(Received for publication, November 11, 1995)
From the
The lymphotoxin (LT) protein complex is a heteromer of
(LT-
, also called tumor necrosis factor (TNF)-
) and
(LT-
) chains anchored to the membrane surface by the transmembrane
domain of the LT-
portion. Both proteins belong to the TNF family
of ligands and receptors that regulate aspects of the immune and
inflammatory systems. The LT complex is found on activated lymphocytes
and binds to the lymphotoxin-
receptor, which is generally present
on nonlymphoid cells. The signaling function of this receptor-ligand
pair is not precisely known but is believed to be involved in the
development of the peripheral lymphoid organs. To analyze the
properties of this complex, a soluble, biologically active form of the
surface complex was desired. The LT-
molecule was engineered into
a secreted form and co-expressed with LT-
using baculovirus/insect
cell technology. By exploiting receptor affinity columns, the
LT-
3, LT-
2/
1, and LT-
1/
2 forms were purified.
All three molecules were trimers, and their biochemical properties are
described. The level of LT-
3-like components in the
LT-
1/
2 preparation was found to be 0.02% by following the
activity of the preparation in a WEHI 164 cytotoxicity assay. LT-
3
with an asparagine 50 mutation (D50N) cannot bind the TNF receptors.
Heteromeric LT complexes were prepared with this mutant LT-
form,
allowing a precise delineation of the extent of biological activity
mediated by the TNF receptors. A LT-
3 based cytotoxic activity was
used to show that the LT-
1/
2 form cannot readily scramble
into a mixture of forms following various treatments and storage
periods. This biochemical characterization of the LT heteromeric
ligands and the demonstration of their stability provides a solid
foundation for both biological studies and an analysis of the
specificity of the LT-
and TNF receptors for the various LT forms.
Recently, a family of receptors and ligands having structural
homology to TNF ()and its receptors was elucidated at the
molecular level(1, 2) . This family includes the TNF,
LT-
, LT-
, Fas, CD27, CD30, CD40, OX-40, 4-1BB, and NGF
systems. TNF and LT-
were the first members of this family to be
defined, and because they exhibited similar biological activity and
receptor binding properties, LT-
was originally believed to be
functionally identical to TNF. Surprisingly, biochemical
characterization of a surface form of LT-
showed that it was a
complex of two proteins(3, 4) . Surface LT-
is
distinguished from surface TNF because LT-
does not retain the
transmembrane region but rather is tethered to the surface via
complexation with a related gene called
LT-
(5, 6) . In addition to differences in
expression patterns, this observation was one of the first indications
that a separate biological role for LT may exist(7) . A
separate receptor specifically recognizing surface LT was identified
validating the hypothesis that the LT system was not simply a redundant
aspect of TNF function(8) . Furthermore, disruption of the
LT-
gene in mice leads to loss of lymph node development further
reinforcing the concept of unique biological functions for the LT
pathway distinct from those of TNF(9, 10) .
Each of
the ligands in the TNF family has now been shown to possess a
corresponding unique receptor. The TNF system is complex because there
are two receptors, i.e. the 55-60- and 75-80-kDa
forms (referred to here as the TNF-R55 and TNF-R75 but also called
TNF-R1 and TNF-R2, respectively) recognizing both LT- and
TNF(11) . The interactions of these receptors has been studied
and both TNF-Rs can bind to a minor form of surface LT that has an
apparent LT-
2/
1 composition(8) . The primary surface
LT form, which is expected to be LT-
1/
2, binds to a new
receptor termed the LT-
receptor (LT-
-R)(8) . The
LT-
-R is structurally related to both the TNF-R55 and TNF-R75 yet
does not recognize either soluble LT-
or TNF. Signalling in this
family is believed to occur when the trivalent ligand is able to bring
together two or more receptors. The oligomerization of receptor
extracellular domains presumably alters the arrangement of the
intracellular domains, which is in turn translated into some further
signal(12) . The major question that becomes apparent upon
consideration of the LT system is how does receptor signal transduction
occur? In the case of LT-
1/
2 on a cell surface, the three
receptor binding clefts are not equivalent and therefore the
delineation of which receptors bind to which clefts will help to
explain how signaling can occur. A precise characterization of the LT
ligand forms would form the basis for such studies.
