| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 10, 7766-7775, March 8, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
§,§,
, and**
From the Immunology Laboratory, Institute of
Microbial Technology, the Department of Experimental Medicine,
Postgraduate Institute of Medical Education & Research, Chandigarh
160036, and the ¶ Centre for DNA Fingerprinting and Diagnostics,
Hyderabad 500 076, India
Received for publication, June 25, 2001, and in revised form, November 27, 2001
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
To date, not much has been known regarding the
role of CD80 and CD86 molecules in signaling of B cells. The CD28/CTLA4
ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on the surface of
freshly isolated splenic B cells, and their expression is up-regulated by lipopolysaccharides. In the present study, we have investigated whether signaling via CD80/CD86 could alter the proliferation and
immunoglobulin synthesis of B cells. Splenic B cells were stimulated
with lipopolysaccharides in the presence of anti-B7-1 (16-10A1) and
anti-B7-2 (GL1) monoclonal antibodies (mAbs). Exciting features
observed during the study were that cross-linking of CD86 with GL1
enhanced the proliferation and production of IgG1 and IgG2a isotypes.
In contrast, anti-B7-1 (16-10A1) mAb could efficiently block the
proliferation and production of IgG1 and IgG2a. Furthermore, GL1 mAb
could also induce the secretion of IgG isotypes from B cell lymphomas.
Importantly, 16-10A1 could retard the growth of lymphomas and favored
the up-regulation of pro-apoptotic molecules caspase-3, caspase-8, Fas,
FasL, Bak, and Bax and down-regulation of anti-apoptotic molecule
Bcl-x(L). In contrast, GL1 augmented the level of anti-apoptotic
molecules Bcl-w and Bcl-x(L) and decreased the levels of pro-apoptotic
molecule caspase-8, thereby providing a novel insight into the
mechanism whereby triggering through CD80 and CD86 could deliver
regulatory signals. Thus, this study is the first demonstration of a
distinct signaling event induced by CD80 and CD86 molecules in B cell
lymphoma. Finally, the significance of the finding is that CD80
provided negative signal for the proliferation and IgG secretion of
normal B cells and B cell lymphomas. In contrast, CD86 encouraged the activity of B cells.
Evidence from a variety of studies has suggested that the B cell
contact with T helper cells is important for its optimal activation and
responsiveness to cytokines during Ig secretion (1-3). Contact between
B and T cells can be mediated by antigen presentation, as well as
antigen-independent cell interaction by molecules known as adhesion
(LFA-1, LFA-3, ICAM-1, etc.) and costimulatory molecules (CD80, CD86,
CD40, etc.) (3-7). Signals from T cells induce two opposite fates in B
cells: clonal expansion of B cells that bind specifically to foreign
antigens and clonal deletion of equivalent B cells that bind
self-antigen. The role of costimulatory molecules is very well
established in the activation of T cells (3), but nothing
has been determined definitively about how these molecules operate in
the activation and differentiation of B cells (8-10).
The best defined co-stimulators to date are two structurally related
proteins, CD80 and CD86 (11-14). Both of these play a major role in
providing costimulation to T cells, leading to their proliferation,
cytokine production, and development of effector functions. CD80 and
CD86 could also serve as counter-receptors that transduce distinct
signal to the antigen-presenting cells upon engagement by CD28
or CTLA-4. The intracellular domains of CD80 and CD86 are quite
distinct and could mediate differential signal transduction. Such
signaling could alter the B cells' ability to function as effector
cells. The ability of the B cells to deliver the costimulatory signal
to T cells by B7 molecules is very well established (15-17). In
contrast, whether the engagement of CD80 and CD86 molecules by CD28 and
CTLA-4 affect the function of the B cells is very poorly documented (9,
10). Moreover, nothing is known precisely in the case of B cell
proliferation and differentiation by the engagement of CD80 and CD86
molecules. However, there is indirect evidence reported earlier that
CD28-CTLA4/B7-signaling pathways may affect B cell responses and the
regulation of immunoglobulin (Ig) synthesis (18-20). To compare the
regulatory role of CD80 (B7-1) and CD86 (B7-2) molecules in humoral
immune responses, B7-1 Thus, the aim of our study was to evaluate for the first time the role
of CD80- and CD86-mediated signal transduction in the costimulation of
B cells. The results show that triggering through CD80 specifically
inhibits the proliferation and IgG secretion by
LPS1-stimulated B cells. By
contrast, CD86 chiefly augments the B cell activity. Further, the
growth of B cell lymphomas could also be retarded by CD80
cross-linking. Signaling through CD80 inhibited the growth of the B
cell lymphoma by up-regulating the expression of pro-apoptotic
molecules caspase-3, caspase-8, Fas, FasL, Bak, and Bax and
down-regulating the levels of anti-apoptotic molecule Bcl-x(L). In
contrast, triggering through CD86 augmented the level of anti-apoptotic
molecules Bcl-w and Bcl-x(L) and decreased the levels of caspase-8.
