Portal de Periódicos da CAPES

A Newly Identified Member of the Tumor Necrosis Factor Receptor Superfamily with a Wide Tissue Distribution and Involvement in Lymphocyte Activation

1997; Elsevier BV; Volume: 272; Issue: 22 Linguagem: Inglês

10.1074/jbc.272.22.14272

1083-351X

Byoung S. Kwon, K. B. Tan, Jian Ni, Zang H Lee Kwi-Ok-Oh, Kack K. Kim, Young-J. Kim, Sa Wang, Reiner Gentz, Guoliang Yu, Jeremy Harrop, Sally D. Lyn, Carol Silverman, Terence G. Porter, Alem Truneh, Peter R. Young,

interferon and immune responses

The tumor necrosis factor receptor (TNFR) superfamily consists of approximately 10 characterized members of human proteins. We have identified a new member of the TNFR superfamily, TR2, from a search of an expressed sequence tag data base. cDNA cloning and Northern blot hybridization demonstrated multiple mRNA species, of which a 1.7-kilobase form was most abundant. However, TR2 is encoded by a single gene which, maps to chromosome 1p36.22–36.3, in the same region as several other members of the TNFR superfamily. The most abundant TR2 open reading frame encodes a 283-amino acid single transmembrane protein with a 36-residue signal sequence, two perfect and two imperfect TNFR-like cysteine-rich domains, and a short cytoplasmic tail with some similarity to 4–1BB and CD40. TR2 mRNA is expressed in multiple human tissues and cell lines and shows a constitutive and relatively high expression in peripheral blood T cells, B cells, and monocytes. A TR2-Fc fusion protein inhibited a mixed lymphocyte reaction-mediated proliferation suggesting that the receptor and/or its ligand play a role in T cell stimulation. The tumor necrosis factor receptor (TNFR) superfamily consists of approximately 10 characterized members of human proteins. We have identified a new member of the TNFR superfamily, TR2, from a search of an expressed sequence tag data base. cDNA cloning and Northern blot hybridization demonstrated multiple mRNA species, of which a 1.7-kilobase form was most abundant. However, TR2 is encoded by a single gene which, maps to chromosome 1p36.22–36.3, in the same region as several other members of the TNFR superfamily. The most abundant TR2 open reading frame encodes a 283-amino acid single transmembrane protein with a 36-residue signal sequence, two perfect and two imperfect TNFR-like cysteine-rich domains, and a short cytoplasmic tail with some similarity to 4–1BB and CD40. TR2 mRNA is expressed in multiple human tissues and cell lines and shows a constitutive and relatively high expression in peripheral blood T cells, B cells, and monocytes. A TR2-Fc fusion protein inhibited a mixed lymphocyte reaction-mediated proliferation suggesting that the receptor and/or its ligand play a role in T cell stimulation. The members of the tumor necrosis factor receptor (TNFR) 1The abbreviations used are: TNFR, tumor necrosis factor receptor; NGFR, nerve growth factor receptor; CHO, Chinese hamster ovary; DAPI, 4,6-diamidino-2-phenylindole; EST, expressed sequence tag; FISH, fluorescein in situ hybridization; MLR, mixed lymphocyte reaction; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; PMA, phorbol myristic acetate; RACE, rapid amplication of cDNA ends; mAb, monoclonal antibody; HVEM, herpesvirus entry mediator./nerve growth factor receptor (NGFR) superfamily are characterized by the presence of three to six repeats of a cysteine-rich motif that consists of approximately 30–40 amino acids in the extracellular part of the molecule (1Mallett S. Barclay A.N. Immunol. Today. 1991; 12: 220-224Abstract Full Text PDF PubMed Scopus (170) Google Scholar). The crystal structure of TNFR-I complexed with its ligand showed that a cysteine-rich motif (TNFR domain) was composed of three elongated strands of residues held together by a twisted ladder of disulfide bonds (2Banner D.W. D'Arcy A. Jones W. Gentz R. Schoenfeld H.