CD40 Ligand
Edward A. Clark*
Primate Center, University of Washington, Box 357330, Seattle, WA 98195-0001, USA
* corresponding author tel: 206-543-8706, fax: 206-685-0305, e-mail: eclark@bart.rprc.washington.edu
DOI: 10.1006/rwcy.2000.05006.
SUMMARY
CD40L is a member of the tumor necrosis factor
(TNF) family expressed on activated T cells after
T cell antigen receptor ligation. It binds to the CD40
receptor on B cells and dendritic cells and thereby
provides a critical `helper T cell' signal necessary for
germinal center formation, isotype class switching,
and production of, for example, IgG and IgE anti-
bodies. CD40L may also transmit a signal back to
T cells, to promote short-term T cell proliferation.
CD40L and CD40 are not simply restricted to cells
regulating immune responses. The CD40LÿCD40
signaling pathway may also play an important role in
the regulation of epithelial cell, fibroblast, and smooth
muscle cell proliferation.
BACKGROUND
The MedLine database from 1990 to May 1999
listed over 800 references for CD40L, and in 1998
there were over 180 studies published in which
CD40L was mentioned. This chapter will, therefore,
simply summarize key information about CD40L
(CD154) and the most exciting recent studies on it.
There are several excellent reviews discussing CD40L
and its historical background (Callard et al., 1993;
Banchereau et al., 1994; Foy et al., 1996; Grewal and
Flavell, 1998). This chapter emphasizes the expres-
sion of CD40L and the biologic consequences of
CD40L deficiency or interfering with CD40L bind-
ing to CD40. In contrast, the chapter on CD40
principally deals with the expression of CD40 and
the signaling pathways and biologic functions of this
receptor.
Discovery
CD40L/CD154 is a 33±35 kDa type 2 membrane
glycoprotein expressed primarily on activated CD4+
T cells but also on CD8+ T cells. It is a member of
the TNF family of ligands. The cDNA encoding
CD40L was initially described by Armitage et al.
(1992), while the CD40L protein was identified by two
groups using monoclonal antibody to mouse CD40L/
gp39 (Noelle et al., 1992) and human CD40L/T-BAM
(5C8, Lederman et al., 1992) respectively. The
CD40L+ T cells are found principally in the outer
zone of germinal centers (GCs) and within T zones
that are rich in dendritic cells (Casamajor-Palleja
et al., 1995). Mature T cells and not thymocytes
express CD40L (Fuleihan et al., 1995).
Alternative names
CD40L (CD154) is also called gp39, T-BAM or
TRAP.
Main activities and
pathophysiological roles
Patients with X-linked hyper-IgM syndrome (HIM)
have mutations in a gene mapping to Xq24 and
encoding for CD40L (Callard et al., 1993; DiSanto
et al., 1993; Korthauer et al., 1993). Patients
458 Edward A. Clark

with HIM do not isotype class-switch and produce GENE AND GENE REGULATION
IgG antibodies, and have few or no GCs in their
lymphoid tissues. Similarly, CD40L-deficient mice
fail to mount secondary antibody responses to T cell- Accession numbers
dependent antigens and undergo isotype class-switch-
ing (Xu et al., 1994; Renshaw et al., 1994). GenBank:
CD40L-deficient individuals have not only deficient Mouse CD40L: CAA46448
antibody responses, but also defective antigen-specific Human CD40L: NP_00065, P29965
T cell responses (Grewel et al., 1995). The T cell The CD40L gene has also been cloned and
defect is apparently not caused simply by a lack of sequenced in cattle (Mertens et al., 1995; GenBank
signaling to antigen-presenting cells (APCs), but is accession numbers P51749 and Q28203) and in dogs
also due to lack of signaling directly to the T cell via (GenBank accession number AAD04375).
CD40L (Armitage et al., 1993; Blair et al., 2000,
see below). These findings, together with those of
a large series of in vitro studies, have shown PROTEIN
that CD40LÿCD40 interactions are usually but not
invariably essential for isotype class-switching and Sequence
germinal center development in responses to T cell-
dependent antigens. See Figure 1.

