(Received for publication, March 26, 1997)
From the § Division of Molecular Immunology, La Jolla
Institute for Allergy and Immunology, San Diego, California 92121, the
Department of Biochemistry, University of California,
Riverside, California 92021, and the ¶ Institute of Molecular and
Cell Biology, National University of Singapore, 10 Kent Ridge Crescent
S., 119260 Republic of Singapore
The lymphotoxins (LT) and
, members of the
tumor necrosis factor (TNF) cytokine superfamily, are implicated as
important regulators and developmental factors for the immune system.
LT
is secreted as a homotrimer and signals through two TNF receptors of 55-60 kDa (TNFR60) or 75-80 kDa (TNFR80). LT
also assembles with LT
into a membrane-anchored, heterotrimeric LT
1
2 complex that engages a distinct cognate receptor, the LT
receptor (LT
R). To investigate the role of the LT
subunit in the function of the
membrane LT
1
2 complex, gene transfer via baculovirus was used to
assemble LT
and -
complexes in insect cells. LT
containing mutations at D50N or Y108F are secreted as homotrimers that fail to
bind either TNF receptor and are functionally inactive in triggering cell death of the HT29 adenocarcinoma cell line. In contrast, these
mutant LT
proteins retain the ability to co-assemble with LT
into
membrane-anchored LT
1
2 complexes that engage the LT
R and
trigger the death of HT29 cells. Membrane-anchored LT
expressed on
the cell surface in absence of the LT
subunit binds the LT
R but
is functionally inactive in the cell death assay. These results indicate that the TNF receptor-binding regions of the LT
subunit are
not necessary for engagement of the LT
R, but the LT
subunit is
required for the assembly of LT
into a functional heteromeric ligand.
Lymphotoxins (LT)1 and
are structurally related to TNF, the prototypical member of a
superfamily of type II transmembrane glycoproteins (1, 2). These
cytokines also exist in soluble forms, although distinct mechanisms
generate secreted and membrane-bound LT
and TNF. Secreted TNF is
generated by proteolysis of the transmembrane protein (3-5), whereas
LT
lacks a transmembrane domain and is exclusively secreted as a
homotrimer (and in this form is also known as TNF
). Unlike TNF,
LT
also assembles with LT
into heteromeric complexes and is
consequently localized to the cell surface by the transmembrane domain
of LT
(6, 7). Substantial evidence indicates that membrane LT exists
in two trimeric forms with either an
1
2 or
2
1 stoichiometry
(6, 8). The secreted and membrane-bound forms of LT are further
distinguished by their distinct specificities for cell surface
receptors. LT
and TNF both bind and signal through two receptors,
the 55-60-kDa TNF receptor (TNFR60; CD120a or type 1) (9, 10) and the
75-80-kDa TNFR (TNFR80; type 2 or CD120b) (11). By contrast, the
surface LT
1
2 complex binds a related but distinct receptor,
termed LT
R, that does not bind either LT
or TNF, whereas both
TNFRs bind the LT
2
1 heterotrimer (8, 12). The LT
1
2 complex
is the most abundant form expressed by activated T cells (13), and
unlike TNF, it is not produced naturally in soluble form (8, 12). The
existence of a LT
homotrimer is uncertain, since LT
protein is
apparently always associated with LT
in T cells, and a direct
assessment has been hindered by unsuccessful attempts at stable
expression of membrane-bound LT
in mammalian cells (8).
In tissue culture systems, TNF and LT homotrimers are well
recognized for their abilities to elicit a similar but not identical spectrum of cellular responses, including apoptosis and proinflammatory activities (14). Purified soluble recombinant LT
1
2 (15) exhibits
the ability to induce tumor cell death (16) and chemokine secretion
(17) and activate NF-
B, a transcription factor that regulates
inflammatory gene expression through the LT
R (18, 19), but may be
less potent than LT
and TNF. Interestingly, membrane-anchored TNF is
more active in signaling via TNFR80 than soluble TNF (20), raising the
possibility that membrane-bound and soluble ligands may diverge in some
of their functions. This possibility was suspected for the different
forms of LT (7) and was brought into acute focus by the
characterization of mice with an inactivated LT
gene (21, 22).
