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(Received for publication, October 23, 1996, and in revised form, December 23, 1996)
From Bristol-Myers Squibb Pharmaceutical Research Institute,
Seattle, Washington 98121
ABSTRACT The T cell activation antigen 4-1BB (CDw137) is
a distantly related member of the tumor necrosis factor receptor family
of cell surface receptors. We previously reported that murine 4-1BB (m4-1BB) bound to extracellular matrix (ECM) proteins. Recently, a
tumor necrosis factor-like ligand of m4-1BB, m4-1BBL, as well as the
human counterparts of 4-1BB (ILA) and 4-1BBL (h4-1BB and h4-1BBL,
respectively) have been cloned. No information is currently available
on how binding of m4-1BB to ECM proteins affects its binding to
m4-1BBL and vice versa and if the ability of m4-1BB to
bind ECM proteins is conserved across species. We report that binding
of m4-1BBL to m4-1BB blocked its ability to bind laminin (LN), while
binding of m4-1BB to LN did not block its ability to bind m4-1BBL.
Furthermore, binding of m4-1BBL to the m4-1BB·LN complex did not
displace LN. These findings suggest the two ligands bind to proximal
but distinct sites on m4-1BB. This is supported by the observation
that six of eight anti-m4-1BB monoclonal antibodies blocked the
interaction between 4-1BB and 4-1BBL, while seven blocked LN binding.
Ligand and monoclonal antibody binding studies with a truncated protein
lacking the amino-terminal LN-homologous domain of m4-1BB demonstrated
that regions downstream of the LN-homologous domain participate in LN
binding and that the intact protein is required for m4-1BBL binding.
Studies with h4-1BB showed that h4-1BB only bound h4-1BBL,
indicating that the ECM binding activity of 4-1BB is not conserved
across species. This finding allowed the construction of murine/human
4-1BB chimeras, which permitted further dissection of the regions of
4-1BB involved in LN and 4-1BBL binding and suggests that sequence
differences in the LN-homologous domain of h4-1BB in part account for
the inability of h4-1BB to bind ECM proteins.
INTRODUCTION The T cell receptor-dependent activation of T cells is
tightly regulated by a discreet number of cell surface receptors,
including members of the tumor necrosis factor receptor
(TNFR)1 family. 4-1BB (CDw137) is a type I
membrane protein whose extracellular domain is related to the members
of the TNFR family (1, 2) and has low level homology to the
extracellular matrix protein laminin (LN) (3, 4). Early studies with an
immunoglobulin chimera of murine 4-1BB (m4-1BB-Ig) and with COS and L
cell transfectants expressing full-length m4-1BB showed that this
protein was able to bind extracellular matrix proteins including LN and
vitronectin (3). Later, similar m4-1BB-Ig and human 4-1BB-Ig
(h4-1BB-Ig) fusion proteins were used to isolate cDNAs encoding
the high affinity TNF-like ligands m4-1BBL and h4-1BBL, respectively
(5, 6).
M4-1BB was initially identified as a protein whose expression was
up-regulated following T cell activation (1). M4-1BB is predominantly
expressed by activated thymocytes and T cells but not by resting or
lipopolysaccharide-activated B cells (7). This contrasts with the
expression of mRNA encoding h4-1BB in human peripheral blood
mononuclear cells. Analogous to what has been observed in murine T
cells, h4-1BB is expressed by activated T cells; however, unlike
m4-1BB, transcripts encoding h4-1BB can be detected in activated
human peripheral blood B cells (8). RNA encoding h4-1BB is also
detected in interleukin-1 Early evidence for a role of m4-1BB in the regulation of the immune
response came from mAb cross-linking studies, which showed that
co-immobilized suboptimal concentrations of an anti-CD3 mAb with the
anti-m4-1BB mAb 53A2 could efficiently drive resting T cells to
proliferate (7). These studies were extended by Goodwin et
al. (5), who showed that CV-1 cells expressing m4-1BBL could
significantly enhance the proliferation of IL-2- and concanavalin A-stimulated thymocytes. M4-1BBL-expressing CV-1 cells could also significantly enhance the proliferation of
phytohemagglutanin-stimulated splenic T cells (5). Independent evidence
for a role of 4-1BB in T cell activation came from studies which
sought to identify the molecules responsible for the T cell
costimulatory activity of the B cell lymphoma K46J. These studies
showed that the costimulatory activity of K46J B cells could be blocked
with a soluble alkaline phosphatase chimera of m4-1BB (m4-1BB-AP) but
not with a soluble immunoglobulin chimera of CTLA4 (CTLA4-Ig) or mAbs
against B7-1, B7-2, intercellular adhesion molecule-1, or heat-stable
antigen (10). This is consistent with the results of experiments
showing that stimulation of purified resting T cells by anti-CD3 mAb
and fixed antigen presenting cells could be partially blocked with an
immunoglobulin chimera of m4-1BB (m4-1BB/Fc) (11). In addition, m4-1BB/Fc was capable of blocking IL-2 production by purified T cells
coactivated with anti-CD3 mAb and accessory cells (11). Similar studies
with an anti-h4-1BB mAb (M14), or CV-1 cells expressing the h4-1BBL,
showed that h4-1BB can function as a costimulatory molecule for
resting human T cells and enhance the anti-CD3 mAb-driven apoptosis of
chronically activated T cells (6). Consistent with a role of 4-1BB in
T cell signaling, the cytoplasmic domain of m4-1BB contains a
CXCXCP motif recognized by protein-tyrosine kinases of the Src family. Indeed, p56lck has been found to
associate with m4-1BB in the CD8+ T cell line CTLL-2
(12).