-J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (984) Google Scholar). These receptors contain a hinge-like region immediately adjacent to the transmembrane domain, characterized by a lack of cysteine residues and a high proportion of serine, threonine, and proline, which are likely to be glycosylated with O-linked sugars. A cytoplasmic part of these molecules shows limited sequence similarities, a finding that may be the basis for diverse cellular signaling. At present, the members identified from human cells include CD40 (3Banchereau J. Bazan F. Blanchard D. Briere F. Galizzi J.P. van Kooten C. Liu Y.-J. Rousset F. Saeland S. Annu. Rev. Immunol. 1994; 12: 881-922Crossref PubMed Google Scholar, 4Stamenkovic I. Clark E.A. Seed B. EMBO J. 1989; 8: 1403-1410Crossref PubMed Scopus (492) Google Scholar), 4–1BB (5Kwon B.S. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1963-1967Crossref PubMed Scopus (325) Google Scholar), OX-40 (6Mallett S. Fossum S. Barclay A.N. EMBO J. 1990; 9: 1063-1068Crossref PubMed Scopus (328) Google Scholar), TNFR-I (7Loetscher H. Pan Y.-C. Lohm H.W. Gentz R. Brockhaus M. Tabuchi H. Lesslauer W. Cell. 1990; 61: 351-359Abstract Full Text PDF PubMed Scopus (770) Google Scholar, 8Schall T.J. Lewis M. Koller K.J. Lee A. Rice G.C. Wong G.H. Gatanaga T. Granger G.A. Lentz R. Raab R. Kohr W.J. Goeddel D.V.. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (846) Google Scholar), TNFR-II (9Smith C.A. Davis T. Anderson D. Solam L. Beckmann M.P. Jerzy R. Dower S.K. Cosman D. Goodwin R.G.. Science. 1990; 248: 1019-1023Crossref PubMed Scopus (852) Google Scholar), CD27 (10Van Lier R.A. Borst J. Vroom T.M. Klein H. Van Mourik P. Zeijlemaker W.P. Melief C.J. J. Immunol. 1987; 139: 1589-1596PubMed Google Scholar), Fas (11Itoh N. Yonehara S. Ishii A. Yonehoro M. Mizushima S.I. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Abstract Full Text PDF PubMed Scopus (2676) Google Scholar), NGFR (12Johnson D. Lanahan A. Buck C.R. Sehgal A. Morgan C. Mercer E. Bothwell M. Chao M. Cell. 1986; 47: 545-554Abstract Full Text PDF PubMed Scopus (745) Google Scholar), CD30 (13Durkop H. Latza U. Hummel M. Eitelbach F. Seed B. Stein H. Cell. 1992; 68: 421-427Abstract Full Text PDF PubMed Scopus (602) Google Scholar), and LTBR (14Baens M. Chaffanet M. Cassiman J.J. van den Berghe H. Marymen P. Genomics. 1993; 16: 214-218Crossref PubMed Scopus (54) Google Scholar). Viral open reading frames encoding soluble TNFRs have also been identified, such as SFV-T2 (9Smith C.A. Davis T. Anderson D. Solam L. Beckmann M.P. Jerzy R. Dower S.K. Cosman D. Goodwin R.G.. Science. 1990; 248: 1019-1023Crossref PubMed Scopus (852) Google Scholar), Va53 (15Smith C.A. Davis T. Anderson D. Solam L. Beckmann M.P. Jerzy R. Dowey S.K. Cosman D. Goodwin R.G. Science. 1990; 248: 1019-1023Crossref PubMed Google Scholar), G4RG (16Howard S.T. Chan Y.S. Smith G.L.. Virology. 1991; 180: 633-647Crossref PubMed Scopus (117) Google Scholar), and crmB (17Hu F.-Q. Smith C.A. Pickup D.J. Virology. 1994; 204: 343-356Crossref PubMed Scopus (162) Google Scholar). Recent studies have shown that these molecules are involved in diverse biological activities such as immunoregulation (18Smith G.L. J. Gen. Viol. 1993; 74: 1725-1740Crossref PubMed Scopus (76) Google Scholar, 19Armitage R.J. Curr. Opin. Immunol. 1994; 6: 407-413Crossref PubMed Scopus (287) Google Scholar), by regulating cell proliferation (20Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 75: 959-962Abstract Full Text PDF Scopus (1838) Google Scholar, 21Banchereau J. de Paoli P. Valle A. Garcia E. Rousset P. Science. 1991; 251: 70-72Crossref PubMed Scopus (531) Google Scholar, 22Pollok K.E. Kim Y.-J. Zhou A. Hurtado J. Kim K.-K. Pickard R.T. Kwon B.S. J. Immunol. 1993; 150: 771-781PubMed Google Scholar), cell survival (23Baum P.R. Gayle III, R.B. Ramsdell F. Srinivasan S. Sorensen R.A. Watson M.L. Seldin M.F. Baker E. Sutherland G.R. Clifford K.N. Alderson M.R. Goodwin R.G. Fanslow W.C. EMBO J. 1994; 13: 3992-4001Crossref PubMed Scopus (262) Google Scholar, 24Grass H.-J. Boiani N. Williams D.E. Armitage R.J. Smith C.A. Goodwin R.G. Blood. 