Ox40

Ox40 Dass S. Vinay1 and Byoung S. Kwon2,3,* 1

Department of General Surgery, University of Michigan Medical School, 1516 MSRB I, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA 2 The Immunomodulation Research Center, University of Ulsan, Ulsan, Korea 3 Department of Ophthalmology, LSUMC, 2020 Gravier Street Suite B, New Orleans, LA 70112, USA * corresponding author tel: 504-412-1200 ex 1379, fax: 504-412-1315, e-mail: [email protected] DOI: 10.1006/rwcy.2000.16014.

SUMMARY Ox40 is a member of TNF receptor superfamily and is found on activated T cells (Watts and DeBenedette, 1999) but there is a difference in their expression patterns on T cell subsets. While in vitro studies showed the expression peaks at 48 hours following activation on both CD4+ and CD8+ T cells, the vivo experiments, on the other hand, demonstrate a selective expression on CD4+ T cells upon immunizing with adjuvant or under specific clinical conditions (Weinberg, 1998; Weinberg et al., 1998; Higgins et al., 1999). Ox40 is an activation-dependent molecule with a broad tissue distribution, and has been localized on activated T cells (Baum et al., 1994), B cells (Calderhead et al., 1993), dendritic cells (Ohshima et al., 1997), and vascular endothelial cells (Imura et al., 1996). Data available indicate that both Ox40±Ox40L interactions and antiOx40±anti-CD3 combinations provide costimulation to T cells, resulting in elevated cytokine secretion (Weinberg et al., 1998). The authenticity of these findings was further substantiated by the findings that in Ox40-deficient mice the T helper responses were greatly diminished, while the B cell and CTL responses remain unaffected (Kopf et al., 1999). Also, studies with Ox40±Ig fusion protein demonstrate decreased T cell responses under the conditions tested (Weinberg et al., 1999). Ox40 has also been shown to play an important role in various disease conditions (Kaleeba et al., 1998). Detailed functional outcomes of Ox40-mediated immune reactions are beginning to be appreciated. Reports suggest that ligation of Ox40 promotes the differentiation of naõÈ ve CD4+ T cells into TH2 cells,

producing IL-4 in vitro (Ohshima et al., 1998; Flynn et al., 1998) indicating that Ox40 may be involved in the development of TH1 and TH2 in vivo. Interestingly, Ox40 expression on CD4+ T cells and Ox40L expression on CD11c+ dendritic cells (DC) in popliteal lymph nodes of Leishmania major-infected mice has been noted. This indicates a crucial role of Ox40±Ox40L interaction in the development of TH2 cells in vivo, possibly through T cell±DC interaction in the draining lymph nodes (Akibaa et al., 2000). In addition, Ox40-positive T cells have been noted in vivo in several inflammatory states, including experimental autoimmune encephalomyelitis, rheumatoid arthritis, and graft-versus-host disease (Weinberg et al., 1996a, 1996b; Tittle et al., 1997). The clinical importance of Ox40-expressing T cells has also been highlighted in experimental autoimmune encephalomyelitis in which disease prevention was achieved by selective depletion with an immunotoxin-conjugated antibody to Ox40 (Weinberg et al., 1996a, 1996b). Besides its role in costimulation of CD4 cells, evolving results point out that Ox40/Ox40L interactions are closely involved in effector functions as well. For example, Ox40L crosslinking has been reported to support B cell stimulation and antibody production (Stuber et al., 1995) and elevated dendritic cell effector functions (Ohshima et al., 1997) and interfering with this association can lead to the inhibition of both primary and secondary IgG responses (Stuber and Strober, 1996). Also, the Ox40L-associated B lymphocyte and dendritic cell activation appear to depend on the strength of CD40 signaling, thereby leading to the assumption that Ox40/Ox40L association may initially

1750 Dass S. Vinay and Byoung S. Kwon appear during later phase responses after engagement of one or more other ligand±receptor pairs (Gramaglia et al., 1998). Members of TNF family are known to bring about intricate signaling mechanisms in interacting cells. However, the signal transductory pathways traversed by Ox40 in regulating T cell-dependent immune regulation are still at an infant stage. The few available data suggest that Ox40, like some of the other TNFR superfamily members, is closely linked to the TRAF family of signaling molecules, especially TRAF2, TRAF3, and TRAF5, resulting in NFB activation (Arch and Thompson, 1998; Kawamata et al., 1998).