Figure 1 Amino acid sequences for mouse CD40L (Armitage et al., 1992) and
human CD40L (Hollenbaugh et al., 1992).

Gene

Mouse CD40 (Armitage et al., 1992, Genebank Assession number CAA46448):

1 MIETYSQPSP RSVATGLPAS MKIFMYLLTV FLITQMIGSV LFAVYLHRRL DKVEEEVNLH

61 EDFVFIKKLK RCNKGEGSLS LLNCEEMRRQ FEDLVKDITL NKEEKKENSF EMQRGDEDPQ

121 IAAHVVSEAN SNAASVLQWA KKGYYTMKSN LVMLENGKQL TVKREGLYYV YTQVTFCSNR

181 EPSSQRPFIV GLWLKPSIGS ERILLKAANT HSSSQLCEQQ SVHLGGVFEL QAGASVFVNV

241 TEASQVIHRV GFSSFGLLKL

Human CD40 (Hollenbaugh et al., 1992, Genebank Accession number NP_00065

or P29965):

1 MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL DKIEDERNLH

61 EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ FEGFVKDIML NKEETKKENS FEMQKGDQNP

121 QIAAHVISEA SSKTTSVLQW AEKGYYTMSN NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN

181 REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN

241 VTDPSQVSHG TGFTSFGLLK L


Discussion of crystal structure
A crystal structure of the extracellular region of
CD40L has been described (Karpusas et al., 1995),
and a three-dimensional structure has been produced
showing how CD40L interacts with the CD40 recep-
tor (Singh et al., 1998).
CELLULAR SOURCES AND
TISSUE EXPRESSION
Cellular sources that produce
Although the expression of CD40L on activated T
cells is emphasized in the literature, CD40L is in fact
expressed on a number of cell types, including
 T via their so-called ligands, CD40L and CD80/CD86
cells, macrophages, Kupffer cells in the liver, den-
dritic cells, eosinophils, endothelial cells, and smooth
muscle cells (Armitage et al., 1992; Gauchat et al.,
1995; Horner et al., 1995; Pinchuk et al., 1996; Mach
et al., 1997; Gaweco et al., 1999). Some human B cell
lines (Grammer et al., 1995), chronic lymphocytic
leukemias (Schattner et al., 1998), and B cells from
autoimmune (Blossom et al., 1997) or normal mice
(Wykes et al., 1998) express CD40L. Ligating CD40
itself can upregulate the expression of its ligand on
dendritic cells (Pinchuk et al., 1996) and induce the
release of soluble CD40L from mouse B cells expres-
sing cytosolic CD40L (Wykes et al., 1998). Activated
NK cells also express CD40L, which may in fact
function to facilitate the NK-mediated killing of
CD40+ target cells (Carbone et al., 1997). Thus, the
`T cell centric' models in the literature almost cer-
tainly oversimplify how CD154ÿCD40 interactions
are regulated in vivo.
Eliciting and inhibitory stimuli,
including exogenous and
endogenous modulators
Again, most of the studies on CD40L expression have
been carried out using T cells. Ligating the TCR is the
major means by which CD40L is induced on T cells
(e.g. de Boer et al., 1993; Roy et al., 1995; Jaiswal
et al., 1996), but costimulatory signals such as those
via CD28 or cytokines such as IL-2 can sustain
CD40L expression on T cells (de Boer et al., 1993;
Klaus et al., 1994; Johnson-L'eger et al., 1998a).
CD40L expression is reduced on T cells upon contact
with CD40+ B cells. This may be induced by
CD40 Ligand 459
decreasing CD40L mRNA expression, soluble CD40
binding CD40L or the rapid internalization of CD40L
(van Kooten et al., 1994; Yellin et al., 1994). A subset
of CD4+ memory T cells, like some B cells
(Wykes et al., 1998), have preformed cytosolic
