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Acta Biochim Biophys Sin 2005,37:694-701

doi:10.1111/j.1745-7270.2005.00097.x

Cloning, Structural Organization and Chromosomal Mapping of Rat Costimulatory Molecule 4-1BBL

 

Qiu-Ming DONG1, Li-Jie MA2, Guang-Bo ZHANG1, Ya-Fang WU3, Jia-Yao SHEN1, Ying CHEN1, Yong-Jing CHEN1, Xiang-Ke PU1, Sai-Yu HANG1, and Xue-Guang ZHANG1*

 

1 Biotechnology Institute, Soochow University, Suzhou 215007, China;

2 Genetics Institute, Fudan University, Shanghai 200433, China;

3 First Affiliated Hospital of Soochow University, Suzhou 215006, China

 

Received: April 22, 2005

Accepted: July 18, 2005

*Corresponding author: Tel, 86-512-65104909; Fax, 86-512-65104908; E-mail, [email protected]

 

Abstract         4-1BBL (TNFSF9) is a member of the tumor necrosis factor (TNF) ligand superfamily, which is expressed on some activated antigen presenting cells and B cells. We isolated a rat cDNA clone encoding the rat homologue of the human 4-1BBL (GenBank accession No. AY259541). The deduced rat 4-1BBL protein, consisting of 308 amino acids with a molecular weight of 33,469 Da, was a typical type II ­transmembrane glycoprotein, the same as human and murine 4-1BBL. "SDAA" in the cytoplasmic domain of rat 4-1BBL was deduced to act as the phosphorylation site for casein kinase I ("SXXS" motif), which is present in the cytoplasmic domains of human and murine 4-1BBL, and all other TNF ligand family members known to utilize reverse signaling. The two introns of 4-1BBL were also cloned (GenBank accession No. AY332409). Rat 4-1BBL is much more homologous with murine 4-1BBL than with human 4-1BBL, in both nucleotide and amino acid sequences. Rat 4-1BBL was expressed in all tested tissues: brain, lung, colon, liver, thymus, testicle, kidney, adrenal, stomach, spleen and heart. The chromosomal location of rat 4-1BBL was first identified by bioinformatics, then by fluorescence in situ hybridization at 9q11 (GenBank accession UniGene No. Rn.46783). Rat, murine and human 4-1BBL genes are evolved from a common gene. The identification and characterization of the rat counterpart of human 4-1BBL will facilitate studies of the biological­ function of this molecule.

 

Key words        TNF superfamily; rat; 4-1BBL; cloning; mapping

 

Over the past several years, costimulatory molecules belonging to the tumor necrosis factor (TNF) receptor and TNF ligand superfamilies have been identified and ­characterized [1,2]. These include CD27/CD27L, CD30/CD30L (CD153), CD40/CD40L (CD154), OX40 (CD134)/OX40L and 4-1BB (CD137)/4-1BBL. These molecules are type II transmembrane glycoproteins, except for LT-a which is the only entirely secreted tumor necrosis factor-like protein. The members of the TNF ligand superfamily share common biological activities, but some properties are shared by only some ligands, while others are unique. The diverse biological activities triggered through tumor necrosis factor receptor (TNFR) are mainly related to the regulation of cellular activation, including immune ­responses and inflammatory reactions, as well as the ­pathology of a series of human diseases.

The murine 4-1BBL cDNA was isolated from a murine thymoma cell line in 1993 using an expression screening approach [3]. In 1994, Alderson et al. [4] utilized cross-species hybridization to isolate a cDNA encoding human 4-1BB (hu4-1BB). A fusion protein of the extracellular portion of hu4-1BB coupled to the Fc region of human IgG1 (hu4-1BBL.Fc) was then used to identify and clone the gene for human 4-1BBL from an activated CD4+ T cell clone using a direct expression cloning strategy. Human and murine 4-1BBL genes are located on human ­chromosome 19p13 and mouse chromosome 17D, respectively. Human 4-1BBL is expressed primarily on professional antigen presenting cells (APCs), such as ­mature dendritic cells, activated B cells and activated ­macrophages [5,6], and is also expressed inducibly in T lymphocytes [7] and constitutively in monocytes [4]. 4-1BB is expressed on CD4 and CD8 T cell lines [8]. ­Agonistic antibodies and the ligand of 4-1BB enhance ­lymphocyte activation [6,7,9-11], and the 4-1BB protein has an opposite effect in that it inhibits proliferation of activated T lymphocytes and induces programmed cell death [3,6,7,9,10]. This means that a reverse signal is transmissive through the ligand [12].

