation patterns for Gici-CD83 were then library. Nested PCR was performed using two gene-speciﬁc primers cor- compared with previously identiﬁed patterns for the nurse shark MHC (23). responding to the putative 5-untranslated region (UTR) (forward, 5-CAT GATGCTTTAAACGTG-3) and ATG codon (forward 2, 5-AACGTGT Onmy-CD83 in situ hybridization and karyotyping TCGAAAGGATGATG-3) with the anchored T7 primer corresponding to the 3 region of all inserts within the library. Full-length cDNAs were The V region of trout CD83 was used as a probe to screen high-density ampliﬁed and cloned into pTOPO and sequenced in both directions (Ap- ﬁlters corresponding to 4.5 times coverage of a bacterial artiﬁcial chromo- plied Biosystems 377). some (BAC) genomic library from the OSU-142 all-female clonal line of Rainbow trout CD83 was obtained via a tBlastN search of the trout trout. DNA was isolated from positive BAC clones using the Qiagen (Va- expressed sequence tags (ESTs) within GenBank using sequence informa- lencia, CA) columns. Both PCR and direct sequencing were used to con- tion from the Ginglymostoma cirratum (Gici)-CD83 open reading frame ﬁrm positive BAC clones harboring Onmy-CD83. Peripheral blood was (ORF). The EST clone was completely sequenced and found to lack an cultured from the Donaldson strain of rainbow trout using standard meth- initiation codon. The 5 end of Oncorhynchus mykiss (Onmy)-CD83 was ods (25). This strain has the same chromosome number as the OSU clonal ampliﬁed from a directional PBL cDNA library (UniZap; Stratagene) using line (n 30). Onmy-CD83 BAC DNA was labeled with Spectrum Orange a vector (T3) and an Onmy-CD83 gene-speciﬁc primer (CD83-E3R1; 5- (Vysis, Downers Grove, IL) using nick translation kit (Vysis). Human pla- CCAGGAGACACTTGTACCTT-3) in anchored PCR. Products were cental DNA (0.2 g) and Cot-1 DNA (1 g; prepared from rainbow trout cloned and sequenced to derive a consensus 5 ATG primer. Primers gDNA) were added to the probe mixture for blocking. Hybridizations were (Onmy-CD83.ATG.F, 5-CATTGCTGTAGTTCTACAAATATG-3; and conducted at 37°C overnight and posthybridization washes were as rec- Onmy-CD83-STOP.R, 5-TTCCTCTTTATGTTCAAGTATAC-3) were ommended by the manufacturer (Vysis) with minor modiﬁcations (26). then used to amplify full-length clones of CD83 cDNA from splenic cDNA Abs to Spectrum Orange (Molecular Probes, Eugene, OR) were used to derived from the homozygous strains of trout, OSU-142, HotCreek, and amplify the signal in some cases. Slides were counterstained with 4,6- Arlee (22). Naive spleens from the OSU-142, HotCreek, and Arlee clonal diamidino-2-phenylindole at a concentration of 125 ng of 4,6-diamidino- lines were generously provided by Dr. G. Thorgaard (Washington State 2-phenylindole in 1 ml of antifade solution. Images were captured with a University). Products were cloned (pTOPO) and sequenced. Sensys camera (Photometrics, Tucson, AZ) and analyzed with Cytovision The following accession numbers were used for assembling the ﬂoun- Genus (Applied Imaging, San Jose, CA) software. Chromosomes were der, zebraﬁsh, chicken, rat, and bovine CD83-like sequences: ﬂounder, classiﬁed using a combination of relative size, chromosome arm ratios, and AU091120; zebraﬁsh, BI671547/BM157226; chicken, BI384417/BU457418/ the intensity of 4,6-diamidino-2-phenylindole bands at the centromeres BU452135/BM440129/AI981465; rat, XM_225224; and bovine, BE755445/ (27). It should be mentioned that the Onmy-CD83 BAC contained two BI539602. ESTs were assembled using AssemblyLine, version 1.0.9, within Onmy-CD83 genes as determined by direct sequencing. MacVector (Accelrys, San Diego, CA). The V domains (amino acid se- Northern blot hybridization and RT-PCR quences) from the various ESTs and full-length clones were used for phylo- genetic analysis as previously described (15). The human CD83 pseudogene RNA isolation and Northern blotting protocols have been previously de- was found using the human CD83 protein (NM_004233) in conjunction with scribed (24, 28). Brieﬂy, 15 g of total RNA was electrophoresed under the BLAT server (http://genome.ucsc.edu/cgi-bin/hgBlat). alkaline conditions and transferred to Nytran (Fisher Scientiﬁc, Atlanta, GA) using 20 standard saline citrate phosphate/EDTA. Two different Genomic structure probes were ampliﬁed from the pGici-CD83 plasmid to conﬁrm the ex- Long-range PCR (Elongase; Invitrogen Life Technologies, Carlsbad, CA) pression pattern for Gici-CD83, one corresponding to the V domain exon was used to deduce the exon/intron structure for Gici-CD83. PCR condi- 2F.21455 and exon 3R.21699 (see genomic structure for details) and the tions consisted of the following proﬁle: 2 min at 95°C followed by 35 other to the cytoplasmic domain plus the 