(Received for publication, October 23, 1996, and in revised form, December 23, 1996)
From Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121
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.
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 (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).
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.
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).
Construction of Full-length 4-1BB cDNAComplementary
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-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
.
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-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
.
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.
Binding of 4-1BB-Ig to s4-1BBLThe 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.
Binding of Rat Anti-m4-1BB Monoclonal Antibodies to 4-1BB-Ig Fusion Proteins and Blocking of Ligand BindingThe 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.
Binding Assays in COS CellsThe 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.
Sequence AlignmentsInitially, 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.
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.
Ligand Blocking Studies with Anti-m4-1BB mAbs
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.
4-1BB Extracellular Domain Organization and Sequence Alignment
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.
Ligand and mAb Binding to a Panel of Truncated m4-1BB Proteins
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.
Binding of h4-1BB to h4-1BBL and LN
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.
Preparation and Binding Studies with Chimeric 4-1BB ProteinsThe 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.
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.
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.