1994; 83: 2045-2056Crossref PubMed Google Scholar, 25Torcia M. Bracci-Laudiero L. Lucibello M. Nencioni L. Labardi D. Rubartelli A. Cozzolino F. Aloi L. Garaci E. Cell. 1996; 85: 345-356Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), and cell death (26Tartaglia L.A. Ayres T.M. Wong G.H. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1169) Google Scholar, 27Gillette-Ferguson I. Sidman C.L. Eur. J. Immunol. 1994; 24: 1181-1185Crossref PubMed Scopus (79) Google Scholar, 28Krammer P.H. Behrmann I. Daniel P. Dhein J. Debatin K.M. Curr. Opin. Immunol. 1994; 6: 279-289Crossref PubMed Scopus (317) Google Scholar). Because of their biological significance and the diverse membership of this superfamily, we predicted that there would be further members of the superfamily. By searching an EST data base, we identified a new member of the TNFR superfamily. We report here the initial characterization of the molecule called TR2. An EST cDNA data base, obtained from over 500 different cDNA libraries (29Adams M.D. Kelley J.M. Gocayne J.D. Dubnick M. Polymeropoulos M.H. Xiao H. Meril C.R. Wu A. Olde B. Moreno R.F. Science. 1991; 252: 1651-1656Crossref PubMed Scopus (1861) Google Scholar, 30Adams M.D. Dubnick M. Kerlavage A.R. Moreno R.F. Kelley J.M. Utterback T.R. Nagle J.W. Fields C. Venter J.C. Nature. 1992; 355: 632-634Crossref PubMed Scopus (726) Google Scholar), was screened for sequence homology with cysteine-rich motif of the TNFR superfamily, using the blastn and tblastn algorithms (31Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70762) Google Scholar). One EST was identified in a human T cell line library, which showed significant sequence identity to TNFR-II at the amino acid level. This sequence was used to clone the missing 5′ end by RACE (rapid amplification of cDNA ends) using a 5′-RACE ends-ready cDNA from human leukocytes (Clontech, Palo Alto, CA). This sequence matched three further ESTs (HTOBH42, HTOAU65, and HLHA49). Complete sequencing of these and other cDNAs indicated that they contained an identical open reading frame homologous to the TNFR superfamily, and it was named TR2. Analysis of several other ESTs and cDNAs indicated that some cDNAs had additional sequences inserted into the open reading frame identified above and might represent various partially spliced mRNAs. The myeloid and B cell lines studied represent cell types at different stages of the differentiation pathway. KG1a and PLB 985 (32Koeffler H.P. Billing R. Lusis A.J. Sparkes R. Golde D.W. Blood. 1980; 56: 265-273Crossref PubMed Google Scholar, 33Tucker K.A. Lilly M.B. Heck L. Rado T.A. Blood. 1987; 70: 372-378Crossref PubMed Google Scholar) were obtained from Phillip Koeffler (UCLA School of Medicine), BJA-B was from Z Jonak (SmithKline Beecham), and TF 274, a stromal cell line exhibiting osteoblastic features, was generated from the bone marrow of a healthy male donor. 2K. B. Tan and Z. Jonak, unpublished data.All of the other cell lines were obtained from the American Type Culture Collection (Rockville, MD). Monocytes were prepared by differential centrifugation of peripheral blood mononuclear cells (PBMC) and adhesion to tissue culture dish. CD19+, CD4+, and CD8+ were isolated from PBMC by immunomagnetic beads (Dynal, Lake Success, NY). Endothelial cells from human coronary artery were purchased from Clonetics (San Diego, CA). Total RNA of adult tissues was purchased from CLONTECH or extracted from primary cells and cell lines with TriReagent (Molecular Research Center, Inc., Cincinnati, OH). 5–7.5 μg of total RNA was fractionated in a 1% agarose gel containing formaldehyde, as described (34Sambrook J. Fritsch E.F. Maniatis J. Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory Cold Spring Harbor, NY. 1989; Google Scholar), and transferred quantitatively to Zeta-Probe nylon membrane (Bio-Rad) by vacuum blotting. The blots were prehybridized, hybridized with 32P-labeled Xho l/Eco RI fragment of TR2 or OX-40 probe, washed under high stringency conditions, and exposed to x-ray films. High molecular weight human DNA was digested with various restriction enzymes and fractionated in 0.8% agarose gel. The DNA was denatured, neutralized, and transferred to nylon membrane and hybridized to32P-labeled TR2 or its variant cDNA. The in situ hybridization and FISH detection of TR2 location in human chromosomes were performed as described previously (35Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar, 36Heng H.H.Q. Xiao H. Shi S.