T helper cell activation is accomplished by recognition of
antigen-Ia complex, expressed by antigen presenting cell (APC), (
)via a clonally restricted heterodimeric receptor
(TcR)(1, 2, 3) . The precise mechanism by
which APCs activate T cells is quite complex and not fully understood.
The current dogma is that at least two signals are required. The first
signal is provided by the occupancy of TcR, which is major
histocompatibility complex restricted(1) , and the second
non-major histocompatibility complex restricted signal (costimulatory
signal) is delivered by certain molecules present on the surface of
APCs(4, 5, 6) . The participation of
costimulatory signal in T cell activation is of paramount importance as
it results in two potential outcomes, activation or clonal
anergy(7, 8) . The two different outcomes of antigen
recognition, by T cells, is first explained by the dual signal model of
T cell activation by Bretscher and Cohn (9) and updated
recently by Jenkins and Schwartz(10) . Since then, efforts of
numerous researchers have culminated in the identification of various
molecules capable of providing costimulatory signal(11) . The
list of these second signal generating molecules, however, is still far
from complete as reports are rapidly appearing in the literature
regarding the possible existence of certain hitherto unknown molecules
with costimulatory
properties(12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) .
To identify additional cell surface-associated molecules that
provide costimulatory signals to T cells, we have isolated proteins
from lipopolysaccharide (LPS)-activated B cell membrane. When
reconstituted into lipid bilayer, at least three proteins (B1, B2, and
B3) gave differential levels of costimulatory help to primary T cell
activation. The present document presents the results obtained with one
of the above proteins with a molecular mass range of 38-42 kDa
(B3) (the data of B1 and B2 have been communicated elsewhere) and its
relation to primary T cell activation and differentiation.
EXPERIMENTAL PROCEDURES
Animals
Female inbred BALB/C mice, 8-10 weeks old, were
obtained from the National Institute of Nutrition, Hyderabad, India,
and from our conventional conditions and were allowed free access to
food and water.
Cell Lines and Hybridomas Used
All the cell lines and hybridomas used in this study, viz. anti-Thy 1 (TIB 99), anti-L3T4 (TIB 207), anti-CD8 (TIB
150), anti-Mac2 (TIB 166), anti-Mac3 (TIB 168), 33D1 (anti-dendritic
cell Ab; TIB 227), anti-IL-2 R (CRL 1698), HT-2 (CRL 1841),
anti-Ia
(HB3), anti-IFN-
(HB 170), anti-IL-2 (HB
8794), anti-IL-4 (HB 188), anti-LFA-1 (TIB 217), and anti-ICAM-1 (CRL
1878) were procured from the American Type Culture Collection (ATCC),
Rockville, MD. Anti-CD3 (145.2C11) was a kind gift from Dr. Charles A.
Janeway Jr., Howard Hughes Medical Institute, New Haven, CT. WEHI-279
(CRL 1704), A20 (TIB 208), and anti-HSA (TIB 183) were kind gifts from
Dr. Satyajit Rath, National Institute of Immunology, New Delhi, India.
Primary T Cells
CD4
T cells were prepared from mice spleens
as follows. Briefly, a single cell suspension of spleens was prepared
in balanced salt solution (pH 7.2). Red cells were lysed using
hemolytic Gey's solution. Non-adherent cells, collected by
allowing cells to adhere to plastic Petri plates (Nunc, Denmark) at 37
°C with 7% CO
for 2 h, were treated sequentially with a
mixture of anti-Mac2 and anti-Mac3 (45 min on ice), 33D1 (45 min on
ice), and anti-Ia
(45 min on ice). The cells were then
washed and incubated with two rounds of anti-Lyt-2.2 (Cedarlane,
Ontario, Canada) with 45 min each on ice followed by treatment with low
toxicity baby rabbit complement. CD4
T cells were
enriched by passing through nylon wool column. The cells were collected
after five to six washes with prewarmed RPMI, 10% FCS (37 °C) and
plated on Petri plates, previously coated with goat anti-mouse IgM, for
1 h at 37 °C. The non-adherent cells were used as a source of
CD4
T cells and the purity of such cell population
routinely exceeded 98% as estimated by FACScan (Beckton Dickinson) in
preparations stained with anti-L3T4.
Preparation of Resting B Lymphocytes
Resting B cells from mice spleens were prepared as follows.
Briefly, a single cell suspension of spleens was prepared in balanced
salt solution (pH 7.2). The red blood cells were removed by treatment
with Gey's solution. After removing the macrophages by allowing
them to adhere twice to plastic surface (1 h each at 37 °C and 7%
CO
), the cells were treated twice (45 min each on ice) with
a mixture of anti-Mac2, anti-Mac3, and 33D1 and a mixture of anti-Thy1,
anti-L3T4, and anti-CD8 followed by labeling (30 min at 37 °C) with
low toxicity baby rabbit complement. The cells obtained were then
loaded on a discontinuous Percoll gradient (100, 70, and 50%) and
centrifuged at 1600
g for 30 min at 4 °C. The
cells collected from 100-70% interface layer were considered as
resting B cells.