Recently, the
size of TNF family of receptors and ligands has increased dramatically
leading to an enhanced appreciation of the complexity of the control
mechanisms governing the immune system. The types of communication
occurring between cells in this system have been reasonably defined in
the TNF, CD40, and Fas signaling systems. Understanding of the function
of CD40 and Fas was aided by the linkage of these molecules to the
profound phenotypes accompanying the lpr mouse in the Fas case (13) and the hyper IgM globulinemia associated with CD40 ligand
defects in humans(14) . TNF, on the other hand, benefitted from
its natural existence as a soluble mediator and the ready availability
of a recombinant form. We have taken the approach of converting the
surface LT heteromeric complexes into a TNF-like secreted form. The
generation of the surface LT complexes in soluble forms has allowed us
to examine several key facets of this unusual structure. First, the
LT- and LT-
stoichiometry in the soluble complexes could be
unambiguously determined. Next we were able to assess the stability of
the trimers and ascertain whether scrambling could occur. Such
interconversions would vastly complicate any biological studies
performed using the soluble molecules as mimics of the surface forms.
The receptor binding properties of these ligands will be analyzed in a
subsequent paper. (
)
To create a mutated
version of LT- in which Asp
was replaced by Asn, a NotI fragment containing the entire human LT-
gene (5) was subcloned into a pUC8 derivative, pNN11. Unique site
elimination mutagenesis (Pharmacia Biotech Inc.) was used to alter the
Asp
codon, GAC, to AAC (Asn). Plasmids containing the
mutation were screened for the loss of a RsrII site and then
confirmed by DNA sequence analysis. The entire NotI fragment
was sequenced to ensure that extraneous mutations were not introduced
during the new strand synthesis reaction. For expression of the mutant
protein in insect cells, the gel purified NotI fragment was
ligated into NotI linearized, dephosphorylated BlueBacIII.
Recombinant virus were produced and plaque purified as described by
Invitrogen. For homo- or heterotrimer production, the LT
D50N
virus was used to infect High Five(TM) (Invitrogen) cells alone or
in combination with the VCAM/c-myc-tagged wild type LT-
virus.
Figure 1:
Schematic diagram of the LT-
construct used to convert membrane LT-
into the soluble myc and myc-less LT-
forms. The indicated cleavage sites
demarcate the actual processing. The myc peptide tag is underlined.
To express
soluble LT-/
complexes, High Five(TM) insect cells were
infected in late log phase with a mixture of two baculovirus stocks
coding for LT-
and LT-
, respectively. Kinetic analysis of
insect cell supernatant by Western blotting revealed maximal expression
of both LT forms at about 60 h post infection. However, degradation
products of LT-
in the culture medium could be detected as early
as 25 h post infection. In contrast, LT-
appeared to be stable in
the culture supernatant well after peak expression was reached (data
not shown). To avoid excessive degradation of LT-
during the
production phase, all subsequent cultures were harvested between 40 and
50 h post infection. The cells were immediately removed by
centrifugation, phenylmethylsulfonyl fluoride and EDTA were added as
protease inhibitors prior to ultrafiltration, and the concentrate was
stored frozen at -70 °C.
Figure 2:
Gel exclusion chromatography analysis of
LT- produced by insect cells. Cell supernatant was chromatographed
on a Superose(TM) 6 column, and LT-
was assessed in a dot
blot/Western using the anti-myc mA6 9E10. The figure shows the A
tracing of the fractionated cell supernatant,
and overlaid is a scanned image of the developed dot
blot.
By
analogy with other members of the TNF family, LT-/
heteromeric forms were expected to be trimers. Therefore, LT-
and
LT-
co-infection of insect cells could theoretically produce
LT-
3, LT-
2/
1, LT-
1/
2, and LT-
3 forms, and
the separation of these related LT complexes presented an interesting
challenge. Initially, we explored affinity chromatography using
immobilized mAbs specific for LT-
and LT-
. However, the
affinity of the LT-
specific mAbs was high, and it was difficult
to eluate bound material from the resin without destroying the column.
Attempts to take advantage of the ``FACS dull'' and
``FACS bright'' groups of mAbs specific for LT-
(15) as an affinity purification strategy for LT-
1/
2
were equally unsuccessful. Thus, we abandoned mAb-based affinity
columns for purification purposes.