Although extracellular matrix proteins and the high affinity TNF-like
ligand have been shown to bind to m4-1BB, the relationship between the
two ligand binding sites on m4-1BB has not been explored. It is also
not clear whether h4-1BB is also able to bind extracellular matrix
proteins. In this study, we report on the results of experiments designed to investigate the relationship between the m4-1BB binding sites for its two ligands. Ligand cross-blocking studies were carried
out to investigate if the m4-1BBL and extracellular matrix binding
sites are distinct or not and to examine if this molecule can
simultaneously bind to both ligands. These studies were extended using
a series of truncated m4-1BB proteins and a panel of anti-m4-1BB mAbs
to further define the m4-1BBL and extracellular matrix binding sites
and their relative proximities. The recent cloning of the human
homologues of 4-1BB and 4-1BBL allowed us to test the ligand binding
properties of h4-1BB and to prepare murine/human 4-1BB chimeric
proteins to further examine the two ligand binding sites on
m4-1BB.
EXPERIMENTAL PROCEDURES COS cells were grown and
maintained in DMEM (Life Technologies, Inc.) supplemented with 10%
fetal bovine serum (FBS) and 100 units/ml penicillin and 100 µg/ml
streptomycin. Generation of the rat anti-m4-1BB monoclonal antibodies
is described elsewhere.2 The anti-Lyt2a mAb
53-6, directed against CD8, was a gift from Dr. J. Ledbetter
(Bristol-Myers Squibb, Seattle, WA).
Complementary
DNA (cDNA) encoding the full-length m4-1BB and CD40 molecules was
generated and subcloned as described previously (3). The full-length
h4-1BB cDNA was similarly generated by polymerase chain reaction
(PCR) using the following primers: 5 cDNA fragments encoding the extracellular region of
murine and human 4-1BB and 4-1BBL were obtained by PCR. The soluble
m4-1BB-Ig fusion protein, containing the CD5 signal sequence, was
constructed and prepared as described (3). The soluble h4-1BB-Ig
fusion protein, including its native signal sequence, was constructed and prepared as described (3) using the following primers: 5 The binding of purified
m4-1BB-Ig to rat laminin (Life Technologies, Inc.) was tested using an
ELISA assay as described (3). Blocking by sm4-1BBL was
tested by adding neat sm4-1BBL supernatant to the wells
for 1 h at 22 °C, followed by washing, and then the addition of
biotinylated rat laminin.
The binding of
purified 4-1BB-Ig to s4-1BBL was tested using an ELISA
assay. 96-well plates (Immulon-2, Dynatech, Chantilly, VA) were coated
with the anti-Lyt2a mAb 53-6 diluted to 1 µg/ml in PBS for 1 h
at 22 °C. The wells were blocked for 1 h at 22 °C. Wells
were washed, and serial dilutions of s4-1BBL supernatants were added and incubated for 1 h at 22 °C. Wells were washed, and purified 4-1BB-Ig or CD40-Ig was added at 2.5 µg/ml in PBS and
incubated 1 h at 22 °C. Wells were washed, and
peroxidase-conjugated goat anti-human IgG (1:10,000; Jackson
Immunoresearch, West Grove, PA) was added and incubated 1 h at
22 °C. Wells were washed and treated with the EIA chromogen
reagent (Genetics Systems, Seattle, WA) for 15 min at 22 °C. The
colorimetric reaction was quenched with 1 N
H2SO4, and the optical density was measured on
an ELISA plate reader at dual wavelengths of 450 and 630 nm. Blocking
by rat laminin was tested by preincubating 4-1BB-Ig with rat laminin at 10 µg/ml.
As an alternate format, purified 4-1BB-Ig (2 µg/ml) was captured on
goat anti-human IgG Fc-coated wells as described previously (3).
Following washing, serial dilutions of s4-1BBL
supernatants or purified s4-1BBL were added to the wells
and incubated 1 h at 22 °C. Wells were washed and biotinylated
anti-Ly-2 (CD8a) mAb (Caltag Laboratories, South San Francisco, CA)
diluted 1:200 in 1 × specimen diluent (SD, Genetics Systems,
Seattle, WA) was added and incubated 1 h at 22 °C. Wells were
washed, and horseradish peroxidase-conjugated streptavidin (Vector
Laboratories, Inc., Burlingame, CA) diluted 1:2000 in 1 × SD was
added to the wells and incubated 1 h at 22 °C. Wells were
washed and treated with the EIA chromogen reagent as described above.
Blocking by rat laminin was tested by incubating the wells with LN (10 µg/ml) for 1 h at 22 °C, followed by washing, then the
addition of s4-1BBL.
The binding of rat
anti-m4-1BB mAbs to the various 4-1BB-Ig fusion proteins was tested
using an ELISA assay. Purified 4-1BB-Ig (2 µg/ml) was captured on
goat anti-human IgG Fc-coated wells as described previously (3). The
wells were then blocked for 1 h at 22 °C. The wells were
washed, and purified rat anti-m4-1BB mAbs were added at 10 µg/ml in
1 × SD and incubated for 2 h at 22 °C. After washing,
horseradish peroxidase-conjugated goat anti-human IgG (Jackson
Immunoresearch, West Grove, PA) diluted 1:3000 in 1 × SD was
added and incubated 1 h at 22 °C. Wells were washed and treated
with the EIA chromogen reagent as described above. To examine the
ability of the rat anti-m4-1BB mAbs to block laminin and m4-1BBL
binding to m4-1BB-Ig fusion protein, an analogous ELISA assay was
performed. Following incubation of the rat anti-m4-1BB mAbs (10 µg/ml in 1 × SD) on m4-1BB-Ig fusion protein-coated wells for
1 h at 22 °C, biotinylated rat laminin (5 µg/ml) or purified sm4-1BBL (0.05 µg/ml) was added to antibody-containing
wells and incubated 1 h at 22 °C. Wells were washed, and
sm4-1BBL was detected with biotinylated anti-Ly-2 (CD8a)
mAb, followed by horseradish peroxidase-conjugated streptavidin.