Preparation of LPS-activated B Lymphocytes
Activated B cells were prepared as follows. Briefly, a single
cell suspension of mice spleens was prepared in balanced salt solution
(pH 7.2). The red blood cell were depleted by treatment with hemeolytic
Gey's solution. The cells were then plated on plastic Petri
plates (Nunc, Denmark) for 2 h at 37 °C and 7% CO
. The
non-adherent cells were treated sequentially on ice for 45 min each
with a mixture of anti-Mac2 and anti-Mac3, and a mixture containing
anti-Thy1, anti-L3T4, and anti-CD8 antibodies followed by complement
mediated killing. The cells were then incubated at a concentration of 4
10
/10 ml/pertiplate with 10 µg/ml LPS (from Salmonella typhosa; Sigma) for 72 h at 37 °C and 7%
CO
. The purity of such cells was over 98% as analyzed by
FACscan (Beckton Dickinson).
Isolation of LPS-activated B Cell Surface Proteins
The LPS-activated B cells were harvested and washed three
times with PBS (pH. 7.2) and frozen overnight at -70 °C. The
cells were thawed and homogenized in the presence of 0.25 M
sucrose, 20 mM Tris-HCl (pH 7.4), and 1 mM EDTA along
with a protease inhibitor mixture (leupeptin 10 µg/ml, aprotinin 10
µg/ml, iodoacetamide 10 mM, antipain 10 µg/ml,
pepstatin 10 µg/ml, chymostatin 10 µg/ml, and
phenylmethylsulfonyl fluoride (1 mM). The nuclear fraction was
removed by centrifugation for 10 min at 700
g at 4
°C. The supernatant (S1) was collected and kept aside. The pellet
was rehomogenized and spun as above. The supernatant (S2) was
collected, and the pellet was discarded. S1 and S2 were mixed and
subjected to centrifugation at 1,10,000
g for 2 h at 4
°C. The supernatant was discarded, and the pellet was solubilized
in 1% Triton X-100, 20% glycerol, and 20 mM Tris-HCl (pH 7.5)
and protease inhibitor mixture (composition as mentioned above) and
agitated overnight at 4 °C followed by centrifugation at 100,000
g for 1 h at 4 °C. The proteins, from the
supernatant, were separated using 10% preparative polyacrylamide gel
electrophoresis according to Laemmli(15) . After
electrophoresis, the protein bands were located by staining a strip of
the gel with Coomassie Blue, and the appropriate unstained regions were
crushed and eluted with 1% SDS, 100 mM
NH
HCO
,50 mM Tris-HCl, 0.1 mM EDTA, and 0.15 M NaCl (pH 8.0) at 37 °C for 48 h.
After filtration and centrifugation to remove polyacrylamide particles,
the solution was dialyzed against 0.1% SDS, 10 mM NH
HCO
, 10 mM Tris-HCl, 0.01
mM EDTA, 50 mM NaCl (pH 8.0) for 24 h at 4 °C.
SDS Removal, Estimation, and Partial Protein Renaturation
To the protein solution was added Lubrol Px (a neutral
detergent) to effect a final 10% of the detergent concentration, and it
was kept at 37 °C for 6 h to enable a micelle mixture of SDS-Lubrol
Px. This was followed by dialysis against 10 mM Tris-HCl (pH
8.0), 1% Lubrol Px, 0.01 mM EDTA, 50 mM NaCl at 4
°C for 96 h. The dialysis buffer was replaced by a fresh one after
every 8 h. The extent of SDS removal, from the protein in the dialyzing
bag, was estimated with a basic fuschin method(16) .
Reconstitution of Proteins into Liposomes
Preparation of Liposomes
These were prepared by
a reverse phase evaporation method using DL-
-phosphatidylcholine, dipalmitoyl, Sigma) in 1:1
chloroform/methanol ratio. The lipid film was left in vacuum for 2 h at
room temperature. The film was then hydrated in 10 mM Tris-HCl
(pH 8.0), 60 mM NaCl and sonicated in a bath type sonicator
(Bransonic, model B 2200 E4, Danbury, CT) to clarity at 4 °C for 30
min. The liposomes were sequentially sized through 0.4 and 0.2 µm
polycarbonate membranes before use.
Protein-Liposome Coupling
A 1 to 500 ratio of
SDS-depleted and partially renatured protein sample to liposomes was
placed in a dialysis bag and dialyzed for 96 h at 4 °C against 10
mM Tris (pH 8.0), 0.01 mM EDTA, and 10 mM NaCl with at least 12 changes of dialysis buffer. The contents of
the dialyzing bag were spun down for 2 h at 4 °C at 178,000
g. The supernatant was discarded, and the pellet was dissolved
in physiological saline and later passed through a Sephadex G-50
minicolumn according to Fry et al.(17) to remove
unliposomized protein.
Density Gradient Centrifugation
2 ml of the above
sample was layered on top of a discontinuous gradient of 5-40%
sucrose (w/v) in 10 mM Tris-HCl (6.8), 0.15 M NaCl,
and 0.1 mM EDTA. The sample was centrifuged overnight at
98,000
g in a Beckman SW28 rotor at 4 °C. 2-ml
samples were collected from the 5 and 10% interface, washed in 5
volumes of physiological saline by centrifuging at 178,000
g for 2 h at 4 °C. The pellet was dissolved in
physiological saline and stored sterile for not more than 3 months at
-20 °C. The protein content, coupled to liposomes, was
estimated after lysing with 1% SDS by the BCA method(18) . The
lipid phosphorous was estimated as per the method of Ames and Dubin (19) .