Receptor-based affinity columns
provided an alternate method that exploited the expected specificity of
the TNF-R55 for LT forms containing a LT-/LT-
cleft, i.e. LT-
3 and LT-
2/
1 and the specificity of the
LT-
-R for LT forms containing LT-
/LT-
or
LT-
/LT-
clefts, i.e. LT-
1/
2 and
LT-
2/
1. Shown in Fig. 3A is a schematic
diagram of the purification strategy that yielded essentially pure
LT-
1/
2 and LT-
2/
1 (Fig. 3B).
Conditioned medium from dually infected insect cells was passed several
times over a TNF-R55-Fc affinity column to remove both LT-
3 and
LT-
2/
1. The depleted flow-through from this step was loaded
onto a LT-
-R affinity column, and the eluate contained pure
LT-
1/
2. The eluate from the original p55 TNF-R column was
loaded onto a LT-
-R affinity column, and LT-
2/
1 was
eluted. The preparation obtained from the TNF-R55-Fc flow-through
LT-
-R elution arm is referred to as LT-
1/
2; the material
prepared from the TNF-R55-Fc eluate that was further fractionated on
the LT-
-R affinity column is referred to as LT-
2/
1.
These preparations are used in the analytical evaluations. Switching
the order of the receptor affinity columns to using the LT-
-R
column first and the TNF-R55-Fc column second also results in the
successful separation of the two heteromers where LT-
1/
2 is
contained in the flow-through and LT-
2/
1 in the elution pool
of the TNF-R55-Fc column.
Figure 3:
Purification of LT heteromeric forms. A schematically shows the purification strategy using a
sequence of TNF-R55-Fc and LT--R-Fc affinity columns and the
various fractions obtained at each step. B shows a nonreducing
SDS-PAGE analysis of the final LT-
1/
2 (lane 1) and
LT-
2/
1 (lane 2) eluates. The upper band in
this analysis is the secreted myc-tagged LT-
, and the lower band is LT-
.
A receptor-based ELISA was developed to
aid in the purification and provided our first insight into the
receptor specificities of the various ligand forms. The
LT-1/
2 form was found to bind selectively to the LT-
-R
and poorly to the TNF-R55, whereas the LT-
2/
1 form bound well
to both receptors. As expected, LT-
3 only bound to the TNF-R55,
and no appreciable binding could be detected to LT-
-R (Table 1). The ELISA was especially useful for optimizing the
affinity column elution pH, the buffer compositions, and storage
temperatures used during the purification. The conditions described
under ``Materials and Methods'' are optimized for ease of
elution, stability, and purity of the eluted material.
Figure 4: Analysis of purified LT forms by C4 reversed phase HPLC. Purified LT forms were eluted with a 0-75% acetonitrile gradient over 30 min. The y axis depicts the absorbance at 280 nm.
To determine which of the various
heteromeric forms were present in the preparations derived from the
affinity columns, cation exchange chromatography was found to resolve
the LT-3, LT-
2/
1, and LT-
1/
2 forms (Fig. 5). Judged on this basis, the purity of these preparations
was greater than 95%. It was difficult using biochemical methods to
ascertain more accurately the levels of contamination by other forms.
The ion exchange data suggested that the LT-
1/
2 preparation
was contaminated with about 5% LT-
2/
1, whereas the
LT-
2/
1 preparation lacked obvious LT-
3 or
LT-
1/
2 contaminants. Based on the receptor binding data
obtained in the ELISA (Table 1) and preliminary BIAcore(TM)
data,
it is clear that the LT-
1/
2 is unable to
bind with reasonable affinity to the TNF-R55 and that LT-
3 cannot
bind to the LT-
-R. On this basis one would expect a high level of
purity in the preparations obtained using combinations of TNF-R55-Fc
and LT-
-R-Fc affinity columns. It is possible that the
preparations contain different glycoforms of the two LT-
/
or
the LT-
3 forms with isoelectric points similar to the bulk
LT-
1/
2 or LT-
2/
1. The occurrence of minor forms
with different oligosaccharides may result in contaminating species and
would underlie the parent LT-
1/
2 and LT-
2/
1 peaks
in the cation exchange chromatographic analysis and lead to a potential
overestimation of the purity of the preparations. However, it is likely
that such cross-contamination is only present at low levels if at all
because the LT-
to LT-
ratios determined by SDS-PAGE and C4
reversed phase analysis suggest a precise stoichiometry of 2:1.