Thus, the signals delivered by anti-B7-1 and -B7-2 antibodies can
differentially regulate the activity of B cell and its lymphoma.
Animals
Female inbred BALB/c mice (8-10 weeks old) were obtained from
the National Institute of Immunology (New Delhi, India).
Antigens, Antibodies, Lymphokines, and Reagents
Fetal calf serum was purchased from Sera-Lab (Crawley
Down, United Kingdom), RPMI 1640 was from Invitrogen,
L-glutamine and streptomycin were from Serva (Heidelberg,
Germany), and recombinant IL-4 and IFN- Cell Lines and Hybridomas
The cell lines and hybridomas used in this study,
i.e. CRL 1704, TIB 208, HB 188, and HB 170, were procured
from American Type Culture Collection (ATCC; Rockville, MD).
Medium
Cells were cultured in RPMI 1640 medium supplemented with 10%
FCS, L-glutamine (2 mM), penicillin (50 µg/ml), streptomycin (50 µg/ml), and 2-mercaptoethanol (0.05 mM).
Cell Preparation
B Cells--
B cells were prepared as reported previously (2).
Briefly, a single cell suspension of BALB/c mice spleens was prepared in balanced salt solution. The red blood cells 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. Nonadherent
cells were incubated in nylon wool column for 1 h at 37 °C in
7% CO2. The adherent cells eluted from the column were
treated sequentially on ice for 45 min each with a mixture of anti-Mac2
and anti-Mac3 Abs and a mixture containing anti-Thy1, L3T4, and CD8
Abs, followed by complement-mediated killing. The purity of cells
stained with anti-IgM Ab was over 95%, as analyzed by FACS (Becton
Dickinson, Mountain View, CA).
Expression of B7-1 and B7-2 on B Cells and WEHI-279--
FACS
analysis was done as mentioned previously (2). Briefly, 1,000,000 B
cells (stimulated with 20 µg/ml LPS for 48 h) and WEHI-279 cells
were incubated with the anti-B7-1, anti-B7-2, and CD40 Abs (1.0 µg)
diluted in 100 µl of PBS plus 2% BSA for 1 h at 4 °C. The
cells were then washed three times with PBS plus 2% BSA. Anti-rat FITC
(1:135 dilution) was then added and the cells were further incubated
for 1 h at 4 °C. The cells were washed five times with PBS and
fixed in 1% paraformaldehyde and analyzed by FACS for the expression
of B7-1 and B7-2 molecules. As a control for anti-B7-1 and anti-B7-2
Abs, the cells were also incubated with RtIg.
The cells from each suspension were acquired on CellQuest software for
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 B cells. The analysis for the mean fluorescence
intensity (MFI) was done on histograms, where abscissa and ordinate
denote log FITC fluorescence and relative cell count, respectively.
