©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Recombinant Soluble Trimeric CD40 Ligand Is Biologically Active (*)

(Received for publication, November 17, 1994; and in revised form, January 26, 1995)

Gonzalo J. Mazzei (§) Michael D. Edgerton Christophe Losberger Sybille Lecoanet-Henchoz Pierre Graber Anne Durandy (1) Jean-Francois Gauchat Alain Bernard Bernard Allet Jean-Yves Bonnefoy (¶)

From the Glaxo Institute for Molecular Biology, 14 Chemin des Aulx, 1228 Plan les Ouates, Geneva, Switzerland and INSERM Unité 132, Hopital Necker-Enfants Malades, 149 Chemin de Sevres, 75743 Paris Cedex 15, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD40 ligand (CD40L) is expressed on the surface of activated CD4 T cells, basophils, and mast cells. Binding of C40L to its receptor, CD40, on the surface of B cells stimulates B cell proliferation, adhesion and differentiation. A preparation of soluble, recombinant CD40L (Tyr-45 to Leu-261), containing the full-length 29-kDa protein and two smaller fragments of 18 and 14 kDa, has been shown to induce differentiation of B cells derived either from normal donors or from patients with X-linked hyper-IgM syndrome (Durandy, A., Schiff, C., Bonnefoy, J.-Y., Forveille, M., Rousset, F., Mazzei, G., Milili, M., and Fischer, A. (1993) Eur. J. Immunol. 23, 2294-2299). We have now purified each of these fragments to homogeneity and show that only the 18-kDa fragment (identified as Glu-108 to Leu-261) is biologically active. When expressed in recombinant form, the 18-kDa protein exhibited full activity in B cell proliferation and differentiation assays, was able to rescue of B cells from apoptosis, and bound soluble CD40. Sucrose gradient sedimentation shows that the 18-kDa protein sediments as an apparent homotrimer, a result consistent with the proposed trimeric structure of CD40L. This demonstrates that a soluble CD40L can stimulate CD40 in a manner indistinguishable from the membrane-bound form of the protein.


INTRODUCTION

CD40 ligand (CD40L, TRAP, or gp39) is a 39-kDa glycoprotein expressed as a type II integral membrane protein on the surface of T cells, basophils, and mast cells(1, 2, 3, 4, 5) . The interaction of CD40L with CD40 in association with different cytokines (for reviews see Refs. 6 and 7) is required for B cell proliferation and for production of immunoglobulins. Mutations or deletions in the CD40L gene cause X-linked hyper-IgM syndrome, HIGM1 (^1)(for review, see (8) ). The majority of the CD40L mutations in HIGM1 patients are located in the extracellular domain of the CD40L, resulting in a failure of the ligand to bind CD40. Purified B cells from patients with X-linked hyper-IgM syndrome respond normally to agonistic anti-CD40 antibodies and recombinant CD40 ligand, indicating the lack of an active CD40L in vivo(9, 10, 11) . The role of CD40L in vivo is supported as well by animal model studies using neutralizing antibodies specific for murine CD40L (muCD40L) that block both primary and secondary humoral response to T cell-dependent antigen(12, 13) . Together, these experimental studies and clinical results demonstrate the pivotal role of the CD40L-CD40 interaction in the regulation of humoral immunity(8, 12, 13) .

Based on its structural homology with TNF(14) , CD40L has been predicted to exist as a homotrimer in the cell membrane(15) . The interaction of CD40L with B cells can be more readily investigated if the active site of CD40L can be produced in a soluble form. To obtain such molecule, we expressed in Escherichia coli the extracellular domain of CD40L (shuCD40L-EC: Tyr-45 to Leu-261). In addition to the expected full-length extracellular domain (29 kDa), two other CD40L fragments of 18 and 14 kDa were observed in E. coli extracts. This mixture of the CD40L fragments was shown to be active in a B cell differentiation assay. Following purification of the 29-, 18-, and 14-kDa fragments, only the 18-kDa fragment was able to recognize CD40, both in solution and on the surface of human B cells. The 18-kDa fragment showed biological activities similar to those described for the membrane-bound form of CD40L, including induction of B cell proliferation and differentiation, rescue of B cells from apoptosis, and binding to soluble CD40. The amino acid sequence of the 18-kDa fragment coincides in size and homology with the mature TNF molecule and behaves as a homotrimeric molecule.


