Affinity and Kinetics of the Interaction between Soluble Trimeric OX40 Ligand, a Member of the Tumor Necrosis Factor Superfamily, and Its Receptor OX40 on Activated T Cells
1997; Elsevier BV; Volume: 272; Issue: 8 Linguagem: Inglês
10.1074/jbc.272.8.5275
ISSN1083-351X
AutoresAymen Al‐Shamkhani, Susan Mallett, Marion H. Brown, William James, A. Neil Barclay,
Tópico(s)Cell Adhesion Molecules Research
ResumoOX40 ligand (OX40L) and OX40 are members of the tumor necrosis factor and tumor necrosis factor receptor superfamilies, respectively. OX40L is expressed on activated B and T cells and endothelial cell lines, whereas OX40 is expressed on activated T cells. A construct for mouse OX40L was expressed as a soluble protein with domains 3 and 4 of rat CD4 as a tag (sCD4-OX40L). It formed a homotrimer as assessed by chemical cross-linking and gel filtration chromatography. Radiolabeled sCD4-OX40L bound to activated mouse T cells with a high affinity (KD = 0.2-0.4 nM) and dissociated slowly (koff = 4 × 10−5 s−1). The affinity and kinetics of the OX40L/OX40 interactions were studied using the BIAcore™ biosensor, which measures macromolecular interactions in real time. The extracellular part of the OX40 antigen was expressed as a soluble monomeric protein and immobilized on the BIAcore sensor chip. sCD4-OX40L bound the OX40 with a high affinity (KD = 3.8 nM), although this was lower than that determined on the surface of activated T cells (KD = 0.2-0.4 nM), where there is likely to be less restriction in mobility of the receptor. In the reverse orientation, sOX40 bound to immobilized sCD4-OX40L with a stoichiometry of 3.1 receptors to one ligand, with low affinity (KD = 190 nM) and had a relatively fast dissociation rate constant (koff = 2 × 10−2 s−1). Thus if the OX40 receptor is cleaved by proteolysis, it will release any bound ligand and is unlikely to block re-binding of ligand to cell surface OX40 because of the low monomeric affinity. OX40 ligand (OX40L) and OX40 are members of the tumor necrosis factor and tumor necrosis factor receptor superfamilies, respectively. OX40L is expressed on activated B and T cells and endothelial cell lines, whereas OX40 is expressed on activated T cells. A construct for mouse OX40L was expressed as a soluble protein with domains 3 and 4 of rat CD4 as a tag (sCD4-OX40L). It formed a homotrimer as assessed by chemical cross-linking and gel filtration chromatography. Radiolabeled sCD4-OX40L bound to activated mouse T cells with a high affinity (KD = 0.2-0.4 nM) and dissociated slowly (koff = 4 × 10−5 s−1). The affinity and kinetics of the OX40L/OX40 interactions were studied using the BIAcore™ biosensor, which measures macromolecular interactions in real time. The extracellular part of the OX40 antigen was expressed as a soluble monomeric protein and immobilized on the BIAcore sensor chip. sCD4-OX40L bound the OX40 with a high affinity (KD = 3.8 nM), although this was lower than that determined on the surface of activated T cells (KD = 0.2-0.4 nM), where there is likely to be less restriction in mobility of the receptor. In the reverse orientation, sOX40 bound to immobilized sCD4-OX40L with a stoichiometry of 3.1 receptors to one ligand, with low affinity (KD = 190 nM) and had a relatively fast dissociation rate constant (koff = 2 × 10−2 s−1). Thus if the OX40 receptor is cleaved by proteolysis, it will release any bound ligand and is unlikely to block re-binding of ligand to cell surface OX40 because of the low monomeric affinity. INTRODUCTIONThe OX40 antigen was defined in the rat as an antigen with a highly restricted distribution being present only on activated rat CD4+ T lymphocytes and absent from resting lymphocytes and other tissues (1Paterson D.J. Jefferies W.A. Green J.R. Brandon M.R. Corthesy P. Puklavec M. Williams A.F. Mol. Immunol. 