B Cell Proliferation--
B cells activated with 20 µg/ml
lipopolysaccharide (5 × 103 to 1 × 105 cells/well) were incubated with anti-B7-1 and -B7-2
mAbs at a concentration of 0.01-1.0 µg/ml in a total volume of 200 µl of RPMI, 10% FCS at different (24-96 h) time intervals. The
LPS-activated B cells (1 × 105/well) were also
cultured with optimum dose of IFN- IgG1 and IgG2a Isotypes
The cultures were set as mentioned in B cell proliferation
assays. The SNs were collected on day 6 from the experimental as well
as control wells and were analyzed for IgG1 and IgG2a by enzyme-linked
immunosorbent assay (2). Briefly, triplicate wells were coated
overnight at 4 °C with 5 µg/ml goat anti-mouse IgG1 and IgG2a Abs
in carbonate-bicarbonate buffer, pH 9.6. The unbound sites were blocked
with 2% BSA, and then log 6 dilutions of culture SNs were added for
2 h at 37 °C. The plates were then incubated with rabbit
anti-mouse IgG horseradish peroxidase-labeled antibody at 37 °C for
2 h. The usual steps of washings with PBS-Tween 20 were carried at
each step. The color developed because of the substrate OPD was
visualized at 492 nm. The concentration of Abs is represented as
nanograms/ml, as computed by using standard mouse IgG1 and IgG2a isotypes.
Flow Cytometric Analysis of Intracellular IgG1 and IgG2a
Synthesis
The B cells (5 × 10 5/ml) stimulated
with LPS (20 µg/ml) were cultured with anti-B7-1 and -B7-2 mAbs (1.0 µg/ml) for 72 h. Four hours before harvesting the cells,
brefeldin A (10 µg/ml) was added. The cells were harvested and
resuspended in PBS containing brefeldin A and fixed by adding equal
volume of 4% paraformaldehyde for 20 min. The cells were incubated for
10 min in PBS/BSA/saponin to permeabilize the cells. The cells were
blocked with 0.5% rabbit sera and saponin and stained immediately for
intracellular expression of IgG1 and IgG2a using a modified method
described by Murphy et al. (22). The cells were incubated
with anti-IgG1-biotin and anti-IgG2a-biotin (2 µg/ml) for 30 min and
then with avidin-FITC (1:1000 dilution). The usual steps of washings
were followed at each step using 1% BSA and 0.5% saponin. The last
washing was done in PBS/BSA without saponin to allow membrane closure.
The samples were analyzed by FACScan flow aptometer (Becton Dickinson). Results were analyzed by CellQuest software.
The Proliferation of WEHI-279 Lymphoma and the Secretion of IgG1
and IgG2a Isotypes
Different concentrations of WEHI-279 cells (5 × 102 to 5 × 103/well) were cultured with
anti-B7-1, B7-2, and CD40 mAbs (0.01-1.0 µg/ml) in a total volume of
200 µl of RPMI, 10% FCS. The suitable controls containing WEHI-279
cells cultured in the presence or absence of LPS, control RtIg, or
without antibodies were also kept. The cells were incubated for 72 h, pulsed with 0.5 µCi of [3H]thymidine for another
4 h, and then processed for The Expression of Fas and FasL Demonstrated by FACScan on the
Surface of B Cells and WEHI-279 Lymphoma Stimulated with Anti-B7-1,
B7-2, and CD40 Abs
B cells (LPS-stimulated) and WEHI-279 cells were cultured for
48 h with 1.0 µg/ml anti-B7-1, B7-2, and CD40 Abs and control RtIg in 200 µl of RPMI 1640 and 10% FCS. The cells were washed and
labeled with anti-Fas-FITC and anti-FasL-biotin/avidin-FITC. As
mentioned above, the expression was demonstrated by FACScan.
Demonstration of Apoptosis on B Cells and WEHI-279 Cells Induced
by Anti-B7-1 and -B7-2 Abs
Apoptosis was detected by utilizing the method developed by
Nicoletti (23). Briefly, B cells and WEHI-279 cells were stimulated with anti-B7-1 and -B7-2 Abs (1.0 µg/ml) for 48 h. The cells
were further incubated with anti-Fas Ab (1.0 µg/ml) for a period of 14 h. The cells harvested from the cultures were centrifuged; the
cell pellet was fixed in 0.5 ml of cold 70% ethanol at 4 °C for
1 h. The cells were centrifuged again and resuspended in 0.5 ml of
RNase (1.0 mg/ml) and incubated at room temperature for 20 min.