In exploring the physiological and pathological functions­ of 4-1BBL, the rat system has some advantages over the human and murine systems. Superior models of autoimmune­ diseases, such as collagen-induced arthritis, experimental autoimmune uveoretinitis and experimental allergic encephalomyelitis, have been developed [13,14]. Some models of human diseases, such as the human T-cell lymphotropic virus-1-associated pathologies [15], have only been set up in the rat system.

In order to facilitate the study of 4-1BBL, we cloned rat 4-1BBL using Internet resources and characterized its expression and chromosome mapping in the rat system.

 

 

Materials and Methods

 

Animal and cDNA library

 

Six-week-old male Sprague-Dawley (SD) rats (Rattus norvegicus) were purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). cDNA library of rat fetal brain was kindly provided by Dr. Long Yu (Fudan University, Shanghai, China).

 

Rat 4-1BBL cDNA cloning and sequence analysis

 

Murine 4-1BBL mRNA was used to blast rat expressed sequence tags (ESTs) in GenBank (http://www.ncbi.nlm.nih.gov/blastn). Primers were designed by comparing murine 4-1BBL mRNA and rat ESTs. Polymerase chain reaction (PCR) was performed using a cDNA library of rat fetal brain and PCR product was purified using a PCR product purification kit (Promega, Madison, USA), then ligated to the pMD18-T vector (TaKaRa, Japan). The cDNA insert was sequenced on both strands using ­synthetic oligonucleotide primers and dye-labeled terminator/Taq DNA polymerase on automated fluorescent DNA sequence (Sangon, Shanghai, China). Alignment of amino acid ­sequences and estimation of homology were performed using DNAtool 6.0.122 (http://www.crc.dk/dnatools) and ClustalW (http://www.ebi.ac.uk/clustalw) softwares.

 

Intron cloning

 

Rat genome DNA was extracted from adult rat brain using phenol-chloroform. Primers were designed by ­comparing rat 4-1BBL cDNA with the murine 4-1BBL genome. PCR products were ligated to the pMD18-T vector and were sequenced.

 

Expression of rat 4-1BBL

 

Total RNA was extracted from 11 rat tissues using Trizol (Invitrogen, Carlsbad, USA) and cDNA synthesis was performed using RevertAid M-MuLV reverse transcriptase (Fermentas, Lithuania). Reverse transcription (RT)-PCR was performed with rat 4-1BBL cDNA primers and rat b-actin primers to quantify the relative abundance of 4-1BBL mRNA in each tissue. Primers of rat b-actin are as follows: 5'-TAAAGACCTCTATGCCAACAC-3', 5'-TAAAGCCATGCCAAATGTCTC-3'.

 

Chromosome mapping by UniGene

 

Human chromosome 19 and murine chromosome 17 were used to blast the rat genome in GenBank, then UniGenes were analyzed (http://www.ncbi.nlm.nih.gov/blast/unigene).