1987; 24: 1281-1290Google Scholar). In the mouse OX40 is present on both CD4+- and CD8+-activated T cells (2Calderhead D.M. Buhlmann J.E. Van den Eertwegh A. Claassen E. Noelle R.J. Fell H.P. J. Immunol. 1993; 151: 5261-5271Google Scholar, 3Al-Shamkhani A. Birkeland M.L. Puklavec M. Brown M.H. James W. Barclay A.N. Eur. J. Immunol. 1996; 26: 1695-1699Google Scholar). It is a transmembrane glycoprotein whose extracellular portion contains three cysteine-rich repeats of approximately 40 amino acids (4Mallett S. Fossum S. Barclay A.N. EMBO J. 1990; 9: 1063-1068Google Scholar). Similar repeats are found in the extracellular parts of several other membrane glycoproteins, including the low affinity nerve growth factor receptor, two receptors for tumor necrosis factor (TNFR) 1The abbreviations used are: TNFRtumor necrosis factor receptorTNFtumor necrosis factorOX40LOX40 ligandsOX40soluble OX40CHOChinese hamster ovarymAbmonoclonal antibodyPBSphosphate-buffered salineBSAbovine serum albuminPAGEpolyacrylamide gel electrophoresisRUresponse units. and the leukocyte antigens CD40, CD27, CD30, 4-1BB, and Fas (CD95) that make up the TNFR superfamily (reviewed in Refs. 5Barclay A.N. Beyers A. Birkeland M.L. Brown M.H. Davis S.J. Somoza C. Williams A.F. Leucocyte Antigens Factsbook. Academic Press, London1993Google Scholar and 6Armitage R.J. Curr. Opin. Immunol. 1994; 6: 407-413Google Scholar). The structure of the TNFR I shows that the cysteine-rich repeats form a linear array of small domains, which comprise the binding site for TNF (7Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Google Scholar).The ligands of the TNFR superfamily members, with the exception of nerve growth factor and other neurotrophins, also share sequence similarity (∼15-36%) in what is now known as the TNF superfamily. These proteins are type II membrane proteins, and their similarity is confined to the COOH-terminal extracellular domains, which are often released as soluble proteins by proteolysis (reviewed in Refs. 6Armitage R.J. Curr. Opin. Immunol. 1994; 6: 407-413Google Scholar and 8Gruss H.J. Dower S.K. Blood. 1995; 85: 3378-3404Google Scholar). Structural studies on TNF-α (9Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Google Scholar, 10Jones E.Y. Stuart D.I. Walker N. Nature. 1989; 338: 225-228Google Scholar), TNF-β (7Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Google Scholar), and CD40 ligand (11Karpusas M. Hsu Y.-M. Wang J.-H. Thompson J. Lederman S. Chess L. Thomas D. Structure (Lond.). 1995; 3: 1031-1039Google Scholar) show that they form homotrimers with a characteristic "jelly roll"β-sandwich. The stoichiometry of the interaction between TNFR I and TNF-β trimer is three to one (7Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Google Scholar).The OX40 ligand (OX40L) is expressed on the surface of activated B (2Calderhead D.M. Buhlmann J.E. Van den Eertwegh A. Claassen E. Noelle R.J. Fell H.P. J. Immunol. 1993; 151: 5261-5271Google Scholar) and T (12Baum P.R. Gayle 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: 3992-4001Google Scholar) lymphocytes and has been shown recently to be present on endothelial cell lines (13Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Google Scholar). The OX40L is involved in T cell help for B cells in the development of IgG responses (14Stuber E. Neurath M. Calderhead D. Fell H.P. Strober W. Immunity. 