MATERIALS AND METHODS

Construction of the Extracellular Domain of CD40L

The extracellular domains of human and murine CD40L were amplified by polymerase chain reaction from the full-length genes(1, 16) , digested with NdeI and XbaI and cloned into p`236cIts vector containing p(L)(17) for expression in E. coli. All constructs were verified by DNA sequencing.

Purification of the Recombinant 29-kDa shuCD40L-EC and Fragments

The purification was performed as follows. Fifteen g of cell paste was resuspended in 50 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 25% (w/v) sucrose, 1 mM EDTA, 1 mM EGTA, 5 mM benzamidine-HCl, 1.5 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml lysozyme, and 0.1% sodium azide) and incubated at room temperature for 60 min under mixing. The lysate was centrifuged at 27,000 times g for 60 min. CD40L-EC was recovered as an insoluble pellet. It was washed seven times with 200 ml of 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 1% (v/v) Triton X-100, 2 mM benzamidine-HCl, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 0.2 M NaCl. The washed pellet was solubilized in 20 ml of 25 mM triethylamine (pH 11.5) containing 7.5 M urea and 10 mM CHAPS, spun, and supernatant was loaded to a Q-Hyper D Column (2.6 times 5 cm, Sepracor S.A., France) preequilibrated with solubilization buffer. Bound proteins were eluted stepwise with a pH gradient of 25 mM triethylamine at pH 11, 25 mM triethylamine at pH 10, and 25 mM Tris-HCl, pH 8.5, containing 7.5 M urea (deionized with Amberlite AG501-X8) and 10 mM CHAPS. Protein peaks were analyzed by SDS-PAGE, pooled, and dialyzed against 5 liters of 0.5% (v/v) acetic acid.

Purification of the 18-kDa shuCD40L

Recombinant 18-kDa shuCD40L was purified as described above from 78 g of cell paste with minor modifications to ensure proper renaturation; washed pellet was extracted with 100 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM dithiothreitol, and 6 M guanidinium HCl, the 18-kDa monomers were separated on a Sephacryl S-200 (5 times 90 cm) equilibrated with 100 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM dithiothreitol, and 6 M guanidinium HCl. Monomers were pooled and desalted on a Sephadex G-25 (5 times 50 cm) equilibrated with 50 mM Tris-HCl, pH 9.8, and 7 M urea. The protein pool was loaded onto a Q-Hyper D column (5 times 4 cm) equilibrated with same buffer. The 18-kDa protein was eluted with the equilibration buffer containing 100 mM NaCl, and the buffer was exchanged on a Sephadex G-25 (5 times 50 cm) to 50 mM sodium phosphate, pH 6.5, containing 6 M urea. Renaturation was done by diluting the protein pool to 20 µg/ml and to 4 M urea with 50 mM phosphate buffer, pH 6.5, and then added dropwise to an equal volume of 50 mM sodium phosphate, pH 6.5, containing 0.1 mM reduced glutathione and 0.01 mM glutathione disulfite (redox buffer) plus 4 M urea and 2.5% (w/v) ammonium sulfate. Urea was dialyzed out stepwise by two subsequent changes of 60 liters of redox buffer. Glutathione was then removed by dialysis against several changes of 25 mM sodium phosphate, pH 6.5. Renaturated shuCD40L was concentrated and clarified by centrifugation, filtered through Millex 0.22-µm sterile filters (Millipore), and stored at -80 °C. All manipulations were done at 4 °C or as otherwise indicated.

sCD40/sCD40L Binding Assay

Purified 18-kDa shuCD40L was coated on a 96-well plate (Maxisorb, Nunc) and detected by incubating with sCD40-Fc (1) and then developed with goat anti-mouse Fc peroxidase-conjugated antibody. Specificity of binding was determined by competition with an excess of 18-kDa shuCD40L added with the soluble CD40-Fc. The binding assay is briefly as follows. The 18-kDa shuCD40L was diluted to a concentration of 60 µg/ml in 0.2 M sodium phosphate buffer, pH 7. Serial dilutions containing 100 µl/well were made and incubated overnight at 4 °C. The wells were then washed with phosphate-buffered saline, 0.05% (v/v) Tween 20 (TPBS) and blocked with 150 µl of PBS, 1% (v/v) normal goat serum and 2% (v/v) bovine serum albumin (NBPBS, for 1 h at 37 °C). After washing with TPBS, 100 µl of sCD40-Fc at (2 µg/ml diluted in NBPBS) was added. Following a 1-h incubation at 4 °C, bound CD40-Fc was detected by peroxidase-conjugated goat anti-mouse antibody.