3-UTR (exon 5F.21800, 5- cycles of 94°C for 30 s, 57°C for 30 s, and 68°C for either 3, 6, or 9 min GTACAAGCAACCCAATGTTC-3 and 3-UTR-R.21801, 5-GTTTAT depending on the primer pairs. Products were then cloned into pBLUNT TTGAAATCACAAGCT-3). Ampliﬁed fragments were puriﬁed and then (Invitrogen Life Technologies) for verifying the sequence and to determine randomly primed (Invitrogen Life Technologies) with [ P]dCTP (Amer- the exon/intron splice site and phase. Intron size estimates were based upon sham Biosciences, Piscataway, NJ). Nonincorporated nucleotides were re- standard 1% agarose gel electrophoresis of PCR products minus exon con- moved using G-50 spin columns (Invitrogen Life Technologies) before tributions. The following primers were used: exon 1 to exon 2: exon hybridization. The Gici-MHC class IIA and NDPK probes have been pre- 1F.21789, 5-ATGTTTCACCTTAAGAAATGT-3, and exon 2R.21790, viously described. For Onmy-CD83 Northern blotting, 12 g of total RNA 5-TTCTCCACACTTTACTGTAAC-3; exon 2 to exon 3: exon 2F.21455, was electrophoresed, transferred to nylon (Nytran), and hybridized over- 5-TCCGAAGTTACAGTAAAGTGT-3, and exon 3R.21700, 5-TTGA night using an Onmy-CD83 V domain probe (VaVb). The blot was washed ACACTGATACTTTCCAA-3, plus nested exon 3R.21699, 5-ACATTGA at high stringency and exposed for 6 days whereupon the ﬁlter was stripped ACACTGATACTTTC-3; exon 3 to exon 4: exon 3F.21457, 5-GACTT and reprobed sequentially with trout MHC class IIA (exon 2, 28-h expo- TGGAAAGTATCAGTGT-3, and exon 4R.21788, 5-TCAGAACAACT sure) and then with EfTu-1 (12-h exposure). The protocols for acute in- GGTAAGATAG-3; and exon 4 to exon 5, exon 4F.21787, 5-CTAT fectious hemopoietic necrosis virus (IHNV) infection have been described CTTACCAGTTGTTCTGA-3, and exon 5R.21701, 5-GAACATTGGG previously (29). Brieﬂy, trout weighing 200 g were infected by injection TTGCTTGTACT-3. (i.p.) with 1 10 PFU of IHNV, and tissues were harvested on speciﬁed Primers within the A (Onmy-CD83-E2F1.5-GTCAGTTTGTGGAGA days. Controls consisted of saline-injected ﬁsh. GGATTC-3)andF(Onmy-CD83-E3R1.5-CCAGGAGACACTTGTAC Promoter and reporter assays CTT-3) strand of the trout CD83 V domain were used in PCR to determine whether the Onmy-CD83 V domain is encoded by a single or multiple The promoter region for Onmy-CD83 was obtained using the promoter exon(s) using OSU-142 genomic DNA (gDNA) as the template. Products trapping kit (Genome Walking; Bio S&T, Montreal, Quebec, Canada) in were cloned (pTOPO2.1) and sequenced to identify splice site location and conjunction with speciﬁc reverse primers found within exon 1 (exon 1R, phase. 5-CATTGCACGGAGGCAGCTAG-3)of Onmy-CD83. Two different promoters were ampliﬁed from the OSU-142 Onmy-CD83 BAC clone that Southern blotting for Onmy-CD83 was used for physical mapping. Products were cloned into pBlunt (Invitro- For Southern blotting, 15 g of rainbow trout gDNA was digested, elec- gen Life Technologies), sequenced, and assessed for potential transcription trophoresed, and transferred to nylon as previously described (15). The blot factor binding sites using the MatInspector Professional software suite Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 The Journal of Immunology 4555 (www.genomatix.de/index.html). The following settings were used: core An amino acid alignment (Fig. 1A) was generated to display similarity, 0.85; and matrix similarity, 0.90, against the Transfac database features that have been conserved during vertebrate evolution for (version 7.4). Four different versions of the Onmy-CD83 promoter were CD83. The leaders for all sequences are predicted to be 19 –20 ampliﬁed from the longest promoter clone (956 bp) by PCR and then aa via pSort (http://.psort.nibb.ac.jp/). The N-terminal cleavage site cloned into XhoI/BglII sites of pGL3-Basic. All constructs were sequence (TK) for the Gici-CD83 leader was conﬁrmed using N-terminal veriﬁed before transfection. The four different Onmy-CD83-pGL3 (A–D) constructs were cotransfected (0.2 g) with the pRNL (Renilla coreporter) amino sequencing of Gici-CD83-Ig fusion proteins (J. D. Hansen, to normalize transfection efﬁciencies (i.e., pGL3-OmCD83-full plus unpublished data). The Gici-CD83 mature ORF displays conser- pRNL). Deletion constructs were ampliﬁed by PCR from the 956-bp vation of critical residues found within CD83, and overall, the cloned promoter using forward 1/reverse 1 (Om-CD83-full), forward 2/re- sequences display 20%. Both trout and ﬂounder share 37% sim- verse 1 (Om-CD83-2), forward 3/reverse 1 (Om-CD83-3), and forward 1/reverse 2 (Om-CD83-4), digested, and cloned into the XhoI/HindIII-cut ilarity with Gici-CD83 and 63% similarity (45% identity) between pGLL3. Forward 1 (5-GAGAGCTCGAGGAAGCATCGTTACCCATCG themselves for