Biotinylated laminin was detected with horseradish
peroxidase-conjugated streptavidin directly. Wells were washed and
treated with the EIA chromogen reagent as described above.
The binding of
sm4-1BBL, sh4-1BBL, sgp39, and LN
to m4-1BB, h4-1BB, and CD40 was tested by staining transfected COS
cells. COS cells were either mock-transfected or transfected with
full-length m4-1BB, h4-1BB, or CD40 cDNAs using DEAE-dextran
(15). One day after transfection, cells were trypsinized and replated.
Two days later, cells were washed with PBS then recovered following
incubation with Ca2+/Mg2+-free PBS containing
0.5 mM EDTA. Cells were stained with sm4-1BBL, sh4-1BBL, or sgp39 supernatants (1:10 in DMEM,
2% FBS) for 1 h on ice, followed by incubation with biotinylated
anti-Ly-2 (CD8a) mAb (1:200 in DMEM, 2% FBS) for 45 min on ice, then
avidin-FITC (1:500 in DMEM, 2% FBS; Molecular Probes, Eugene, OR) for
45 min on ice. Cells were stained with biotinylated LN (10 µg/ml in
DMEM, 2% FBS) for 2 h on ice followed by avidin-FITC (1:500 in
DMEM, 2% FBS) for 45 min on ice. Flow cytometry was performed with a FACScan unit (Becton Dickinson, San Jose, CA). At least 5000 viable cells were analyzed in each sample.
Initially, segments of the 4-1BB
extracellular region sequence were aligned manually against sequences
of members of the TNFR family to identify TNFR repeat domains in
4-1BB. Due to the low sequence similarities, these alignments were
guided by structural consensus residues of TNFR (18). 4-1BB sequence
segments outside identified TNFR-like domains were then searched
against the SwissProt and GenPept data bases using GCG programs.
Obtained alignments for these regions were manually modified to adjust
gaps.
RESULTS Our
initial attempts to identify a ligand for m4-1BB showed that a
m4-1BB-Ig fusion protein and COS and L cell transfectants expressing
m4-1BB were able to bind to extracellular matrix proteins. Subsequently, using a similar m4-1BB-Ig fusion protein, another group
identified and cloned a second m4-1BB ligand, which they named
m4-1BBL (5). This molecule, which is expressed by activated T cells,
is homologous to TNF. Although other lymphocyte cell surface receptors
such as CD2, CD28, and integrins (for example lymphocyte
function-associated molecule-1 and very late antigen-4 (VLA-4)) have
more than one ligand, 4-1BB is the first member of the TNFR family
known to bind both a TNF-like molecule and extracellular matrix
proteins.
To gain a better understanding of the interaction of m4-1BB with its
ligands, we carried out a series of binding and cross-blocking studies
using m4-1BB-Ig, LN, and a soluble recombinant form of m4-1BBL,
sm4-1BBL. As shown in Fig. 1A,
sm4-1BBL bound to m4-1BB-Ig but not to the immunoglobulin
fusion of the related receptor CD40, CD40-Ig. Ligand cross-blocking
studies showed that the interaction between m4-1BB and
sm4-1BBL was not blocked by preincubating m4-1BB with a
high concentration of LN (10 µg/ml). Furthermore, the binding of
sm4-1BBL to the LN-m4-1BB complex did not result in the
displacement of LN, indicating that m4-1BB was capable of
simultaneously binding to both ligands (Fig. 1C). However,
preincubating m4-1BB with a high concentration of
sm4-1BBL prevented LN from binding m4-1BB-Ig (Fig.
1B). In these studies, CD40-Ig binding to
sm4-1BBL and LN was carried out to evaluate nonspecific
binding. As reported previously, CD40-Ig was unable to bind to either
m4-1BBL or LN (Fig. 1, A and B). These studies
suggest that the interaction of m4-1BB with m4-1BBL and LN is
specific, and that m4-1BBL and LN bind to distinct but proximal sites
on m4-1BB.
To further
characterize the LN and m4-1BBL binding sites on m4-1BB, eight
anti-m4-1BB mAbs were examined for their ability to block the binding
of m4-1BB to either m4-1BBL or LN. As shown in Fig.
2A, six of the eight anti-m4-1BB mAbs
effectively blocked the binding of m4-1BB to sm4-1BBL. On
the other hand, seven of the mAbs were able to effectively block the
interaction between m4-1BB and LN (Fig. 2B). As
expected the interaction of m4-1BB with either m4-1BBL or LN could
not be blocked with a mAb directed to gp39, the TNF-like ligand of CD40
(14) (Fig. 2). These findings provide further evidence that the
m4-1BBL and LN binding sites of m4-1BB are distinct.