LT
-deficient mice lack most lymph nodes and Peyer's patches, a
phenotype not associated with deletions of TNF (23) or either of the
TNF receptor genes (24-27). Placental transfer of an LT
1
2
antagonist constructed as a fusion protein between LT
R extracellular
domain and the Fc region of IgG (LT
R-Fc) results in lymph
node-deficient offspring, which established a role for membrane-bound
LT
1
2 distinct from the LT
trimer (28). In addition, the
formation of germinal centers during an immune response, a process
critical for efficient antibody class switching, is also dramatically
altered in mice that lack LT
(21, 22), TNF (23), TNFR60 (23, 29), or LT
1
2 (29, 30) or express LT
R-Fc as a transgene (31). Thus,
characterization of the membrane-anchored LT ligands will help
elucidate their physiologic functions.
Here, we employ recombinant baculovirus to reconstitute LT and LT
homo- and heteromeric complexes in insect cells to investigate the
roles of the LT
and LT
subunits in activation of the cell death
response in tumor cells. Using two loss of function mutations in LT
(32), aspartic acid 50 to asparagine (D50N) and tyrosine 108 to
phenylalanine (Y108F), we show that these LT
mutant proteins co-assemble with LT
to form a ligand that binds LT
R but not TNFR.
The membrane-bound mutant LT
·LT
complexes, but not the secreted
homotrimers, are active at inducing death of the HT29 colon carcinoma
cell line. By contrast, LT
when expressed alone as a membrane
protein binds the LT
R but is functionally inactive in inducing cell
death. These results demonstrate that the LT
subunit is necessary
for the LT
1
2 complex to activate the LT
R cell death
pathway.
The insect cell line BTI Tn5B1-4
(Tn5B1-4), kindly provided by JRH Biosciences (Lenexa, KS), was
cultured at 27 °C in ExCell 401 serum-free medium containing 10 µg/ml gentamycin. The human adenocarcinoma cell line, HT29.14S, was
cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics (penicillin and streptomycin,
100 µg/ml) (16). The anti-human LT mAb, 9B9 (mouse IgG1), was
purchased from Boehringer Mannheim. The anti-human LT
mAb, NC2
(mouse IgG2a), and anti-human LT
mAbs, B9 and B27 (mouse IgG1) (8),
were generous gifts from Jeffrey Browning (Biogen, Inc.), as was the
recombinant human LT
produced in Chinese hamster ovary cells and
rabbit anti-LT
polyclonal serum (33). Construction, expression, and
purification of the bivalent chimeric proteins formed with the Fc
region of human IgG1 and the ligand binding domains of LT
R (12),
TNFR60 (34), and TNFR80 (35) have been previously described.
The construction
of recombinant baculoviruses expressing LT or soluble LT
tagged
with a Myc epitope (sLT
myc) has been described (12, 34). A cDNA
encoding full-length membrane-bound LT
was isolated as an 860-base
pair HindIII fragment from pCDM8/LT
(7), and
HindIII/BamHI linkers were added. After
restriction with BamHI, the LT
cDNA was ligated into
the baculovirus transfer vector, pVL1393. Recombinant baculoviruses
were produced by coinfection of pVL1393/LT
with baculovirus DNA as
described (34). LT
Y108F and LT
D50N mutant cDNAs as originally
constructed for expression in bacteria (32) lack the LT
signal
sequence required for export; therefore, a 300-base pair
Nsi1/Pfl M1 cassette, containing the Y108F or
D50N mutation, was isolated from p8/3 and p11A/20, respectively, and
then used to replace the corresponding region in wild-type LT
. The
resulting mutant cDNAs containing the LT
signal sequence were
isolated as NotI fragments and ligated into pVL1393. The mutant constructs were confirmed by sequence analysis of the
baculovirus vector (U.S. Biochemical Corp. Sequenase version 2.0 sequencing kit).
Recombinant baculoviruses containing LTY108F and LT
D50N were
generated as described for wild-type LT
(34). At 6 days post-infection, supernatants from Tn5B1-4 insect cells infected with
LT
, LT
Y108F, or LT
D50N recombinant baculovirus were harvested and clarified by centrifugation; protease inhibitors (2 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml
aprotinin, and 0.8 mg/ml benzamidine HCl) and fetal bovine serum (10%)
were added before dialysis against Hanks' balanced salt solution. The
supernatants were filter-sterilized before testing in cytotoxicity
assays.
The concentration of LT was determined
by competitive radioimmunoassay using the anti-LT
mAbs NC2 and
125I-LT
. Anti-LT
NC2 was bound (50 ng/well) to
plastic snap wells (Immulon 2, Dynatech, Chantilly, VA) precoated with
goat anti-mouse Ig (500 ng/well) to capture 125I-LT
. The
standard curve was generated with purified recombinant LT
(33)
diluted in 100 µl of phosphate-buffered saline with 1% bovine serum
albumin with a 30-min binding interval. LT
was radioiodinated to a
specific activity of 126 µCi/µg by the IodoGen method (36).