the entire ORF. The cysteine residues in strands B CGCCACAA-3), forward 2 (5-GAGAGCTCGAGACTAATAAACTGC and F that forms a disulﬁde bond within most Ig domains, as well TAAATAAAGTATTGAGT-3), forward 3 (5-GAGAGCTCGAGTTCA as the invariant tryptophan in the C strand, are present in CD83. ATGAGATCACATTAACACCTCAT-3), reverse 1 (5-GAGAGAAGC One unique feature shared by all CD83 sequences is the presence TTTTGTAGAACTACAGCAATGAAGATGAT-3) and reverse 2 (5- GAGAGAAGCTTTGCTTCGTCACAGTGGGTGTAGATGGA-3) XhoI of a cysteine residue (also found in CD90) within the A strand of and HindIII restriction sites are underlined. Plasmids were cotransfected the IgSf V domain. However, Gici-CD83 does not possess the (1.2 g: 0.6 g of pGL3 constructs and 0.6 g of pcDNA3.1 Onmy-IFN conserved cysteine residue between the G strand and the trans- regulatory factor (IRF)-1 and/or IRF-2) with Lipofectin (Invitrogen Life membrane region. The relevance of this additional cysteine residue Technologies) using standard protocols for Chinese hamster ovary (CHO) cell transfection in 24-well format. Cells were passively lysed (Promega, is not known, but mammalian CD83 is monomeric, and thus, the Madison, WI), and luciferase activity (ﬁreﬂy and Renilla) was measured additional cysteine residue does not play a role in dimerization. (MicroLumat; Berthold Technologies, Oak Ridge, TN) using the dual-lu- Immunoprecipitation of human and murine CD83 under both ciferase reporter system (Stop-n-Glow; Promega). Two wells for each reducing and nonreducing conditions demonstrated that CD83 pro- transfection situation were read in triplicate (n 6 per sample) and were teins are heavily glycosylated (broad 45-kDa band), because the consistent between two independent experiments. Values are represented as fold induction over luciferase activity for the complete CD83 promoter predicted molecular masses for the mature proteins are roughly alone (i.e., CD83-full alone set to 1). only 20 kDa (3). The relative locations of two of the three N-linked glycosylation sites (N-X-S/T) are conserved in evolution, suggest- ing that ﬁsh CD83 proteins are also heavily glycosylated. The third Results and Discussion mammalian N-glycosylation site in the G strand is absent in ﬁsh. Cloning and sequence analysis of Gici- and Onmy-CD83 Conserved O-linked glycosylation sites (XPXX, glycosylated if We used a degenerate PCR primer strategy that incorporates fea- X S or T) were not observed within extracellular domain for any tures of the V domain F strand in conjunction with a unidirectional of the CD83 sequences. The transmembrane and cytoplasmic do- cDNA library for the ampliﬁcation of new V domain sequences mains display 17– 41% amino acid identity among the various from the nurse shark Gici. Sixty random products ﬁtting the proper CD83-like sequences and teleost, rat, and avian cytoplasmic do- size range for V domains plus 5-UTR (300 – 450 bp) were se- mains are much longer than those of the other vertebrates. The quenced and subjected to BlastX/N searches of the National Center trout, ﬂounder, and chicken cytoplasmic domains had to be aligned for Biotechnology Information vertebrate database. One fragment separately from the other sequences due to their low sequence (413 bp) showed strong similarity to both mouse and human CD83 identity. A role for the cytoplasmic domain has yet to be reported (BlastX 7e-04/2e-03) and to Ig L chains (1e-02) using the entire for CD83, but it has been shown that the conserved serine and vertebrate database. Because this fragment likely corresponded to threonine residues in mice and humans are not phosphorylated (3). the 5 end of the putative Gici-CD83 cDNA, we used 3 anchored Neither ITIM nor ITAM motifs are found in any of the sequences, PCR to amplify the full-length sequence from the nurse shark although several tyrosine residues are found especially within the cDNA library. Gici-CD83 (accession no. AY183667) contains 86 chicken cytoplasmic domain. Finally, cysteine residues present in bp of 5-UTR followed by single ORF coding for 194 aa and a the ﬁsh CD83 (also in rat and chicken) cytoplasmic domains war- short 3-UTR (137 bp) that includes a consensus polyadenylation sig- rant further investigation. Cysteine residues were also found in the nal and tail. BlastX analysis of the ORF again showed strongest sim- human CD83 pseudogene (6p22:27636584 –27637508)) upon ilarity to both human and mouse CD83 (8e-08/7e-07, respectively) BLAT (http://genome.ucsc.edu/cgi-bin/hgBlat) inspection. followed by V domains for Ig chains (7e-04) and TCR (1e-03). Phylogenetic analysis of the CD83 V domains Gici-CD83 was then used a template for tBlastN analysis of the public domain databases resulting in potential matches for trout, To compare the ﬁsh