Differential Effect of Anti-B7-1 and Anti-M150 Antibodies in
Restricting the Delivery of Costimulatory Signals from B Cells
Javed N. Agrewala,1* Susmit Suvas,* Rakesh K. Verma,* and Gyan C. Mishra†
B7-1 and M150 are potent costimulatory molecules expressed on B cells and macrophages. We have examined the capacity of Abs
against B7-1 and M150 in differentially inhibiting the costimulatory signals delivered by macrophages and B cells to OVA-specific
CD4?T cells. The anti-B7-1 Ab significantly blocked the proliferation of Th cells, MLR, T cell help to B cells, and secretion of
IFN-? when B cells were used to provide costimulation, but not when macrophages were used. In contrast, anti-M150 Ab
significantly decreased the proliferation of Th cells, MLR, and production of IFN-?, when macrophages were utilized to provide
costimulatory signals, but not when B cells were used as APC. However, when macrophages activated with IFN-? were used as
a source of costimulation, like anti-M150 Ab, Ab to B7-1 also down-regulated the activation of Th cells. The significance of this
finding is that M150 is a potent first costimulatory signal for initiating proliferation and secretion of IFN-? and providing cognate
help for B cells by Th cells when the macrophage is used as an accessory cell. M150-induced IFN-? production induces the
expression of B7-1 on the surface of macrophages, which then delivers a second cosignal for Th cells. B7-1 works efficiently when
B cell provides cosignal. Both of the molecules promote Th1 activity, as evidenced by the inhibition of the secretion of IFN-? but
not IL-4 by Th cells with anti-M150 and B7-1 Abs. The Journal of Immunology, 1998, 160: 1067–1077.
APC (1–8). On the basis of lymphokine profile Th cells have
been divided into Th1 and Th2 subtypes. Th1 cells secrete IL-2,
IFN-?, lymphotoxin, etc., and are mainly involved in the gen-
eration of cell-mediated immunity (CMI)2responses. Th2 cells
secrete IL-4, IL-5, IL-6, etc., and are generally involved in hu-
moral immunity (9–11). Both subsets recognize foreign Ags in
association with MHC class II molecules. It appears that these
two distinct Th cells are not only functionally different but also
require discreet costimulatory signals for their optimum activa-
tion (12, 13). Although, the controversy still exists, it seems
that B7-1 predominantly activates Th1 cells (14–16). Its other
isoform, B7-2, stimulates Th2 cells (17–18).
B7-1 plays a major role in providing costimulation to T cells,
leading to their proliferation, cytokine production, and develop-
ment of effector functions. The fact that, in addition to B7-1, APCs
are endowed with a large number of costimulatory molecules (19–
25), raises a possibility of existence of multimolecular pathways
for T cell activation and their clonal amplification (26, 27). The
strongest evidence to date for the existence of non-B7-mediated
costimulation has been reported from studies with CD28-deficient
(CD28?/?) mice (28, 29). Moreover, the resting macrophages do
he optimum activation of Th cells requires not only
TCR occupancy by presented MHC-Ag complex, but
also a set of other costimulatory signals provided by
not express B7-1. However, the expression of B7-1 is induced on
the macrophages on activation with IFN-? or LPS (30–32).
In our previous study, we provided evidence that a 150 kDa
(M150) molecule associated with the membrane of macrophage
activates Th cells leading to their proliferation and release of lym-
phokines representative of Th1 subtype (7). In the present study,
we have compared the costimulatory activity of B7-1 and M150.
By using Abs against B7-1 and M150, we have demonstrated that
B cells activated with LPS primarily employ B7-1 and macro-
phages utilize predominantly M150 to costimulate Th cells to pro-
liferate and secrete IFN-?.
Materials and Methods
Female inbred BALB/c, C3He, and C57BL/6 mice, 8 to 10 wk old, were
obtained from the Institute’s Animals House Facility.
Ags, Abs, lymphokines, and reagents
OVA, phosphatidylcholine (PC), cholesterol, penicillin, gangliosides, and
rabbit anti-hamster (Ham) Abs were purchased from Sigma Chemical Co.
(St. Louis, MO). FCS was from Sera Lab (Crawley Down, U.K.); RPMI
1640 was purchased from Life Technologies (Grand Island, NY); and L-
glutamine and streptomycin were from Serva (Heidelberg, Germany);
rIL-4, rIFN-?, rIL-12, and anti-IL-12 Abs were the products of Genzyme
(Boston, MA); whereas Abs to IL-4 and IFN-? were obtained from Texstar
and Holland Biotechnology (Leiden, The Netherlands), respectively. Anti-
IL-2 and anti-IL-2R Abs were used as culture supernatants (SN) from TIB
222 (PC 61.53), CRL 1698 (7D4), and HB 8794 (S4B6). Anti-B7-1 mAb
was purchased from PharMingen (San Diego, CA); rabbit anti-mouse
FITC-labeled Ab was procured from the Binding Site, Birmingham, U.K.
Cell lines and hybridomas
The cell lines and hybridomas used in this study, HT-2 (CRL-1841), TIB-
222, CRL-1698, CRL 1878, TIB 217, and HB 8794, were procured from
American Type Culture Collection (Rockville, MD). WEHI-279 and TIB
183 were gifts from Dr. S. Rath, National Institute of Immunology, New
Delhi, India and 145.2C11 was from Prof. C. A. Janeway, Jr. (Yale Uni-
versity, New Haven, CT).
*Institute of Microbial Technology, Chandigarh, India, and†National Centre for Cell
Sciences, Pune, India
Received for publication March 27, 1997. Accepted for publication October 9, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Address correspondence and reprint requests to Dr. Javed N. Agrewala, Institute of
Microbial Technology, Sector 39A, Chandigarh-160 036, India.
2Abbreviations used in this paper: CMI, cell mediated immunity; PC, phosphatidyl-
choline; PEC, peritoneal exudate cells; BSS, balanced salt solution; SN, supernatant;
Ham, hamster; Rt, rat; MFI, mean fluorescence intensity; M*, macrophages activated
Copyright © 1998 by The American Association of Immunologists0022-1767/98/$02.00
Cells were cultured in RPMI 1640 medium supplemented with 10% FCS,
L-glutamine (2 mM), penicillin (50 ?g/ml), streptomycin (50 ?g/ml), and
2-ME (0.05 mM).
Before immunization, the purity of each batch of M150 was established by
two-dimensional gel electrophoresis as mentioned earlier (7). The isolated
M150 contained only a single protein without any detectable contamina-
tion, demonstrated by the fact that in two-dimensional gel electrophoresis
M150 appeared as a single spot (7). Purified M150 (100 ?g) was emulsified
in CFA. Syrian hamsters were immunized s.c. Later, 3 to 4 boosters of
M150 (50 ?g) emulsified in IFA were given after intervals of 21 days. The
animals were bled for Ab when the titers were positive by ELISA and
Purification of anti-M150 on protein A-Sepharose column
Protein A-Sepharose column was equilibrated with 10 mM Tris-HCl
buffer, pH 8.0. Ham Ig obtained from precipitation of serum by ammonium
sulfate was passed through the column. The column was washed exten-
sively with 100 mM Tris-HCl buffer. The bound Ab was eluted with 100
mM glycine-HCl buffer, pH 3.0, and collected in tubes containing 1 M
Tris-HCl to neutralize the pH.