Figure 5: Analytical cation exchange ion exchange chromatography of LT forms. Samples were applied to a carboxymethyl ion exchange column and eluted with a 0-1 M NaCl salt buffer. The y axis depicts the absorbance at 280 nm.
Gel
exclusion chromatography of the purified fractions showed that each of
the three forms eluted in the size range of 40-70 kDa with the
LT- containing forms being larger (Fig. 6). Trimeric TNF
migrates as a compact 40-kDa molecule that is smaller than its actual
molecular mass of about 51 kDa. Because these structures are also
expected to be relatively compact, we interpret the LT sizing results
to indicate that all three forms are trimers. If the sizing results
were representative of dimers, it would be impossible to reconcile the
SDS-PAGE and reversed phase HPLC ratio analyses. Taking together, the
ratio analyses and the sizing results show that the LT-
1/
2
and LT-
2/
1 heteromers form trimers. There was some concern
that the myc tag attached to the N terminus of LT-
may
affect the properties of the complexes. A soluble LT-
construct
essentially identical to the myc-tagged version was prepared
without the myc tag. Heteromeric complexes prepared by the
same route described above yet lacking the myc-tagged LT-
had identical biochemical properties.
Figure 6: Gel exclusion sizing chromatography of LT forms. Samples were loaded onto a TSK G3000 column and eluted in PBS buffer. The y axis depicts the absorbance at 280 nm.
Figure 7:
Comparison of cytotoxic properties of wild
type (solid symbols) and the D50N mutant (open
symbols) forms of LT-3, LT-
2/
1, and
LT-
1/
2 on WEHI 164 cells as assessed using a 3-day growth
assay. Shown is the absorbance of reduced MTT, which is proportional to
viable cell number.
To more carefully
address the issue of contaminating LT-3 versus direct
signaling by LT-
1/
2 forms on LT-
-R, we prepared a
mutated form of LT-
. Mutation of Asp
to Asn
was shown to disrupt the activity of human LT-
in the mouse
assay system(19) . Preparation of this mutant and expression in
the baculovirus/insect cell system confirmed a 100,000-fold reduction
in cytotoxic activity both in the mouse WEHI 164 (Fig. 7) and in
a human TNF-R55-based cytotoxic system using WiDr cells (data not
shown). LT-
1/
2 and LT-
2/
1 heteromeric forms
containing D50N LT-
were prepared by first selecting all forms
capable of binding to the LT-
-R affinity resin, i.e. LT-
1/
2 and LT-
2/
1. These two forms were then
resolved by ion exchange chromatography. All three mutated forms,
LT-
3, LT-
2/
1, and LT-
1/
2 were subjected to the
same biochemical tests described for wild type LT preparations, which
confirmed that the D50N mutation in LT-
allowed trimer formation
with the expected LT-
:LT-
ratios. All WEHI 164 activity in
the heteromeric forms was eliminated by the D50N mutation, strongly
indicating that the residual WEHI 164 activity in the heteromeric forms
could be accounted for by minor amounts of LT-
3 contaminates. It
is possible that other receptor components are involved in
LT-
1/
2 signaling, e.g. a putative LT-
/LT-
cleft specific receptor, and that the D50N mutation also affects this
interaction resulting in the loss of activity. At this point, however,
the presence of low level contaminating LT-
3 represents the
simplest explanation of the data. Based on the ability to purify the
D50N mutated form of LT-
1/
2 by LT-
-R-Fc affinity
chromatography, the mutation does not grossly affect binding to the
LT-
-R. Human LT-
1/
2 is cytotoxic to the human
adenocarcinoma cell line HT-29, and this event is mediated by the
LT-
-R(20) . In this assay, the D50N LT-
1/
2 form
retained essentially full specific activity. (
)LT-
1/
2 lacking the myc tag was also
fully active in the HT-29 assay.
Figure 8:
WEHI 164 cytotoxicity assay used to follow
the generation of LT-3-like forms in solutions of
LT-
1/
2. A 400 ng/ml solution of LT-
1/
2 was stored
for 0 (
), 2 (
), or 7 (
) days in minimum essential
medium with 10% fetal bovine serum at either 4 °C in air or at 37
°C in a CO
incubator. Samples were assayed in a 24-h
cytotoxicity assay in the presence of 10 µg/ml cycloheximide. A
fresh dilution of insect cell derived recombinant LT-
3 is included
for comparison (
). Plotted is the absorbance of reduced
MTT.