CD83 V domains with other members of this ﬂounder, zebraﬁsh, bovine, and chicken CD83 homologs. The family present as cell surface proteins on blood cells, we con- trout clone was completely sequenced and used to design primers ducted a phylogenetic analysis of CD83, CD7, CD8, CD28, CD83, for the ampliﬁcation of full-length versions of Onmy-CD83 from and CD152. As shown in the V region neighbor-joining tree (Fig. splenic cDNA from OSU-142 (AY263793), Arlee (AY263794/ 2), all of the CD83 sequences cluster as one group as supported by 795), and Hotcreek (AY263796/797) clonal trout. Two different bootstrap analysis. In addition, the ﬁsh and higher vertebrates form cDNAs were ampliﬁed from Arlee and Hotcreek and one from their own separate clusters within the CD83 branch, with the OSU-142, indicating that Onmy-CD83 is not a single-copy gene, chicken sequence found between the two groups, proving that because these sequences were derived from homozygous ﬁsh. A these sequences represent homologs. second OSU-142 CD83 gene (partial gDNA sequence) was ob- Genomic structure tained by PCR ampliﬁcation using an OSU-142 CD83 BAC clone as the template. The majority of amino acid differences were We used long-range PCR to determine the exon/intron organiza- focused in the leader and within the A, B, and C strands for the V tion for Gici-CD83 and found that Gici-CD83 possesses the same domain (Fig. 1B). Differing from Gici-CD83, the trout ORF is exon/intron pattern as mammalian CD83. The Gici-CD83 gene is larger (218 aa) but still possesses essential features that are shared composed of ﬁve exons and spans roughly 18 kbp from the ATG between the vertebrate CD83 genes. initiation codon through the polyadenylation site (Fig. 3). Exon 1 Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 4556 HOMOLOGS OF CD83 FROM ELASMOBRANCH AND TELEOST FISH FIGURE 1. CD83 protein alignment. Amino acid sequences were aligned using ClustalX and by manual adjustment. A, Alignment of CD83 genes from various ver- tebrate species. Solid vertical lines represent the borders between the leader, extracellular domain, transmembrane region, and the cy- toplasmic domain. Presumed strands (A–G) are indicated with horizontal lines above the amino acid translation. The loca- tions of exon/intron boundaries are denoted by underlining adjacent amino acids (i.e., GL in the nurse shark). Conserved cysteine res- idues involved in the V domain structure are shaded, and the location of conserved N-linked glycosylation sites are boxed. Iden- tical () and similar (:) amino acids are also shown within the alignment. B, Amino acid alignment of Onmy-CD83 from three differ- ent homozygous trout. Two different Onmy- CD83 genes were found in the Arlee and Hotcreek clonal lines. A second partial (2P) OSU-142 CD83 gene was obtained from the OSU-142 CD83 BAC clone (AY437982). encodes the 5-UTR and the ﬁrst 13 aa of the leader; exons 2 and (exons Va and Vb) and used it as a probe for Southern blot analysis 3 encode the IgSf V domain; exon 4 encodes the remainder of the (Fig. 4). Four to seven strong and two to four weak bands can be V domain (last 10 residues) and the transmembrane domain; and seen for each individual (outbred) and digest (EcoRV and HindIII). exon 5 encodes the cytoplasmic domain and 3-UTR. In all cases, Computer-based restriction analysis of sequenced genomic clones splice junctions correlated with consensus 5 and 3 donor and for the V region, indicated the presence of one HindIII and one acceptor splice sites. Overall, the Gici-CD83 genomic structure is EcoRV site within this region for Onmy-CD83. Thus, our Southern nearly identical with that of mammals including conservation of blot analysis is in agreement with Fig. 1B in which two different phase for the four intron splice sites. Finally, we also determined Onmy-CD83 genes were found in the Arlee and Hotcreek homozy- that the Onmy-CD83 V region is composed of two exons, but the gous, clonal rainbow trout. Furthermore, based upon the presence intron between the Onmy-CD83 Va and Vb exons is much smaller of distinct RFLPs among the different individuals, it appears that (150 or 763 bp, phase 0) than that for Gici or murine CD83. polymorphic variants for Onmy-CD83 may exist. Probably the most interesting and conﬁrmatory aspect from pre- MHC linkage analysis vious genomic analysis of CD83 is that the V domain gene struc- ture is composed of two split exons (phase 0 splice site), Va and In mammals, amphibians, and birds, MHC class I, II, and III re- Vb, with the splice location occurring in the C strand of the V gions are closely linked. However, in all teleost ﬁsh, the MHC domain (3, 6). To date, only a few other genes aside from CD83 class I and II regions are not linked and