CD40L (Casamajor-Pallaja et al., 1995). After TCR
ligation, this preformed CD40L is rapidly expressed
on the cell surface.
Based on a combination of findings suggesting
that, for example, (a) CD40 ligation upregulates
the CD28 ligands CD80 and CD86, and (b) CD28
stimulation promotes the expression of CD40L, we
proposed a `reciprocal dialogue' model for the acti-
vation of T cells and APCs (Clark and Ledbetter,
1994). This model emphasizes the interrelationship
between these two receptor/ligand systems and shows
that the signaling pathways are reciprocal, i.e.
transmitted not only via CD40 and CD28 but also
(Figure 2).
Figure 2 The `reciprocal dialogue' model for the recipro-
cal activation of antigen-presenting cells (APCs) and T cells
(Clark and Ledbetter, 1994). The engagement of MHC plus
peptide on the APC by the TCR complex on a T cell
activates APCs to express CD80/CD86, and the ligation of
TCRs on T cells induces the expression of CD40L. The
engagement of CD28 on T cells or CD40 on APCs induces
a similar activation pathway including the activation of
NFB and pro-growth signals in T and B cells. The reci-
procal engagement of CD40L on T cells or CD80 and CD86
on APCs also transmits key signals to cells to promote
sustained T cell/APC activation. An additional receptor for
CD80/CD86, CTLA4, is also induced via T cell activation
and functions in a feedback inhibitory pathway. Another
receptor ligand pair, ICOS and ICOSL, which are related
to CD28 and CD80 respectively, are also involved in T cell/
APC activation and IL-10 regulation.
• proliferation
• cytokines for
isotype switch
T
CD40L CD28
CD40 CD80/86
B or DC
• proliferation
• isotype switch
460 Edward A. Clark
As described below, this model has been sub-
stantiated in a number of studies both in vivo and
in vitro. However, although CD40L expression is
further increased principally by ligating CD28
(Klaus et al., 1994), it is also regulated via CD28-
independent signals from APCs (Ding et al., 1995).
Furthermore, several studies have shown that CD40L/
CD40 signaling is not essential for T cell-dependent
B cell responses. For example, Life et al. (1994)
found that T cells from HIM patients can stimulate
IgE production via a CD40L-independent mechan-
ism; similarly, Lane et al. (1995) showed that CD40L-
negative T cells can still induce human B cells to
proliferate and become plasma cells. Wu et al. (1995)
also found that CD40L T cells can induce the
expression of CD80 and CD86 on APCs. More
recently, Yu et al. (1999) reported that CD40-
deficient mice can be induced to class-switch their
B cells and produce IgG and IgE. Although the
CD40L/CD40 and CD28/CD80ÿCD86 systems play
a key role in T and APC activation and B cell
. However, whether this
development, they are under some conditions not
essential.
The induction of CD40L on CD3-stimulated T cells
can be blocked by inhibitors of phosphatidylinositol
3-kinase (PI-3 kinase; Aagaard-Tillery and Jelinek,
1996), indicating that this kinase plays a key role in
the regulation of CD40L. It is also blocked by
cyclosporin A (Klaus et al., 1994), suggesting that two
distinct pathways via calcineurin and PI-3 kinase
work together to promote CD40L expression. IL-12
upregulates CD40L on T cells (Peng et al., 1998),