BACKGROUND

Discovery The Ox40 molecule was originally described as a cell surface antigen found on activated rat T cells (Paterson et al., 1987).

Alternative names The human homolog of Ox40 is called ACT35 antigen.

Structure Northern analysis showed that the Ox40 cDNA reacted with a single mRNA of 1.4 kb present in both Con A-activated thymocytes and lymph node cells. The predicted molecular weight of the monomeric protein, based on the amino acid sequence alone, is  49,000.

GENE

Accession numbers Murine Ox40: X85214 Human Ox40: X75962

Sequence The cDNA of 1.2 kb contains a translational site, ATC, surrounded by residues consistent with the consensus sequence found around initiator ATGs. This is followed by an open reading frame (ORF) coding for 271 amino acids. The 30 noncoding

sequence contains a typical polyadenylation site 33 residues from a poly(A) tail of more than 80 residues (see Mallet et al., 1990).

PROTEIN

Accession numbers The sequence data are available from EMBL/ GenBank/DDBJ under accession number X75962 (human Ox40), Z21674 (murine Ox40), X79929 (human Ox40 ligand), and U12763 (murine Ox40 ligand).

Sequence The initiator methionine is followed by a sequence containing a high proportion of hydrophobic residues typical of a signal sequence (Figure 1). The most likely signal cleavage point, predicted on the basis of other signal sequence cleavage sites (von Heijine, 1986), is after residue 19 which could give a mature protein of 252 amino acids. There is a single putative transmembrane domain of 25 predominantly hydrophobic amino acids, dividing the protein into an extracellular domain of 191 amino acids and a cytoplasmic region of 36 amino acids. The extracellular part contains a cysteine-rich region in which 18 out of 140 residues are cysteines and, adjacent to the membrane, a 46 amino acid hinge-like region which includes 25 residues that are threonine, serine, or proline. This latter type of sequence is often glycosylated with O-linked sugars. This seems likely to be the case for Ox40 as the apparent Mr on SDS-PAGE is 47±51 kDa (Paterson et al., 1987) but the Mr predicted for the mature protein from the cDNA sequence is 27,777 and there are only two potential sites for N-linked glycosylation. One of these is NCTP and may not be glycosylated as the sequence NTTP and NCTP in an Ig and the leukocyte common antigen, respectively, have been shown not to be glycosylated (Barclay et al., 1987) presumably due to the juxtaposition of the proline residue and the NXS/T sequence.

Relevant homologies and species differences The murine Ox40 was cloned from mouse TH cell line TH2 D.10. Comparison of the mouse sequence with the rat revealed greater than 90% homology between

Ox40 1751 Figure 1 Homology between the amino acid sequences of human, mouse, and rat species. Identical amino acids are shown in red. (Full colour figure may be viewed online.) 1 1 1

MCVGARRLGRGPCAALLLLGLGLS-TVTGLHCVGDTYPSN MYV----WVQQP-TALLLLGLTLGVTARRLNCVKHTYPSG MYV----WVQQP-TAFLLLGLSLGVTVK-LNCVKDTYPSG

0x40-Human 0x40-Mouse 0x40-Rat

40 36 35

DRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSK HKCCRECQPGHGMVSRCDHTRDTLCHPCETGFYNEAVNYD HKCCRECQPGHGMVSRCDHTRDTVCHPCEPGFYNEAVNYD

0x40-Human 0x40-Mouse Ox40-Rat

80 76 75

PCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGRQPLD-TCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDS TCKQCTQCNHRSGSELKQNTPTTEDTVCQCRPGTQPRQDS

Ox40-Human 0x40-Mouse Ox40-Rat

118 116 115

SYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQP GYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHP SHKLGVDCVPCPPGHFSPGSNQACKPWTNCTLSGKQIRHP

0x40-Human 0x40-Mouse 0x40-Rat

158 156 155

ASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPR ASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPR ASNSLDTVCEDRSLLATLLWETQRTTFRPTTVPSTTVWPR

0x40-Human 0x40-Mouse 0x40-Rat

198 196 195

TSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLAL TSELPSPPTLVTPEGPAFAVLLGLG--LGLLAPLTVLLAL TSQLPSTPTLVAPEGPAFAVILGLG--LGLLAPLTVLLAL