Isolation and reconstitution of M150
The peritoneal exudate cells (PEC) were harvested from BALB/c mice
inoculated 4 days previously with 2 to 3 ml of thioglycollate. The PEC
were washed with cold balanced salt solution (BSS). The macrophages
were obtained by adhering for 1 h at 37°C on plastic petri dishes, followed
by washing at least three times in cold BSS and freezing overnight at
?37°C. Membrane proteins were isolated as described elsewhere (33).
Briefly, the cells were thawed and homogenized in the presence of 0.25 M
sucrose, 10 mM Tris-HCl, and 1 mM EDTA (pH 7.4) along with pro-
tease inhibitor cocktail. The cells debris was removed by spinning at 600 ?
g for 15 min at 4°C. The SN obtained was centrifuged at 110,000 ? g for
2 h. The pellet was solubilized in 1% Triton X-100, 20% glycerol, 10 mM
Tris-HCl (pH 7.5), and agitated overnight at 4°C. The insoluble ma-
terial was removed by centrifugation at 100,000 ? g for 1 h at 4°C. The
proteins from SN were separated by SDS-PAGE (34) using 10% poly-
After electrophoresis, M150 was located by staining a strip of the gel
with Coomassie blue, and the appropriate unstained regions were crushed
and eluted with 0.1% SDS, 0.05 M Tris-HCl (pH 8.0), 0.1 mM EDTA, and
0.15 M NaCl at 37°C for 24 h (15). After filtration and centrifugation to
remove polyacrylamide particles. The protein content was estimated es-
sentially according to Bradford (35). The SDS was removed from the
eluted M150 by Extracti D gel column (Pierce, Rockford, IL). The column
was equilibrated with 0.05 M Tris-buffer (pH 9.0). Before loading M150,
1 mg/ml BSA in elution buffer was passed through the column and then
thoroughly washed to remove it. The M150 was loaded on the column and
eluted. Positive fractions obtained were pooled and the protein content was
Reconstitution of M150 into lipid bilayers
Liposomal vesicles were prepared by dissolving L-?-PC (Sigma) in a 1:1
ratio of chloroform and methanol and evaporated under N2gas while ro-
tating the vial to deposit a thin film. The lipids were dried under vacuum
for 2 h and were then dissolved in 10 mM Tris-HCl, (pH 8.0), 0.5% Lubrol
Px, 140 mM NaCl, and 0.1 mM EDTA. This was followed by sonication
at 4°C in a bath-type sonicator for 30 min. A 500:1 ratio of vesicle prep-
aration and M150 protein, isolated from SDS-PAGE, or control OVA, were
mixed and vortexed properly (36). This mixture was then dialyzed at 4°C
for 4 days against 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, and 0.1 mM
EDTA with at least eight changes of buffer. The contents of the dialyzing
bag were centrifuged for 2 h at 4°C at 178,000 ? g. The SN was discarded
and the pellet was dissolved in 0.9% NaCl. The unbound protein was
eliminated by passing the solution through Sephadex-G10 mini-columns.
A 2-ml sample was layered on the top of the discontinuous gradient of 5 to
40% sucrose in 10 mM Tris-HCl (pH 6.8), 0.15 M NaCl, and 0.1 mM
EDTA. The sample was centrifuged at 9800 ? g overnight at 4°C. The
2-ml samples were collected from the 10% interface layer and washed in
0.9% NaCl at 178,000 ? g for 2 h. The pellet was dissolved in 0.9% NaCl,
passed initially through both 0.45-?m and 0.22-?m sterile filters, and
stored at ?20°C until further use. Similarly, all the 21 proteins of macro-
phage membrane visualized by Coomassie blue staining on SDS-PAGE
were also purified.
Samples containing macrophage membrane were transferred from SDS-
PAGE on nitrocellulose membrane. Electrophoretic transfer of proteins
was carried out as described by Towbin et al. (37) in 10 mM 3-(cyclo-
hexylamino)-1-propanesulfonic acid) buffer (pH 11.0) at 200 mA for 2 h at
4°C and stained in Ponceau S to ensure that the proteins were transferred.
After blocking overnight in PBS (pH 7.4) containing 3% skimmed milk,
the blots were exposed for 1 h at 37°C to hamster (Ham) anti-M150 Ab and
control Ham Ig (1:10 dilution) with gentle rocking. The blots were then
incubated with rabbit anti-Ham IgG Ab (1:1000 dilution) and later on with
anti-rabbit IgG-peroxidase-labeled Ab (1:15,000). The bound Ab was vi-
sualized using substrate diaminobenzidine prepared in 50 mM Tris (pH
7.6) containing 0.01% H2O2. The usual steps of washings using PBS-
Tween-20 were maintained after every incubation. The macrophage mem-
brane lysate and m.w. markers were also run simultaneously in appropriate
lanes and were stained with Coomassie blue.
Ag-specific Th cells. Ag-specific Th cells were prepared as described ear-
lier (38). OVA-specific Th cells were obtained from mice immunized s.c.
in the footpad with 100 ?g of Ag, emulsified in CFA. After 7 days, inguinal
and para-aortic lymph nodes were removed and a single-cell suspension
was cultured in 24-well plates. Th cells (5 ? 105/well) were cultured with
6 ? 106mitomycin C (50 ?g/ml)-treated feeder cells in the presence of 1.5
ml RPMI 1640 containing 10% FCS and 100 ?g OVA, in 7% CO2atmo-
sphere at 37°C. On the fourth day, the cells were harvested and centrifuged
on Ficoll-Hypaque at 22°C for 20 min at 1170 ? g. The cells at the in-
terface were collected and recultured with syngeneic spleen cells treated
with mitomycin C, as described earlier. This cycle of activation and resting
was repeated at least three or four times before the cells were utilized for
the experiments. The cells rested for 10 to 14 days were used in the study.
These cells were of CD4?Th phenotype, as revealed by FACS analysis,
and produced IL-4, IFN-?, and IL-2, and responded to the Ag OVA in a
dose-dependent manner (data not shown).
B cells. A single-cell suspension of mice spleens was prepared in balanced
salt solution (BSS). The RBCs were depleted by treatment with hemolytic
Gey’s solution. The adherent cells were removed by plating on the plastic
petri plates (Nunc, Roskilde, Denmark) for 2 h at 37°C and 7% CO2. The
nonadherent cells were treated sequentially on ice for 45 min, each with a
mixture of anti-Mac2 and 3 Abs, and a mixture containing anti-Thy1, anti-
L3T4, and anti-CD8 Abs followed by complement-mediated killing. The
cells were then incubated at a concentration of 4 ? 107/10 ml/Petriplate
with 10 ?g/ml LPS (from Salmonella typhosa) (Sigma) for 48 h at 37°C
and 7% CO2. The purity of cells stained with anti-IgM Abs by indirect
immunofluorescence was over 85% as analyzed by FACS (Becton Dick-
inson, Mountain View, CA).
Macrophages. The PEC were harvested from BALB/c mice inoculated 4
days previously with 2 to 3 ml of thioglycollate. The cells were washed
with cold BSS. The macrophages were obtained by adhering for 1 h at
37°C on plastic petri dishes, followed by washing several times in cold
BSS. The purity of cells was ?98%.