A preliminary comparison of the amino acid sequence of
the extracellular domain of m4-1BB with that of other proteins
revealed the presence of regions homologous to both members of the TNFR family and LN. This observation led us to reexamine in more detail the
extracellular domain organization of m4-1BB and its homology to other
proteins. Consistent with an earlier proposal (2), only two TNFR
domains could be identified with certainty in the extracellular region
of m4-1BB (Fig. 3). The two TNFR-like domains in
m4-1BB are best aligned with TNFR domains 1 and 3, respectively. These
two TNFR-like domains are not adjacent, but separated by a short
cysteine-rich segment. Data base searches with the sequence of this
short segment revealed significant matches of its cysteine pattern to
that found in metallothionein (MT; Fig. 3). MT is a small cysteine-rich
cytoplasmic protein, which binds divalent cations using clusters of
cysteine residues (16) and is not related to TNFR molecules. However,
on the cell surface, a motif with cysteine spacing similar to MT may
form a small and highly disulfide bond constrained domain. The region
of m4-1BB/MT homology overlaps with the COOH-terminal region of the
first TNFR-like domain of m4-1BB (Fig. 3). Although the structural
significance of this overlap remains unclear, the finding that the two
TNFR-like domains in m4-1BB may be connected by a constrained
structural motif suggests that this domain organization may result in a
"fusion" of the two TNFR domains. In contrast, the
NH2-terminal region in m4-1BB displays sequence
similarities to LN (Fig. 3). The x-ray structure of three consecutive
LN-type epidermal growth factor-like modules was reported recently
(17). These modules were arranged in a linear "stacked" fashion,
reminiscent of the gross domain arrangement of TNFR (18). Based on this
structure and the amino acid sequence alignment (Fig. 3), it appears
that the LN-homologous domain of m4-1BB includes about half of a LN
repeat module, corresponding to the disulfide stabilized "loops" d
and c (17). Taken together, these observations suggest the possibility
of a combined arrangement of a LN-type and two TNFR-type domains.
To begin investigating if the different ligand binding
properties of m4-1BB resided within discrete domains or required
multiple domains of the extracellular region of m4-1BB, we prepared a
series of DNA constructs encoding truncated forms of m4-1BB (a total of nine constructs) lacking various NH2- and COOH-terminal
extracellular domains and domain fragments. The initial design of these
constructs was based on early sequence alignment studies, which
predicted that 4-1BB contained one LN-homologous domain and three
TNFR-like domains. Subsequent sequence alignment of 4-1BB, as
described herein, predicted that the majority of these truncated
proteins contained incomplete domains. Most of the proteins encoded by these constructs were unable to bind to our panel of anti-m4-1BB mAbs,
m4-1BBL, and LN and thus were not further studied (data not shown).
However one truncated protein, m4-1BBdLN (Fig.
4A), which lacked only the amino-terminal
LN-homologous domain of m4-1BB was recognized by the anti-m4-1BB mAb
1D8, the mAb that blocked LN binding but not m4-1BBL binding (Fig.
4B). This protein was capable of weakly binding to LN but
was no longer able to bind m4-1BBL (Fig. 4, C and
D). These findings suggest that some of the binding
determinants responsible for m4-1BB-LN interactions reside downstream
from the LN homologous domain of m4-1BB and that the presence of an
intact LN-homologous domain is required for m4-1BBL binding.
Recently, cDNA
clones encoding the putative human homologues of m4-1BB (19) and
m4-1BBL (6) were isolated and characterized. To determine if the
interaction between m4-1BB and extracellular matrix ligands was
conserved in different species, we isolated cDNA clones encoding
h4-1BB and h4-1BBL. COS cell transfectants expressing h4-1BB bound
to sh4-1BBL but were unable to bind LN (Fig.
5A). These findings contrast with those using
COS cell transfectants expressing m4-1BB, which were capable of
binding efficiently to sm4-1BBL and LN (Fig.
5B). COS cell transfectants expressing either h4-1BB or
m4-1BB were unable to bind sgp39. COS cell transfectants expressing CD40 bound to gp39, but were unable to bind to m4-1BBL, h4-1BBL, or LN (Fig. 5C). To ensure that the inability of
h4-1BB to bind extracellular matrix proteins was not due to our assay format, we prepared a chimeric gene encoding an h4-1BB immunoglobulin fusion protein, h4-1BB-Ig. As shown in Fig. 6,
h4-1BB-Ig bound to sh4-1BBL but not LN. These findings
suggest that the extracellular matrix binding activity of 4-1BB is not
conserved in different species.
Comparison of the amino acid sequence of h4-1BB with m4-1BB, other
TNFR molecules, LN and MT suggested equivalent domain organization in
m4-1BB and h4-1BB. Like m4-1BB, the amino-terminal region of h4-1BB
is homologous to LN. However, the c loop of LN is more closely mimicked
by murine than by human 4-1BB sequences (Fig. 3). Presently it is
unclear if these gross sequence differences are sufficient to account
for the differences in the ability of human and murine 4-1BB to bind
LN.
The isolation of cDNA clones encoding h4-1BB opened
a different avenue to study the structural organization and function of 4-1BB. The finding that h4-1BB was unable to bind to LN permitted further investigation as to which regions of m4-1BB are involved in
m4-1BBL and LN binding through the generation of human/mouse 4-1BB
chimeric proteins. In one construct, termed m/h4-1BB, the LN-homologous domain of m4-1BB replaced the equivalent domain of
h4-1BB (Fig. 7A). In the converse construct,
termed h/m4-1BB, the LN-homologous domain of h4-1BB replaced the
equivalent domain of m4-1BB (Fig. 7A). Ligand binding
studies showed that replacement of the h4-1BB LN-homologous domain
with the equivalent m4-1BB domain conferred weak binding to LN (Fig.
7B). The m/h4-1BB chimeric protein displayed reduced
binding to sh4-1BBL (Fig. 7C) and was unable to
bind sm4-1BBL (data not shown). Conversely, replacement of
the m4-1BB LN-homologous domain with the equivalent h4-1BB domain
abolished LN binding, while it did not alter binding to sm4-1BBL (Fig. 7, D and E). The
h/m4-1BB chimera was unable to bind to sh4-1BBL (data not
shown). CD40-Ig was used as a control in these experiments and, as
shown previously, was unable to bind LN, sm4-1BB, or
sh4-1BB (Fig. 7). These findings suggest that a portion of
the LN binding determinants of m4-1BB are located in the LN-homologous
region of m4-1BB and that some of the binding determinants
required by 4-1BBL ligand reside in the TNFR homologous region of
4-1BB. In addition, these findings indicate that the inability of
h4-1BB to bind LN is in part due to amino acid differences in
the LN-homologous domain of m4-1BB and h4-1BB.