T Cell Proliferation Assay
CD4
T
cells were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented
with penicillin (70 µg/ml), streptomycin (100 µg/ml), glutamine
(4 mM), 2-mercaptoethanol (50 µM) sodium pyruvate
(1 mM), HEPES (20 µM), and 10% heat-inactivated
FCS (Sera Laboratories, Crawley Down, Sussex, United Kingdom). Affinity
purified anti-CD3 (145.2C11) (10 µg/ml), diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6), was immobilized on the
surface of 96-well flat-bottomed (Costar, Cambridge, MA) culture plates
by overnight incubation at 4 °C. The wells were washed three times
with PBS (pH 7.2) before adding the cells along with varying
concentrations of experimental, reconstituted, and control proteins.
Phorbol myristate acetate (PMA) (Sigma) was used at a concentration of
10 ng/ml. The proliferation was assessed after 72 h of culture with 1
µCi [
H]thymidine added during the last 16 h
of culture. Cells were harvested on a multiple sample harvestor
(Skatron, Norway), and the incorportated radioactivity was assessed in
a scintillation counter.
Lymphokine Bioassay
CD4
T cells
were cultured in 24-well plates at a density of 0.25
10
/well with different stimuli. The culture supernatants
were collected after 22 h for lymphokines assay.
Interleukin-2 and -4 Assay
Interleukin-2 and
interleukin-4, in culture supernatants, were determined by the
induction of HT-2 proliferation as described by Fernandez-Bortan et
al.(2) . Briefly, HT-2 cells (1
10
/well) were cultured in 96-well flat bottomed plates,
containing RPMI 10% FCS and various concentrations of culture
supernatants from control and experimental wells. Since HT-2 cells are
responsive to both these lymphokines, therefore, while measuring IL-2
level, the activity of IL-4 was neutralized using anti-murine IL-4
antibody (600 ng/ml). Likewise, for IL-4 assay, the activity of IL-2
was inhibited by adding anti-IL-2 and anti-IL-2 receptor antibodies.
The cultures were incubated for 24 h at 37 °C/7% CO
followed by pulsing with 1 µCi
[
H]thymidine during the last 6 h of culture. The
cells were harvested, and the incorporated radioactivity was measured
by liquid scintillation counting. The lymphokines activity, in terms of
units, was derived by extrapolating the standard curve values obtained
by using recombinant interleukin-2 and -4 (Genzyme).
Interferon-
Assay
Interferon-
was
assayed by its ability to inhibit the proliferation of WEHI-279 cells.
Cells were cultured in 96-well flat bottomed plates with different
concentrations of culture supernatants from control as well as
experimental wells. The cultures were pulsed with
[
H]thymidine and the incorporated radioactivity
was measured as above. Recombinant IFN-
served as standard to
extrapolate the lymphokine activity in terms of units.
Interleukin-5 Assay
The activity of IL-5 was
assayed by the induction of proliferation of mouse splenic resting B
cells using dextran sulfate as a comitogen as described by
Swain(21) . Resting B cells were obtained from splenocyte
suspension as mentioned above. After washing, 1
10
cells/well were added into individual wells of 96-well flat
bottomed microtiter plate. 100-µl aliquots of a range of dilutions
of the culture supernatants under test were added in triplicate wells
along with 50 µg/ml dextran sulfate. Murine recombinant IL-5 added
in different dilutions to obtain a standard curve whose specificity was
cross-checked with anti-IL-5 (500 ng/ml). The cultures were pulsed with
1 µCi of [
H]thymidine after 72 h and
harvested 16 h later. The incorporated radioactivity was determined by
liquid scintillation counting. IL-5 activity, expressed in terms of
units/ml, was obtained from the standard values.
Northern Blotting of mRNA
CD4
T
cells (5
10
/ml) were cultured in 24-well plate
(Costar, MA) for 8 h at 37 °C, 7% CO
in the presence of
previously immobilized anti-CD3 (10 µg/ml), PMA (10 ng/ml), and B3
(0.01 µg/ml) in separate sets of experiments. Thereafter, the cells
were harvested and washed repeatedly in cold PBS (pH 7.2) and stored at
-70 °C in pellets until RNA extraction was performed. Total
cytoplasmic RNA was prepared according to White and Bancroft (22) and blotted over to Immobilon-N (Millipore, MA) and
cross-linked to the membranes by Stratalinker 1800 (Stratagene, La
Jolla, CA). Membranes were washed with 1
SSC and 0.1% SDS for 1
h at 65 °C and then prehybridized in a solution containing 50%
formamide, 5
SSPE (20
SSPE = 3 M NaCl,
0.2 M Na
HPO
, and 20 mM EDTA)
and 5
Denhardt's reagent (50
Denhardt's
= 1% each of Ficoll, BSA fraction V, polyvinylpyrrolidone, 0.1%
SDS) overnight at 42 °C. Specific RNA was detected by probing the
membranes with [
-
P]ATP-labeled cDNA probes
for murine IL-2, IL-4, IL-5, and IFN-
(Amgen Biological, Thousand
Oaks, CA) in fresh prehybridization buffer for 24 h at 42 °C. After
washing, the membranes were exposed to x-ray film with an intensifying
screen at -70 °C for 48 h.