125I-LT
in 10 µl was added to a final concentration of
0.2 nM and allowed to bind for an additional 30 min. Each
well was washed five times, and the bound 125I-LT
in
individual wells was detected using a
-counter. Each data point is
the mean of duplicate wells from which the LT
concentration in
supernatants was determined from the mean of four dilutions using the
radioimmune assay template in Prism (GrapdPAD Software, San Diego, CA).
The range was less than 5% for duplicate determinations.
Receptor binding activity of LT and mutant proteins was assessed
using a solid phase competitive radioligand binding assay with
TNFR60-Fc as a surrogate receptor. The format was identical to the
radioimmunoassay described above except that purified TNFR60-Fc was
bound at 50 ng/well to wells previously coated with goat anti-human Ig
at 500 ng/well.
Baculovirus-infected insect cells were
labeled with [35S]methionine and
[35S]cysteine as described (34). Briefly, 24 h after
infection (multiplicity of infection was 10 at 105
cells/cm2), the cells were washed with buffered saline and
incubated in medium deficient in methionine for 2 h before adding
[35S]methionine and [35S]cysteine labeling
mixture at 0.2 mCi/ml. After 20 h, the supernatants were cleared
by centrifugation for 15 min at 23,000 × g, treated with protease inhibitors, and dialyzed against saline. Protein cross-linking was carried out by the addition of 10 µl of BSCOES, freshly dissolved in Me2SO at 100 mM, to 1.0 ml
of serum-free culture supernatants from baculovirus-infected Tn5B1-4
cells (37). After a 30-min incubation on ice, the reaction was stopped
by the addition of 25 µl of glycine (1 M) and subjected
to immunoprecipitation as described below. The cellular fraction was
extracted with Nonidet P-40 (1%) nonionic detergent in buffer with 50 mM Tris, pH 7.4, containing 10 mM iodoacetamide
and protease inhibitors. The detergent-soluble fraction, obtained after
centrifugation, was subjected to immunoprecipitation as described (13).
Briefly, detergent extracts were precleared by the addition of 10 µg
of normal mouse or rabbit IgG and 20 µl of protein G-Sepharose beads
followed by the addition of 10 µg of either mouse anti-LT or LT
antibodies or polyclonal rabbit anti-LT
and protein G beads.
Proteins were resolved by SDS-polyacrylamide gel electrophoresis (12%
acrylamide) and detected by PhosphorImager analysis (Molecular
Dynamics).
Tn5B1-4 cells were harvested, washed, and incubated on ice in Hanks' balanced salt solution with 10% bovine calf serum, 0.1% sodium azide containing the antibodies or TNFR-Fc chimeras at 10 µg/ml. Phycoerythrin-conjugated affinity-purified goat anti-mouse or anti-human IgG (5 µg/ml) was used to stain for mAb or TNFR-Fc, respectively. Controls for nonspecific binding included normal mouse or human IgG and inclusion in the buffer of human (or mouse) heat-aggregated IgG at 10 µg/ml to block nonspecific binding when staining for mouse IgG. Immunofluorescence staining was detected by flow cytometry (FACScan, Becton-Dickenson) using forward and side scatter parameters to identify infected and noninfected cells. Each fluorescence histogram represents 1 × 104 events gated on infected cells. Fluorescence intensity = (mean fluorescent channel) × (percentage of positive fluorescent events), where a positive event has a fluorescence value >98% of the value for normal IgG. Specific fluorescence intensity represents the fluorescence intensity after subtraction of the value for normal IgG.
Cytotoxicity AssaysCytotoxicity of soluble LT produced
by insect cells was determined using a colorimetric assay with
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
as described (38). Briefly, the HT29.14S (a TNF/LT-sensitive subclone
of HT29 human adenocarcinoma) (16) or murine L929 fibrosarcoma cells
(104 cells/well in 96-well flat bottom microtiter plates)
were incubated in medium with serial dilutions of supernatants from
infected insect cells. For HT29.14S cells, human interferon-
was
included in the medium at 80 units/ml. After 3 days of incubation,
viable cells were detected by the addition of the MTT dye. Cytotoxicity assays with insect cells were performed using paraformaldehyde-fixed insect cells. Tn5B1-4 cells were infected with LT
and/or LT
recombinant baculoviruses at a multiplicity of infection of 10 for each
virus. After 2 days, Tn5B1-4 cells were harvested, washed twice in
phosphate-buffered saline, and incubated for 15 min on ice with 1%
paraformaldehyde in phosphate-buffered saline and then washed four
times with RPMI 1640 containing 10% fetal bovine serum. The fixed
insect cells were added at various ratios to HT29.14S cells
(104 cells/well) and incubated for 3 days, and viability
was detected by reduced MTT dye. The percentage of cell viability was
calculated as a ratio of the absorbance of reduced MTT dye at 570 nm
for cytokine (or insect cell)-treated cells to the absorbance of dye by
cells in medium (with interferon-
) times 100. Each data point represents the mean ± S.D. of triplicate wells.