During these storage experiments, LT-1/
2 activity was also
analyzed in another cytotoxicity assay. LT-
1/
2 is cytotoxic
to the HT-29 cell line via a mechanism that involves the LT-
receptor (20) . Solutions of LT-
1/
2 that had
undergone high levels of conversion such as shown for the sample stored
for 7 days (Fig. 8) did not have altered activity in the HT-29
assay. Therefore, even preparations that contained 1-3% of
LT-
3-like activity as determined by the WEHI 164 cytotoxicity
assay showed no substantial change in the properties of the bulk
LT-
1/
2 as assessed with the HT-29 cytotoxicity assay. Lastly
we attempted to force scrambling by repeated freeze-thawing and even
following multiple rounds; there was no increase in LT-
3-like
activity whether measured by the cytotoxicity assay or biochemically by
ion exchange chromatography. Taken together, these experiments show
that the LT-
1/
2 form is stable and does not readily scramble.
To study the function of surface LT heteromers we chose to
express and characterize soluble recombinant versions of the
LT-/
complexes. Recombinant LT-
has been expressed in
bacteria(21, 22, 23) or secreted from
Chinese hamster ovary cells (16, 24, 25) and
insect cells(17, 26) . In view of the efficient
expression observed in the baculovirus system of LT-
, we
concentrated on the simultaneous expression of LT-
and LT-
in
that system. The co-infection of insect cells with baculoviruses
encoding the LT-
and a re-engineered secreted LT-
protein led
to secretion of complexes of LT-
/
, and the LT-
2/
1
and LT-
1/
2 forms could be selectively purified using receptor
based affinity resins. We wished to address the following questions:
(i) Was there sufficient discrimination by the TNF-R55-Fc and
LT-
-R-Fc affinity columns to purify the expected forms? (ii) What
stoichiometric ratios of LT-
and LT-
were present in the
heteromers? (iii) Were the heteromers trimers? (iv) Could the defined
heteromeric forms eventually scramble into a mixture of all four
conceivable forms?
The data presented here show that soluble
LT- was highly aggregated when expressed by itself and only by
co-expression with LT-
did proper trimeric forms result.
Presumably, LT-
is incapable of packing into a stable homomeric
trimers and inclusion of LT-
compensates for the incorrect
geometry. We were unable to purify a pure LT-
form using the
LT-
-R-Fc or TNF-R55-Fc affinity columns even when LT-
was
expressed in the absence of LT-
. In contrast, the purification
scheme described here led to pure preparations of LT-
1/
2 and
LT-
2/
1. The combination of an approximately trimeric size on
gel exclusion chromatography coupled with the 2:1 ratios observed by
SDS-PAGE and in the reversed phase HPLC analyses definitively
demonstrates the trimeric nature of these soluble forms. The dramatic
conversion of the LT-
aggregates into LT-
/
trimers when
LT-
is co-expressed with LT-
is interpreted as strong
biochemical evidence favoring the heteromeric complex as the
physiological LT ligand. Previous studies on cell surface LT were also
consistent with a complex that was at least as large as the trimeric
LT
1
2 structure(4, 7) . Human TNF and
LT-
have been shown to exist as trimers by a number of
techniques(7) . Likewise, recent crystallographic analysis of
the CD40 ligand also revealed it to be a trimer(27) . The
structural homology among the members of the family occurs in the
regions of the molecule known to be involved in intersubunit contacts,
and therefore, one would expect that the trimer packing would be
conserved as a fundamental feature of the entire family. The
delineation of the soluble LT forms as trimers strengthens the
hypothesis that the cell surface forms are similar trimeric forms.
Moreover, the ability to separate the two LT-
/
complexes
rather than obtaining heterogeneic mixed aggregates indicates that
assembly into trimeric structures is definitely preferred. Therefore,
the interactions between LT-
and LT-
subunits must lend
considerable stability to the trimeric structure.