 

Fluorescence in situ hybridization

 

Rat G-band metaphase chromosomes were prepared using­ marrow from an SD rat by cell cycle synchronization­ [16]. G-banded metaphases were located and photographed. Giemsa stain was removed with ethanol­ and the slides were treated with DNase-free RNase (100 mg/ml) at 37 ºC for 1 h in 2´SSC. Before hybridization the chromosomes were dehydrated through an ethanol series (70%, 95%, 100%). The 2.0 kb 4-1BBL genomic DNA fragment was generated with cDNA primers and subcloned into pMD18-T vector. DNA probes were labeled­ with ­biotin-14-dATP (Gibco BRL, Carlsbad, USA) by ­nick-translation, then the unincorporated nucleotides were ­removed by ethanol precipitation in the presence of 20 mg glycogen and stored at -20 ºC. In situ hybridization and detection were performed as described previously [17] with a slight modification. In brief, 50-100 ng of biotinylated probe was applied to each slide under sealed glass coverslips. Hybridization buffer contained 1.5 ml probe (50-100 ng), 1.5 ml 100 mg/ml denatured salmon sperm DNA (100 mg/ml) (Sigma, St. Louis, USA), and 12 ml hybridization solution VII (Oncor, Gaithersburg, USA). The chromosome DNA was denatured in 70% formamide in 2´SSC for 3 min, and probe was denatured at 72 ºC for 10 min. Hybridization was performed in a humidified box at 42 ºC for approximately 16 h. Subsequently the slides were washed in 2´SSC at 72 ºC for 5 min, in 1´PBD for 2 min and in 100 ml blocking solution at 37 ºC for 10 min. The biotin-labeled probe DNA was visualized and amplified­ by the fluorescein-isothiocyanate (FITC)-conjugated­ avidin­ and biotinylated anti-avidin system (biotin-FITC detection­ kit; Oncor). Individual chromosomes­ were identified­ by counterstaining with 4,6-diamidino-2-phenylindoledihydrochloride (DAPI; Sigma). Separated images of DAPI-banded chromosomes were obtained and merged electronically using microscope Leica Q550 CW (Leica, Wetzlar, Germany) and image analysis software Leica QFISH V2.1 (Leica).

 

 

Results

 

Cloning of rat 4-1BBL cDNA

 

In order to identify rat 4-1BBL, murine 4-1BBL mRNA was used to blast the rat ESTs in GenBank. Two highly homologous rat ESTs were obtained (GenBank accession No. BF565084 and BF394106) and coincidently the two ESTs properly spanned the murine 4-1BBL coding region. Primers possibly spanning the possible coding region were designed according to the two ESTs. Electrophoresis of the PCR product on 1% agarose gel showed a 1000-bp band as expected. The sequencing result was then linked with the two ESTs, and a 1328-bp fragment of rat 4-1BBL cDNA was obtained. The sequence of the cDNA clone shown in Fig. 1(A) contained a single large open reading frame (ORF) encoding a protein of 308 amino acid residues. Hydrophilicity analysis predicted a single hydrophobic ­domain encompassing AA83 to AA102 and no signal sequence. Thus, like murine and human 4-1BBL, this ­protein was predicted to be a type II membrane protein with its carboxy-terminal domain extracellular. Like ­murine 4-1BBL (containing three potential N-linked glycosylation sites: AA139, AA161, AA293), the extracellular domain of rat 4-1BBL also contains three potential N-linked glycosylation sites: AA138, AA160 and AA292, while ­human 4-1BBL does not. The nucleotides of the possible coding region of rat 4-1BBL has 88% and 37% homology with that of murine and human 4-1BBL, respectively, in which the cytoplasmic domain is 85%, 30%; the transmembrane domain is 81%, 33% and the extracellular ­domain is 89%, 46% homology with murine and human 4-1BBL, respectively. Alignment of the predicted protein product of this rat cDNA with that of murine 4-1BBL and human 4-1BBL is shown in Fig. 1(B). Rat 4-1BBL is more closely related to murine 4-1BBL with 83% homology and to a lesser extent to human 4-1BBL with 31% homology, in which the cytoplasmic domain is 76%, 32%, the transmembrane domain is 45%, 40% and the extracellular ­domain is 87%, 28% homology with murine and human 4-1BBL, respectively. The molecular weight of the ­deduced rat 4-1BBL protein is 33,469 Da, and its isoelectric point is 5.82.