Biotinylation of anti-M150 Abs
Anti-M150 Ab was dialyzed against 0.1 M NaHCO3for 4 h. NHS-biotin
(Pierce) (1 mg/ml) was dissolved in DMSO and added to proteins and
mixed for 4 h at room temperature and then dialyzed against PBS contain-
ing 0.02% NaN3.
Formation of complexes with M150
M150 (0.1 ?g/ml) was incubated to form complexes with different con-
centrations (0.00001–10 ?g/ml) of anti-M150 Ab, anti-IL-12 Ab, Ham Ig,
and rat (Rt) Ig at 37°C for 1 h. Similarly, the complexes of IL-12 (10
pg/ml) were made using anti-IL-12 and anti-M150 Abs. These complexes
were used in cultures to monitor the proliferation and secretion of IFN-? by
Expression of B7-1 and M150 on macrophages and B cells
One million thioglycollate-stimulated macrophages were incubated with
rIFN-? (100 U/ml) for 24 h at 37°C/7% CO2. Both IFN-? activated and
nonactivated macrophages and LPS-activated B cells were incubated with
the biotinylated anti-M150 (1:100 dilution) and anti-B7-1 Abs (1 ?g/100
?l) diluted in PBS-FCS 2% for 1 h at 4°C. The cells were then washed
three times with PBS-1% FCS. Streptavidin-FITC (1:10,000 dilution) was
added and the cells were further incubated for 1 h at 4°C. The cells were
then washed five times with PBS and fixed in 1% paraformaldehyde and
analyzed by FACS for the expression of M150 and B7-1. As a control for
1068 ANTI-B7-1 AND M150 Abs INHIBIT COSTIMULATORY SIGNALS
anti-M150 and anti-B7 Abs, the cells were also incubated with Ham Ig and
The cells from each suspension were acquired on Lysis II software of
FACScan (Becton Dickinson). Debris in the cell suspension was excluded
from the analysis by suitable gating that allowed the collection of data only
from those light-scattering events (i.e., cells) of a size consistent with mac-
rophages or B cells. The analysis for the mean fluorescence intensity (MFI)
was done on histograms in which the abscissa and the ordinate denote log
FITC fluorescence and relative cell count, respectively.
The proliferation of Th cells
Inhibition of the proliferation of Th cells by anti-B7-1 and anti-M150
Abs when B cells and macrophages were used to deliver costimulatory
signals. The 96-W microtiter plates (Costar, Cambridge, MA) were
coated overnight at 4°C with 50 ?l of 10 ?g/ml of anti-CD3 Ab in car-
bonate-bicarbonate buffer, pH 9.2. The wells were washed three times with
BSS. The OVA-specific CD4?Th cells rested for 10 to 14 days and were
used in the assays after separating the splenic feeder cells on Ficoll-
Hypaque gradient. The LPS-activated B cells (1 ? 105/well) or thiogly-
collate-stimulated macrophages (1 ? 105/well) or macrophages activated
for 24 h with IFN-? (100 U/ml) were incubated with anti-B7-1 and M150
Abs for 1 h at 4°C. APCs preincubated with the Abs were then cultured
with anti-CD3 Ab-stimulated Th cells (2 ? 104/well). Anti-M150 and B7-1
Abs were also present throughout culture conditions. In control wells, anti-
M150 and anti-B7-1 Abs and control Ham and Rt Igs were also added to
the cultures in which Th cells were cultured with anti-CD3 Ab only. The
cultures were also set when Th cells were incubated with M150 without
anti-CD3 Ab. Th cells incubated with only anti-CD3 Ab or in the presence
of PMA (10 ng/ml); B cells unstimulated with LPS were also taken as
controls. The cultures were incubated at 37°C in a humidified atmosphere
containing 7% CO2. After 48 h, the cultures were pulsed with 1 ?Ci of
[3H]thymidine. After 16 h, the plates were harvested and incorporation of
thymidine was determined by using an automatic cell harvester (Skatron,
Tranby, Norway) and liquid scintillation counting.
The proliferation and secretion of IFN-? by Th cells stimulated with
M150 and IL-12 complexes. Anti-CD3 Ab-stimulated Th cells were in-
cubated with M150 (0.01 ?g/ml) complexed with different concentrations
(0.00001–10 ?g/ml) of anti-M150 Ab, anti-IL-12 Ab, Ham Ig, and Rt Ig.
Similarly, Th cells were also cultured with the complexes of IL-12 (10
pg/ml) prepared with different concentrations of anti-IL-12 and anti-M150
Abs. The control cultures were also set where anti-CD3 Ab-activated Th
cells were either incubated with medium only or control liposomes or OVA
entrapped in liposomes. The cultures were incubated at 37°C in a humid-
ified atmosphere containing 7% CO2. After 48 h, the culture SNs were
collected from the experimental and control wells to monitor IFN-?. The
cultures were pulsed 72 h later with 1 ?Ci of [3H]thymidine. After 16 h, the
plates were harvested and incorporation of thymidine was determined us-
ing an automatic cell harvester (Skatron) and liquid scintillation counting.
The proliferation of Th cells using different proteins isolated from the
membrane of macrophage. Twenty-one proteins were isolated from the
SDS-PAGE and were tested for their ability to proliferate anti-CD3 Ab-
activated Th cells. The cultures conditions were the same as mentioned in
the case of the proliferation of Th cells. Th cells were incubated with the
isolated proteins (0.01 ?g/ml) and anti-M150 Ab (10 ?g/ml). The control
cultures were also set where anti-CD3 Ab-stimulated Th cells were incu-
bated either with control Ham Ig or with anti-M150 Ab. The cultures were
incubated at 37°C in a humidified atmosphere containing 7% CO2. After
72 h, the cultures were pulsed with 1 ?Ci of [3H]thymidine. After 16 h, the
plates were harvested and incorporation of thymidine was determined by
using an automatic cell harvester (Skatron) and liquid scintillation
The cultures were set as mentioned in Th cell proliferation using macro-
phages and B cells as a source of costimulation. The cultures SN from the
experimental and control wells were collected after 24 to 48 h and IL-4 was
measured by its ability to induce the proliferation of HT-2 cells as de-
scribed earlier (39). Briefly, 1 ? 104/well of HT-2 cells were cultured in
96-well plates containing medium and various concentrations of culture SN
obtained from the control and experimental wells. For the selective inhi-
bition of IL-2, Abs to IL-2 and its receptor (culture SN in 1:12 dilution of
CRL 1698, TIB 222, and HB 8794) were used. The cells were incubated for
16 h at 37°C, pulsed with 1 ?Ci of [3H]thymidine, and harvested 8 h later.
The [3H]thymidine incorporation was measured by liquid scintillation
spectrometry. IFN-? was assayed by its ability to inhibit the proliferation
of WEHI-279 cells (40). WEHI-279 cells were cultured in 96-well plates
at a density of 1 ? 105cells/ml with different dilutions of cultured SNs
harvested from control and experimental wells. [3H]Thymidine (1 ?Ci/
well) was added and, after 24 h of incubation, the cells were harvested and
counted 6 h later. For the specificity of the lymphokines, the inhibitory
activity of IFN-? and IL-4 was neutralized with anti-IFN-? (4 ?g/ml) and
11B11 (1 ?g/ml) Abs, respectively. The lymphokine data were calculated
from the mean cpm of triplicate determinations and expressed as picograms
per milliliter in the culture SN as computed by comparison with the stan-
dard curve plotted using rIL-4 and rIFN-? (Genzyme).