DISCUSSION There are multiple examples of leukocyte cell surface receptors
with more than one ligand; however, to date 4-1BB is the only member
of the TNFR supergene family that has been shown to bind to both a
TNF-like protein and a protein that is not related to TNF. The finding
that m4-1BB is capable of binding to both LN and m4-1BBL suggests
that m4-1BB has a dual function as a cell adhesion and activation
molecule. A number of studies have shown that the interaction between
m4-1BBL and m4-1BB results in m4-1BB-mediated intracellular
signaling. By analogy to other members of the TNFR/TNF family of
receptor/ligands, this likely occurs via a m4-1BBL-driven receptor
oligomerization, which is mediated by the trimeric nature of the
TNF-like ligand. The observation that m4-1BBL is capable of binding to
the m4-1BB-LN complex suggests that m4-1BBL-m4-1BB signaling is
likely to take place even if m4-1BB is binding extracellular matrix
proteins. This is significant since extracellular matrix proteins such
as LN are very abundant and widely distributed and are likely to bind
m4-1BB shortly after it is expressed on the surface of activated T
cells. Presently there is no information on the signaling effects that
might result from the formation of molecular complexes between m4-1BB
and extracellular matrix or on the effect that extracellular matrix
protein binding might have on m4-1BBL-mediated signaling.
The finding that m4-1BB was capable of simultaneously binding to
m4-1BBL and LN provides evidence that the binding sites for these two
ligand are distinct. Additional evidence for this is provided by the
identification of an anti-m4-1BB mAb, which is capable of blocking LN
binding to m4-1BB but was unable to effectively block the interaction
between m4-1BB and m4-1BBL. The dual ligand binding activity of
m4-1BB is not unique. The leukocyte antigen VLA-4 is capable of
binding to vascular cell adhesion molecule-1 (VCAM-1) (20), a type I
cell surface protein of the Ig supergene family, and fibronectin (21).
Similar to our observations with m4-1BB, VLA-4 is able to bind VCAM-1
and fibronectin via distinct sites. In this system it appears that the
function of binding to the different ligands is overlapping, with both
interactions being involved in VLA-4-mediated signaling and cell
adhesion. The availability of the anti-m4-1BB mAb 1D8, which is
capable of blocking extracellular matrix protein binding without
affecting m4-1BBL binding, as well as other anti-m4-1BB mAbs, which
are capable of blocking the interaction between m4-1BB and its
two ligands, provides an avenue for the investigation of the role of
extracellular matrix protein binding on m4-1BB signaling.
Unexpectedly, we found that h4-1BB was unable to bind the
extracellular matrix protein LN. Although we cannot rule out the possibility that expression of h4-1BB in a heterologous mammalian expression system (COS cells instead of T cells) selectively affected the ability of h4-1BB to bind extracellular matrix proteins, this is
unlikely. It is more likely that this function of 4-1BB is not
conserved. This is not unique to 4-1BB and has been observed with the
T cell antigen CD2, which has been reported to bind both CD48 (22) and
CD58 (23) in the murine system. However, human CD2 is only able to bind
CD58 with high affinity (24). Based on our data, the function of 4-1BB
in the regulation of the immune system in humans and mice may be
different. This suggests that data obtained from in vitro
and in vivo experiments investigating the function of 4-1BB
in murine systems may not be fully predictive of the function of 4-1BB
in the regulation of the human immune system.
A detailed comparison of the amino acid sequence of both human and
murine 4-1BB with that of other members of the TNFR family and with
other published proteins clearly shows that the domain organization of
4-1BB differs from other members of the family. In particular, 4-1BB
contains at its amino terminus a LN-homologous domain and has only two
extracellular TNFR-like domains. Additionally, in a region upstream of
the second TNFR-like domain of 4-1BB, which overlaps the COOH-terminal
portion of the first TNFR-like domain of 4-1BB, we identified a
cysteine-rich motif that is homologous to MT. The availability of
crystallographic data on the structure of a LN fragment (17) and the
TNFR (18) has shown that these proteins are elongated structures built
by the "stacking" of either LN-like or TNFR-like domains,
respectively. This suggests that LN-like and TNFR-like subdomains of
4-1BB may be combined. These observation, in conjunction with the
results of our sequence alignment studies, led us to investigate
whether the different ligand binding activities of m4-1BB could be
segregated into discreet subdomains or groups of domains. Most of the
data obtained from these studies was not readily interpretable, since
most of the m4-1BB proteins lacking one or more extracellular domains
failed to bind m4-1BBL and LN, and were no longer recognized by any of
our anti-m4-1BB mAbs. This further suggests an intimate association of
these domains. There was one exception, the m4-1BBdLN-Ig protein,
which lacked the LN-like domain of m4-1BB. This m4-1BBdLN-Ig protein
was recognized by the anti-m4-1BB mAb 1D8 and was able to weakly bind
to LN. The latter finding, in conjunction with the observation that mAb 1D8 is capable of blocking the interaction between m4-1BB and LN
without affecting m4-1BBL binding, clearly indicates that regions located carboxyl-terminal to the LN-like domain of m4-1BB are involved
in LN binding. This finding was surprising since we had hypothesized,
based on the observation that LN can form homoaggregates, that the
m4-1BB-LN interaction was mediated via interactions between the
LN-like domain of m4-1BB and LN. However, the finding that the
m/h4-1BB chimera, which contains the LN-like domain from m4-1BB and
the TNFR-like regions of h4-1BB, is capable of weakly binding to LN,
while the h/m4-1BB chimera does not bind LN, indicates that the
LN-like motif of m4-1BB also contributes to LN binding.