1995; 2: 507-521Google Scholar, 15Stuber E. Strober W. J. Exp. Med. 1996; 183: 979-989Google Scholar). The quaternary organization of OX40L is unknown, although sequence similarity with other members of the TNF superfamily suggests that it is likely to form a homotrimer. However, another member of this superfamily, 4-1BB ligand, forms a disulfide-linked homodimer (16Goodwin R.G. Din W.S. DavisSmith T. Anderson D.M. Gimpel S.D. Sato T.A. Maliszewski C.R. Brannan C.I. Copeland N.G. Jenkins N.A. Farrah T. Armitage R.J. Fanslow W.C. Smith C.A. Eur. J. Immunol. 1993; 23: 2631-2641Google Scholar), indicating that there exists heterogeneity in the quaternary structure among members of this superfamily.A recombinant soluble OX40L-Fc fusion protein binds OX40 on activated T cells (3Al-Shamkhani A. Birkeland M.L. Puklavec M. Brown M.H. James W. Barclay A.N. Eur. J. Immunol. 1996; 26: 1695-1699Google Scholar, 12Baum P.R. Gayle 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: 3992-4001Google Scholar), but the strength of binding could not be quantified, because the effect on the avidity brought about by the dimeric Fc portion of the OX40L-Fc construct could not be estimated. We have expressed a soluble recombinant protein containing the COOH-terminal extracellular domain of OX40L fused to domains 3 and 4 of rat CD4. The CD4 portion of the fusion protein has been used previously as a tag to generate monomeric fusion proteins, including several different domain types (17Brown M.H. Barclay A.N. Protein Eng. 1994; 7: 515-521Google Scholar). Using the soluble CD4-OX40L (sCD4-OX40L) fusion protein, we have studied the affinity and kinetics of the OX40L binding to OX40 using (i) conventional radiolabeled ligand binding studies to activated T cells and (ii) the BIAcore™ biosensor, which detects macromolecular interactions in real time using the phenomenon of surface plasmon resonance (18Johnsson B. Löfås S. Lindquist G. Anal. Biochem. 1991; 198: 268-277Google Scholar, 19Karlsson R. Michaelsson A. Mattsson L. J. Immunol. Methods. 1991; 145: 229-240Google Scholar, 20van der Merwe P.A. Brown M.H. Davis S.J. Barclay A.N. EMBO J. 1993; 12: 4945-4954Google Scholar). We have also investigated the affinity and kinetics of sOX40 binding to immobilized OX40L, as soluble forms of several TNFR superfamily members, including both TNFR I and TNFR II (21Engelmann H. Novick D. Wallach D. J. Biol. Chem. 1990; 265: 1531-1536Google Scholar, 22Porteu F. Nathan C. J. Exp. Med. 1990; 172: 599-607Google Scholar), CD27 (23Hintzen R.Q. De Jong R. Hack C.E. Chamuleau M. De Vries E. Ten Berge I. Borst J. Van Lier R. J. Immunol. 1991; 147: 29-35Google Scholar), and CD30 (24Del Prete G. Maggi E. Pizzolo G. Romagnani S. Immunol. Today. 1995; 16: 76-80Google Scholar) are released from the cell surface by proteolysis, and any functional effects of the soluble receptors will be limited by their monomeric affinities.DISCUSSIONWe have studied the interaction between OX40 and its ligand using soluble recombinant proteins and a combination of biosensor technology and conventional radioligand binding studies. The finding that sCD4-OX40L is a trimer suggests that membrane-bound OX40L and native soluble OX40L, if it exists, will also form trimers. This is consistent with the determined stoichiometry of 3.1:1 for the interaction of sOX40 with immobilized trimeric sCD4-OX40L. The fact that sCD4-OX40L binds to immobilized sOX40 and to OX40 expressed on activated T cells with a much higher apparent affinity than when monomeric sOX40 binds immobilized sCD4-OX40L is also consistent with the existence of a trimeric form of the OX40L. The increase in the overall affinity of the trimeric sCD4-OX40L is primarily due to a decrease (∼500-fold) in the koff. The apparent affinity of the interaction between sCD4-OX40L and OX40 expressed on the surface of activated T cells (KD∼ 0.2-0.4 nM) obtained by either equilibrium binding or kinetic measurements at 4°C is very similar to that of the TNF-α interaction with TNFR I/II (34Loetscher H. Pan Y.