Mixed lymphocyte reaction
Thioglycollate-stimulated macrophages and LPS-activated B cells pre-
pared from C57BL/6 mice were used as stimulator cells. These cells were
treated with mitomycin C and were cultured in 1:1 ratio with 106spleen
cells of BALB/c mice in 3 ml complete medium. On day 4, T cell blasts
were isolated by the Ficoll-density gradient method. The cultures for Th
cell proliferation were set using different concentrations (1 ? 103–105
cells/well) of stimulator cells (macrophages untreated or treated with
IFN-? and LPS-activated B cells) and T cells (2 ? 104/well) in the pres-
ence or absence of anti-M150 and B7-1 Abs. The cultures were also set
using Igs of Ham and Rt as controls for anti-M150 and anti-B7-1 Abs. On
day 5 of MLR cultures, [3H]thymidine was added and the cultures were
harvested after 16 h and assayed for DNA synthesis by beta scintillation
B cell proliferation
As described above, anti-CD3 Ab-stimulated and mitomycin C-treated Th
cells (2 ? 104/well) were cultured with LPS-activated B cells (1 ? 105/
well) either in the presence of M150 (0.01 ?g/ml) or Abs to M150 and
B7-1 molecules at a concentration of 1 ?g/ml in a total volume of 200 ?l
of RPMI 1640-FCS 10%. The cells were incubated for 48 h, pulsed with 1
?Ci [3H]thymidine for a further 16 h, and then processed for beta scintil-
lation counting. The suitable controls containing anti-CD3 Ab-stimulated
Th cells incubated with control liposomes, Igs of Ham and Rt, or without
Abs were also kept.
IgG1 and IgG2a isotypes
Cultures were set as mentioned in B cell proliferation assays. Supernatants
were collected on day 6 from experimental as well as control wells and
were analyzed for IgG1 and IgG2a by ELISA. Briefly, triplicate wells were
coated overnight at 4°C with 5 ?g/ml of rabbit anti-mouse IgG in carbon-
ate-bicarbonate buffer, pH 9.6. The unbound sites were blocked with 2%
BSA and then log10dilutions of culture supernatants were added for 2 h at
37°C. IgG1 and IgG2a were detected using biotinylated goat anti-mouse
IgG1 and IgG2a Ab, respectively. After incubating the plates at 37°C for
2 h, streptavidin-horseradish peroxidase was added. The usual steps of
washings using PBS-Tween-20 were carried out at each step. Color devel-
oped due to the substrate OPD (orthophenylene diamine-2HCl) was visu-
alized at 492 nm. The results are represented as the OD of a single dilution
(1:100) of mean absorbance of triplicate wells after subtracting the absor-
bance obtained with control wells (i.e., SN obtained from LPS-stimulated
MHC restriction assay
Cultures were kept as mentioned in the case of the proliferation of Th cells.
OVA-specific Th cells (2 ? 104/well) generated from BALB/c, C57BL/6,
and C3He mice were stimulated with plate-bound anti-CD3 Ab and M150
derived from BALB/c mice. In control cultures, Th cells were incubated
with M150 only or with anti-CD3 Ab and medium alone. After 72 h,
1 ?Ci/well of [3H]thymidine was added. The cultures were harvested after
16 h using an automatic cell harvester and beta emission were counted
using liquid scintillation counting.
Anti-M150 Ab recognizes M150 on Western blots
The Western blot analysis has revealed that the membrane of mac-
rophages probed with anti-M150 Ab could specifically identify
M150 only. Anti-IL-12 Ab, however, failed to recognize M150
(data not shown). The control Ham Ig also could not detect M150
Expression of M150 and B7-1
The macrophages before and after treatment with IFN-? were
tested for the expression of M150 and B7-1 by FACS. LPS-acti-
vated B cells were also analyzed for the expression of M150. It
1069 The Journal of Immunology
was observed that macrophages expressed a higher level of M150
(Fig. 2A) as compared with the B cells (Fig. 2C). In contrast, the
expression of B7-1 was not observed on the macrophages (Fig.
2B). However, macrophages incubated with IFN-? exhibited sig-
nificantly higher expression of M150 as well as B7-1 (Fig. 2, A and
B). M150 was detected on resident macrophages as well, but not
on unactivated B cells (data not shown). The control Igs of Ham
and Rt failed to show any shift in MFI in FACScan.
Anti-M150 and anti-B7-1 Abs neutralize costimulatory signals
delivered by macrophages and B cells, respectively
In our earlier studies, we have reported that M150 costimulates Th
cells resulting in their proliferation and lymphokine secretion (7).
In the present study, we raised Ab against this molecule and an-
alyzed the ability of this Ab to inhibit M150-induced proliferation
of Th cells. Our study revealed that, compared with B cells, mac-
rophages display a higher level of M150. Other workers have re-
ported that unlike macrophages, B cells express a significant level
of B7-1. Therefore we next performed experiments to determine if
there was selective utilization of costimulatory molecules by mac-
rophages and B cells. Our assay system consisted of Th cells ac-
tivated with anti-CD3 Ab and costimulated with either macro-
phages or LPS-activated B cells. A profound decline (82.70%) in
the growth of Th cells was found in the cultures in which anti-
M150 Ab was used to inhibit the costimulatory signals delivered
by the macrophages (Fig. 3A). In a similar condition, the prolif-
erative response of Th cells remained grossly unchanged when
anti-B7-1 Ab was used. Like anti-M150 Ab, anti-B7-1 Abs could
also significantly obstruct the proliferation of Th cells costimulated
with macrophages activated with IFN-? (or SN generated by cul-
turing anti-CD3 Ab-stimulated Th cells with M150) (data not
shown). It may be pointed out that it has been reported earlier that
IFN-? induces the expression of B7 on macrophages (30, 31). In
contrast, when B cells were used as a source to provide costimu-
latory activity to Th cells, anti-B7-1 Ab significantly inhibited
(54.38%) the proliferation of Th cells. Abs against M150, how-
ever, could only partially check the growth of Th cells (Fig. 3B).
The Abs used in the study worked in a dose-dependent manner. To
ascertain the specificity of anti-M150 Ab, Th cells were also co-
stimulated with anti-CD3Ab and PMA. PMA-activated Th cell
proliferation (123,966 ? 14,247 cpm) could not be blocked with
anti-M150 Ab (142,031 ? 6,539 cpm). Furthermore, M150 could
not induce the proliferation of PHA-activated human lymphoblasts
(data not shown). The control cultures containing anti-CD3 acti-
vated Th cells incubated either with Ham Ig or Rt Ig, and M150
without anti-CD3 Ab did not show any effect on Th cell activity.