while glucocorticoids reduce its expression (Bischof
, as well as of TH2 cytokines such as IL-4,
and Melms, 1998). Whereas IFN
can inhibit the IL-5, and IL-10, by a direct effect on T cells. While it
expression of CD40L on both TH1 and TH2 T cells,
RECEPTOR UTILIZATION
CD40L has just one receptor, CD40, a member of
the TNF receptor family expressed on B cells,
dendritic cells, activated macrophages, follicular
dendritic cells, epithelial cells, and smooth muscle
cells.
IN VITRO ACTIVITIES
In vitro findings
Signaling via CD40L on Activated T Cells
There is now good evidence that CD40L can both
receive and transmit signals to CD40L+ cells as
predicted by the `reciprocal dialogue' model (Clark
and Ledbetter, 1994). Armitage et al. (1993) initially
reported that CD40L can stimulate T cells to make
more IL-2, TNF and IFN
occurred via a direct signal or an indirect effect was
not clear. Then Cayabyab et al. (1994) found that
CD40+ transfectants can augment anti-CD3-induced
T cell proliferation via an IL-2-dependent mechanism.
The crosslinking of CD40L and CD28 may also
promote thymocyte proliferation (Ruggerio et al.,
1996). Just as ligating CD40 on B cells activates JNK
and p38 MAP kinase so too does ligating CD40L on
T cells activate these kinases (Brenner et al., 1997).
Peng et al. (1996) found that CD40L/CD40
interactions can upregulate the expression of IL-2
and IFN
remains possible that some of the effects observed
TGF effectively blocks CD40L expression only on
TH2 cells (Roy et al., 1993).
Studies using TCR transgenic mice suggest that
the induction of CD40L does not require costi-
mulatory molecules on APCs, or at least is less
dependent on costimulation than is IL-2 production
in T cells (Jaiswal et al., 1996). However, Croft et al.
(1997) found, in a screen of a number of different T
cell-associated molecules, that CD40L and IL-2
expression in particular were dependent on costimu-
lation. Furthermore, Johnson-L'eger et al. (1998b)
reported that CD3-stimulated T cells, although
initially expressing CD40L, do not produce sufficient
IL-2 to sustain CD40L expression; these authors
propose that CD4+ helper T cells may need to
encounter APCs expressing CD80/86 so that suffi-
cient costimulation occurs, leading to the
stable expression of CD40L and maximal secretion
of IL-2.
required signaling via CD28 on the T cells, van Essen
et al. (1995) found that rudimentary GCs can develop
in CD40ÿ/ÿ mice that are injected with a CD40-
immunoglobulin fusion protein. This suggests that
CD40L is essential for transducing a signal to T cells
in vivo that is important for the process of GC
formation. Likewise, Grewal et al. (1995) found that
CD40L was required to induce antigen-specific helper
T cells to proliferate; this was probably the result of a
direct effect on the T cells as well as via the indirect
regulation of `costimulation' on the APCs (Grewal
and Flavell, 1998). Blotta et al. (1996) found that
crosslinking CD40L on T cells upregulates IL-4 syn-
thesis, and Poudrier et al. (1998) found that CD40L
costimulation promotes IL-4 production by T cells
in vivo. Most recently, Blair et al. (2000) showed that
ligating CD40L promoted CD3-induced T cell
proliferation. However, unlike CD28 costimulation,
CD40L costimulation does not upregulate Bcl-X and