reside on different chro- have this particular genomic architecture including members of the mosomes (31). Recently, the genomic architecture for the elasmo- greater cortical thymocyte marker in Xenopus (CTX) family (30) branch MHC was examined. In two different shark species, class I, (CTX, CTH, A33, and one JAM member), all of which are poten- II, and III genes were shown to be linked, strongly suggesting that tial adhesion and receptor molecules involved in the cellular im- the primordial organization of the MHC was similar to that found mune response. for all other vertebrates, and the lack of linkage for the bony ﬁsh MHC is a derived characteristic (23, 32, 33). Southern blot analysis of Onmy-CD83 The human CD83 gene maps near the human MHC (6p23, te- Based upon the genomic organization of the Onmy-CD83 V do- lomeric of myelin oligodendrocyte glycoprotein) (6, 13). In mice, main, we ampliﬁed a cDNA probe corresponding to the V region this association is not found, because CD83 maps outside of the Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 The Journal of Immunology 4557 FIGURE 3. The genomic structure for human and nurse shark CD83 is evolutionarily conserved. Exons are boxed (exon 1 encodes the leader, exons 2 and 3 code for IgSf V domain, exon 4 encodes the transmembrane domain, and exon 5 codes for the cytoplasmic domain and 3-UTR). In- tronic distances (in kilobase pairs) are found between the exons with the phase of the given intron in parentheses. The partial genomic structure for Onmy-CD83 is also shown (based upon AY437982 and AY437983). highest levels within the brain, epigonal tissue, gills, intestine, PBLs, spleen, and testis (Fig. 7A). A nurse shark MHC class IIA probe was then used as a comparative marker for the expression FIGURE 2. Fish CD83 proteins are homologous to mouse and human analysis, because some DC lineages express high levels of MHC CD83. Phylogenetic tree of IgSf V domains from proteins with similar class II, consistent with their role in Ag presentation. As expected, structures as CD83. An amino acid alignment (ClustalX) of the V domains nurse shark MHC class IIA also displayed strongest expression was used for the generation of an unrooted neighbor-joining tree. Values at within the gills, intestine, PBLs, and spleen. Transient transfection the tree nodes represent bootstrap values from 1000 replicants. of N terminus Flag-tagged Gici-CD83 and subsequent FACS anal- ysis, demonstrated surface expression on CHO cells (data not shown), thus supporting that Gici-CD83 is a type-1 transmembrane MHC on chromosome 13 (6, 13). To determine whether the Gici- protein. We then examined the expression of Onmy-CD83 in se- CD83 gene is linked to the shark MHC, we studied segregation of lected trout tissues. Onmy-CD83 (Fig. 7B) was primarily expressed Gici-CD83 in a nurse shark family of 39 pups. According to RFLP within the spleen and testis by Northern blotting (V domain probe) analysis with a classical class IA probe, pups were sorted into at at much lower levels in comparison to MHC class IIA (DAA). least 13 groups (A–M). These groups represent the combination of maternal and paternal alleles; therefore, a maximum of four groups can be obtained from a single-copy gene. The increased number of groups is due to multiple paternities (at least ﬁve fathers in this family) (23). Because most of the groups contain only a few pups, we used groups of more than three pups (A, D, and H) for the linkage analysis. Furthermore, groups A and D appear to share the same paternal allele p2. In Fig. 5, a 6.6-kb band is absent in the maternal lane; therefore, the 6.6-kb band represents a paternal band. In all cases, only 50% of the pups carry the 6.6-kb band (i.e., 50% recombinant between two paternal chromosomes), strongly suggesting that Gici-CD83 is not linked to the shark MHC. Recently, the rainbow trout chromosomes and linkage groups harboring the trout MHC have been determined (34). Interestingly, a duplication event has led to the development of two class I re- gions in trout on chromosomes 14 and 18, whereas a compact class II region containing both DAA and DAB was found on chromo- some 17. In addition, ABCB2 (TAP1), a transporter gene located in mammalian class II region, was found on chromosome 3. In this study, we determined that Onmy-CD83 is found near the centro- mere on the short arm of chromosome 9 (9p) using in situ hybrid- ization (Fig. 6). Therefore, as in sharks and mice, Onmy-CD83 is not linked to the trout MHC, and thus the linkage of the two human CD83 genes (CD83 and the CD83 pseudogene) to the MHC is most likely a derived feature. Tissue-speciﬁc expression of Gici- and Onmy-CD83 We then examined the tissue-speciﬁc expression of Gici-CD83 by FIGURE 4. Onmy-CD83 is not a single-copy gene as assessed by Southern Northern blotting using two probes, one designed for the V domain blotting. gDNA from four different individual trout (outbred) were digested and the other encompassing the cytoplasmic tail and 3-UTR. Both with EcoRV and HindIII, blotted, and hybridized with a cDNA probe corre- probes showed identical results with ubiquitous expression and sponding to the Onmy-CD83 IgSf V domain (exons Va and Vb). Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 4558 HOMOLOGS OF CD83 FROM ELASMOBRANCH AND TELEOST FISH FIGURE 5. The nurse shark CD83 gene is not linked to the MHC. M, Maternal RFLP pattern. Individual pups (numbered) corresponding to MHC segregating groups H, D, and A are shown. Recombinants are noted by the presence or absence of the 6.6-kbp band. Expression of Onmy-CD83 was also found in the thymus, prone- phros (bone marrow equivalent), and mesonephros. RT-PCR dem- onstrated that all tissues examined were positive for Onmy-CD83, including skin (data not shown). Finally, we determined that two Onmy-CD83 genes are up-regulated during acute IHNV (a ﬁsh rhabdovirus) infection in the spleen, pronephros, and intestine at day 6 postinfection (Fig. 7C). Mammalian CD83 gene expression is largely controlled by SP1 and NF-B(7–9), with no apparent involvement of IRFs. Onmy-CD83 up-regulation occurred as early as 24 h postinfection and remained induced up to 192 h postin- fection (data not shown). Recently, we showed that STAT-1 and members of the class I pathway are also up-regulated during acute IHNV infection (29). In that study, STAT-1, ABCB2 (TAP1), and PSMB9A (LMP2) were clearly induced by 24 h postinfection, im- FIGURE 7. Tissue-speciﬁc mRNA expression for Gici- and Onmy- plicating the involvement of type-I IFNs. CD83. A, Northern blot hybridization of nurse shark tissues (Br, brain; Ep, In mice, CD83 tissue mRNA expression was found in the heart, epigonal; Gi, gills; Hr, heart; Int, intestine; Kd, kidney; Lv, liver; Pa, pan- brain (high), spleen (high), lung, and muscle, and weakly within creas; PBLs; Sp, spleen; Ts, testis; and Thy, thymus) with the Gici-CD83 the kidney, but similar to Gici-CD83 and Onmy-CD83, prolonged probe (V domain) demonstrates that a single transcript for Gici-CD83 exposure or RT-PCR demonstrated expression within all tissues (900 bp) is expressed at highest levels within the brain, epigonal tissue, examined (6). In humans, CD83 mRNA expression was also de- gills, intestine, PBLs, spleen, and testis (6-day exposure). Strong signals for Gici-MHC class IIA transcription were found in gills, intestine, and spleen (3-day exposure). The Gici-NPDK housekeeping probe was used to show relative equivalency in loading (overnight exposure). B, Onmy-CD83 is expressed mainly in trout lymphoid tissues (9-mo-old trout), including the thymus, pronephros (bone marrow equivalent), and spleen (7-day expo- sure) at lower levels in comparison to MHC class IIA (36-h exposure). Thy, Thymus; PN, pronephros; MN, mesonephros; Sp, spleen; Lv, liver; Int, Intestine; Ms, muscle; Hr, heart; Ts, testis. EfTu-1 was used to show relative equivalency in loading (overnight exposure). C, Onmy-CD83 mRNA expression is up-regulated in the spleen, pronephros, and intestine during acute IHNV infection (day 6 postinfection). Two CD83 messages are induced upon infection. CD83 blots were exposed for 5 days, and EfTu-1 served as a control for loading (overnight exposure). tected in the brain, lungs, and mitogen-activated B lymphocytes, and within some T cell populations. Flow cytometric analysis has shown that CD83 expression is limited to cells of hemopoietic lineages, especially to cells of DC lineages (3, 35). Interestingly, CD83 expression has also been associated with a rather unique murine cell type, one that possesses both B cell CD markers and DC morphology and function, because they were found to be po- tent allo-APCs in MLRs (36). Thus, it will be interesting to see whether a similar cell type can be found in ﬁsh. Because DCs have a diverse tissue distribution, the above results (Fig. 6) are consis- tent with CD83 expression by DC lineages in the nurse shark. Recently, the primary and secondary lymphoid tissues of the nurse shark have been examined (37). Based upon expression of key FIGURE 6. Fish CD83 is not linked to the MHC. Onmy-CD83 resides markers including RAG1 and TdT, the epigonal tissue and thymus on the short arm of rainbow trout chromosome 9 as visualized by in situ were found to be the primary lymphoid tissues throughout the life hybridization using CD83 BAC clones as probes. The trout MHC class I and II regions (boxed) are found on chromosomes 3, 14, 17, and 18 (34). of the nurse shark. Interestingly, the nurse shark