The D50N and Y108F
mutations in LT were identified as cytotoxicity loss mutants that
failed to bind to L929 cells (32). Both LT
D50N and Y108F mutants,
like wild type LT
, are secreted by Tn5B1-4 cells, typically to
20-40 µg/ml (34). When treated with BSOCOES (1 mM), a
homobifunctional protein cross-linking reagent, LT
and the two
mutant proteins formed a ladder of three bands consistent with
predicted sizes for trimers, dimers, and monomers of LT
(Fig.
1a). The ladder is created by
the incomplete cross-linking of LT
subunits by BSCOES (37). The less
selective cross-linker glutaraldehyde (0.1%) forms a 65-70-kDa adduct
in similar preparations (data not shown, and see Ref. 33), indicating that the majority of the LT
subunits exist as trimers. Both LT
mutants were ineffective as competitors for binding to TNFR60-Fc (Ki = >700 nM) when compared with wild
type LT
(Ki = 10 nM) (Fig.
1b). Wild type LT
induces death in HT29 adenocarcinoma cells (IC50 = 100-200 pM), but both mutants
were inactive when tested on HT29 cells (Fig. 1c) or L929
cells (data not shown).
Reconstitution of LT
The D50N and Y108F LT mutants were tested for their
ability to assemble into membrane LT
complexes by coinfection of
Tn5B1-4 cells with recombinant LT
baculovirus. Reciprocal
co-immunoprecipitations with antibodies to individual
and
subunits were used to detect the formation of LT
complexes.
Anti-LT
specifically immunoprecipitated major bands at 18, 21, and
22-23 kDa, consistent with precursor and glycosylated forms of LT
from baculovirus-infected Tn5B1-4 cells labeled with
[35S]methionine and [35S]cysteine (Fig.
2, lanes 1-4). A band at
31-33 kDa expected for LT
was also immunoprecipitated by
anti-LT
. Similarly, anti-LT
co-immunoprecipitated two major
bands: LT
at 22-23 kDa and LT
at 31-33 kDa (Fig. 2, lanes
5-7). The LT
mutants associated with LT
equally as well as
wild type LT
as judged by the volume-density of the phosphor image.
These results indicate that these mutations do not disrupt assembly of
LT
heteromers. Also, note that LT
is not associated with the
secreted form of LT
(see Fig. 1a), indicating that insect
cells, like mammalian T lymphocytes, do not cleave LT
.
In the Tn5B1-4 cells, anti-LT primarily immunoprecipitated the
22-23-kDa mature form of LT
and not the smaller 18- and 21-kDa forms recognized by anti-LT
(Fig. 2, lanes 4-7). These
two smaller LT
bands match reasonably well with the predicted sizes
of the nascent and signal peptidase-cleaved LT
polypeptides of 22.2 and 18.6 kDa, respectively. LT
produced by insect cells is
glycosylated (39), which indicates that LT
assembles with LT
soon
after the initial processing steps. Precursor-product analysis of
LT
synthesis in T-lymphocytes by pulse-chase methods revealed
that LT
initially associates with a 21-22-kDa LT
precursor that
matures to a 25-kDa form (6, 8). Glycosylation (N- and
O-linked) of LT
also occurs in mammalian cells (40),
although in T lymphocytes LT
shows more extensive change in
molecular mass compared with the protein produced by Chinese hamster
ovary cells (21 kDa).
Tn5B1-4 cells singly infected with LT baculovirus express LT
protein on the cell surface as detected by immunofluorescence staining
with anti-LT
, whereas anti-LT
did not stain (Fig.
3, a and b).