-M. Greenblatt J. Tsui L.-C. Hum. Mol. Genet. 1994; 3: 61-64Crossref PubMed Scopus (33) Google Scholar). FISH signals and the DAPI banding pattern were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with a DAPI-banded chromosome (37Heng H.H.Q. Tsui L.-C. Chromosoma ( Berl .). 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). The 5′ portion of the TR2 containing the entire putative open reading frame of extracellular domain was amplified by polymerase chain reaction (38Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13496) Google Scholar). For correctly oriented cloning, a Hin dIII site on the 5′ end of the forward primer and a Bgl II site on the 5′ end of the reverse primer were created. The Fc portion of human IgG1was PCR-amplified from ARH-77 (ATCC) cell RNA and cloned in theSma I site of the pGem7 vector (Promega, Madison, WI). The Fc fragment, including hinge, CH2, and CH3 domain sequences, contained a Bgl II site at its 5′ end and anXho I site at its 3′ end. TheHin dIII-Bgl II fragment of TR2 cDNA was inserted upstream of human IgG1Fc and an in-frame fusion was confirmed by sequencing. The TR2-Fc fragment was released by digesting the plasmid with Hin dIII-Xho I and cloned into pcDNA3 expression plasmid. The TR2-Fc plasmid, linearized with Pvu I, was transfected into NIH 3T3 by the calcium phosphate co-precipitation method. After selection in 400 μg/ml G418, neomycin-resistant colonies were picked and expanded. Enzyme-linked immunosorbent assay with anti-human IgG1 and Northern analysis with 32P-labeled TR2 probe were used to select for clones that produce high levels of TR2-Fc in the supernatant. In some experiments, a slightly differently engineered TR2-Fc produced in Chinese hamster ovary (CHO) cells was used. The TR2-Fc was purified by protein G chromatography, and the amino acid sequence of the N terminus was determined by automatic peptide sequencer (ABI). The full-length TR2 cDNA was inserted into Hin dIII-Xho I sites of pcDNA 3 vector (Invitrogen, San Diego, CA). TNT-coupled reticulocyte lysate system (Promega) was used to in vitro transcribe and translate the TR2 cDNA in pcDNA 3. The35S-labeled reaction product was fractionated on a 5–15% gradient SDS-polyacrylamide gel, transferred onto an Immobilon membrane (Millipore, Bedford, MA), and exposed to x-ray film. PBMC were isolated from three healthy adult volunteers by Ficoll gradient centrifugation at 400 × g for 30 min. PBMCs were recovered, washed in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 300 μg/ml l-glutamine, and 50 μg/ml gentamycin, and adjusted to 1 × 106 cells/ml for two donors and to 2 × 105 cells/ml for the third donor. Fifty μl of each cell suspension was added to 96-well (round bottom) plates (Falcon, Franklin Lakes, NJ) together with 50 μl of TR2-Fc, IL-5R-Fc, anti-CD4 mAb, or control mAb. Plates were incubated at 37 °C in 5% CO2 for 96 h. One μCi of [3H]methylthymidine (ICN Biomedicals, Costa Mesa, CA) was then added for an additional 16 h. Cells were harvested, and radioactivity was counted. Fig.1 a shows the amino acid sequence of TR2 deduced from the longest open reading frame of one of the isolated cDNAs (HLHA49). Comparison with other sequenced cDNAs and with ESTs in the data base indicated potential allelic variants that resulted in amino acid changes at positions 17 (either Arg or Lys) and 41 (either Ser or Phe) of the protein sequence. The open reading frame encodes 283 amino acids with a calculated molecular weight of 30,417. The TR2 protein was expected to be a receptor. Therefore, the potential signal sequence and transmembrane domain were sought. A hydrophobic stretch of 23 amino acids toward the C terminus (amino acids 203–225) was