The observation that the m4-1BBdLN-Ig protein was unable to bind to
m4-1BBL suggests that the interaction between 4-1BB and its TNF-like
ligand requires that the extracellular domain of 4-1BB be intact.
Interestingly, the m/h4-1BB chimera displayed reduced binding to
h4-1BBL, while the h/m4-1BB chimera displayed binding to m4-1BBL
equivalent to that of the native m4-1BB protein. This suggests that
the 4-1BBL binding determinants contributed by the LN-homologous
domain of h4-1BB are capable of supporting m4-1BBL binding but not
vice versa. This one-sided interaction between ligands and
receptors across species has been observed in other systems, including
interleukin-2 (25) and interleukin-6 (26). To date there are no reports
describing the results of experiments in which the extracellular
domains of other members of the TNFR family have been truncated to map
the interaction between this receptor family and their respective
ligands. However, a subset of human patients with human autoimmune
lymphoproliferative syndrome have defects in Fas expression and
function (27). One of these patients was shown to have a mutation that
led to aberrant splicing, resulting in the deletion of a portion of the
extracellular domain of Fas. T cells from this patient were unable to
respond to Fas, suggesting that this truncation was either not
expressed or, if expressed, was unable to bind Fas. Our in
vitro studies with truncated forms of m4-1BB are consistent with
the second explanation. Although studies with truncated forms of
m4-1BB and with the m/h4-1BB chimera provide some information of the
molecular mechanisms that allow 4-1BB to interact with its ligands, in
general we found that manipulation of the extracellular region of this protein was difficult and resulted in structural perturbations that
compromised its binding functions.
In summary, this study provides the first in-depth analysis of the
molecular basis of the interaction of 4-1BB with its different ligands
in different species, and allow the identification of a mAb that is
capable of blocking the interaction between m4-1BB and extracellular
matrix proteins but not m4-1BBL. Thus, the design of functional
experiments to shed light on the role of the interaction between
m4-1BB and its different ligands in regulating the immune response may
now be possible. Recently, molecules in this family of proteins have
been targeted for the development of novel drugs to treat immunological
diseases in humans. The development of these drugs has relied upon the
use of murine models of human disease. The finding that the
extracellular matrix binding activity of m4-1BB is not conserved in
humans indicates that in vivo studies in mice designed to
evaluate the efficacy of targeting 4-1BB function to treat human
disease should be interpreted with caution in light of the different
binding functions of this receptor in these two species.
FOOTNOTES Acknowledgments We thank Bill Bear for preparation of
oligonucleotides; Mike Neubauer, Joe Cook, and Trent Youngman for DNA
sequencing; Dr. James McCarthy and Dr. Amy Skubitz for helpful
discussions; and Debby Baxter for assistance in preparation of the
manuscript.
REFERENCES
Volume 272, Number 10,
Issue of March 7, 1997
pp. 6448-6456
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
§,¶,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
(IL-1)-activated or
phorbol-12-myristate-13-acetate-activated peripheral blood monocytes
(8). In addition, transcripts encoding h4-1BB can be detected in an
activated human T cell line (Jurkat), in resting human B cell lines
(Raji and Epstein-Barr virus transformed B cells from normal donors),
and in a number of unstimulated and IL-1 stimulated nonlymphoid cell
lines (8). Expression of 4-1BB by activated murine and human T cells
increases gradually over time. Studies using anti-CD3 monoclonal
antibodies (mAb) or concanavalin A-activated murine T cells showed that
following activation the level of m4-1BB detectable on the surface of
T cells steadily increases over 3 days (9). Additionally, h4-1BB mRNA expression by anti-CD3 mAb-activated peripheral blood
mononuclear cells can be detected at 4 h and remains strongly
expressed at 48 h (8).
-CCG AAG CTT GCT TTG CTA GTA TCA
TAC CTG TGC-3 and 5-CCG CTG CAG GCT GTG ATA GCG GAT GAC TCA-3.