Surface expression of LT
requires infection with both LT
and
LT
recombinant baculoviruses (Fig. 3c). The level of LT
protein is approximately the same on both singly and coinfected cells, indicating that the presence of the LT
subunit does not modify surface expression of LT
protein. Receptor binding function, assessed by staining with Fc fusion proteins as surrogate receptors, revealed that LT
R-Fc, but not TNFR60-Fc, stained cells expressing LT
(Fig. 3d). However, coinfection with the LT
baculovirus dramatically increased the LT
R-Fc-specific fluorescence
staining. Half-maximal binding of the LT
R-Fc to LT
-expressing
cells occurred at 0.8 µg/ml (~6 nM), similar to the
binding to LT
alone (Fig. 4). However, the total LT
R-Fc bound is substantially greater (~100-fold) in cells coinfected with LT
and LT
. This result would be consistent with LT
increasing the number of binding sites for LT
R. Specific binding of TNFR60-Fc occurred only with LT
coinfection, a result consistent with the formation of the LT
2
1 ligand (Figs.
3e and 4) with half-maximum binding at 0.6 µg/ml, although
the total TNFR60-Fc bound was substantially less (~10-fold) than the
LT
R-Fc.
As expected, both the LTD50N and Y108F mutant proteins were retained
on the surface of insect cells coinfected with LT
baculovirus, consistent with the ability of these mutants to assemble with LT
into heteromers (Fig. 5, a and
b). Tn5B1-4 insect cells infected with mutant LT
baculoviruses specifically bound to the LT
R-Fc fusion protein (Fig.
5, d and f). However, binding interactions with
TNFR60-Fc were dramatically reduced by the LT
mutants (Fig. 5,
e and g). The D50N mutant retained a some
capacity to bind TNFR60-Fc compared with Y108F, with a half-maximum
binding at 80-100 nM for D50N and >200 nM for
Y108F (data not shown). TNFR80-Fc binding to insect cells expressing
either LT
mutants was also decreased (data not shown).
These results indicated that the D50N and Y108F residues in the LT
subunit are not directly involved in interactions with the LT
R,
although the LT
subunit dramatically enhanced binding of the
LT
R-Fc. The binding of LT
R-Fc to LT
expressed alone suggested
the possibility that, as a resident membrane protein, LT
could
activate the LT
R.
To investigate the role of the LT and LT
subunits
in the activation of the LT
R, insect cells infected with LT
or
coinfected with LT
baculoviruses were fixed and used as effector
cells in cytotoxicity assays to measure the functional capacity of the surface ligands. LT
-infected cells displayed no significant
cytotoxic activity for HT29.14S cells when compared with uninfected
Tn5B1-4 cells (Fig. 6a). By
contrast, LT
and LT
co-expressing cells were highly effective at
killing HT29.14S cells, typically with a 50% reduction in viability at
an effector:target cell ratio of 0.5 (Fig. 6b). Supernatants
from fixed cells were not active in this assay, demonstrating that cell
death requires cell contact. In striking contrast to the soluble LT
mutants, insect cells infected with either D50N or Y108F mutants and
LT
were completely functional in this assay (Fig. 6, c
and d). That the effect of cell death was mediated by
LT
1
2 is indicated by the ability of the LT
R-Fc, but not
TNFR60-Fc, to block the death-inducing activity of these killer insect
cells. Together, these results indicate that the LT
subunit is
required for functional conformation of the LT
1
2 but not for
specificity of binding to the LT
R.
The crystal structures of LT and TNFR60 provide a conceptual
framework to model interactions between LT
ligands and their receptors (41, 42). Aspartic acid 50 located in the A-A" loop and Y108
in the D-E loop are solvent-exposed residues positioned on opposite
sides of the LT
monomer, although in the native trimer both residues
from different subunits localize to the same receptor binding site
(Fig. 7, a and b).
Our results indicate that the D50N and Y108F mutations probably cause a
local distortion of the TNFR binding site, and not disruption of
trimeric architecture, that results in the loss of cytotoxic activity.
Additional support for this conclusion is seen in the ability of these
LT
mutants to assemble with membrane-bound or soluble forms of LT
(15). Furthermore, these LT
mutants form a functional ligand with
LT
that activates the LT
R cell death pathway.
The TNFR60 binding site lies along the cleft formed by adjacent LT
subunits, an "
" cleft (42). Based on this model, the interface between two adjacent LT
subunits is hypothesized to create
an analogous
(z-x) site that forms the major LT
R binding site
within the LT
1
2 complex (Fig. 7c). This model is
consistent with high affinity binding observed between the LT
R and
the LT
1
2 complex and LT
but not the LT
2
1 complex, which
lacks a
interface. Similarly, the major TNFR60 binding site on
LT
2
1 would be at the
interface (Fig. 7d). The
single LT
subunit within the LT
1
2 complex creates two
nonequivalent
interfaces (x-Y108 and z-D50), where the D50N and
Y108F mutations reside in different
clefts.
