(Received for publication, October 16, 1995; and in revised form, January 16, 1996)
From the
CD40 ligand (CD40L) is a glycoprotein expressed on the surface of activated helper T cells, basophils, mast cells, and eosinophils. Binding of CD40L to its receptor CD40 on the B cell surface induces B cell proliferation, adhesion, and immunoglobulin class switching. We have identified soluble cleavage products of human CD40L in the supernatant of a stimulated human T cell clone. Subcellular fractionation experiments have shown that the transmembrane CD40L is processed inside the microsomes and that its cleavage is stimulation-dependent. The native human soluble CD40L is trimeric and, when used in conjunction with interleukin-4, induces B cell proliferation.
CD40L is a type II surface protein expressed by T cells,
basophils, mast cells, and
eosinophils(1, 2, 3, 4, 5) .
CD40L can induce B cell proliferation and is involved in the control of
immunoglobulin (Ig) class
switching(1, 2, 3, 6, 7) .
Patients expressing mutated forms of CD40L (hyper-IgM syndrome) have
lymph nodes without germinal centers and fail to produce the Ig
isotypes requiring a class
switch(8, 9, 10, 11, 12) .
CD40L belongs to a family of surface proteins that exist in soluble and
membrane-bound forms such as TNF- (
)and Fas
ligand(13, 14) . We previously observed that
recombinant, truncated forms of CD40L could be trimeric and
biologically active(15) . We therefore examined if activated T
cells could release soluble forms of CD40L. We detected soluble forms
of CD40L similar to the ones recently described by Graf et
al.(16) . These forms behaved as trimers and resulted, at
least partially, from an intracellular processing. Soluble CD40L was
active in B cell proliferation assays in the presence of IL-4. This
suggests that both the soluble and membrane-bound forms of CD40L share
biological activities and that cleavage of the membrane form might not
simply represent an alternative way to down-regulate CD40L expression.
Figure 1:
CD40L surface expression. 2
10
JF7 cells were unstimulated (A) or stimulated (B) for 16 h using ionomycin and PMA. Cells were incubated
with the human anti-CD40L mAb or with IgG2a, then stained with
fluorescein isothiocyanate-conjugated anti-mouse antibodies, and
analyzed by flow cytometry.
Figure 2:
The native human soluble CD40L is
processed inside the microsomes. After 16 h with (lane 2) or
without (lane 1) PMA and ionomycin, supernatants of JF7 cells
were 200 concentrated. In parallel, 2
10
cells were used to prepare microsomes from control (lane
3) and activated (lane 4) JF7 cells, and 2
10
cells were used to prepare total cell extract from the
control (lane 6) and activated (lane 7) JF7 cells.
Proteins from each fraction were titrated, and 30 µg/ml was
subjected to SDS-PAGE analysis, transferred on nitrocellulose, and
blotted with 5 µg/ml the human anti-CD40L mAb. The molecular mass
marker is shown on lane 5.
To determine
whether the soluble forms corresponded to a cleavage of the membrane
form on the T cell surface or to an intracellular processing, we
isolated microsomal fractions from unstimulated and stimulated JF7
cells and analyzed the fractions by Western blot assays with anti-CD40L
mAb (Fig. 2). Whereas in the total cell extract and the
microsomal fractions of the unstimulated T cell clone only the 33-kDa
membrane form of CD40L could be detected, in activated cells the
membrane-bound form and the 18- and 15-kDa forms were detectable (Fig. 2). These data suggest that the soluble forms released
into the supernatant are the result of an intracellular cleavage event
dependent on an enzymatic activity only present in stimulated cells.
Based on the known IL-1 and the TNF-
processing, one can
imagine that the enzyme responsible for the CD40L cleavage belongs to a
convertase family of enzymes(13, 20) . The existence
of a soluble form of CD40L allowed us to postulate two different
pathways of CD40L regulation in the immune response. These soluble
forms could represent inactive by-products generated during the
down-regulation of CD40L expression. On the other hand, they could
represent alternative forms of CD40L displaying biological activities.
Previous work has shown that the soluble recombinant form of CD40L is
trimeric(15) . We therefore examined whether CD40L released by
activated T cells was monomeric or multimeric.
Figure 3: Sucrose gradient sedimentation of the human native soluble CD40L. An aliquot of 100 µl of concentrated, stimulated JF7 supernatant was mixed with 100 µl of biotinylated protein standards and layered onto a 5-20% sucrose gradient in PBS. After centrifugation for 42 h at 40,000 rpm in a SW 41 Ti rotor, fractions were collected, trichloroacetic acid-precipitated, and divided into two pools for analysis with the anti-CD40L mAb to detect CD40L and with the streptavidin horseradish peroxidase-conjugated antibody to reveal the biotinylated molecular mass marker. A, molecular mass of globular protein standards was plotted against fraction number. Sedimentation points of the 18- and 15-kDa native soluble CD40L are indicated. B, fraction from representative gradient was separated by SDS-PAGE, transferred onto nitrocellulose, and blotted with 5 µg/ml human anti-CD40L mAb. Positions of the 18- and 15-kDa CD40L cleavage products are indicated by arrows.
Figure 4:
Biological activity of the native human
soluble CD40L. Purified tonsillar B cells were incubated either alone (lane 1), with rIL-4 (lane 2), with rIL-4 and
anti-CD40 mAb (lane 3), or with rIL-4 and aliquots of either
sucrose gradient fraction 3 or fraction 19 (from lane 4 to 7), and then the [H]deoxythymidine
incorporation was measured. Results are expressed in counts/min (mean
± S.D.). Lanes 4 and 5, B cells were incubated
with 20 µl of the sucrose gradient fraction 19 in the absence or
presence of 10 µg/ml CD40-Fc, respectively. Lanes 6 and 7, B cells were incubated with 20 µl of the sucrose
gradient fraction 3 in the absence or presence of 10 µg/ml CD40-Fc,
respectively.
Whereas it remains to be determined if all the identified soluble
forms of CD40L are biologically active, our data suggest that cleavage
of CD40L does not simply represent an alternative way to down-regulate
the expression of this surface molecule by T cells. It seems unlikely
that one of the two forms of the soluble CD40L acts as an antagonist
since we have identified them as trimers. Soluble forms produced by
intracellular cleavage share activities with the membrane form of CD40L
and might therefore be involved in the control of B cell activation by
helper T cells. Whereas the 33-kDa membrane form could be detected
intracellularly on unstimulated CD4 T cell clone cells
by Western blotting assays, surface expression was undetectable by flow
cytometry, indicating a post-transcriptional control of surface
expression(22) . This would appear to suggest that a preformed
CD40L is stored inside unstimulated cells to be readily available in
case of an immediate need. The absence of surface expression was
paralleled by the CD40L cleavage, suggesting that the release of
soluble CD40L by T cells is also tightly regulated.
CD40L is involved in the induction of a large variety of events in the immune system as indicated by the pleiotropic effects of the mutations observed in hyper-IgM syndrome patients(8, 23) . The complex physiology of CD40L might be partially linked to the existence of multiple forms of the protein.
The existence of a membrane-bound and a soluble form of CD40L suggests that this molecule might transmit signals in two different ways. The two different forms could share some activities, but they could also display specific functions. The membrane molecule could be implicated in a cell-cell interaction process, which presents some physical limits of the signal propagation. In contrast the soluble form with its ``cytokine-like activity'' could represent a quick and diffusible way for T cells to transmit the signal.