Gel Electrophoretic and Lectin Gel Binding
Analysis
SDS-PAGE was performed in 0.5-mm thick slab gels
containing 4% acrylamide in stacking gel and a 10% acrylamide in
separating gel, with a buffer system according to Laemmli(15) .
The lane containing B3 was transferred on to Immobilon-P (Millipore,
CA), washed, and visualized by staining with 0.2% aqueous Ponceau S,
destained, and its ability to bind
I-ConA was tested in
conjunction with autoradiography on x-ray film.
Phosphorylation Procedure
The assay mixture (final
volume, 0.2 ml) contained 50 mM MOPS/KOH (pH 6.0),
[
-
P]ATP (0.2 mCi/ml), 0.3 M
MgCl
, and approximately 75 µg protein of LPS-activated
B cell membrane lysate. The reaction carried out at room temperature
was started by addition of the proteins and stopped by adding 50 µl
of 50% trichloroacetic acid, 0.3 M unlabeled ATP, and 0.3 M MgCl
. All the following operations were carried
out at 4 °C. The proteins were washed four times. The pellet,
extracted by 1 ml of diethylether, was further processed and
electrophoresed according to Amory et al.(23) After
electrophoresis, the gel was stained with Coomassie Blue, destained,
and B3 was located, excised, dried, and exposed to x-ray film.
Iodination and Competitive Binding
Assay
I-Labeling of B3 was performed using the
IODO-bead method with PD-10 column (Pharmacia, Sweden). Iodinated
samples were trichloroacetic acid- precipitated and an equivalent
fraction containing 2 ng of protein was allowed to bind the
CD4
T cells preactivated with anti-CD3 (10 µg/ml)
or PMA (10 ng/ml) and/or both as the case may be. The reaction was
carried out for 30 min at 37 °C in RPMI containing 10% FCS, 0.2%
sodium azide, 20 mM HEPES (pH 7.0). For competition binding, a
range of (5-100 times the protein concentration) non-radiolabeled
dilutions of B3 was used. The reaction mixture was incubated at 4
°C for 2 h on an orbital shaker and mixed with a vortex mixer at
15-min intervals. After extensive washing, the cell pellets were
subjected to gamma counting (Beckmann).
Electron Microscopy
Negative Staining of Liposomes
A 20-µl
droplet of aqueous suspension of liposomes was placed on a fresh piece
of parafilm, and a sample droplet was picked up by touching a
carbon-coated grid to it. After allowing the excess liquid to drain
off, the grid was gently dipped in 1% phosphotungstic acid (pH 7.0) and
dried by blotting on a filter paper. Grids were observed in a
transmission electron microscope (JEOL 1200 EXII, Japan) and
representative fields were photographed.
Electron Microscopic Autoradiography
The
B3
liposome complex as well as goat anti-mouse IgM (Sigma) were
I-labeled by IODO-bead method essentially as described in
the previous section. The radiolabeled samples were incubated with
CD4
T cells (previously activated at 37 °C for 30
min with 10 µg/ml anti-CD3) for 30 min at 37 °C. After
incubation, the cells were washed thrice with PBS to remove unbound
material by centrifuging for 10 min at 500
g at 4
°C. To the pellet was added an equal volume of low melting agarose
(Sigma) and allowed to gel, and the latter was cut into 1-mm
pieces. Trapped cells were fixed in 1% paraformaldehyde, 1%
glutaraldehyde in PBS at 4 °C for 1 h. After washing with PBS, the
cells were post-fixed in 1% osmium tetraoxide for 90 min at 4 °C in
the dark. After washing with PBS and passing through graded acetone
series, the samples were embedded in epoxy resin (Bio-Rad). Ultrathin
sections, cut on Ultracut S (Richert-Jung, Austria), were coated in
dark with photographic emulsion (Ilford nuclear L4 emulsion) and
incubated in the dark for 2-3 weeks in a dessicator at 4 °C.
The autoradiographs were developed and stained with aqueous uranyl
acetate and lead citrate and observed in a transmission electron
microsope (JEOL 1200 EXII, Japan).
Western Blotting
Western immunoblots were made
from SDS-PAGE(24) . Samples were electrophoresed for 90 min
with constant amperage of 14 mA in a minigel apparatus (Atto, Japan).
The proteins were transferred to Immobilon-P polyvinylidine difluoride
membranes (Millipore, MA) using a Bio-Rad electrophoretic transfer unit
with 10 mM CAPS buffer (pH 11.0). After washing in PBS, pH.
7.2, for 15 min, the membranes were blocked with 3% nonfat dry milk and
0.2% Tween-20 in PBS for 2 h at 37 °C with gentle agitation. After
washing in PBS, the membranes were incubated overnight at 4 °C with
appropriate antibody in PBS-Tween, washed, and incubated with anti-rat
horseradish peroxidase-conjugated IgG for 2 h at room temperature on an
orbital shaker. Bound antibody was detected with metal ion enhanced
diaminobenzidine.
Reverse Phase-HPLC
B3 was located, isolated, and
eluted as mentioned earlier in this section. The protein sample was
then diluted with 0.5% trifluoroacetic acid and loaded on to a
microbore HPLC column (C8) (Aquapore RP 300, Brownlee columns, ABI) and
monitored.