spleen strongly Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 The Journal of Immunology 4559 resembles the mammalian splenic architecture in possessing dis- tinct/organized T and B cell zones and the presence of MHC class II DC networks. In salmonids, MHC class IIA and -B mRNA and protein expression are mainly limited to lymphoid tissues including the thymus, kidney, spleen, and intestine (28, 38). Additionally, At- lantic salmon class IIB protein expression was primarily localized to cells of the white pulp with a few positive cells (likely melanomac- rophages) in the red pulp. This suggests that salmonid splenic APCs are found in the same location as that for sharks and mammals. Promoter analysis of Onmy-CD83 In this study, we found that Onmy-CD83 is up-regulated during acute rhabdoviral infection similar to that for the trout MHC class I pathway (29). Based on this result, we cloned the putative promoter region for Onmy-CD83 to assess the presence of transcription factor motifs that may be involved in regulating Onmy-CD83. Two different sequences were obtained (AY650049 and AY650050, 98% identity) from the OSU-142 clonal line cor- responding to the 5-ﬂanking region of Onmy-CD83. Upon MatInspector analysis, transcription factor binding motifs known to be implicated in the regulation of immune relevant genes were found, including NF-B, IRF-7, STAT6, PU.1, IFN-stimulated regulatory element (ISRE), in addition to a consensus TATA-box (Fig. 8A). The location of the TATA-box is 28 bp upstream of the initiation site of Onmy-CD83 as judged by 5-RACE analysis, which conforms to consensus location for TATA-boxes. Peak promoter activity for the human CD83 promoter (7) was conﬁned to a 261 fragment relative to the ATG containing four SP1 and a single NF-B element. Putative SP1 sites were not found in the Onmy-CD83 promoter, but interestingly, a consensus NF-B site (100) was found in the same overall location as that for the human CD83 promoter (123) relative to the ATG. To initially characterize the Onmy-CD83 promoter, four different versions (Fig. 8B)ofthe Onmy-CD83 promoter were cloned into the pGL3- Basic luciferase reporter vector to assess their activity in CHO cells upon cotransfection with trout IRF-1 and/or IRF-2 (39) expression constructs. Upon single transfection of the four Onmy-CD83 pGL3 luciferase constructs, only pOm-CD83-4 (minus NF-B/ISRE/TATA) showed slightly diminished activity in comparison to the pOm-CD83.1-3 pGL3 constructs alone, sug- gesting that this region (103 to 3) may be involved in basal transcription. Poly(I:C) treatment of CHO cells transfected with the various pGL3-OmCD83 constructs did not result in enhanced FIGURE 8. Deletion analysis of the Onmy-CD83 promoter. A,5- luciferase activity (data not shown), suggesting that hamster- Flanking region for Onmy-CD83. Numbers on the left and right borders are derived IRFs and NF-B do not physically interact with sites relative to the inferred ATG for Onmy-CD83. Putative transcription factor found within the Omny-CD83 promoter. In direct contrast, binding sites are in bold with the name of site above. Numbers in paren- enhanced luciferase activity (6- to 7-fold induction) was found for theses reﬂect MatInstpector Professional values for the matrix and core, pGL3-OmCD83-1-3 constructs cotransfected with the IRF-1 where 1 is a perfect match. The potential interaction site for Onmy IRF-1 expression construct but not IRF-2 (Fig. 8C). The enhanced is indicated with asterisks underneath. The TATA-box is underlined. B, luciferase activity was not found upon cotransfection of pGL3- Schematic depiction of the four Onmy-CD83 pGL3 reporter constructs. Om-CD83-4 with IRF-1, indicating that trout IRF-1 interacts Om-CD83-4 represents a deletion from 102 to 4, thereby deleting the with a site located between 103 to 3 relative to the Onmy-C second NF-B and IRF potential binding sites as well as the TATA-box. C, D83 ATG. A true consensus IRF-1 site (SAAAGYGAAAC; Luciferase activity of various reporter constructs for the trout CD83 pro- moter transfected alone or cotransfected with Omny-IRF-1 and/or Onmy- www.gene-regulation.com/) is not found in the 103 to 3 IRF-2 expression constructs in CHO cells. Samples indicated as “none” are region, but a TTTCNNTT motif (83) found within the putative single transfectants. All transfections were performed in the presence of ISRE site (85) is identical with the IRF-1 and -2 physical pRL (Renilla luciferase) for the normalization of transfection efﬁciencies. recognition site as determined by crystallography (40, 41). This Samples were lysed and ﬁreﬂy (pGL3) and Renilla (pRL) luciferase ac- implies a direct interaction of trout IRF-1 with the TTTCACTT tivities were using the Stop-n-Glow substrate