Inhibition of the secretion of IFN-? by anti-M150 and anti-B7-1
Abs by Th cells, when macrophages and B cells were used as a
source to deliver cosignals
Our experiments demonstrate that anti-M150 and anti-B7-1 Abs
selectively blocked the costimulatory activity of macrophages and
B cells. Literature also suggests that M150 and B7-1 molecules
possibly participate primarily in the activation of Th-1 like cells (7,
14). Keeping these points in view, we next performed experiments
phage membrane proteins. Western blots show membrane of macrophages
probed with anti-M150 Ab (lane a) and control Ham Ig (lane b). The
molecular masses indicated in kDa were estimated from standards electro-
phoresed in a parallel lane.
Anti-M150 Ab recognizes only M150 but not other macro-
anti-M150 Ab (A), and anti-B7-1 Ab (B). The LPS-activated B cells were also stained with anti-M150 Ab (C) and analyzed by FACS. Igs of Ham and Rt
were used as controls for anti-M150 and anti-B7-1 Abs. The asterisk (*) indicates macrophages activated with IFN-?. The data shown in the figures
Expression of M150 and B7-1. Thioglycollate-stimulated peritoneal macrophages before and after IFN-? treatment were stained with
1070 ANTI-B7-1 AND M150 Abs INHIBIT COSTIMULATORY SIGNALS
to analyze the secretion of IFN-? and IL-4 by Th cells costimu-
lated by macrophages or B cells in the presence or absence of
anti-M150 and B7-1 Abs. Interestingly, anti-M150 Ab could ab-
rogate the secretion of IFN-? by 97.28% when macrophages were
used as APC but not when B cells were utilized to generate co-
stimulatory signals. Anti-B7-1 Ab could only decrease the produc-
tion of IFN-? by 1.39%. In comparison, when macrophages stim-
ulated with IFN-? were used, the production of IFN-? was
inhibited by 46.25% and 88.72% by anti-B7-1 and anti-M150 Abs,
respectively (Fig. 4A). In contrast, anti-B7-1 Ab nearly blocked
(97.45%) the production of IFN-? when B cells were used to co-
stimulate Th cells. Anti-B7-2 Ab, however, inhibited the secretion
of both IFN-? and IL-4 when either macrophages or B cells were
used as APC (data not shown). Blocking with both anti-M150 and
anti-B7-1 Abs, however, enhanced the secretion of IL-4 by Th
cells, costimulated with macrophages and B cells, respectively
(Fig. 4B). The control cultures consisting of Igs of either Ham or
Rt could not influence the secretion of lymphokines. The specific-
ity of assays for monitoring IFN-? and IL-4 was ensured by neu-
tralizing the activity of these cytokines by their respective Abs.
Anti-M150 and anti-B7-1 Abs blocks the signals delivered by
macrophages and B cells in MLR
Another interesting feature we observed during the study was that
anti-M150 Ab inhibited MLR by 69.89% when macrophages were
used as stimulator cells (Fig. 5). Similarly, anti-B7-1 Ab blocked
MLR by 72.66% when B cells were used as stimulator cells. Fur-
thermore, Abs against M150 and B7-1 molecules did not signifi-
cantly block the alloreactivity of B cells and macrophages, respec-
tively. However, as the observation mentioned in Figure 3A, when
macrophages activated with IFN-? were used as allostimulators,
anti-B7-1 Ab could also significantly inhibit (62.23%) the allo-
stimulation. The proliferation of Th cells remained grossly un-
changed when control Igs of Ham and Rt were used.
Anti-B7-1 Ab, but not anti-M150 Ab, could inhibit the
proliferation of B cells and the secretion of IgG2a Abs
Since it has been reported in the literature that LPS-activated B
cells express B7-1 and since our FACScan data also suggest the
expression of M150 molecules, we therefore next determined
whether blocking of these molecules would effect the proliferation
and differentiation of B cells. For this, LPS-activated B cells were
cultured with mitomycin C-treated and anti-CD3 Ab-activated Th
cells. It was found that in such a culture system there was a sig-
nificant proliferation of B cells. This proliferation was not inhib-
ited by the addition of anti-M150 Ab (Fig. 6A). In contrast, anti-
B7-1 Ab showed significant
proliferation of B cells. We also evaluated the capacity acquired by
anti-CD3 Ab- and M150-stimulated Th cells to provide help to B
cells. It was observed that Th cells stimulated with M150 induced
significant increase in the proliferation of B cells (78,235 ? 8,018
cpm) as compared with the Th cells cultured without M150
(22,304 ? 6,032 cpm). No proliferation of B cells was noticed in
the control cultures containing Ham Ig, Rt Ig and liposomes incu-
bated with Th cells.
We next measured the yield of IgG1 and IgG2a isotype during
the coculture of T and B cells. We found that LPS activated B
cells, when cultured with anti-CD3 Ab-stimulated Th cells, se-
creted both IgG1 and IgG2a isotypes. The secretion of IgG2a was
only inhibited in the cultures when T and B cells were incubated
with anti-B7-1 Ab. Anti-M150 Abs did not show any noticeable
effect in the secretion of either IgG1 or IgG2a istotypes (Fig. 6B).
Interestingly, when anti-CD3 Ab-stimulated Th cells were incu-
bated with M150, there was augmentation in the secretion of
rophages were incubated with different concentrations of anti-M150 and anti-B7-1 Abs at 4°C for 1 h and then cultured with Th cells stimulated with
anti-CD3 Ab. The macrophages activated with IFN-? (M*) were also used for costimulation. Anti-M150 and anti-B7-1 Abs were also present throughout
the culture period. The proliferation was monitored by [3H]thymidine incorporation. As a control, anti-CD3 Ab-activated Th cells cultured either with
thioglycollate-activated macrophages or M* showed 122,353 ? 9,862 and 82,937 ? 3,352 cpm, respectively. Anti-CD3 Ab-stimulated Th cells cultured
with PMA and with or without anti-M150 Ab showed 142,031 ? 6,539 and 123,966 ? 14,247 cpm, respectively. Th cells cultured with anti-CD3 Ab
generated ?5000 cpm. Th cells cultured either with medium alone or macrophages without anti-CD3 Ab could not generate more than 2000 cpm. B, The
B cells were cultured as mentioned in the legend to A. As a control, anti-CD3 Ab-activated Th cells cultured either with medium alone or with LPS-activated
B cells showed 5,017 ? 1,372 and 32,568 ? 3,715 cpm, respectively. Th cells cultured with either anti-B7-1 or anti-M150 Abs or cultured with medium
alone or with B cells in the absence of anti-CD3 Ab could not generate more than 2000 cpm. The Igs of Ham and Rt used as controls for anti-M150 and
anti-B7-1 Abs, respectively, did not block the Th cell response. The data are the mean ? SD of duplicate experiments.
Differential capacity of anti-M150 and B7-1 Abs to block the costimulatory signals delivered by macrophages and B cells. A, The mac-
1071 The Journal of Immunology
IgG2a Abs. The control Igs of Ham and Rt used in the cultures
could not influence any change in B cell activity.
Anti-M150 Ab, but not anti-IL-12 Ab, inhibits the proliferation
of Th cells mediated by M150
We next monitored the specificity of the proliferation of Th cells
induced by M150 by blocking its activity with anti-M150 Ab.
M150 was complexed with different concentrations of anti-M150
and anti-IL-12 Abs. M150-induced proliferation of anti-CD3 Ab-
stimulated Th cells was inhibited specifically with anti-M150 Ab
but not with anti-IL-12 Ab (Fig. 7A). Ten micrograms of anti-
M150 Ab complexed with 0.01 ?g/ml of M150 neutralized 90% of
the M150-induced proliferation of Th cells. Anti-IL-12 Ab did not
have any impact on the inhibition of the proliferation of Th cells.