Protein Sequencing
B3 isolated from activated B
cell membranes, as described above, was purified by 10%
SDS-PAGE(15) . After the electrophoresis, B3 was blotted on to
``ProtBlot'' (Applied Biosystems) in CAPS buffer (pH 11.0)
followed by staining in 0.2% Ponceau S, 1% acetic acid and washed with
PBS. The protein was then digested with trypsin as its N terminus was
found to be blocked and a sample equivalent of 2 pmol, from one of the
digested fractions, was subjected to internal sequencing up to 15
residues on Applied Biosystem (model 492 A) Procise Sequencer (at The
Protein Sequencing Facility, Worcester Foundation for Experimental
Biology, Shrewsbury, MA). The repetitive yields of the first 15 amino
acids sequenced were found to be at least 90%.
Two-dimensional Electrophoresis
The reagents for
this purpose were prepared essentially according to Amory et al.(23) and performed as advocated by Penin et
al.(25) .
RESULTS
Identification and Partial Characterization of
B3
The membranes of LPS-activated murine splenic B cells, when
subjected to SDS-PAGE analysis, revealed about 15 major protein bands
when stained with Coomassie Brilliant Blue (Fig. 1a).
B3 was localized, crushed, and eluted as described under
``Experimental Procedures.'' The accuracy with which B3 was
isolated was checked by rerunning the protein on SDS-PAGE. When stained
with Coomassie Brilliant Blue, it demonstrated a single band both under
non-reducing (Fig. 1b), reducing (Fig. 1c), and as a single spot in two-dimensional gel
electrophoretic (Fig. 1e) conditions. When B3 was
subjected to lectin gel binding (Fig. 1f) and
phosphorylation (Fig. 1g) assays it was noticed that B3
binds
I-ConA and incorporates radiolabeled phosphate
indicating the possibility that it is a phosphoglycoprotein. The
results of our attempts to localize B3 on the surface of resting B
cells demonstrated that it is hardly detectable even when probed by
silver stain of SDS-gel possibly indicating that its high expression is
induced when pretreated with LPS (Fig. 1d).
Figure 1:
SDS-PAGE analysis of LPS-activated B
cell membrane, stained with Coomassie blue, revealing protein
bands. a, the samples were analyzed on a 10% gel under
non-reducing conditions. The arrowhead denotes the position of
B3. b and c, one-dimensional profile of B3 under
non-reducing (b) and reducing (c) conditions (see arrowheads). d, SDS-PAGE analysis of resting B cell
membrane. The sample was run on 10% gel under non-reducing conditions
and probed with silver stain. The arrowhead denotes the
possible position of B3. e, two-dimensional non-reducing
(SDS-PAGE)/reducing (TDAB-PAGE) electrophoretic pattern of B3. Gel
electrophoresis was performed on 10% gels in both the dimensions and
stained with Coomassie Blue. e, lectin gel binding analysis of
B3. This was performed with
I-ConA as outlined under
``Experimental Procedures.'' The arrowhead indicates
the position of B3. f, phosphorylation assay of B3. The B cell
membrane proteins were subjected to phosphorylation analysis with
[
-
P]ATP. B3 was located, isolated by PAGE,
and analyzed by autoradiography.
B3 Binds to T Cell Surface
B3, before checking for
its costimulatory ability, was reconstituted into lipid bilayers. Prior
to reconstituting the protein, it was ensured that a fairly homogeneous
preparation of liposomes was obtained. Fig. 2a reveals
the negative staining of liposomes indicating a near homogeneous
preparation of lipid vesicles. In order to demonstrate the protein
reconstitution in lipid vesicles, the aid of electron microscopic (EM)
autoradiography was employed. Since B3 is a membrane protein,
presumably with a hydrophobic stretch, it was expected to be inserted
into the lipid bilayer. In order to illustrate the liposome-protein
coupling, the reconstituted protein was iodinated and processed for EM
autoradiography. Fig. 2b (arrows) shows the
predominant presence of iodinated protein on the vesicle surface
suggesting by that each of the liposome has a fairly even distribution
of B3. Such
I-B3-bearing liposomes when incubated with
anti-CD3 activated T cells, and it was observed that B3 was distributed
uniformly all along their surface (arrows, Fig. 2c).
Figure 2:
Binding and distribution of B3 on T cell
surface. B3 coupled to liposomes was labeled with
I and
incubated with anti-CD3-activated CD4
T cells and EM
autoradiography was performed as mentioned under ``Experimental
Procedures.'' Cells were examined by transmission electron
microscopy. a, negative staining of liposomes by
phosphotungstic acid (
10,000, bar = 200 nm). b,
I-labeled B3 coupled to liposomes. Arrows show
the liposomes frequently bearing the iodinated B3 (
20,000, bar
= 200 nm) c, CD4
T cells labeled with
iodinated B3 (arrows;
20,000; bar = 200 nm).
Note the distribution of B3 only on the T cell
surface.
That the binding of B3 to T cell surface
was specific was demonstrated in experiments with nonspecific control
like murine anti-IgM. The data obtained clearly shows that there was no
binding of liposome-coupled
I-anti-IgM on the T cell
surface presumably because T cells do not possess receptors for IgM (Fig. 3a). On the other hand, when liposomized
I-anti-IgM was incubated with A20 cells, as a positive
control, the radiolabeled antibody was seen to be evenly distributed on
the plasma membrane (PM, arrows, Fig. 3b) and also localized in the interior of the cell (arrows, Fig. 3c).