Transfection of the Human CD40L in COS-7 Cells

COS-7 cells were transfected by electroporation with either empty pcDL-SRalpha296 vector (18) or pcDL-SRalpha296 containing CD40L cDNA (ATCC 79814). Transfected COS-7 cells were harvested 48 h post-transfection and fixed with 0.5% paraformaldehyde(16) . Membrane CD40L expression was assessed by fluorescence-activated cell sorting using soluble CD40-Fc (1) . The fixed cell preparation was able to stimulate B cell proliferation and IgE production in a dose-dependent manner (data not shown).

B Cell Proliferation Assay

Purified tonsillar B cells were stimulated for 3 days with IL-4 and various concentrations of recombinant soluble CD40L, anti-CD40 antibody (0.1 µg/ml, B-B20, Serotec, Oxford, United Kingdom) or various number of fixed CD40L-transfected COS-7 cells. Proliferation was assessed by incorporation of [^3H]thymidine for 6 h(1, 2) .

B Cell Differentiation Assay

The assay was performed with purified tonsillar B cells stimulated with IL-4 plus anti-CD40 and recombinant soluble CD40L. Immunoglobulins E, G, A, and M were quantified by specific enzyme-linked immunosorbent assay as described previously(19) .

Apoptosis Assay

Recombinant soluble CD40L and anti-CD40 were tested for the ability to rescue purified germinal center B cells from apoptosis as described(20) .

The sucrose gradient was performed as described for TNF(21) .


RESULTS AND DISCUSSION

Extracellular Domain of the Recombinant CD40L

The extracellular domain of human CD40L (sCD40L-EC, Tyr-45 to Leu-261) expressed in E. coli, was found as an insoluble protein. In addition to the full-length form (29 kDa), two smaller fragments of 18 and 14 kDa were found and identified by N-terminal sequencing as beginning at ENSFEMQ and SNNLVT, respectively ( Fig. 1and Fig. 2). The 18-kDa fragment of sCD40L-EC is probably derived by processing of the 29-kDa form by an endogenous E. coli protease, since the fragment is preceded by two lysines. The observed proportions of the 18-kDa fragment varied in an E. coli strain-dependent manner (data not shown). The 14-kDa fragment may be due to an internal start as suggested by the presence of a Shine-Dalgarno-like sequence close to Met-148, 5`-AAA GGA TAC TAC ACC ATG. A partially purified mixture of these three proteins (Fig. 2B, lane 2), when added together with IL-10 or IL-4, was active in both a human B cell proliferation assay (data not shown) and an immunoglobulin switching assay using B cells derived from normal individuals and from patients with X-linked hyper-IgM syndrome ( Table 1and (9) ), indicating that the recombinant sCD40L-EC was able to trigger B cell activation through binding to the membrane CD40 on B cells. To understand which of the fragments of the extracellular domain of the CD40L trigger the biological activity, the recombinant extracellular CD40L form and the two fragments were further purified to homogeneity.


Figure 1: Schematic representation of extracellular domain of human and murine CD40L constructs and fragments. The initial construct of the extracellular domain of the human CD40L encodes Tyr-44 to Leu-261 (shuCD40L-EC). Expression in E. coli gave the full-length (29 kDa) and two fragments of 18 and 14 kDa, which began at Glu-108 and Ser-149, respectively. Constructs were made encoding the human 18-kDa fragment of CD40L (sHuCD40L) and two murine constructs encoding Met-87 to Leu-260 (sMuCD40L-1) and Met-112 to Leu-260 (sMuCD40L-2), corresponding to the TNFalpha homologous domain, from Val-77 to Leu-233(15) .




Figure 2: Purification of sCD40L-EC and its fragments. Soluble CD40L-EC was purified on a Q-Hyper D anion exchanger as described under ``Materials and Methods.'' PanelA shows elution profile. Fractions were analyzed by SDS-PAGE and purest fractions of 29, 18, and 14 kDa were each pooled and concentrated to 1 mg/ml. PanelB shows the SDS-PAGE analysis of the total washed pellet on lane2, and the 14-, 18-, and 29-kDa shuCD40L protein pools on lanes3, 4, and 5, respectively, obtained from elution at pH 11, 10, and 8.5 as shown in panelA. Lane1 corresponds to protein standards from Pharmacia.