In contrast, anti-IL-12 Ab but not anti-M150 Ab, however, could
efficiently block the proliferation of Th cells activated with anti-
CD3 Ab in the presence of rIL-12. The Th cell activity was not
affected by the control liposomes or OVA-entrapped liposomes.
The Ham Ig and Rt Ig could not induce any change in M150-
mediated proliferation of Th cells. Furthermore, M150 failed to
induce the growth of Th cells in the absence of anti-CD3 Ab. Th
cells activated with anti-CD3 Ab showed less than 5000 cpm.
Anti-M150 Ab, but not anti-IL-12 Ab, inhibits the secretion of
IFN-? by Th cells stimulated with M150
We also evaluated the role of anti-M150 and anti-IL-12 Abs in
inhibiting the secretion of IFN-? by Th cells activated either with
M150 or rIL-12 (Fig. 7B). M150-induced secretion of IFN-? was
inhibited by anti-M150 Ab, but not with anti-IL-12 Ab. Similarly,
IL-12-induced production of IFN-? was blocked by anti-IL-12 Ab,
but not with anti-M150 Ab. The control cultures kept as mentioned
in Figure 7A did not show detectable levels of IFN-?.
M150, but not other macrophage membrane proteins, induce the
proliferation of Th cells
We did more experiments to establish the specificity of anti-M150
Ab. The SDS-PAGE analysis of the membrane of macrophage
revealed 21 major bands when stained with Coomassie blue (7).
All the proteins were isolated and tested for their ability to stim-
ulate anti-CD3 Ab-incubated Th cells (Fig. 8). Only M150 induced
a significant level of Th cell proliferation. The 59-, 75-, and 85-
kDa proteins could induce only minor change in the proliferation
of Th cells (?7100 cpm). The rest of the tested macrophage mem-
brane proteins failed to stimulate Th cells (?5000 cpm). Anti-
M150 Ab could block the proliferation mediated by M150 only,
but not by other macrophage membrane proteins (Fig. 8). The
control cultures containing Th cells and medium or anti-CD3 Ab-
stimulated Th cells incubated either with medium alone or Ham Ig
induced proliferation less than 5000 cpm.
M150 is not an alloantigen but a costimulatory molecule and
works in an MHC-nonrestricted manner
OVA-specific Th cell lines were generated from BALB/c (IAd),
C57BL/6 (IAb), and C3He (IAk) haplotype of mice. M150 was
isolated from the macrophages of BALB/c (IAd) mice. Figure 9
shows that M150 induced significant proliferation of anti-CD3 Ab-
activated Th cells generated from BALB/c, C57BL/6, and C3He
mice. M150, however, did not exhibit any effect on Th cells of
syngeneic or allogeneic strains without the occupancy of TCR.
Anti-CD3 Ab-stimulated Th cells could not generate more than
cultures were set as described in the legend to Figure 3, A and B. The culture SN from the experimental and control wells were collected after 24 to 48 h
for the estimation of IL-4 and IFN-?, respectively. M* indicates macrophages activated with IFN-?. A, IFN-? was assayed by its ability to inhibit the
proliferation of WEHI-279 cells. The specificity of the inhibition induced by IFN-? was further established by the restoration of proliferation of WEHI-279
cells with anti-IFN-? Abs (4 ?g/ml). B, IL-4 was measured by its ability to induce the proliferation of HT-2 cells as described in Materials and Methods.
The specificity of IL-4 was established by blocking the proliferation of HT-2 cells by anti-IL-4 Ab (11B11, 1 ?g/ml). After 24 h of incubation, [3H]thy-
midine (1 ?Ci/well) was added and the cells were harvested and radioactivity incorporated was counted 6 h later. Ham Ig and Rt Ig used as controls could
not induce the secretion of lymphokines. All the data were calculated from the mean cpm of triplicate determinations.
Anti-M150 and B7-1 Abs inhibits the secretion of IFN-? but not of IL-4 when macrophages and B cells provide costimulus to Th cells. The
1072 ANTI-B7-1 AND M150 Abs INHIBIT COSTIMULATORY SIGNALS
5802 ? 1641 background cpm as compared with 907 ? 439 cpm
in the case of unstimulated Th cells.
CD4?T cells need at least two stimuli provided by APC for their
ultimate activation. The first signal is provided by the engagement
of clonotypic TCR by a complex of MHC class II molecules and
peptide. The second signal, known as costimulatory signal, is also
accessory cell derived (1–3). The significance of accessory cell
molecules in Th cell activation has gained considerable impetus
following the observation that occupancy of TCR alone is gener-
ally inadequate for exerting complete T cell activation. Several
reports are available in the literature highlighting the potential role
of a number of stimuli necessary for the initiation of the differen-
tiation events occurring during T cell-APC interactions. An array
of costimulatory molecules, e.g., ICAM-1, LFA-3, VCAM-1, IL-1,
heat-stable Ag CD40, M150, and B7 (4–8) are expressed on the
surface of APC. The exact mechanism of sequence of signals pro-
vided by different costimulatory molecules in stimulating T cells is
The best defined costimulators to date are two structurally re-
lated proteins, B7-1 and B7-2, both of which have been well doc-
umented in providing a critical costimulatory signal for T cell ac-
tivation by interacting with their specific receptor, CD28.
Although one might assume that CD28?/?mice should have ab-
sent T cell proliferation, cytokine production, and their responses
to mitogen or alloantigens, instead, it has been reported that only
30% responses were reduced (29). Furthermore, significant levels
of mRNA for IL-2, IL-4, and IFN-? can be detected at early time
points, presumably before the engagement of B7 and CD28,
needed for T cell activation in vivo and in vitro (29, 41), and
significant proliferative responses to soluble Ags can be detected.
by macrophages and B cells, respectively, for the proliferation of Th cells
in MLR. Alloreactive T cells were generated against macrophages and B
cells of C57BL/6 mice. The inhibition of the proliferation of T cells by
anti-M150 and anti-B7-1 Abs was monitored in the presence of macro-
phages, M*, and B cells as stimulator cells. The control cultures containing
T cells plus medium or mitomycin C-treated macrophages and B cells did
not show any significant radioisotype incorporation (cpm ? 1000). The
control Igs of Ham and Rt did not show any decrease in T cell proliferation.
Results represent the mean ? SD of data from triplicate cultures.
Anti-M150 and anti-B7-1 Abs block the signals generated
and anti-CD3 Ab-stimulated Th cells were cultured with LPS-activated B cells either in the presence of M150 (0.01 ?g/ml) or Abs to M150 and B7-1 (10
?g/ml). The control cultures were also kept using Ham Ig, Rt Ig, and control liposomes. After 48 h, 1 ?Ci [3H]thymidine was incorporated. The plates were
harvested 16 h later and processed for beta scintillation counting. B, For the estimation of IgG1 and IgG2a isotypes, the cultures were set as mentioned
in the legend to Figure 6A. SN were collected from triplicate wells of control and experimental cultures on day 6, pooled, and analyzed for IgG1 and IgG2a
by ELISA. The results are represented as OD of a single dilution (1:100) of mean absorbance of triplicate wells after subtracting the absorbance obtained
with control wells (i.e., SN obtained from LPS-stimulated B cells). Results are expressed as mean ? SD.