long-term proliferation, suggesting that CD40L may
function as a short-term or effector cell costimulator.
PATHOPHYSIOLOGICAL ROLES
IN NORMAL HUMANS AND
DISEASE STATES AND
DIAGNOSTIC UTILITY
Role in experiments of nature and
disease states
Clinical Effects of Blocking the CD40L/CD40
Pathway In Vivo
Grewal et al. (1996) found that CD40Lÿ/ÿ myelin
basic protein (MBP) transgenic mice cannot be
induced to make IFN
or develop experimental proteinases and promotes angiogenic functions
allergic encephalomyelitis with MBP unless they are
given CD80+ APCs. The lack of CD40L apparently
leads to an insufficient induction of functional APCs
so that autoimmune disease cannot be induced. The
acceptance of allografts in anti-CD40L-treated mice
requires the presence of IFN
(Markees et al., factors, including the animal's underlying condition.
1998), but the mechanism behind this requirement
is unknown. Antigen-induced airway inflammatory
responses are dramatically reduced in CD40Lÿ/ÿ
mice, perhaps because IL-4 and TNF levels are
reduced (Lei et al., 1998).
Given that CD40L/CD40 interactions are essential
for many T cell-dependent processes, including the
formation of certain autoantibodies, blockade of the
CD40L/CD40 pathway with monoclonal antibody or
soluble fusion proteins is an attractive immuno-
therapeutic strategy. Indeed, CD40L (gp39) mono-
clonal antibodies have been shown in a number of
murine models to block the development of auto-
immune diseases, especially if given prior to onset of
is required for CD40/CD40L and CD28/
the disease, including collagen-induced arthritis (Durie
et al., 1993), spontaneous lupus nephritis (Mohan et al.,
1995), autoimmune oophoritis (Griggs et al., 1996), and
insulitis/diabetes (Balasa et al., 1997).
The effect of anti-CD154 on disease progression is
more variable. Whereas the progression of diabetes in
nonobese mice is not blocked by anti-CD154 (Balasa
et al., 1997), anti-CD40L can block the progression of
relapsing experimental autoimmune encephalomyelitis
in mice when given either at the peak of the acute
disease or during remission (Howard et al., 1999).
Furthermore, Larsen et al. (1996a) blocked murine
cardiac allograft rejection with anti-CD40L mono-
clonal antibody in both unimmunized and sensitized
mice. Thus, CD40L blockage can be efficacious even
CD40 Ligand 461
in already sensitized individuals. Balashov et al.
(1997) reported that CD40L is expressed at higher
levels on activated T cells from patients with multiple
sclerosis compared with controls. They suggest that
CD40L-induced IL-12 production by these cells may
play a key role in the pathogenesis and disease
progression of multiple sclerosis.
CD40L-positive T cells accumulate in atheroma,
and ligating CD40 on atheroma-associated cells
induces proinflammatory responses. Thus, Mach
et al. (1998) tested whether CD154 monoclonal
antibody could influence atherogenesis in vivo; they
found that CD40L monoclonal antibody treatment
reduced the size of aortic atherosclerotic lesions in
mice developing atherosclerosis. Consistent with this,
the same group reported later that the CD40L-
mediated ligation of CD40 on endothelial cells
induces the de novo expression of matrix metallo-
(Mach et al., 1999).
Noelle's group, although pioneers in the area of
CD154 monoclonal antibody therapy, have also
emphasized that the effect of CD154 monoclonal
antibody in vivo may vary depending on a number of
Autoimmune CD95-deficient mice, for example,
treated with anti-CD40L have accelerated renal
disease and lymphadenopathy (Russell et al., 1998).
Ligating CD40L on lpr/lpr thymocytes apparently
inhibits apoptosis.
A combination of CD40/CD40L and CD28/
CD80ÿCD86 blockade, as predicted by the model in
Figure 2, has been found to be specially effective in
blocking deleterious immune responses. Larsen et al.
(1996b) found that combined blockade promotes
the long-term survival of fully allogeneic skin grafts
and vascularized cardiac allografts. This has been
substantiated by others (Sun et al., 1997), and again
IFN
CD80±CD86 blockade in order to prevent allograft
rejection (Konieczny et al., 1998). This combined
blockade can also prevent the development of
murine lupus (Daikh et al., 1997).
However, the additive or synergistic effects of
CD40/CD40L and CD28/CD80±CD86 blockade are
not universal. For instance, Bumgardner et al.
(1998) suggest that allogeneic responses to hepato-
cytes require CD40L/CD40 interactions but are not
affected by an CTLA4-Ig blockade of CD28
signaling. Furthermore, the CD154 and CD28 block-
ades may work by affecting independent pathways
rather than by simply affecting each other (Judge
et al., 1999). Clearly, much remains to be learned
about how the selective disruption of CD40L/CD40
462 Edward A. Clark
interactions, either alone or in combination with
blockade or other receptor systems (e.g. ICOS/
ICOSL, 41BB/41BBL, CD30/CD30L, RANK/
RANKL, etc.), affect T cell anergy or the quality of
in vivo immune responses.
Knockout mouse phenotypes
CD40L-deficient mice fail to mount secondary
antibody responses to T cell-dependent antigens and
undergo isotype class-switching (Xu et al., 1994;
Renshaw et al., 1994).
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