 

Cloning of rat 4-1BBL introns

 

Comparing the rat 4-1BBL cDNA and the murine 4-1BBL genomic gene, the rat 4-1BBL gene was deduced to have two introns just the same as murine 4-1BBL. Sequencing results displayed two sequences of 335 bp and 687 bp, respectively, corresponding to murine intron 1 (389 bp) and intron 2 (754 bp), whereas those of human are 1489 bp and 1783 bp, respectively. Two rat 4-1BBL introns followed the gt-ag rule (Fig. 2). To test the accuracy of the results, PCR was performed with the primers which were used in cloning rat 4-1BBL cDNA and with rat ­genome DNA as the model. PCR results showed a band of about 2000 bp as expected (389+754+1000=2043). The intron nucleotide sequences showed 86% and 85% ­identity in intron 1 and intron 2, respectively, between rat and ­murine 4-1BBL; and 8% (intron 1) and 26% (intron 2) between rat and human. The identity from start codon to stop codon was 86% between rat 4-1BBL (1946 bp) and murine 4-1BBL (2073 bp), and 13% between rat 4-1BBL and ­human 4-1BBL (4027 bp). So rat 4-1BBL is more homologous with murine 4-1BBL than with human 4-1BBL, in both nucleotide and amino acid sequences. Human 4-1BBL ­protein is poorly homologous with rat 4-1BBL protein and murine 4-1BBL protein (both 31%). Their genes are all composed of three exons and two introns, in which the lengths of exon 2 are short: 31 bp (human), 37 bp (rat and murine); and the lengths of exon 3 are similar: 461 bp (rat), 464 bp (human and murine). The exon 2 is 50%-60% idential among the three genes, the same as exon 3. Exon 2 and exon 3 mainly encode the transmembrane and the extracellular domains. Compared with the identities of nucleotide­ sequences of the cytoplasmic domain of the three molecules, which is encoded by exon 1 (34% between human and murine, 22% between human and rat), the ­identities of nucleotide sequences of transmembrane and extracellular domains are higher.

 

mRNA expression of rat 4-1BBL

 

RT-PCR results showed that 4-1BBL mRNA was widely but differently expressed in tested tissues (Fig. 3). The 4-1BBL mRNA level in each tissue, compared with that of rat b-actin mRNA, was analyzed using bandleader 3.0 (http://www.bio-soft.net/draw.html); and the relative ­abundance from high level to low level was as follows: brain, colon, lung, thymus, adrenal, liver, testicle, kidney, stomach, spleen and heart.

 

Rat 4-1BBL chromosomal mapping by bioinformatics

 

