(Received for publication, November 17, 1994; and in revised form, January 26, 1995)
From the
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.
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 ()(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.
The sucrose gradient was performed as described for TNF(21) .
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 TNF 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.
Figure 3:
Biological activity of sCD40L-EC and its
fragments. Panel A shows the binding of shuCD40L-EC 29-kDa
(), 18-kDa (
), and 14-kDa (
) 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 (
), 18-kDa (
), or 14-kDa
(
) 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 () and smuCD40L-1 (
). Control without
CD40-Fc (
) 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 TNF 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.