system. Fireﬂy luciferase motif. Cotransfection of IRF-1 and IRF-2 together with the pGL3- values (reporter constructs) are presented as fold induction: averaged sam- OmCD83 constructs resulted in lower activities in comparison to ple value over the averaged value of Om-CD83-full alone (i.e., Om-CD83- IRF-1 alone, suggesting that IRF-2 likely competes for the IRF-1 full “none”). The data are shown from a representative experiment reported binding site as is found in higher vertebrates (41, 42). as the mean (n 6) SEM. Considering the presence of a consensus NF-B site at position 100, and the interaction of trout IRFs with the Onmy-CD83 Downloaded from http://journals.aai.org/jimmunol/article-pdf/173/7/4553/1188498/4553.pdf by guest on 29 April 2024 4560 HOMOLOGS OF CD83 FROM ELASMOBRANCH AND TELEOST FISH 17. Qian, Y., A. J. Ainsworth, and M. Noya. 1999. Identiﬁcation of a 2 (CD18) promoter, our results imply that Onmy-CD83 mRNA expression molecule in a teleost species, Ictalurus punctatus Raﬁnesque. Dev. Comp. Im- is likely controlled by innate mechanisms including NF-B and munol. 23:571. type-I IFNs. Taken together, the expression patterns for Gici-C 18. Nagata, T., T. Suzuki, Y. Ohta, M. F. Flajnik, and M. Kasahara. 2002. The leukocyte common antigen (CD45) of the Paciﬁc hagﬁsh, Eptatretus stoutii: im- D83 and Onmy-83 are consistent with the expression of CD83 plications for the primordial function of CD45. Immunogenetics 54:286. in mammals. 19. Okumura, M., R. J. Matthews, B. Robb, G. W. Litman, P. Bork, and M. L. Thomas. 1996. Comparison of CD45 extracellular domain sequences from Concluding remarks divergent vertebrate species suggests the conservation of three ﬁbronectin type III domains. J. Immunol. 157:1569. We have a used a degenerate primer strategy for the isolation of a 20. Uinuk-Ool, T., W. E. Mayer, A. Sato, R. Dongak, M. D. Cooper, and J. Klein. primordial gene that, based upon our analysis, corresponds to a 2002. Lamprey lymphocyte-like cells express homologs of genes involved in immunologically relevant activities of mammalian lymphocytes. Proc. Natl. CD83 homolog of the nurse shark. We then performed a broad Acad. Sci. USA 99:14356. comparison of CD83 from a variety of vertebrates that suggest that 21. Yoder, J. A., and G. W. Litman. 2000. The zebraﬁsh fth1, slc3a2, men1, pc, fgf3, all CD83 proteins are heavily glycosylated, consistent with the and cycd1 genes deﬁne two regions of conserved synteny between linkage group 7 and human chromosome 11q13. Gene 261:235. identiﬁcation of CD83 as a sialic acid-binding Ig-like lectin pro- 22. Young, W. P., P. A. Wheeler, R. D. Fields, and G. H. Thorgaard. 1996. DNA tein. Finally, expression analysis indicates that CD83 is largely ﬁngerprinting conﬁrms isogenicity of androgenetically derived rainbow trout expressed within immunologically important tissues in ﬁsh, and lines. J. Hered. 87:77. 23. Ohta, Y., E. C. McKinney, M. F. Criscitiello, and M. F. Flajnik. 2002. Protea- that expression is up-regulated during acute viral infection in trout. some, transporter associated with antigen processing, and class I genes in the This represents the ﬁrst isolation of a putative ﬁsh marker for DC nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages. J. Immunol. 168:771. lineages, thus opening the door for studies examining DC biology 24. Bartl, S., M. A. Baish, M. F. Flajnik, and Y. Ohta. 1997. Identiﬁcation of class in elasmobranchs and teleosts, as well as the evolution of adaptive I genes in cartilaginous ﬁsh, the most ancient group of vertebrates displaying an immunity and Ag presentation. adaptive immune response. J. Immunol. 159:6097. 25. Reed, K. M., and R. B. Phillips. 1995. Molecular cytogenetic analysis of the double CMA3 chromosome in lake trout, Salvelinus namaycush. Cytogenet. Cell Acknowledgments Genet. 70:104. We thank Louis Du Pasquier for his comments in regard to the manuscript 26. Phillips, R. B., and K. M. Reed. 2000. Localization of repetitive DNAs to ze- and David Avila (Roche, Basel, Switzerland) for N-terminal sequencing of braﬁsh (Danio rerio) chromosomes by ﬂuorescence in situ hybridization (FISH). Chromosome Res. 8:27. the Gici-CD83-Ig fusion protein. 27. Reed, K. M., M. O. Dorschner, and R. B. Phillips. 1998. Characteristics of two salmonid repetitive DNA families in rainbow trout (Oncorhynchus mykiss). Cy- References togenetics 79:184. 1. 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Homologs of CD83 from Elasmobranch and Teleost Fish

Homologs of CD83 from Elasmobranch and Teleost Fish

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Homologs of CD83 from Elasmobranch and Teleost Fish

Homologs of CD83 from Elasmobranch and Teleost Fish

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