The very fact that
the receptor-based purification scheme presented here yielded trimeric
forms composed of LT-2/
1 and LT-
1/
2 provides some
information about the nature of the receptor interactions. The crystal
structure of LT-
complexed with the TNF-R55 showed the receptor
nested in the cleft between two LT-
subunits with productive
interactions being made to two separate subunits(28) . If this
binding mechanism is generally valid for the family, which is a
reasonable expectation, then only trimers containing a LT
/LT
cleft should bind TNF-R55. The binding of LT-
3 and
LT-
2/
1 to the TNF-R55-Fc affinity column confirmed this
expectation. Likewise the binding of LT
1
2 to the
LT-
-R-Fc column confirmed that a molecule with a LT-
/LT-
cleft will bind the receptor, which was expected based on the weak
interactions of the LT-
-R with pure LT-
described
earlier(8) . The binding of LT-
2/
1 to the LT-
-R
indicated that a LT-
/LT-
cleft is capable of interacting with
the receptor. Further details of the receptor binding interactions will
be discussed elsewhere.
The primary goal of this work
was to prepare a soluble form of the LT-1/
2 ligand that could
be used in biological studies. Therefore, it was important to ascertain
the composition and purity of the ligand. The final preparations are
greater than 95% pure based on ion exchange resolution. Of paramount
importance was the quantitation of the levels of LT-
3-like
contaminants in the LT-
1/
2 preparation because LT-
3 is
biologically active in many systems and its presence would confuse the
interpretation of any study. The murine WEHI 164 cell dies in the
presence of LT-
3, providing a very sensitive indicator of
LT-
3 contaminating the LT-
1/
2 preparation. In such
assays, the LT-
1/
2 preparation was found to contain
approximately 0.02% LT-
3-like activity. This activity could be
blocked by soluble TNF-R55-Fc or anti-LT-
monoclonal antibodies
and therefore is expected to represent signaling by a LT-
3-like
form. The ion exchange analysis suggested that there was about
2-5% LT-
2/
1 in the LT-
1/
2 preparation and a
BIAcore(TM) analysis
was consistent with such a
contaminant. Therefore, in our best assessment, this preparation
contains greater than 95% LT-
1/
2, less than 5%
LT-
2/
1, and less than 0.02% LT-
3. The purity of the
LT-
2/
1 preparation was more difficult to quantitate using the
cytotoxicity assay; however, by ion exchange analysis it is greater
than 95% pure. We were unable to exactly quantitate LT-
3-like
activity in this preparation because the LT-
2/
1 is an
inhibitor
and obscures possible LT-
3 components.
BIAcore(TM) analysis of the binding of these preparations showed
essentially no binding of LT-
1/
2 to the TNF-R55-Fc and no
binding of LT-
3 to the LT-
-R-Fc. Therefore one would expect
that affinity purifications of both heteromers based on a combination
of these columns to be very pure, and that assumption is supported by
these analyses.
In addition to the actual initial purity of these
heteromers, we were concerned that these complexes would scramble into
a mix of the various forms because there are no covalent interactions
involved in trimerization. Some of the initial work on the quaternary
structure of TNF suggested that there was an equilibrium between
monomers, dimers, and trimers. In the original characterization of the
surface form of LT, we added unlabeled soluble LT- to cells
displaying biosynthetically labeled LT-
/
(3) . The
loss of immunoprecipitable LT-
label that would indicate exchange
of LT-
on and off the surface complex was not observed, suggesting
a relatively stable structure. Here we have shown that the
LT-
1/
2 heteromer is indeed quite stable when stored in
neutral pH buffers, and moreover, attempts to force scrambling by
repeated freeze-thawing of the preparation did not readily lead to
increased amounts of LT-
-like activity. For these reasons, the
heteromeric structures are concluded to be relatively stable and
therefore useful for biological work. Outside the demonstration that
stable LT-
/
complexes exist on the lymphocyte surface, there
is little information on the quaternary state of these TNF family
ligands on cell surfaces. The immunoprecipitation of the surface TNF
form showed the presence of 17 kDa of processed TNF in addition to the
26-kDa form retaining the transmembrane form(29) . This result
was interpreted to mean that some processed TNF chains must be
complexed with unprocessed chains, i.e. oligomeric complexes
are present. Because the soluble LT complexes form stabile trimers, we
conclude that the same structures are present on the actual membrane
surface.
The characterization of these soluble trimeric LT forms
further indicates that the previously proposed model for surface LT
structure is correct, i.e. a surface LT-1/
2 complex.
Additionally, the soluble forms are functionally active (20) and therefore suitable for immunological studies. The
availability of these ligands now opens the way to an analysis of their
binding to the LT-
receptor and elucidation of the signaling
mechanism. A separate paper will describe the binding characteristics
of these proteins to the various receptors in the TNF family.