-C.E. Lahm H.-W. Gentz R. Brockhaus M. Tabuchi H. Lesslauer W. Cell. 1990; 61: 351-359Google Scholar, 35Smith C.A. Davis T. Anderson D. Solam L. Beckmann M.P. Jerzy R. Dower S.K. Cosman D. Goodwin R.G. Science. 1990; 248: 1019-1023Google Scholar), but is approximately 10-fold higher than that obtained from the BIAcore measurements. In the BIAcore experiments the direct immobilization of sOX40 on the dextran matrix may limit the possible orientations of sOX40 and hence the observed kinetics may represent a dimeric interaction. In contrast, the slower dissociation of sCD4-OX40L from cells is consistent with a trimeric interaction.The soluble monomeric form of the rat OX40 molecule, containing the cysteine-rich domains, binds to immobilized trimeric mouse OX40L (sCD4-OX40L) with low affinity (KD = 190 nM) and dissociates relatively quickly (t1/2 = 35 s). To our knowledge, direct affinity and kinetic measurements of the interaction between soluble forms of other members of the TNFR superfamily and their ligands have not been reported, and thus we do not know whether these results are representative of other members of this superfamily. A soluble form of the TNFR II was estimated to be∼ 1000-fold less effective than the dimeric TNFR II-Fc in inhibiting a functional assay for TNF (36Mohler K.M. Torrance D.S. Smith C.A. Goodwin R.G. Stremler K.E. Fung V.P. Madani H. Widmer M.B. J. Immunol. 1993; 151: 1548-1561Google Scholar). The sTNFR was only about 50-fold less effective in an inhibition binding assay although kinetic analysis was not carried out.The low dissociation rate of the trimer from cell surfaces makes reversal of the interaction very slow and it seems likely that a general mechanism for reversal is the cleavage of the receptor, together with any bound ligand, from the cell surface as observed for several members of the TNFR superfamily (21Engelmann H. Novick D. Wallach D. J. Biol. Chem. 1990; 265: 1531-1536Google Scholar–24Del Prete G. Maggi E. Pizzolo G. Romagnani S. Immunol. Today. 1995; 16: 76-80Google Scholar). If OX40 is also released from the cell surface, our results suggest that sOX40 will not act as an antagonist of OX40L because of its low affinity. It is not known whether OX40L acts as a soluble protein and/or as a cell surface protein but this shedding mechanism would also provide a mechanism of terminating the interaction between membrane-bound OX40L and OX40 on activated T cells. The role of natural soluble forms of the TNFR superfamily members in vivo remains unknown but Mohler et al. (36Mohler K.M. Torrance D.S. Smith C.A. Goodwin R.G. Stremler K.E. Fung V.P. Madani H. Widmer M.B. J. Immunol. 1993; 151: 1548-1561Google Scholar) showed that injection of soluble TNFR II into mice did not protect them from the lethal effect of LPS, whereas a dimeric TNFR II-Fc chimeric protein gave good levels of protection. These results suggest that soluble TNFR II does not function as an antagonist in vivo.In conclusion, our results indicate that the OX40/OX40L complex will have a similar overall structure to that of TNFR I/TNF-β complex (7Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Google Scholar), namely, that three OX40 molecules interact with a single OX40L trimer. However, receptor dimerization rather than trimerization was shown to be sufficient for inducing the biological effects of TNF (37Bazzoni F. Alejos E. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5376-5380Google Scholar, 38Adam D. Keßler U. Kronke M. J. Biol. Chem. 1995; 270: 17482-17487Google Scholar). This will probably apply also to OX40 as cross-linking with the MRC OX40 mAb is known to enhance the proliferation of activated T cells in vitro (1Paterson D.J. Jefferies W.A. Green J.R. Brandon M.R. Corthesy P. Puklavec M. Williams A.F. Mol. Immunol. 