-CCG AAG
CTT GCT TTG CTA GTA TCA TAC CTG TGC-3 and 5-GCA GAT CTT GCG GAG AGT
GTC CTG GCT CTC TCG C-3. The soluble m4-1BBL fusion protein
(sm4-1BBL) was constructed using the following primers:
5-GCG GCG GAT CCC CGC ACC GAG CCT CGG CCA GCG-3 and 5-CGC TCT AGA
GGA TAG TTC TCA TTC CCA TGG-3. The m4-1BBL extracellular domain PCR
product was digested with BamHI and XbaI and
ligated into a BamHI/XbaI-cut CDM8 vector
containing the extracellular region of Lyt2a (CD8) (13). The soluble
h4-1BBL fusion protein (sh4-1BBL) was constructed
similarly, using the following primers: 5-CTG GGA TCC AGC TCG CGC CTC
GCC CGG CTC CGC-3 and 5-CCG TCT AGA CCA TGA AGG ATG GAG TAG GAT
TCG-3. The soluble CD40-Ig (sCD40-Ig) and gp39
(sgp39) fusion proteins were constructed and prepared as
described previously (13, 14). The soluble m4-1BBdLN-Ig fusion protein
(sm4-1BBdLN-Ig; containing residues 38-185) in which the
LN-homologous domain is deleted was constructed and prepared as
described (3) using the following primers: 5-CCG CGG GTA CCA AGA AAA
TAC AAT CCA GTC TCC AAG-3 and 5-CCG CTA CGT AGG ATC CTG CAA GGA GTG
CCC TCC TGG-3. Note that the cysteine at residue 44 is changed to a
serine. The soluble chimeric m/h4-1BB-Ig fusion protein, in which the
LN-homologous domain of m4-1BB was put in place of the LN-homologous
domain of h4-1BB was constructed by a two-step "sew" PCR using
plasmids encoding m4-1BB-Ig and h4-1BB-Ig as templates. The m4-1BB
LN-homologous domain (residues 24-44) was generated using the
following primers: 5-AAT ACG ACT CAC TAT AGG-3, which primes in the
vector sequence upstream of m4-1BB, and 5-GCA GAC TGG ATT GTA TTT TCT
GCA GAA GGT ACC AGG-3. The h4-1BB domain (residues 46-187) was
generated using the following primers: 5-CCT GGT ACC TTC TGC AGA AAA
TAC AAT CCA GTC TGC AGT CCC TGT CCT CCA AAT AGT TTG TCC AGC-3 and
5-GCA GAT CTT GCG GAG AGT GTC CTG GCT CTC TCG C-3. The two PCR
products were joined together using the flanking primers 5-AAT ACG ACT
CAC TAT AGG-3, which primes in the vector sequence upstream of
m4-1BB, and 5-GCA GAT CTT GCG GAG AGT GTC CTG GCT CTC TCG C-3. The
soluble chimeric h/m4-1BB-Ig fusion protein, in which the
LN-homologous domain of h4-1BB was put in place of the LN-homologous
domain of m4-1BB was constructed by an analogous two-step "sew"
PCR using plasmids encoding m4-1BB-Ig and h4-1BB-Ig as templates. The
h4-1BB LN-homologous domain (residues 1-45) was generated using the
following primers: 5-CCG AAG CTT GCT TTG CTA GTA TCA TAC CTG TGC-3
and 5-GCA AAT CTG ATT CCT GTT ATT ATC ACA GAA TGT ACC-3. The m4-1BB
domain (residues 45-185) was generated using the following
primers: 5-GGT ACA TTC TGT GAT AAT AAC AGG AAT CAG ATT TGC AAG AGC TGC
CCT CCA AGT ACT TTC TCC AGC-3 and 5-GGC CGG ATC CTG CAA GGA GTG CCC TCC TGG TCC-3. The two PCR products were joined together using the
flanking primers 5-CCG AAG CTT GCT TTG CTA GTA TCA TAC CTG TGC-3
and 5-GGC CGG ATC CTG CAA GGA GTG CCC TCC TGG TCC-3.
Fig. 1.
A, binding of m4-1BBL to m4-1BB-Ig and
blocking by laminin. Serial dilutions of m4-1BBL supernatant were
immobilized on plastic coated with anti-Lyt2A mAb 53-6 and its ability
to bind m4-1BB-Ig was measured by an ELISA. The ability of laminin to
block binding of m4-1BBL to m4-1BB-Ig was tested by preincubating
m4-1BB-Ig with rat laminin (10 µg/ml). As a control, the binding
CD40-Ig to m4-1BBL was monitored. B, binding of laminin to
m4-1BB-Ig and blocking by m4-1BBL. m4-1BB-Ig was immobilized on
plastic coated with affinity-purified goat anti-human IgG antibodies,
and its ability to bind to increasing amounts of biotinylated rat
laminin was assessed by ELISA. The ability of m4-1BBL to block laminin binding to m4-1BB was tested by incubating the wells with m4-1BBL supernatant prior to addition of biotinylated rat laminin. As a
control, the binding CD40-Ig to m4-1BBL was monitored. C,
the displacement of laminin by m4-1BBL was examined by ELISA.
m4-1BB-Ig was immobilized on plastic coated with affinity-purified
goat anti-human IgG antibodies. The ability of m4-1BBL to displace laminin was tested by first incubating the wells with laminin (10 µg/ml), followed by the addition of m4-1BBL supernatant, then staining the wells for laminin (LN/m4-1BBL (LN)). Blocking
of m4-1BBL binding to m4-1BB-Ig by laminin was assessed by first incubating the wells with laminin (10 µg/ml), followed by the addition of m4-1BBL supernatant, then staining the wells for m4-1BBL (LN/m4-1BBL (m4-1BBL)). Blocking of laminin by m4-1BBL
was assessed as in B (m4-1BBL/LN (LN)). For
positive controls, the ability of laminin (LN) or m4-1BBL
(m4-1BBL) alone to bind to m4-1BB-Ig was tested.
m4-1BB-Ig was immobilized on plastic coated with affinity-purified goat anti-human IgG antibodies, and its ability to bind m4-1BBL supernatant or biotinylated rat laminin (10 µg/ml) was assessed by
ELISA as above. Data points represent the mean ± S.E. value (n = 3 for each data point).
[View Larger Version of this Image (14K GIF file)]
Fig. 2.
Blocking of m4-1BBL and laminin binding to
m4-1BB-Ig by rat anti-m4-1BB monoclonal antibodies. m4-1BB-Ig
was immobilized on plastic coated with affinity-purified goat
anti-human IgG antibodies. m4-1BBL (0.05 µg/ml) (A) or
biotinylated rat laminin (5 µg/ml) (B) was added to
antibody-containing wells (10 µg/ml), and ligand binding was measured
using an ELISA. The anti-gp39 antibody, 39.5, represents a negative
control. Data points represent the mean ± S.E. value
(n = 3 for each data point).
[View Larger Version of this Image (20K GIF file)]
Fig. 3.