assigned as a transmembrane domain, because it made a potentially single helical span (Fig.1 a), but the signal sequence was less obvious. The potential ectodomain of TR2 was expressed in NIH 3T3 and CHO cells as a Fc-fusion protein, and the N-terminal amino acid sequence of the recombinant TR2-Fc protein was determined in both cases. The N-terminal sequence of the processed mature TR2 started from amino acid 37, indicating that the first 36 amino acids constituted the signal sequence (Fig.1 a). As shown in Fig. 1 b, the in vitro translation product of TR2 cDNA was 32 kDa in molecular size. Since the first 36 amino acids constituted signal sequence, and its calculated molecular size was ∼4 kDa, the molecular size of the protein backbone of processed TR2 would be approximately 28 kDa. Recently, Montgomeryet al. (39Montgomery R.I. Warner M.S. Lum B.J. Spear P.G. Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (1004) Google Scholar) published a herpesvirus entry mediator (HVEM) whose cDNA sequence was identical to TR2. They found that the transfected HVEM cDNA produced a 32–36-kDa protein. Since it is larger than the in vitro product, this suggests that the protein is modified posttranslationally. Two potential asparagine-linked glycosylation sites are located at amino acid positions 110 and 173, as indicated in Fig. 1 a. Along with the other members of the TNFR family, TR2 contains the characteristic cysteine-rich motifs that have been shown by x-ray crystallography (2Banner D.W. D'Arcy A. Jones W. Gentz R. Schoenfeld H.-J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (984) Google Scholar) to represent a repetitive structural unit. Fig.1 c shows the potential TNFR domain aligned among TR2, TNFR-I, TNFR-II, CD40, and 4–1BB. TR2 contained two perfect TNFR motifs and two imperfect ones. The TR2 cytoplasmic tail (TR-2 cy) does not contain the death domain seen in the Fas and TNFR-I intracellular domains, and appears to be more related to those of CD40cy and 4–1bbcy. Signals through 4–1BB and CD40 have been shown to be co-stimulatory to T cells and B cells, respectively (40Banchereau J. Rousset F. Nature. 1991; 353: 678-679Crossref PubMed Scopus (175) Google Scholar, 41Hurtado J. Kim Y.-J. Kwon B.S. J. Immunol. 1997; 158: 2600-2609PubMed Google Scholar). A human tissue RNA blot was used to determine tissue distribution of TR2 mRNA expression. TR2 mRNA was detected in several tissues with a relatively high level in the lung, spleen, and thymus, but was not found in the brain, liver, or skeletal muscle (Fig. 2 a). TR2 was also expressed in monocytes, CD19+ B cells, and resting or PMA plus PHA-treated CD4+ or CD8+ T cells. It was only weakly expressed in bone marrow and endothelial cells (Fig.2 b), although expression was observed in the hematopoietic cell line KG1a. For comparison, the tissue distribution of OX-40, another member of the TNFR superfamily, was examined. Unlike TR2, OX-40 was not detected in the tissues examined and was detected only in activated T cells and KG1a. Several cell lines were negative for TR2 expression, including TF274 (bone marrow stromal), MG63, TE85 (osteosarcomas), RL 95–2 (endometrial sarcoma), MCF-7, T-47D (breast cancer cells), BE, HT 29 (colon cancer cells), HTB-11, and IMR-32 (neuroblastomas), although TR2 was found in the rhabdosarcoma HTB-82 (data not shown). Several cell lines were examined for inducible TR2 expression. HL60, U937, and THP1, which belong to the myelomonocytic lineage, all increased TR2 expression in response to the differentiating agents PMA or Me2SO (Fig. 2 c). Increases in expression in response to these agents were also observed in KG1a and Jurkat cells. In contrast, PMA did not induce TR2 expression in MG63, but unexpectedly TNF-α did. In almost all cases, the predominant mRNA was approximately 1.7 kilobases in size, although several higher molecular weight species could be detected in some tissues (Fig. 2 a), and many cDNAs and ESTs that were sequenced contained insertions in the coding region indicative of partial splicing. The