1987; 24: 1281-1290Google Scholar). Furthermore, the kinetics of binding and dissociation of sCD4-OX40L are comparable with those of F(ab′)2 fragments of many known mAbs (33Mason D.W. Williams A.F. Weir D.M. Handbook of Experimental Immunology. 1. Blackwell Scientific, Oxford1986: 38.1-38.17Google Scholar), but contrasts with the low affinity of the monomeric receptor binding to ligand. Thus the soluble receptor is unlikely to have functional effects in contrast to the high affinity ligand. INTRODUCTIONThe OX40 antigen was defined in the rat as an antigen with a highly restricted distribution being present only on activated rat CD4+ T lymphocytes and absent from resting lymphocytes and other tissues (1Paterson D.J. Jefferies W.A. Green J.R. Brandon M.R. Corthesy P. Puklavec M. Williams A.F. Mol. Immunol. 1987; 24: 1281-1290Google Scholar). In the mouse OX40 is present on both CD4+- and CD8+-activated T cells (2Calderhead D.M. Buhlmann J.E. Van den Eertwegh A. Claassen E. Noelle R.J. Fell H.P. J. Immunol. 1993; 151: 5261-5271Google Scholar, 3Al-Shamkhani A. Birkeland M.L. Puklavec M. Brown M.H. James W. Barclay A.N. Eur. J. Immunol. 1996; 26: 1695-1699Google Scholar). It is a transmembrane glycoprotein whose extracellular portion contains three cysteine-rich repeats of approximately 40 amino acids (4Mallett S. Fossum S. Barclay A.N. EMBO J. 1990; 9: 1063-1068Google Scholar). Similar repeats are found in the extracellular parts of several other membrane glycoproteins, including the low affinity nerve growth factor receptor, two receptors for tumor necrosis factor (TNFR) 1The abbreviations used are: TNFRtumor necrosis factor receptorTNFtumor necrosis factorOX40LOX40 ligandsOX40soluble OX40CHOChinese hamster ovarymAbmonoclonal antibodyPBSphosphate-buffered salineBSAbovine serum albuminPAGEpolyacrylamide gel electrophoresisRUresponse units. and the leukocyte antigens CD40, CD27, CD30, 4-1BB, and Fas (CD95) that make up the TNFR superfamily (reviewed in Refs. 5Barclay A.N. Beyers A. Birkeland M.L. Brown M.H. Davis S.J. Somoza C. Williams A.F. Leucocyte Antigens Factsbook. Academic Press, London1993Google Scholar and 6Armitage R.J. Curr. Opin. Immunol. 1994; 6: 407-413Google Scholar). The structure of the TNFR I shows that the cysteine-rich repeats form a linear array of small domains, which comprise the binding site for TNF (7Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Google Scholar).The ligands of the TNFR superfamily members, with the exception of nerve growth factor and other neurotrophins, also share sequence similarity (∼15-36%) in what is now known as the TNF superfamily. These proteins are type II membrane proteins, and their similarity is confined to the COOH-terminal extracellular domains, which are often released as soluble proteins by proteolysis (reviewed in Refs. 6Armitage R.J. Curr. Opin. Immunol. 1994; 6: 407-413Google Scholar and 8Gruss H.J. Dower S.K. Blood. 1995; 85: 3378-3404Google Scholar). Structural studies on TNF-α (9Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Google Scholar, 10Jones E.Y. Stuart D.I. Walker N. Nature. 1989; 338: 225-228Google Scholar), TNF-β (7Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Google Scholar), and CD40 ligand (11Karpusas M. Hsu Y.-M. Wang J.-H. Thompson J. Lederman S. Chess L. Thomas D. Structure (Lond.). 