Finally, 30 µl of propidium iodide (0.1 mg/ml) was added into each
sample and incubated for another 10 min in the dark at room temperature
before cell cycle was analyzed by flow cytometry. Apoptosis in the
cells was analyzed by a FACScan with cytomation, data acquisition, and
software (lysis II) for red fluorescence. Initial identification of the
cells was made with the help of FSC/SSC plots. The apoptotic cells were
selected on the basis of the expression of higher SSC because of the
condensation of the nuclear chromatin. The debris was excluded on the
basis of its very low SSC and FSC signals, and normal cells were gated and analyzed.
The Induction of the Expression of Pro-apoptotic and
Anti-apoptotic Molecules after Cross-linking CD80 and CD86 Molecules on
WEHI-279 Cells
Multi-probe Ribonuclease Protection Assay--
The WEHI-279
cells were cultured with anti-CD80 Ab, anti-CD86 Ab, and RtIg Ab and
cross-linked with anti-RtIg Ab as mentioned for the proliferation
assay. Total RNA was extracted from WEHI-279 cells by the guanidinium
isothiocyanate-phenol-chloroform method using Trizol reagent
(Invitrogen). The expression was determined using the RiboQuant
multiprobe RNase protection assay system (PharMingen) following the
manufacturer's instruction (24). Briefly, 5 µg of total RNA was
hybridized to 32P-labeled RNA probes overnight at 56 °C,
followed by treatment with RNase for 45 min at 30 °C. In each set we
have included L32 (which encodes a ribosomal protein) and
glyceraldehyde-3-phosphate dehydrogenase genes to serve as housekeeping
gene controls. The samples were submitted to electrophoresis through a
8 M urea, 5% polyacrylamide gel. The probe lengths are
greater than the "protected" fragment lengths; this is because of
the presence of flanking sequences. The resulting resolved bands were
imaged using a phosphorimager (Bio-Rad Molecular Imager FX). The
normalized quantity for each band was obtained by dividing with L32
housekeeping gene control. The -fold activation (increase or decrease)
was calculated by dividing the value of normalized quantity of the experimental samples with that of WEHI-279 cells cultured with medium alone.
Western Blotting--
WEHI-279 cells were incubated with
anti-B7-1 Ab or RtIg and cross-linked with anti-RtIg as mentioned for
the proliferation assay. The experiment was done as mentioned
previously (25, 26). Briefly, after incubation, cells were washed twice
with ice-cold PBS and re-suspended in lysis buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1.0 µg/ml aprotinin, 1.0 µg/ml
leupeptin, 2 mM dithiothreitol, 10 mM Tris-HCl,
pH 8) for 5 min at 4 °C followed by protein determination with BCA
method (Pierce). The protein (50 µg) was resolved on 12%
polyacrylamide gel and was assayed by Western blotting using anti-caspase-3 Ab. The expression levels were analyzed using NIH Image software.
Expression of CD80 and CD86 on B Cells and WEHI-279 Cells
The FACScan revealed the presence of CD80 (MFI of 106.49) and CD86
(MFI of 155.80) molecules on B cells incubated with LPS for 48 h
(Fig. 1a). We also evaluated
the expression of CD80, CD86, and CD40 molecules on the B cell
lymphoma, WEHI-279. WEHI-279 expressed CD80 (MFI of 190.09), CD86 (MFI
of 111.51), and CD40 (MFI of 125.73) molecules (Fig. 1b).
The RtIg used as control for anti-B7-1 and -B7-2 mAbs failed to show
any significant shift in MFI.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ and B7-2/ mice
were generated and profound deficits in both antigen-specific IgG1
(Th2-dependent) and IgG2a (Th1-dependent)
responses demonstrated that B7 costimulation can influence IgG
production in vivo (21). However, the basis of this abnormal
Ig production was contributed to the absence of Th1- or Th2-specific
cytokines in B7-1/2/ mice that regulate the IgG1 and
IgG2a production.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were the products of Genzyme
(Boston, MA). LPS (from Salmonella typhosa), mIgG1, mIgG2a,
and rabbit anti-rat FITC-labeled Ab were procured from Sigma and goat
anti-rat IgG F(ab)2 from Jackson ImmunoResearch
Laboratories (West Grove, PA). Anti-B7-1, CD40, Fas-FITC, FasL-biotin
mAbs were purchased from PharMingen (San Diego, CA) and anti-B7-2 Ab
was a gift from Dr. Vijay Kuchroo (Boston, MA). Rabbit anti-caspase-3
(CPP32) antibody was purchased from Santa Cruz Biotechnologies. The RPA
kit was purchased from PharMingen. Enhanced chemiluminescence kit (ECL) was a product of Amersham Biosciences, Inc., and polyvinylidene difluoride membrane was from Bio-Rad.