Anti-B7-1 Ab down-regulates whereas anti-M150 Ab shows no effect on B cell proliferation and IgG2a production. A, Mitomycin C-treated
1073 The Journal of Immunology
Furthermore, T cells can also be activated in a CD28-independent
manner (15). These findings, as well as studies demonstrating the
failure to inhibit activation of CD4?T cells in vitro (42) and in
vivo (43) with anti-B7 Abs and the fact that CD28?/?-deficient
mice response normally to nominal Ags, have raised the possibility
to hypothesize the existence of undefined additional cosignals es-
sential for the initiation of Th cell activation.
It is worth mentioning here that on the basis of their lympho-
kines profile CD4?T cells have been divided into Th1 and Th2
subtypes. Th1 cells secrete mainly IFN-?, IL-2, TNF-?, etc. and
are responsible for CMI whereas Th2 cells chiefly produce IL-4,
IL-5, IL-10, etc. and are responsible for humoral immunity (9, 10).
It is still not clear how the differentiation and activation of CD4?
T cells into Th1 and Th2 occurs. It appears that cytokines and
costimulatory signals delivered by APC may be the dominant fac-
tors in controlling the generation of Th1 and Th2 responses. To
date, studies related to the biology of costimulation have largely
been centered around activated B cells. Not many efforts have been
made to analyze the ability of macrophages to mediate costimu-
lation of T cells (12, 13, 18, 32). In this regard, we have recently
identified a 150-kDa surface molecule of macrophage that possibly
promotes Th1 cell activity (7). B7-1 also seems to be involved in
the generation of a similar type of response (14, 15).
In the present study, we have attempted to analyze the relation-
ship between anti-M150 and anti-B7-1 Abs in inhibiting the acti-
vation of Ag-specific Th cells. The following six major findings
have emerged from this study. 1) Anti-M150 Ab significantly
blocked the macrophage-mediated costimulation of Th cells but
not of B cells; 2) anti-B7-1 Ab inhibited B cell-mediated costimu-
lation but failed to check macrophage signals; 3) anti-B7-1 Ab
could also obstruct the cosignal delivered by IFN-?-activated mac-
rophages; 4) anti-M150 and B7-1 Abs blocked the secretion of
IFN-? when costimulated with macrophages and B cells, respec-
tively; 5) alloreactivity of macrophages was greatly checked when
anti-M150 Ab was utilized but not by using anti-B7-1 Ab, whereas
alloreactivity generated by B cells was abrogated by anti-B7-1 Ab
but not by anti-M150 Ab; 6) anti-B7-1 Ab greatly inhibited B cell
proliferation and IgG2a secretion.
It is noteworthy that macrophage-mediated costimulation of Th
cells was inhibited by anti-M150 Ab, but not by anti-B7-1 Ab.
However, anti-B7-1 Ab significantly obstructed the costimulation
when macrophages were preactivated with IFN-?. It may be men-
tioned that macrophages do not express B7-1, which is up-regu-
lated later in immune responses upon stimulation with IFN-? (30–
32). IFN-? appears to be secreted at a fairly late stage (48–72 h)
of Th cell activation (44). Therefore, it is safer to assume that at the
initial stage of macrophage-associated Th cell signaling, it might
be M150 through which macrophages provide accessory help to
Th1 cells. Macrophages do not express B7-1 but express a signif-
icantly higher level of M150. It is likely that the B7-1/CD28 path-
way may not be very crucial in the initiation of early events of Th
cell activation by macrophage; however, once the immune re-
sponse is generated, and IFN-? is available, it induces B7-1 and
up-regulates M150 expression on the macrophages. Furthermore,
the activation and clonal amplification of Th cells may then be
controlled by both M150 and B7-1. The fact that, unlike normal
macrophages, anti-M150 Ab could not sufficiently block Th cell
activation of macrophages activated by IFN-?, and the observation
that anti-B7-1 Ab blocked costimulation only when IFN-? acti-
vated macrophages were used, further strengthens this proposition.
LPS-activated B cells are known to express B7-1. Another in-
teresting feature observed during the study was that anti-B7-1 Ab
could significantly block the signals delivered by B cells for the
proliferation of Th cells. M150 was, however, expressed to a lesser
specifically inhibited by anti-M150 Ab, but not by anti-IL-12 Ab. The
complexes of M150 were prepared as described in Materials and Methods.
The complexes were added to the wells containing anti-CD3 Ab-stimulated
OVA-specific CD4?T cells. The cultures were kept for 72 h. The prolif-
eration of Th cells was monitored the last 16 h of 72-h cultures by [3H]thy-
midine incorporation. The OVA entrapped in liposomes was also used as
a control. The control cultures containing either Th cells plus medium
(1229 ? 1016 cpm) or Th cells stimulated with anti-CD3 Ab (6738 ? 569
cpm) and control liposomes, without M150 (6509 ? 1217 cpm), could not
induce a significant level of Th cell proliferation. A, To measure IFN-?, the
cultures were set as described in the legend to A. The culture SN from the
experimental and control wells were collected after 48 h and the secretion
of IFN-? was measured as mentioned in the legend to Figure 4A. The SN
obtained from the control cultures did not induce the production of IFN-?.
The data are the mean ? SD of triplicate experiments.
The induction of the proliferation of Th cells by M150 is
1074 ANTI-B7-1 AND M150 Abs INHIBIT COSTIMULATORY SIGNALS
extent on B cells. Furthermore, anti-M150 Ab could not signifi-
cantly inhibit the proliferation of Th cells. Thus it can be inferred
that at the initial stage of interaction macrophages and B cells use
different costimulatory signals for the activation of Th cells. One
may wonder why anti-M150 and anti-B7-1 Abs could not com-
pletely inhibit the signals delivered by B cells and macrophages. It
could possibly be because of the involvement of other costimula-
tory molecules like B7-2 and CD40 in providing costimulation to
T cells. It may be mentioned here that B7-2 is also reported on the
resting macrophages (15, 16).
We next performed experiments to evaluate the role of anti-
M150 and anti-B7-1 Abs in restricting the secretion of IFN-? and
IL-4 by Th cells when macrophages and B cells were used as APC.
Interestingly, anti-M150 Ab, but not anti-B7-1 Ab, inhibited the
secretion of IFN-? when Th cells were costimulated with macro-
phages. However, when macrophages activated with IFN-? were
used for costimulation, anti-B7-1 Ab also inhibited the production
of IFN-?. In contrast, anti-B7-1 Ab, but not anti-M150, blocked
the secretion of IFN-? when Th cells were activated with B cells.
None of the Ab could inhibit the secretion of IL-4. It may be
concluded here that the activity of IFN-?-producing Th cells can
be differentially inhibited by both anti-M150 and anti-B7-1 Abs
when macrophages and B cells, respectively, functioned as APC.