Figure 3:
Murine anti-IgM coupled to liposomes and
I-labeled iodinated was incubated with T and A20 cells.
EM autoradiography was performed as described under ``Experimental
Procedures.'' The cells were examined by transmission electron
microscopy. PM, plasma membrane; Cyt, cytoplasm; Nuc, nucleus; EV, endocytotic vesicle. a,
anti-CD3-activated T cells incubated with liposomized and
I-labeled murine anti-IgM (
25,000, bar =
200 nm); b, A20 cells incubated with liposomized and
I-anti-IgM (
40,000, bar = 200 nm). Note the
absence of labeled anti-IgM binding on the T cell membrane. Also note
the distribution of labeled anti-IgM in and on the surface of A20
cells.
To further confirm the
specificity of binding of B3 to T cells, competitive binding assay was
performed. When unreconstituted
I-B3 was incubated with T
cells in the absence of anti-CD3, very less binding (1,250 ± 252
cpm) was noticed. However, the number of receptors for B3 appeared to
be up-regulated when the T cells were preactivated with anti-CD3
(10,509 ± 2,562 cpm). Further, the binding specificity of B3 to
T cells was tested by competing
I-B3 with unreconstituted
and unlabeled B3 to bind the anti-CD3-activated T cells. The data
clearly show that the binding capacity of
I-B3 is
diminished by unlabeled B3 (Fig. 4, a-g).
Figure 4:
Competitive binding assay with B3.
CD4
T cells (1
10
/ml) were
incubated with or without anti-CD3 or unlabeled B3 30 min at 37 °C.
After washing,
I-labeled B3 (2 ng) was added to final a
volume of 200 µl and incubated for 2 h at 4 °C with gentle
agitation. After extensive washing, the incorporated radioactivity in
cell pellets was monitored on a gamma counter. a, cells +
I-B3; b, cells + anti-CD3 +
I-B3; c, cells + anti-CD3 + 100:1
(B3:
I-B3); d, cells + anti-CD3 + 75:1
(B3:
I-B3); e, cells + anti-CD3 + 50:1
(B3:
I-B3); f, cells + anti-CD3 + 25:1
(B3:
I-B3); g, cells + anti-CD3 + 5:1
(B3:
I-B3). The range of dilutions of unlabeled B3 used
was in terms of protein content.
Antibodies against Murine LFA-1
, ICAM-1, HSA-1, B7,
and VCAM-1 Do Not Cross-react with B3
It is known that a
majority of costimulatory molecules identified thus far are adhesive in
nature. In our efforts to rule out the possibility of B3 being a known
costimulatory molecule, a Western analysis was performed. The data
obtained are depicted in Fig. 5which demonstrates that B3 did
not cross-react with any of the antibodies against murine LFA-1-
,
ICAM-1, HSA, B7, and VCAM-1. LPS-activated B cell membrane lysate was
run in appropriate lanes as a positive control for these adhesive
molecules.
Figure 5:
Western blotting. Proteins were run on
SDS-PAGE (10%) and transferred onto Immobilon-P membrane and stained in
0.2% aqueous Ponceau S to ensure that all lanes contained the
transferred protein. After washing and blocking, the membrane was
probed with antibodies against ICAM-1, LFA-1
, HSA, B7, and VCAM-1.
As a positive control, LPS-activated B cell membrane sample was run in
appropriate lanes, and the bound antibody was detected by
diaminobenzidine.
B3 Costimulates Primary T Cells to
Proliferate
Fig. 6a shows that the addition of
B3 to the cultures led to an increase in [
H]TdR
incorporation of T cells in a dose-dependent manner. A maximum
statistically significant (p < 0.05) proliferation (39,426
± 4,214 cpm) was noticed at a B3 concentration of 1 µg/ml as
against the basal value (955 ± 268 cpm) obtained with cells plus
anti-CD3 only. When the concentration of B3 was increased, no further
amplification in T cell proliferation was observed. Thus, in all the
subsequent experiments, only the half-maximal concentration (0.01
µg/ml) of B3, as determined by dose-response pattern, was used.
Further, when the cultures were stimulated with controls like
liposomes, SDS, B3 alone, gel eluate, and LPS, no statistically
significant (p < 0.05) proliferation (<2,000 cpm) of T
cells was noticed (Fig. 6b) pointing out thereby that
the activity elicited by B3 was indeed specific. On the other hand,
when PMA was added to anti-CD3-activated T cells, as a positive
control, a maximum incorporation of [
H]TdR
(10,871 ± 2,827 cpm) was observed.
Figure 6:
Effect of various concentrations of B3 on
the proliferation of CD4
T cells. 1
10
T cells/well were cultured in 0.2 ml of RPMI, 10% FCS for 72 h
with various concentrations of liposomized B3. The cells were pulsed
with 1 µCi [
H]thymidine during the last 16 h,
and the incorporated radioactivity was determined by liquid
scintillation counting. All the values represent the mean cpm ±
S.D. of at least three experiments. a demonstrates the
dose-response profile of B3, and b represents the behavior of
various controls like anti-CD3 (10 µg/ml), PMA (10 ng/ml), B3 (0.01
µg/ml), liposomes (2.326 nmol inorganic phosphorous content), SDS
(0.02 nmol), gel eluate (10 µl/well), and LPS (1 µg/ml). Asterisk denotes statistically significant (p <
0.05) over its respective control (i.e. cells +
anti-CD3-treated group).