(10 units/ml) and IL-4 (100 units/ml) in the presence of different concentration of anti-B7 mAbs.
The suitable controls containing B cells, LPS-activated B cells
incubated with control RtIg, or B cells without antibodies were also
kept. The cells were incubated at 37 °C in a humidified atmosphere
containing 7% CO2. After the specified time period, the
cultures were pulsed with 0.5 µCi of
[3H]thymidine/well. 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.
-scintillation counting.
Similar types of cultures were also kept for 4-5 days, and SNs were
harvested and analyzed for IgG1 and IgG2a secretion as mentioned above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
B cells and WEHI-279 express B7-1 and B7-2
molecules. The B cells (stimulated with LPS) (a) and
WEHI-279 (b) were stained with anti-B7 Abs and secondary
anti-RtIg-FITC. In the case of WEHI-279, the data depict probing of the
cells with RtIg (A), anti-B7-1 Ab (B), anti-B7-2
Ab (C), and anti-CD40 Ab (D). The expression of
the molecules on the cells was analyzed by FACS. The data shown in the
parentheses depict MFI. The figure is representative of at
least three experiments.
Cross-linking of CD80 Inhibits but CD86 Increases the Proliferation of LPS-activated B Cells
We next determined whether triggering of CD80 and CD86 molecules
by their respective mAbs will affect the proliferation and differentiation of LPS-activated B cells. The experiments were performed with different concentrations of responder cells and readout
at different time points after stimulation. Surprisingly, cross-linking
of CD80 with anti-B7-1 mAb (16-10A1) significantly inhibited the
proliferation of different doses (5 × 103 to 1 × 105 cells/well) of LPS stimulated B cells from as early
as 24 h of the cultures. The maximum decline (61.56%) in the
proliferation was observed when the B cells were cultured at the
concentration of 5 × 104 with 16-10A1 (1.0 µg/ml)
for 48 h (Fig. 2A). It is
worth to mention here that 16-10A1 was found to be highly potent for a
range of 5 × 103 to 1 × 105 cells
at a concentration of 1.0 µg/ml in significantly down-regulating the
proliferation of B cells at any time period (24-96 h) of the study.
The response was observed in a dose-dependent manner.
|
Unlike anti-B7-1 mAb, signaling by anti-B7-2 mAb (GL1) significantly enhanced the proliferation of LPS stimulated B cells (Fig. 2B). There was gradual increase in the proliferation of B cells, irrespective of cell concentration (5 × 103, 1 × 104, 5 × 104, and 1 × 105/well). The maximum and significantly high proliferation (97.86%) was observed when 1 × 105 cells/well were cultured with GL1 (1.0 µg/ml) for 48 h. In contrast, only 20.70% enhancement in the growth of B cells was observed when the B cells were cultured for 96 h. On account of overproliferation and the death of the cells, the magnitude of proliferation declined by the end of 96 h.
No spectacular difference in the activity of LPS-activated B cell was noticed in the control cultures incubated with RtIg. B cells incubated with medium and anti-B7-1 and -B7-2 mAbs did not register any proliferation.
Regulation of the Secretion of IgG1 and IgG2a Isotypes by B Cells Stimulated with Anti-B7-1 and -B7-2 mAbs
Anti-B7-1 mAb Inhibits whereas Anti-B7-2 mAb Augments the
Production of IgG Isotypes--
We also evaluated the impact of
signaling through CD80 and CD86 on the secretion of IgG1 and IgG2a
isotypes. We have chosen to measure IgG1 and IgG2a isotypes because of
their production precisely by IL-4 and IFN-, respectively (17).