Theoretically, binding to one or both of the heteromeric
interfaces must occur in order for LT
1
2 to cluster (aggregate) receptors. Receptor clustering is necessary to recruit signaling molecules, such as TRAF3, to activate the cell death pathway (43), or
TRAF5 that can activate NF-
B (18). The degree of receptor clustering
appears to have a profound effect on the type of cellular responses. In
the LT
R system, NF-
B activation, but not cell death, occurs when
bivalent anti-LT
R monoclonal antibody is added to the culture medium
(16, 19), although cell death occurs when the same monoclonal antibody
is immobilized to a surface (16) or the receptor is ligated with
soluble polyclonal antibodies (43). Presumably, a higher ordered
aggregation of receptors, or stabilization of the receptor signaling
complex, sufficient for cell death is achieved with immobilized or
polyclonal antibodies. This implies that occupation of all binding
sites on the LT
1
2 ligand is important to signal cell death and
predicts that both
and the
binding sites are important to
achieve this conformation. Either of these LT
mutants should create
a ligand with two normal binding sites, which might not be sufficient
to form complexes capable of signaling cell death. Our results, in
fact, show that neither mutation affects cell death signaling by
LT
1
2. This indicates that LT
R clustering sufficient to signal
death of HT29.14S cells depends upon contact with LT
subunit, and
not the LT
subunit. An alternate possibility is that residues other
than Asp50 and Tyr108 in LT
might be
involved in binding to LT
R. Further mutational analysis may
distinguish between these possibilities.
The D50N and Y108F mutations dramatically affected the binding of
TNFR60-Fc to secreted LT and membrane-anchored LT
2
1. As a
soluble protein, LT
2
1 does not elicit cellular responses akin to
TNF and LT
; rather, it functions as a weak antagonist for TNF (19).
Presumably, the reason for the inability of LT
2
1 to activate
TNFR60 is that the
interfaces are not sufficient to promote
TNFR60 clustering in the same way as LT
or TNF homotrimers. Our
results show no significant gain (or loss) of cytotoxic activity by
killer insect cells that express the membrane-anchored LT
mutant-LT
complexes. This result indicates that LT
2
1 as a
membrane protein is unlikely to activate the TNFR60 or LT
R cell
death pathways. This result is further supported by the complete
blocking effect of the LT
R-Fc, which should have revealed any
putative killing activity by the LT
2
1 complex if this ligand
could independently activate TNFR60. Thus, the LT
2
1-TNFR60
interaction does not mirror the receptor-activating function of
LT
1
2 binding with LT
R and suggests a significant distinction
in the way these two receptors are activated by their respective
ligands.
The physiologically relevant location of LT1
2 complex is presumed
to be at the cell surface, since soluble forms are not naturally
produced by lymphocytes. Soluble LT
and TNF bind to cell surface
receptors with high affinity, typically with an observed Kd of 10-100 pM (37), whereas
LT
1
2 binding to LT
R is in the 1-10 nM range
(15).2 The restricted
diffusion of a membrane-bound ligand should enhance binding to cell
surface receptors so that relatively weak interactions (Kd = ~10-100 nM) may become highly
relevant in the context of cell to cell contact. The finding that LT
expressed alone does not induce cell death, although it is expressed in
a form capable of binding the surrogate LT
R-Fc, indicates that the
presence of the LT
subunit is critical for the conformation that
activates the LT
R. In previous studies (12), soluble LT
protein
(generated by deletion of the cytosolic and transmembrane domains)
bound to LT
R-Fc, but weakly compared with LT
1
2. Soluble LT
is polydisperse in the absence of LT
, forming aggregates of high
molecular mass based on elution through gel filtration matrix (15) or
by protein cross-linking.3
These biochemical findings suggest that the LT
subunit may restrict the assembly of LT
oligomers to dimers. The critical role of the
LT
subunit is further revealed by the lymph node deficiency and
germinal center failure in mice genetically deficient in LT
subunit
(21, 22). These observations indicate that LT
as a single subunit
ligand is insufficient to signal these developmental and physiologic
processes. Rather, LT
is essential to form the biologically active
LT
1
2 ligand.
We are grateful to Dr. Jeffrey Browning and colleagues at Biogen, Inc. for gifts of reagents.