Purification, Biological, and Physical Characterization of the Active Recombinant Soluble Human CD40L

Protein purification was performed on a Q-Hyper D anion exchanger as described under ``Materials and Methods.'' The sCD40L 14-, 18-, and 29-kDa fragments were eluted from the resin at pH 11, 10, and 8.5, respectively, at constant ionic strength (Fig. 2). The three purified sCD40L fragments were tested in the CD40L-CD40 binding and B cell proliferation assay over a concentration range of 1-100 µg/ml and 1-10 µg/ml, respectively (Fig. 3). The 18-kDa fragment was active in both assays, whereas the 29- and 14-kDa fragments failed to show any activity, indicating that the 18-kDa fragment is the active component of the mixture.


Figure 3: Biological activity of sCD40L-EC and its fragments. Panel A shows the binding of shuCD40L-EC 29-kDa (circle), 18-kDa (bullet), and 14-kDa (box) fragments to soluble CD40-Fc as described under ``Materials and Methods.'' PanelB shows activation of human tonsillar B cells stimulated with IL-4 (100 units/ml) with or without increasing concentrations of the 29-kDa (circle), 18-kDa (bullet), or 14-kDa (box) CD40L fragments. Both bioassays showed a saturable activity for 18-kDa fragment of shuCD40L-EC with EC = 5-10 µg/ml.



To further characterize the active extracellular domain of CD40 ligand, constructs of the 18-kDa soluble human CD40L (shuCD40L: Glu-108 to Leu-261) and the murine sCD40L, encoding homologous residues (smuCD40L-1: Met-87 to Leu-260) were made (Fig. 1). The purified 18-kDa shuCD40L and smuCD40L-1 confirmed that the 18-kDa CD40L fragment derived from the sCD40L-EC was the biologically active protein (Fig. 4). Purified 18-kDa shuCD40L and smuCD40L-1 were able to bind soluble CD40-Fc and stimulate B cell proliferation in combination with IL-4 (Fig. 4, A and B) with an IC of about 10 and 5 µg/ml, respectively. No signal was observed when CD40-Fc was not present in the binding assay (Fig. 4A) or when excess of sCD40L was added to the binding assay (data not shown). The 29-kDa extracellular domain (sCD40L-EC) and the 14-kDa fragment did not compete with 18-kDa shuCD40L for binding to CD40-Fc (data not shown). These results indicate that the 18-kDa domain of CD40L recognizes both the soluble and membrane-bound forms of CD40. A second protein construct of the murine CD40L (smuCD40L-2) that differs by five amino acids at the N-terminal end of the 18-kDa shuCD40L (Fig. 1, 108-ENSFE), was found to be inactive at all concentrations tested (data not shown).


Figure 4: Biological activity of recombinant 18-kDa shuCD40L and smuCD40L. The 18-kDa (shuCD40L) and murine (smuCD40L-1) recombinant extracellular domain constructs (Fig. 1) were expressed and purified as described under ``Materials and Methods.'' PanelA shows the binding of CD40-Fc to 18-kDa shuCD40L (circle) and smuCD40L-1 (bullet). Control without CD40-Fc (box) showed no detectable signal. PanelB shows B cell proliferation induced by stimulation with IL4 (100 units/ml) plus 18-kDa shuCD40L (blackbars) or smuCD40L-1 (openbars) (n = 3). The 18-kDa shuCD40L was also able to induce B cells to differentiate and produce immunoglobulins (panelC) and rescue germinal B cells from apoptosis (panelD) (n = 3). The detection limit of IgE in panelC was around 0.2 ng/ml.



CD40 plays an important role in B cell differentiation and in the survival of germinal center B cells, as shown by the effects of cross-linking CD40 with agonistic anti-CD40 antibodies(22, 23) . Recombinant shuCD40L was tested for its ability to induce B cells to differentiate and to rescue germinal B cells from apoptosis. Addition of 1 and 5 µg/ml shuCD40L to purified human B cells allowed production of 23 ± 1 and 45.5 ± 2 ng/ml IgE (n = 3), respectively (Fig. 4C). Similarly, germinal center B cells were rescued from apoptosis in a dose-response manner by recombinant 18-kDa shuCD40L (Fig. 4D). These values were comparable to those obtained by addition of agonistic anti-CD40 antibodies (Fig. 4, C and D; Refs. 9, 22, and 23). Furthermore, a direct comparison of the 18-kDa shuCD40L with the membrane-bound full-length CD40L expressed in COS-7 showed that the two molecules displayed similar biological activity as shown by the B cell proliferation assay (Table 2).