1995; 3: 1031-1039Google Scholar) show that they form homotrimers with a characteristic "jelly roll"β-sandwich. The stoichiometry of the interaction between TNFR I and TNF-β trimer is three to one (7Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Google Scholar).The OX40 ligand (OX40L) is expressed on the surface of activated B (2Calderhead D.M. Buhlmann J.E. Van den Eertwegh A. Claassen E. Noelle R.J. Fell H.P. J. Immunol. 1993; 151: 5261-5271Google Scholar) and T (12Baum P.R. Gayle 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: 3992-4001Google Scholar) lymphocytes and has been shown recently to be present on endothelial cell lines (13Imura A. Hori T. Imada K. Ishikawa T. Tanaka Y. Maeda M. Imamura S. Uchiyama T. J. Exp. Med. 1996; 183: 2185-2195Google Scholar). The OX40L is involved in T cell help for B cells in the development of IgG responses (14Stuber E. Neurath M. Calderhead D. Fell H.P. Strober W. Immunity. 1995; 2: 507-521Google Scholar, 15Stuber E. Strober W. J. Exp. Med. 1996; 183: 979-989Google Scholar). The quaternary organization of OX40L is unknown, although sequence similarity with other members of the TNF superfamily suggests that it is likely to form a homotrimer. However, another member of this superfamily, 4-1BB ligand, forms a disulfide-linked homodimer (16Goodwin R.G. Din W.S. DavisSmith T. Anderson D.M. Gimpel S.D. Sato T.A. Maliszewski C.R. Brannan C.I. Copeland N.G. Jenkins N.A. Farrah T. Armitage R.J. Fanslow W.C. Smith C.A. Eur. J. Immunol. 1993; 23: 2631-2641Google Scholar), indicating that there exists heterogeneity in the quaternary structure among members of this superfamily.A recombinant soluble OX40L-Fc fusion protein binds OX40 on activated T cells (3Al-Shamkhani A. Birkeland M.L. Puklavec M. Brown M.H. James W. Barclay A.N. Eur. J. Immunol. 1996; 26: 1695-1699Google Scholar, 12Baum P.R. Gayle 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: 3992-4001Google Scholar), but the strength of binding could not be quantified, because the effect on the avidity brought about by the dimeric Fc portion of the OX40L-Fc construct could not be estimated. We have expressed a soluble recombinant protein containing the COOH-terminal extracellular domain of OX40L fused to domains 3 and 4 of rat CD4. The CD4 portion of the fusion protein has been used previously as a tag to generate monomeric fusion proteins, including several different domain types (17Brown M.H. Barclay A.N. Protein Eng. 1994; 7: 515-521Google Scholar). Using the soluble CD4-OX40L (sCD4-OX40L) fusion protein, we have studied the affinity and kinetics of the OX40L binding to OX40 using (i) conventional radiolabeled ligand binding studies to activated T cells and (ii) the BIAcore™ biosensor, which detects macromolecular interactions in real time using the phenomenon of surface plasmon resonance (18Johnsson B. Löfås S. Lindquist G. Anal. Biochem. 1991; 198: 268-277Google Scholar, 19Karlsson R. Michaelsson A. Mattsson L. J. Immunol. Methods. 1991; 145: 229-240Google Scholar, 20van der Merwe P.A. Brown M.H. Davis S.J. Barclay A.N. EMBO J. 1993; 12: 4945-4954Google Scholar). We have also investigated the affinity and kinetics of sOX40 binding to immobilized OX40L, as soluble forms of several TNFR superfamily members, including both TNFR I and TNFR II (21Engelmann H. Novick D. Wallach D. J. Biol. Chem. 1990; 265: 1531-1536Google Scholar, 22Porteu F. Nathan C. J. Exp. Med. 1990; 172: 599-607Google Scholar), CD27 (23Hintzen R.Q. De Jong R. Hack C.E. Chamuleau M. De Vries E. Ten Berge I. Borst J. Van Lier R. J. Immunol. 1991; 147: 29-35Google Scholar), and CD30 (24Del Prete G. Maggi E. Pizzolo G. Romagnani S. Immunol. Today. 1995; 16: 76-80Google Scholar) are released from the cell surface by proteolysis, and any functional effects of the soluble receptors will be limited by their monomeric affinities.
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