Anti-B7-2 Ab nevertheless inhibited the secretion of both IFN-?
and IL-4 when macrophages as well as B cells functioned as APC
(data not shown). Recent studies have suggested different conclu-
sions about the relative roles of B7-1 and B7-2 in mediating co-
stimulatory interactions with CD28/CTLA-4 and subsequent T cell
differentiation. Some reports have suggested that IL-4 production
is particularly dependent on B7-2 signaling. Also, administration
of anti-B7-1 and anti-B7-2 Abs during in vivo immune responses
blocks primarily IFN-? and IL-4 secretion, respectively (14, 24,
25). Furthermore, costimulatory molecules M150 and B7-1 may be
playing a major role in selectively regulating the secretion of
IFN-? and in the development and differentiation of CD4?T cells
subsets. Thus, Th cell differentiation not only depends on the type
of APC but also on the costimulatory molecules being displayed
on its surface (45–49).
We carried out more experiments to generate further evidence in
support of M150 and B7-1 providing costimulatory signals when
macrophages and B cells, respectively, are associated with Th cell
signaling. MLCs were set using macrophages and B cells as allo-
stimulator cells. Our results demonstrate that anti-M150 Ab effec-
tively blocked T cell alloreactivity when macrophages were used
as a source of stimulation, but it blocked partially when B cells
were employed. Conversely, anti-B7-1 Ab effectively blocked the
alloreactivity of B cells but not of macrophages. These experi-
ments further suggest that M150 is a primary costimulatory mol-
ecule on macrophages, while B7-1 functions chiefly from B cells.
We also looked into the role of these Abs in restraining the
proliferation and IgG1 and IgG2a Ab secretion by B cells. We
observed that anti-B7-1 Ab significantly blocked the proliferation
of B cells and the production of IgG2a. This may be because B7-1
probably provides costimulatory signal to Th1 cells (14). More-
over, it is also known that the interaction between Th1 and B cells
can lead to the secretion of IgG2a Ab, whereas cognate recognition
between Th2 and B cells induces the production of IgG1 Ab (50).
Since M150 is primarily expressed on macrophages, anti-M150 Ab
could not significantly effect the secretion of IgG istotypes by B
cells. However, when anti-CD3 Ab-stimulated Th cells were fur-
ther stimulated with M150, the capability of Th cells to provide
signals to B cells were dramatically improved, leading to enhanced
secretion of IgG2a, whereas IgG1 response declined slightly. It
may be recalled here that it has been reported that IFN-? primarily
activate B cells to secrete IgG2a and IL-4 induce the production of
Th cells, but not of other macrophage membrane proteins. Anti-CD3 Ab-
incubated Th cells were cultured with 21 different proteins (0.01 ?g/ml)
isolated from the membrane of macrophages. The proteins were also cul-
tured with anti-M150 Ab (10 ?g/ml). Except M150 and proteins of 59, 75,
and 85 kDa, none of the proteins could enhance Th cell proliferation above
the background level (i.e., anti-CD3 Ab ? Th cells, 4937 ? 613 cpm). The
induction of the proliferation of Th cells by M150 was blocked only by
anti-M150 Ab. After 72 h, 1 ?Ci/well of [3H]thymidine was added. The
cultures were harvested 16 h later, and incorporated radioactivity was mon-
itored. Data are expressed as mean ? SD of three experiments.
Anti-M150 Ab blocks the M150-induced proliferation of
OVA-specific Th cells generated from BALB/c, C57BL/6, and C3He mice
were cultured with M150 isolated from BALB/c mice in the presence or
absence of anti-CD3 Ab. Anti-CD3 Ab-stimulated Th cells showed 5802 ?
1641 background cpm. After 72 h, 1 ?Ci/well of [3H]thymidine was added.
The cultures were harvested 16 h later, and incorporated radioactivity was
monitored. Data are expressed as mean ? SD of three experiments.
M150 is a costimulatory molecule and not an alloantigen.
1075 The Journal of Immunology
IgG1 (51). As mentioned earlier, anti-CD3 Ab stimulated Th cells
secrete IFN-? when activated with M150, which may be stimulat-
ing LPS-activated B cells to produce IgG2a Abs.
The augmentations of the proliferation of Th cells and secretion
of IFN-? and IgG2a directly or indirectly have been reported for
IL-12, which is produced at high levels by macrophages (52, 53).
Although IL-12 (35–40 kDa) is not a 150-kDa protein, it was still
of concern that M150 may be this cytokine. To address this ques-
tion we confirmed that the proliferation of Th cells and the secre-
tion of IFN-? induced by M150 were specifically inhibited by
anti-M150 Ab, but not by anti-IL-12 Ab. Similarly, IL-12-induced
proliferation and production of IFN-? was not blocked by anti-
M150 Ab. Anti-IL-12 Ab also failed to recognize M150 on West-
ern blot (data not shown). It may also be mentioned here that Th
cells activated by M150 secrete IL-2 as well as IFN-? (7), how-
ever, when stimulated with IL-12, they produce IFN-? but not IL-2
(54). Moreover, mouse IL-12 (55) but not M150 can augment the
proliferation of PHA-activated human lymphoblasts.
The specificity of anti-M150 Ab was further established by the
fact that it recognizes only M150 but not other macrophage mem-
brane proteins by Western blot analysis. Furthermore, only M150-
mediated proliferation of Th cells was inhibited by anti-M150 Ab
(Fig. 8). Nevertheless, the isolated M150 contained only a single
protein and perhaps no minor contaminant was established by the
fact that in two-dimensional gel electrophoresis M150 appeared as
a single spot (7). The purity of each batch used in the experiments
or immunization was always checked by two-dimensional gel
The fact that M150 could not stimulate Th cells without TCR
cross-linking by anti-CD3 Ab proves that the molecule is not an
alloantigen. However, it works across the MHC barriers to provide
We conclude from our data that activation of CD4?T cells is
not a mere function of B7-1. Both M150 and B7-1 are potent
costimulatory molecules for IFN-?-producing OVA-specific Th
cells. M150 plays a crucial role in Th cell-macrophage interactions
for the early events of Th cell activation and IFN-? secretion. It is
reasonable to assume that IFN-? produced from this signal then
induces the expression of B7-1, which subsequently provides sig-
nals for further expansion of Th cells and secretion of lympho-
kines. Furthermore, M150 also helps in the augmentation of allo-
reactivity and enhances the ability of Th cells to help B cells and
in the secretion of IgG2a Abs and, to a much lesser extent, in B
cell-mediated activation of Th cells. B7-1 is an effective costimu-
latory molecule when B cells deliver signals to Th cells. Both of
the molecules probably promote Th1-type of cells, as is evident in
the secretion of IFN-? and production of IgG2a Abs by B cells.
Moreover, a possibility that those other costimulatory molecules,
viz., B7-2, CD40, etc., also have an effect in activation of Th cells
cannot be ruled out (Fig. 3, A and B).
The role of M150 and B7-1 in delivering costimulatory signals
from macrophage and B cell, however, needs to be evaluated on
the clonal Th1 cell population; and whether the costimulatory
pathway is mediated by these molecules alone or in concert needs
The authors are grateful to Dr. C. M. Gupta for consistent encouragement,
Professor N. K. Ganguly and Dr. H. Vohra, PGIMER, Chandigarh, India,
for helping with the FACScan, Dr. S. Majumdar for critically evaluating
the manuscript, and Mr. Dinesh Verma for secretarial help.
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1077 The Journal of Immunology