Alignment of the extracellular region
sequences of murine and human 4-1BB (41BB) with members of
the tumor necrosis factor receptor family (TNFR: 55-kDa
form of the tumor necrosis factor receptor, CD40,
andFas) and other molecules (laminin-
(LMA), laminin-1 (LMB1), laminin-2
(LMB2), metallothionein-IB (MT1B), metallothionein-IIB (MT2B), and metallothionein-II
(MT2)) from different species. Only regions with
similarities to 4-1BB sequences are shown. The alignments were
generated as described under "Experimental Procedures." Sequence
numbers are given for the predicted mature murine 4-1BB sequence (28),
but we have included an NH2-terminal sequence extension,
which shows similarity to laminin. D1 and D3
indicate domains 1 and 3 in TNFR. Asterisks mark residues
that are identical in more than half of the compared sequences,
including at least one 4-1BB sequence, or conserved in residue
character (e.g. large hydrophobic/aromatic, charged, large
charged/polar etc.). The so defined sequence similarities are ~50%
for the LN region (residues 
6 to 22), ~36% for TNFR domain 1 (23-64), ~48% for the MT region (55-83), and ~35% for TNFR
domain 3.
[View Larger Version of this Image (59K GIF file)]
Fig. 4.
Binding of rat anti-m4-1BB monoclonal
antibodies, rat laminin, and m4-1BBL to m4-1BB-Ig and m4-1BBdLN-Ig.
A, schematic representation of m4-1BB-Ig and m4-1BBdLN-Ig
immunoglobulin fusion proteins. The laminin-homologous domain
(LN), TNF-like domains (TNFr1 and
TNFr3), and the metallothionein domain (MT) are
shown in the open boxes. The human immunoglobulin Fc region,
containing the hinge and second (CH2) and third
(CH3) constant regions of the human IgG1 are shown in the
shaded box. m4-1BB-Ig or m4-1BBdLN-Ig was immobilized on
plastic coated with affinity-purified goat anti-human IgG antibodies
and their ability to bind rat anti-m4-1BB mAbs (B),
increasing concentrations of biotinylated rat laminin (C),
or increasing concentrations of m4-1BBL (D) was measured using an ELISA. For antibody binding, the anti-gp39 antibody, 39.5, represents a negative control. For ligand binding, CD40-Ig represents a
negative control. Data points represent the mean ± S.E. value
(n = 3 for each data point).
[View Larger Version of this Image (25K GIF file)]
Fig. 5.
Binding of 4-1BBL and laminin to
4-1BB-transfected COS cells. Flow cytometry profiles of the
binding of the indicated ligands to h4-1BB-transfected COS cells
(A), m4-1BB-transfected COS cells (B), and
CD40-transfected COS cells (C) (dark profiles). The empty profiles represent mock-transfected COS
cells.
[View Larger Version of this Image (34K GIF file)]
Fig. 6.
Binding of h4-1BBL and laminin to m4-1BB-Ig
and h4-1BB-Ig. m4-1BB-Ig, h4-1BB-Ig, and CD40-Ig were
immobilized on plastic coated with affinity-purified goat anti-human
IgG antibodies and their ability to bind to increasing concentrations
of h4-1BBL supernatant (A) or increasing concentrations of
biotinylated rat laminin (B) was measured using an ELISA
assay. CD40-Ig represents a negative control. Data points
represent the mean ± S.E. value (n = 3 for each
data point).
[View Larger Version of this Image (13K GIF file)]
Fig. 7.
Binding of m4-1BBL, h4-1BBL, and laminin to
the chimeric m/h4-1BB-Ig. A, schematic representation
of m/h4-1BB-Ig and h/m4-1BB-Ig immunoglobulin fusion proteins. The
murine laminin-homologous domain (LN) is shown in the
open box; the human laminin-homologous domain
(LN) is shown in the lightly shaded box. The
murine TNF-like domains (TNFr1 and TNFr3) and the
metallothionein domain (MT) are shown in the open
boxes; the human TNF-like domains (TNFr1 and
TNFr3) and the metallothionein domain (MT) are
shown in the lightly shaded boxes. The human immunoglobulin
Fc region, containing the hinge and second (CH2) and third
(CH3) constant regions of the human IgG1 are shown in the
darkly shaded box. m4-1BB-Ig, h4-1BB-Ig, m/h4-1BB-Ig,
h/m4-1BB-Ig, and CD40-Ig were immobilized on plastic coated with
affinity-purified goat anti-human IgG antibodies and their ability to
bind to increasing concentrations of biotinylated rat laminin
(B and D), increasing concentrations of h4-1BBL
(C), or increasing concentrations of m4-1BBL was measured
using an ELISA (E). CD40-Ig represents a negative
control. Data points represent the mean ± S.E. value
(n = 3 for each data point).
[View Larger Version of this Image (26K GIF file)]
The contribution of the first two authors should be considered
equal.
§
To whom correspondence should be addressed: BMS-PRI, 3005 First
Ave., Seattle, WA 98121. Tel.: 206-728-4800; Fax: 206-727-3602.
¶
Present address: Fred Hutchinson Cancer Research Center, 1124 Columbia, Seattle, WA 98104.
1
The abbreviations used are: TNFR, tumor necrosis
factor receptor; TNF, tumor necrosis factor; LN, laminin; IL-1,
interleukin-1; mAb, monoclonal antibody; MT, metallothionein; VLA-4,
very late antigen-4; VCAM-1, vascular cell adhesion molecule-1; FBS,
fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PCR,
polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay;
FITC, fluorescein isothiocyanate.
2
W. W. Shuford, K. Klussman, D. D. Tritchler, D. T. Loo, J. Chalupny, A. W. Siadak, T. J. Brown, J. Emswiler, H. Raecho,
C. P. Larsen, T. C. Pearson, J. A. Ledbetter, A. Aruffo, and Robert S. Mittler, manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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