Induction of Secretion of IL-4 and IL-5 by
B3
After confirming that B3 was able to enhance the
proliferation of primary T helper cells, we next determined whether or
not this protein could elicit the secretion of any lymphokine. To
verify this, T cells were cultured with anti-CD3 and/or B3, and the
supernatants collected after 22 h were tested for IL-2, IL-4, IL-5, and
IFN-
levels. The data show that only the cultures stimulated with
anti-CD3 and B3 produced significant levels of IL-4 and IL-5 and a very
poor level of IL-2 and IFN-
(Fig. 7). As seen in the
proliferative studies, cultures containing T cells and B3 did not bring
about any significant secretion of the above lymphokines. The trend
obtained with bioassay of lymphokines was further confirmed by Northern
analysis, the details of which are highlighted in Fig. 7(see inset).
Figure 7:
Induction of lymphokines by B3. Cells were
cultured as described in the legend to Fig. 6. The supernatants
from the control and experimental wells were collected after 22 h and
tested for IL-2 and IL-4 using HT-2 cell line (1
10
cells/well), IFN-
on WEHI-279 cell line (2
10
cells/well), and IL-5 on resting splenic B cells (1
10
cells/well). The cells were incubated in RPMI, 10% FCS
(100 µl/well in the case of IL-2, IL-4, and IFN-
and 200
µl/well in the case of IL-5). The cells were pulsed with 1 µCi
of [
H]thymidine during the last 6 h (for IL-2,
IL-4, and IFN-gamma) and the last 16 h (in the case of IL-5) of
culture. The radioactivity incorporated was monitored by liquid
scintillation counting. The values expressed in terms of
units/milliliter were derived from the standard curve. The Northern
analysis data (see inset) also coincide with the pattern
obtained with the lymphokine bioassays. Student's t test
was performed to calculate the degree of significance. Asterisk denotes the statistically significant values (p <
0.05) over its respective control (i.e. cells +
anti-CD3-treated group).
Reverse Phase-HPLC and Protein Sequence
Analysis
B3, when subjected to reverse phase-HPLC, showed a
single prominent peak (at 18.21 min) in the chromatogram (Fig. 8a) demonstrating thereby that this protein is
homogeneous in its present form of isolation coinciding with our
two-dimensional data. This protein was subjected to internal amino acid
sequence upon tryptic digestion (because of its blocked N terminus) up
to 15 residues, and the details are depicted in Fig. 8b.
Figure 8:
Reverse phase-HPLC and internal sequence
of B3. a, reverse phase-HPLC profile of B3. This was performed
as described under ``Experimental Procedures,'' the summary
of internal sequence of B3. The upper case letters represent
the three-letter code of each of the amino acid identified while the lower case numbers indicate its position. Unknown positions
are indicated by the letter X.
DISCUSSION
Optimal T cell activation depends not only on the occupancy
of TcR, but also on accessory molecules, provided by the APCs, that
function in cell-cell adhesion and/or signal transduction(26) .
During the recent past, several new costimulatory molecules have been
identified(12, 13, 14) , and the list still
appears to be incomplete as the evidence for existence of additional
cell surface proteins involved in signal transduction is being raised
by several workers(27, 28) . It may be mentioned here
that on the basis of their ability to secrete specific lymphokines, T
helper cells have been divided into Th1 and Th2. Th1 cells produce IL-2
and IFN-
, lymphotoxin, etc., and primarily participate in
stimulting the cell-mediated immunity(29, 30) , while
Th2 cells secrete IL-4, IL-5, IL-6, etc., and are involved mainly in
the induction of humoral
immunity(29, 31, 32, 33, 34) .
It has been postulated that these two T helper subsets are not only
functionally different but also need qualitatively and quantitatively
distinct requirements for costimulation(35) . However, to date
and to the best of our knowledge, no costimulatory molecules are
reported which exclusively activate Th2 cells.
In the present study,
we describe the biochemical and functional analysis of a novel
LPS-activated murine splenic B lymphocyte cell surface-associated
costimulatory molecule, provisionally termed B3, that chiefly activates
Th2-like cells. Our data suggest that B3 molecule is specifically
involved in the costimulation of resting T helper cells upon
cross-linking TcR
CD3 complex with anti-CD3 monoclonal antibody
resulting in predominant secretion of IL-4 and IL-5 and very poor
levels of IL-2 and IFN-
. Our rationale for choosing B cell surface
molecules to provide costimulatory signal to T cells lies in the
observation that B lymphocytes are major APCs for the clonal expansion
of normal murine CD4
T cells(36) . Further,
our selection of LPS-activated B cells for identifying the
costimulatory molecules is based on the premise that resting B cells
are poor APCs (37) and do not constitute costimulatory
activity(38, 39) ; only upon treatment with either LPS
or IL-1 or immunoglobulin or IFN-
, or cross-linking surface major
histocompatibility complex class-II molecules or neuraminidase etc. (36, 40, 41, 42) do the B cells
acquire enhanced ability to stimulate T cells. Moreover, cytokine
secretion is induced from naive T cells only when activated B cells are
used as APCs (43) . It may also be stressed here that the
molecule, described in the present study, is bearly detectable on the
membranes of resting B cells even when loaded five times the
concentration of LPS-activated B cell membrane lysate probed with
silver strain.