Chromosome blast results demonstrated that human chromosome 19 matched rat chromosomes 1, 5, 7, 9, 12 and 16, and murine chromosome 17 matched rat ­chro­mosomes 1, 6, 9, 10 and 20 (Fig. 4). Rat 4-1BBL gene was inferred on chromosome 1 or 9. UniGenes on human chromosome 19, murine chromosome 17, and rat chromosome 1, 9 were analyzed. We found that the UniGene of human 4-1BBL locating­ on human Chr.19:38.82cR is Hs.1524, and the Uni­Gene of murine 4-1BBL ­locating on murine Chr.17:799.15cR is Mm.41171. ­Coincidently another UniGene (Mm.23982) was also found on murine Chr.17:799.15cR, and this UniGene ­corresponded to rat UniGene Rn.14560. Rat UniGene Rn.14560 was the location of rat gene crumbs 3 (CRB3), which is on rat chromosome 9q11cM (Chr.09:0cR) (http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Rn&CID=14560). The chromosome locations of human­ CRB3 and murine CRB3 were searched on the National Center for Biotechnology Information (NCBI) website, and it was found that CRB3 was located on ­human 19p13.3cM (GenBank accession No. NM_174881, UniGene No. Hs.150319) and murine 17D (GenBank accession­ No. NM_177638, UniGene No. Mm.260076), respectively, just the same as 4-1BBL. According to the gene linkage rule, rat 4-1BBL may be on rat 9q11cM, just like rat CRB3. To prove the deduction, the chromosome arrangement of genes CRB3, 4-1BBL, C3 and another gene (in murine called Vav, in human and rat called Vav1) were analyzed, which are all located on rat chromosome 9q11, human chromosome 19p13.3, and murine chromosome­ 17. On human chromosome 19, the nucleotide span number­ of the four genes are: CRB3, 6414788-6418232 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt= Graphics&list_uids=92359); 4-1BBL, 6482037-6486933 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=Graphics&list_uids= 8744); C3, 6671660-6628878 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt= Graphics&list_uids=718); Vav1, 6723722-6808371 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db= gene&cmd=Retrieve&dopt=Graphics&list_uids=7409). The order of their locations is CRB3/4-1BBL/C3/Vav1. Because the murine genome was not obtained on the internet, UniGene was used to analyze the locations of the four genes on murine chromosome 17: Vav, murine Chr.17:803.35cR (UniGene No. Mm.57191) (http://rgd.mcw.edu/tools/vcmap/vcmap.cgi?MapName=Murine+VirtualMap+5.0_HS&Chr=17&FirstMarker=C3&FirstPos=795.08&SecondMarker=1700093E07 Rik&SecondPos=859.3); CRB3, murine Chr.17:799.15cR (UniGene No. Mm.23982); 4-1BBL, murine Chr.17:799.15cR (UniGene No. Mm.41171); C3, murine Chr.17:795.08cR (UniGene number Mm.1913). The order of their locations­ is C3/CRB3(4-1BBL)/Vav. Fig. 4(C) shows that the relation­ of the genes on rat chromosome­ 9 is Vav1/UniGene Rn.14560/C3, and UniGene Rn.14560 is CRB3. The three genes are all on rat 9q11, so rat 4-1BBL is also located on rat 9q11, and its UniGene accession No. is Rn.46783.

 

Rat 4-1BBL chromosomal mapping by fluorescence in situ hybridization

 

The biotin-labeled probe DNA was prepared with rat genome 4-1BBL corresponding to the 2.0 kb PCR product. The signal was amplified by immunofluorescence. ­Positive signals were visualized on both homologous chromosomes only on 9q11 in 90% of the chromosome spreads which were investigated (Fig. 5). This is consistent with the ­result of chromosomal mapping by informatics using UniGenes.

 

Discussion

 