The soluble human CD40L corresponds to the region of TNFalpha homology (Val-77 to Leu-233). This region has been used to construct a three-dimensional model for CD40 ligand (15) which predicts a homotrimeric structure for CD40L. To investigate its quaternary structure, 18-kDa shuCD40L was analyzed by gel filtration; the molecule migrates with an apparent molecular mass of about 50 kDa. The 18-kDa shuCD40L isolated by gel filtration was active in the CD40L-CD40 binding assay (data not shown), indicating that the 18-kDa shuCD40L probably associates as a high order molecular complex. Further studies by sucrose gradient sedimentation studies suggest that the 18-kDa shuCD40L is homotrimeric (Fig. 5). Equal amounts of the 29-kDa (shuCD40L-EC) or of the 18-kDa (shuCD40L) fragment were overlaid in a 5-20% sucrose gradient and centrifuged for 40 h at 40,000 rpm in a SW 41 rotor. The 18-kDa sedimented as a unique molecular species, migrating with a molecular mass of 54.8 ± 0.6 kDa, which coincides with the predicted trimeric conformation. In contrast, the 29-kDa sedimented with a molecular mass of 30.8 ± 2 kDa, suggestive of an extended monomeric form (Fig. 5). These results suggest that the trimeric conformation of the ligand may be required for binding to CD40. However, despite extensive refolding studies, we cannot rule out the possibility that the 29-kDa shuCD40L-EC fails to form trimers and to bind CD40 due to improper folding. A CD40L-CD8 chimera of the extracellular domain of human CD40L was reported to be active and trimeric(4) . Thus, the CD8 domain may stabilize the trimeric form of CD40L-EC.


Figure 5: Sucrose gradient sedimentation of soluble CD40L. One hundred µg of either 18-kDa shuCD40L or 29-kDa shuCD40L-EC were mixed with protein standards and layered onto a 5-20% sucrose gradient in PBS. After centrifugation for 40 h at 40,000 rpm in a SW 41 rotor, fractions of 300 µl were collected and analyzed by SDS-PAGE. A, molecular mass of globular protein standards was plotted against fraction number. Sedimentation points of the 18-kDa shuCD40L (54.8 ± 0.6 kDa) and the 29-kDa shuCD40L-EC (30.8 ± 2 kDa) are indicated. Calculated molecular mass of the two proteins was obtained from three independent gradients. B and C, fractions from representative gradients were separated by SDS-PAGE and silver-stained. Positions of the 18-kDa shuCD40L and 29-kDa shuCD40L-EC are indicated by arrows.



We have shown here that CD40L sequences corresponding to the TNF homology region can be expressed as a soluble trimeric molecule with biological activity. Its activity correlates with that of the membrane-bound CD40L; it can replace CD40L-T cells in the activation of B cells. These findings are supported by the fact that the soluble form of CD40L activates B cells derived from HIGM1 patients whose T cells lack active CD40L (Table 1, (9) ). Our results suggest that, if a soluble form of CD40L exists in vivo, it could be active. Armitage et al.(24) described an activity from murine thymoma cell line (EL4) conditioned media that binds CD40 and stimulate human and murine B cells, supporting the possible existence of a soluble form of CD40L.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 22-706-98-35; Fax: 22-794-69-65; gjm2107{at}ggr.co.uk.

To whom requests for recombinant CD40L should be addressed.

(^1)
The abbreviations used are: HIGM1, hyper-IgM syndrome; shuCD40L-EC, soluble human CD40 ligand extracellular domain; 18-kDa shuCD40L, 18-kDa soluble human CD40 ligand; smuCD40L, soluble murine CD40 ligand; TNF, tumor necrosis factor; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; IL, interleukin; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank I. Stamenkovic for kindly providing the construct of CD40-Fc fusion construct, M. Guerrier for DNA sequencing, G. Ayala and D. Bertschy-Meier for oligonucleotide synthesis, E. Magnenat for N-terminal amino acid analysis, E. Sebille for preparing the CD40-Fc, C. Hebert for photographic skills, and Drs. K. Hardy and J. Knowles for continued support and helpful discussion.


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