In biochemical experiments, we have characterized B3
as a single molecule with an approximate molecular mass of 38-42
kDa when analyzed by SDS-PAGE. Upon reducing, this molecule was
recovered as a single sharp band. The reverse phase-HPLC approach to
purify this protein clearly showed a solitary and distinct peak (at
18.21 min) in the chromatogram. This information, in conjunction with
the SDS-PAGE analysis, conclusively proves that this protein is
homogeneous in its present form of isolation. In addition to these
aproaches, the two-dimensional profile of B3 always consistently
yielded a single pattern (in about 12 repetitions) which also
reiterates the absence of any other contaminants sticking either
specifically or nonspecifically to the said protein. As assessed by its
ability to bind
I-ConA, B3 appears to be glycosylated a
fact which was further substantiated by partial digestion of this
protein by N-glycosidase F that resulted in two distinct
fragments of 22 and 18 kDa (data not shown). The phosphorylation assay,
on the other hand, revealed that this molecule is capable of
incorporating radiolabeled phosphate. These results indicate that B3 is
a phosphoglycoprotein.
For an effective signal transduction, a
costimulatory molecule is expected to bind its counter ligand on the
target cell. Studies were undertaken to explore this possibility, and
the results obtained indicate that B3 molecule binds to T cells and
this binding can be diminished by competing with unlabeled B3. This
fact is further strengthened by the results obtained with electron
microscopic autoradiographic studies which show that
I-labeled reconstituted B3 molecule when incubated with
anti-CD3-activated T cells, this protein is found associated with the
surface of the T cell. It is interesting to note that the receptors for
this molecule, on T cells, appear to be significantly up-regulated when
prior activated with anti-CD3. In the light of these observations, it
is safer to assume that T cells bear ligands for B3 molecule.
On the
basis of its molecular mass, B3 is clearly distinct from the most
characterized costimulatory molecules such as ICAM-1 (90-114 kDa) (44) . VCAM-1 (100-110 kDa)(45) , B7 (45-60
kDa)(46) , HSA (35-60 kDa)(47) , and human LFA-3
(60-80 kDa)(48) . Our Western analysis data strengthens
this view by the fact that the antibodies directed against the above
molecules failed to cross-react with B3. As regards to LFA-3, it still
has not been reported in the murine system.
The internal amino acid
sequence was obtained by tryptic digestion (as its N-terminal was found
to be blocked) of the protein blotted onto the polyvinylidene
difluoride membrane. This yielded a single sequence, and at present we
only have a partial sequence of 15 amino acids. A data base
(non-redundant amalgamation data base of SwissProt, PDB, SP update,
PIR, GP update) search of this yielded somewhat surprising results, as
it showed a high homology (95%) with pyruvate kinase and is not a part
of any known surface proteins. The pyruvate kinase is a well known
cytosolic enzyme(49) . In a rare event of misidentifying
pyruvate kinase as the present protein, is not possible due to the
following reasons: 1) this protein is isolated using the well known
procedures for isolation of membranes and was washed extensively to
remove any nonspecific contaminants; 2) the present protein is found to
be heavily glycosylated from the data shown as under
``Results,'' and it is very uncertain for cytosolic protein
be heavily glycosylated; and 3) the molecular mass of pyruvate kinase
is about 56-60 kDa (50) while the protein described in
the present study has an approximate molecular mass of 38-42 kDa.
These points strongly disfavor the argument that B3 is pyruvate kinase.
The fact that B3 showed high homology with pyruvate kinase (PK),
prompted us to investigate as to whether B3 possesses any PK-like
activity. When B3 was allowed to react with phosphoenol pyruvate,
substrate for pyruvate kinase, it did not show any enzymatic activity
thereby further demonstrating that B3 and PK are unrelated entities
(data not shown).
The only known costimulatory molecule closest to
B3, in terms of molecular weight, is B7-2. Like B7 (now called
CD80), B7-2 (a 34-kDa protein) is a counterreceptor for CD28 and
CTLA-4 T cell surface molecules and induces the predominant secretion
of IL-2(51) . In contrast, the molecule described in the
present study activates CD4
Th cells to secrete IL-4
and IL-5 but a very little IL-2 and IFN-
. These observations lend
support to the view that B3 molecule is not a counter ligand for either
CD28 or CTLA-4. Also, when anti-CD3-activated T cells incubated with
anti-CD28 and were allowed to interact with
I-labeled B3,
there was no substantial change in the binding capacity of labeled B3
to T cells. Further on, our experiments to identify the receptor for B3
on T cells, using the homobifunctional cross-linker disuccinimidyl
suberate, clearly demonstrated B3 binds a 60-kDa protein on T cell
surface (data not shown). It may be mentioned here that B7 (CD80) binds
a 44-kDa glycoprotein (CD28) on T cell surface.
Thus, all the
generated evidence favors the conclusion that B3 is a novel
costimulatory molecule that activates Th2-like cells. Using a similar
approach, we have recently demonstrated the presence of a 150-kDa
protein (M150) from the membranes of thioglycollate-elicited murine
peritoneal macrophages that selectively activate Th1 type of cells
leading to the secretion of significant levels of IL-2 and IFN-
but negligible amounts of IL-4(52) .