As other TNFR superfamily members, 4-1BB uses adaptor­ molecules, which are called TNFR-associated ­factors (TRAFs), to transduce a downstream signal. The cytoplasmic domain of 4-1BB is able to bind specifically with TRAF1, TRAF2 and TRAF3 [10,18,19]. Accumulating­ evidence indicates that there exists a signal transduction pathway by way of the 4-1BB ligand as well as the 4-1BB. Cross-linking of 4-1BBL with insect cells, which express 4-1BB, induces strong B-cell proliferation synergistically with anti-IgM antibody [5]. Similarly, cross-linking of 4-1BBL by soluble 4-1BB-Fc fusion protein ­induces monocyte­ activation so that the expression of IL-6, IL-8, TNF-a and ICAM is up-regulated, whereas FcgR III expression­ is down-regulated [20]. Signals by way of 4-1BBL also lead to a strong production of macrophage colony-stimulating factor in monocytes, which in turn functions as a potent survival factor for monocytes [21]. In addition, the "reverse signaling" through 4-1BBL ­mediates monocyte proliferation by an autocrine mechanism­ which is still unknown yet [22]. In T lymphocytes, T-cell proliferation induced by anti-CD3 antibody is inhibited by cross-linking of 4-1BBL [12]. Furthermore, this inhibition of T-cell proliferation is ­accompanied by cell apoptosis. The apoptotic signal ­transmitted by 4-1BBL is independent of Fas [23]. Therefore­ 4-1BBL signaling mediates two opposite ­biological phenomena, cell survival and apoptosis, depending­ on cell type. Even though reverse signaling through TNF ligand family members has been well documented, its molecular mechanism remains undiscovered. Interestingly, a phosphorylation site for casein kinase I (SXXS) is present in the cytoplasmic ­domains of all TNF ligand superfamily members, which were known to utilize the reverse signaling, CD30L, 4-1BBL, CD40L and FasL, except OX40 ligand [24]. In transmembrane­ TNF-a, this motif has been the phosphorylation ­target for casein kinase, and treatment of a macrophage­ cell line with dimeric soluble TNFR leads to the dephosphorylation of total cellular transmembrane TNF-a. One biochemical change following the dephosphorylation is an elevation of intracellular calcium levels [24]. This motif appears as "SDAA" in the deduced rat 4-1BBL amino acid sequence, but appears as "SDAS" in human 4-1BBL and as "SDAA" in murine 4-1BBL. The difference is in that "Ser" contains -OH while "Ala" contains­ -H. Ser is a polar amino acid, while Ala is between polar and non-polar, and they are both uncharged. So rat "SDAA" possibly acts as the phosphorylation target for casein kinase. The ­existence of a phosphorylation site for casein kinase I in the ­cytoplasmic domain of the three molecules probably­ makes them function similarly in "reverse signal" transmission. Like murine 4-1BBL, rat 4-1BBL also contains­ additional 55 N-terminal amino acid residues belonging to the ­cytoplasmic domain, which is not found in the human homologue. In co-stimulation signal transmission, ­especially in "reverse signal" transmission, it is unknown what ­difference was caused by the lack of amino acid residues and potential N-linked glycosylation sites in human 4-1BBL. Study on rat 4-1BBL will make its mechanism of function and of reverse signal transmission understood.

A simple method was used here to compare the cloning of murine and human 4-1BBL. The completion of the ­Human Genome Project has made a lot of data available on gene sequences, location, expression, and function analysis, which provides a short cut to clone genes with ESTs and homology analysis. Compared with murine and human 4-1BBL, the rat 4-1BBL was cloned and mapped by in silicon cloning and chromosome mapping, which is also a short cut. Rat 4-1BBL was firstly identified to locate at 9q11 (GenBank accession UniGene No. Rn.46783), the same as the CRB3 gene, by bioinformatics and FISH methods. These two genes also appeared at the same site both in human (19q13) and murine (17D). The gene ­structure of rat, murine and human 4-1BBL are similar. 4-1BBL, CRB3, C3 and Vav1 (Vav) are all lined at rat ­chromosome 9, murine chromosome 17 and human ­chromosome 19. So rat, murine and human 4-1BBL ­probably evolved from a common ancestor gene. The ­identification and characterization of the rat counterpart of human 4-1BBL will facilitate the study of the biological functions of this molecule.

 

 

References

 

 1    Smith CA, Farrah T, Goodwin RG. The TNF receptor superfamily of cellular and viral proteins: Activation, costimulation, and death. Cell 1994, 76: 959-962

 2    Gruss HJ, Dower SK. Tumor necrosis factor ligand superfamily: Involvement­ in the pathology of malignant lymphomas. Blood 1995, 85: 3378-3404

 3    Goodwin RG, Din WS, Davis-Smith T, Anderson DM, Gimpel SD, Sato TA, Maliszewski CR et al. Molecular cloning of a ligand for the inducible T cell gene 4-1BB: A member of an emerging family of cytokines with homology to tumor necrosis factor. Eur J Immunol 1993, 23: 2631-2641

 4    Alderson MR, Smith CA, Tough TW, Davis-Smith T, Armitage RJ, Falk B, Roux E et al. Molecular and biological characterization of human 4-1BB and its ligand. Eur J Immunol 1994, 24: 2219-2227

 5    Pollok KE, Kim YJ, Hurtado J, Zhou Z, Kim KK, Kwon BS. 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B cells. Eur J Immunol 1994, 24: 367-374