0x40-Human 0x40-Mouse 0x40-Rat

238 234 233

YLLRRDQRLPPDAHKPPGGSFRTPIQEEQADAHSTLAKI YLLRKAWRLP-NTPKPCWGNSFRTPIQEEHTDAHFTLAKYLLRKAWRSP-NTPKPCWGNSFRTPIQEEQTDTHFTLAKI

0x40-Human 0x40-Mouse 0x40-Rat

the two sequences at both the DNA and protein level. Northern analysis found that, as in the rat, Ox40 expression appears to be confined to activated T cells. The most significant difference between rat and mouse Ox40 sequences is the change of the valine to alanine at position 22, insertion of an arginine residue, and the conversion of the neighboring lysine to an arginine at positions 23 and 24 in the proposed mouse sequence. All of the 19 cysteines, including the 18 found in the predicted extracellular domain, are conserved between the rat and mouse. Ox40 has two sites within the putative extracellular domain for Nlinked glycosylation as well as a serine/threonine/ proline-rich region near the proposed transmembrane domain, which may contain sites for O-linked

glycosylation. The intracellular domain possesses two putative protein kinase C phosphorylation sites (S/T-X-K/R), but no other obvious intracellular signaling domain (see Calderhead et al., 1993). A search of the protein databases using the amino acid sequence of the Ox40 shows sequence similarities in the N-terminal cysteine-rich region of both NGFR and CD40. The cytoplasmic regions of the Ox40, NGFR and CD40 proteins are dissimilar in length, spanning 36, 152, and 62 amino acids, respectively. In contrast to CD40 and NGFR, both of which have four repeating sequence homology units that usually contain six cysteine residues, Ox40 contains only three clear repeats as domain 3 is much shorter than the rest.

1752 Dass S. Vinay and Byoung S. Kwon A search of the nucleic acid databases with the ACT35 cDNA sequence revealed a good score with the rat Ox40 (65% identity) and the murine Ox40 (68% identity). However, no conservation exists in the 30 UTR of the rat Ox40 and the ACT35 sequences. The human and rat sequences show an overall identity of 63%. The sequence conservation is more pronounced in the cysteine-rich potential ligand-binding domain than in the intracellular portion, including all 16 conserved cysteine residues. The first putative N-linked glycosylation site in the human sequence corresponds to the most probable N-glycosylation site found in the rat Ox40 antigen. Furthermore, the potential PKC phosphorylation site is conserved in human, rat and mouse. Of the other members of the NGFR/TNFR family, best scores were obtained with the human CD40 (24% identity) and the murine 4-1BB (22% identity) (for details, see Latza et al., 1994).

Cell types and tissues expressing the receptor Murine Ox40 DNA probe recognizes an mRNA species of approximately 1.4 kb that is expressed in splenic T cells as well as thymus and spleen. Ox40 could not be localized in heart, lung, brain, kidney or liver. The level of Ox40 greatly increased after activation with plate-bound anti-CD3 (Calderhead et al., 1993). The expression of Ox40 is restricted to activated T lymphocytes (CD4+ and CD8+ cells).

Regulation of receptor expression The transcription and translation of the Ox40 genes are induced after primary activation of T cells by engagement of the TCR by peptide/MHC complexes and costimulatory signals or by mitogenic stimulation of the cells (Al-Shamkhani et al., 1996).

SIGNAL TRANSDUCTION Utilizing the yeast two-hybrid system with the cytoplasmic domain of Ox40 as baits, TRAF2 and 3 were found to interact with the Ox40 molecule. This was further confirmed by co-immunoprecipitation studies. In contrast to 4-1BB, a single TRAF-binding domain was identified in the cytoplasmic tail of Ox40, leading to the activation of NFB (Arch and Thompson, 1998).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors Signaling through Ox40 has been shown to regulate T cell-dependent immune responses. Human Ox40 has been shown to promote adhesion of T lymphocytes to vascular endothelial cells (Imura et al., 1996). Recent experiments suggest that the expression of Ox40 on T cells specific for myelin basic protein in experimental autoimmune encephalomyelitis (EAE). Experimental data also show that an Ox40 immunotoxin led to specific depletion of these autoreactive lymphocytes and to amelioration of EAE in rats (Weinberg et al., 1996). In addition, OX40-mediated signaling also augments the primary B cell proliferation leading to enhanced humoral responses by binding to its ligand on B cells (Stuber and Strober, 1996).

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ACKNOWLEDGEMENTS SRC funds to IRC from the Korean Ministry of Science and Technology and NIH Grants (AI28125 and DE12156) are greatly appreciated.