abundance of higher molecular weight mRNAs raises the possibility that TR2 may in part be regulated at the level of mRNA maturation. The FISH mapping procedure was applied to localize the TR2 gene to a specific human chromosomal region. The assignment of a hybridization signal to the short arm of chromosome 1 was obtained with the aid of DAP I banding. A total of 10 mitotic figures were photographed, one of which is shown in Fig.3 a. The double fluorescent signals are indicated on the schematic diagram of chromosome 1 as shown in Fig.3 b. This result indicated that the TR2 gene is located on the chromosome 1 region p36.2-p36.3. The TR2 position is in close proximity with CD30 (42Smith C.A. Gruss H.-J. Davis-Smith T. Anderson D. Farrah T. Baker E. Sutherland G.R. Brannan C.I. Copeland N.G. Jenkins N.A. Grabstein K.H. Gliniak B. McAlister I.B. Fanslow W. Alderson M. Falk B. Gimpel S. Gillis S. Din W.S. Goodwin R.G. Armitage R.J. Cell. 1993; 73: 1360-1439Abstract Full Text PDF Scopus (513) Google Scholar), 4–1BB (43Kwon B.S. Kozak C.A. Kim K.K. Pickard R.T. J. Immunol. 1994; 152: 2256-2262PubMed Google Scholar, 44Goodwin R.G. Din W.S. Davis-Smith T. Anderson D.M. Gimpel S.D. Sato T.A. Maliszewski C.R. Brannan C.L. Copeland N.G. Jenkins N.A. Farrah T. Armitage R.J. Fanslow W.C. Smith C.A. Eur. J. Immunol. 1993; 23: 2631-2641Crossref PubMed Scopus (286) Google Scholar), OX-40 (45Birkeland M.L. Copeland N.G. Gilbert D.J. Jenkins N.A. Barclay A.N. Eur. J. Immunol. 1995; 25: 926-930Crossref PubMed Scopus (26) Google Scholar), and TNFR-II (46Baker E. Chen L.Z. Smith C.A. Callen D.F. Goodwin R.G. Sutherland G.R. Cytogenet. Cell Genet. 1991; 57: 117-118Crossref PubMed Scopus (44) Google Scholar), suggesting that it evolved through a localized gene duplication event. Interestingly, all of these receptors have stimulatory phenotypes in T cells in response to cognate ligand binding, in contrast to Fas and TNFR-I, which stimulate apoptosis. This prompted us to test if TR2 might be involved in lymphocyte stimulation. To determine the potential involvement of cell surface TR2 with its ligand in lymphocyte proliferation, we examined allogeneic MLR proliferative responses. As shown in Fig. 4, a andb, when TR2-Fc was added to the culture, a significant reduction of maximal responses was observed (p < 0.05). The addition of TR2-Fc at 100 μg/ml inhibited the proliferation up to 53%. No significant inhibition of proliferation was observed with the control IL-5R-Fc. Surprisingly, at high concentrations (10–100 μg/ml) IL-5R-Fc was shown to enhance proliferation. The concentrations of TR2-Fc required to inhibit MLR proliferation (1–100 μg/ml) are comparable with those of CD40-Fc required for inhibition in other lymphocyte assays (47Grammer A.C. Bergman M.C. Miura V. Fujita K. Davis L.S. Lipsky P.E. J. Immunol. 1995; 154: 4996-5010PubMed Google Scholar, 48Fanslow W.C. Anderson D.M. Grabstein K.H. Clark E.A. Cosman D. Armitage R.J. J. Immunol. 1992; 149: 655-660PubMed Google Scholar, 49Grabstein K.H. Maliszewski C.R. Shanebeck K. Sato T.A. Spriggs M.K. Fanslow W.C. Armitage R.J. J. Immunol. 1993; 150: 3141-3147PubMed Google Scholar, 50Noelle R.J. Roy M. Shepherd D.M. Stamenkovic I. Ledbetter J.A. Aruffo A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6550-6554Crossref PubMed Scopus (795) Google Scholar). An anti-CD4 mAb assayed simultaneously inhibited MLR-mediated proliferation up to 60%, whereas a control anti-IL-5 mAb failed to inhibit the proliferation. It is well known that a major component of the MLR proliferative response is T cell-dependent; hence, it would appear that inhibiting the interaction of TR2 with its ligand prevents optimal T lymphocyte activation and proliferation. Hence, we have identified an additional member of the TNFR superfamily that either plays a direct role in T cell stimulation or binds to a ligand which can stimulate T cell proliferation through one or more receptors, which may include TR2. We are currently trying to identify this ligand to which TR2 binds to clarify its role.