 6    DeBenedette MA, Chu NR, Pollok KE, Hurtado J, Wade WF, Kwon BS, Watts TH. Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP. J Exp Med 1995, 181: 985-992

 7    DeBenedette MA, Shahinian A, Mak TW, Watts TH. Costimulation of CD28- T lymphocytes by 4-1BB ligand. J Immunol 1997, 158: 551-559

 8    Pollok KE, Kim YJ, Zhou Z, Hurtado J, Kim KK, Pickard RT, Kwon BS. Inducible T cell antigen 4-1BB. Analysis of expression and function. J Immunol 1993, 150: 771-781

 9    Hurtado JC, Kim SH, Pollok KE, Lee ZH, Kwon BS. Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28. J Immunol 1995, 155: 3360-3367

10   Saoulli K, Lee SY, Cannons JL, Yeh WC, Santana A, Goldstein MD, Bangia N et al. CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand. J Exp Med 1998, 187: 1849-1862

11   Cannons JL, Lau P, Ghumman B, DeBenedette MA, Yagita H, Okumura K, Watts TH. 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J Immunol 2001, 167: 1313-1324

12   Schwarz H, Blanco FJ, von Kempis VJ, Valbracht J, Lotz M. ILA, a member of the human nerve growth factor/tumor necrosis factor receptor family, regulates T-lymphocyte proliferation and survival. Blood 1996, 87: 2839-2845

13   Knoerzer DB, Karr RW, Schwartz BD, Mengle-Gaw LJ. Collagen-induced arthritis in the BB rat. Prevention of disease by treatment with CTLA-4-Ig. J Clin Invest 1995, 96: 987-993

14   Forrester JV, Liversidge J, Dua HS. Regulation of the local immune response­ by retinal cells. Curr Eye Res 1990, 9Suppl: 183-191

15   Ishiguro N, Abe M, Seto K, Sakurai H, Ikeda H, Wakisaka A, Togashi T et al. A rat model of human T lymphocyte virus type I (HTLV-I) infection. 1. Humoral antibody response, provirus integration, and HTLV-I-associated myelopathy/tropical spastic paraparesis-like myelopathy in seronegative HTLV-I carrier rats. J Exp Med 1992, 176: 981-989

16   Satoh H, Yoshida MC, Sasaki M. High resolution chromosome banding in the Norway rat, Rattus norvegicus. Cytogenet Cell Genet 1989, 50: 151-154

17   Garson JA, van den Berghe JA, Kemshead JT. Novel non-isotopic in situ hybridization technique detects small (1 Kb) unique sequences in routinely G-banded human chromosomes: Fine mapping of N-myc and b-NGF genes. Nucleic Acids Res 1987, 15: 4761-4770

18   Arch RH, Thompson CB. 4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor kB. Mol Cell Biol 1998, 18: 558-565

19   Jang IK, Lee ZH, Kim YJ, Kim SH, Kwon BS. Human 4-1BB (CD137) signals are mediated by TRAF2 and activate nuclear factor-kB. Biochem Biophys Res Commun 1998, 242: 613-620

20   Langstein J, Michel J, Fritsche J, Kreutz M, Andreesen R, Schwarz H. CD137 (ILA/4-1BB), a member of the TNF receptor family regulates monocyte­ activation via bidirectional signaling. J Immunol 1998, 160: 2488-2494

21   Langstein J, Schwarz H. Identification of CD137 as a potent monocyte survival factor. J Leukocyte Biol 1999, 65: 829-833

22   Langstein J, Michel J, Schwarz H. CD137 induces proliferation and endomitosis­ in monocyte. Blood 1999, 94: 3161-3168

23   Michel J, Pauly S, Langstein J, Krammer PH, Schwarz H. CD137-induced apoptosis is independent of CD95. Immunology 1999, 98: 42-46

24   Watts AD, Hunt NH, Wanigaskara Y, Bloomfield G, Wallach Roufogalis BD, Chaudhri G. A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in �reverse signaling. EMBO J 1999, 18: 2119-2126