Interestingly, anti-B7-1 mAb down-regulated the production of both IgG1
and IgG2a (Fig. 3A). The
concentration measured of IgG1 Ab was 114 ng/ml, and that of IgG2a was
258 ng/ml, when the B cells were cultured with LPS. There was
significant decline in IgG1 from 114 to 44 ng/ml and in IgG2a from 258 to 44 ng/ml, when anti-B7-1 Ab was added into the cultures (Fig.
3A). More interestingly, anti-B7-2 Ab critically increased
the production of IgG1 (from 116 to 1750 ng/ml) and IgG2a (from 260 to
1944 ng/ml) isotypes, when cocultured with LPS-stimulated B cells (Fig.
3B).
|
The inhibitory signals delivered by anti-B7-1 mAb (1.0 µg/ml) were
powerful enough to down-regulate the secretion of IgG1 and IgG2a
induced by IL-4 (IgG1; from 610 to 518 ng/ml) and IFN- (IgG2a; from
708 to 258 ng/ml), respectively (Fig. 3A). However, in the
case of anti-B7-2 Ab (1.0 mg/ml) stimulation, IL-4 (100 units/ml) and
IFN- (10 units/ml) worked in a synergistic fashion with the Ab and
augmented the yield of IgG1 (from 610 to 2188 ng/ml) and IgG2a (from
710 to 2482 ng/ml), respectively (Fig. 3B). Anti-IL-4 and
IFN- Abs could not block the production of IgG1 and IgG2a isotypes,
respectively, induced by anti-B7-2 mAb (data not shown). The mAbs
worked in a dose-dependent manner, and the maximum activity
was attained at a concentration of 1.0 µg/ml. The control RtIg used
in the cultures could not influence any change in B cell activity.
Cross-linking of CD80 Down-regulates but Signaling through CD86 Increases the Intracellular Expression of IgG1 and IgG2a Isotypes
We also observed the distinct effect on the intracellular
expression of IgG1 and IgG2a isotypes upon cross-linking CD80 and CD86
molecules with their respective mAbs. Similar results were seen as
observed in the case of IgG secretion. Anti-B7-1 mAb diminished, whereas anti-B7-2 mAb enhanced, the intracellular exhibition of IgG1
and IgG2a-isotypes (Fig. 4).
|
CD80 Cross-linking Fails to Alter the Viability of B Cells
It was of concern for us whether the decrease in the proliferation
in B cells is a direct result of the death of the cells induced by
anti-B7-1 mAb stimulus or the inhibition in the DNA synthesis.
Therefore, we co-cultured LPS-stimulated B cells with anti-B7-1 mAb and
then monitored the viability of cells by propidium iodide staining. It
was ascertained that anti-B7-1 mAb signaling did not alter the
viability of the cells (Fig. 5). No
change in the expression of Fas and FasL was also observed on B cells
(data not shown).
|
Effect of Cross-linking of CD80, CD86, and CD40 Molecules on B Cell Lymphoma
Inhibition of the Proliferation of WEHI-279 by Anti-B7-1 and CD40
Abs--
Because the CD40 molecule has differential effect on normal B
cells and B cell lymphomas (27), we evaluated the signaling capacity of
CD80, CD86, and CD40 molecules in the proliferation of WEHI-279 B cell
lymphoma (Fig. 6). Interestingly,
anti-B7-1 and CD40 mAbs but not anti-B7-2 mAb could significantly
down-regulate the growth of WEHI-279 cells. The dose of 1.0 µg/ml
anti-B7-1 mAb was highly potent in inhibiting the proliferation of 500 and 5000 cells up to 43 and 29%, respectively (Fig. 6, A
and B). Similarly, the extent of anti-CD40 mAb mediated
inhibition was comparable with the effect induced by anti-B7-1 mAb. It
has also been reported previously that anti-CD40 mAb renders WEHI-279
cells sensitive for Fas-mediated killing (14). However, for the first
time we have demonstrated that anti-CD40 and anti-B7-1 mAbs show
synergistic effect. Both the Abs, when added into the cultures,
inhibited the proliferation of WEHI-279 by 83 and 48% for the two
doses of the cells used in the experiments, respectively (Fig. 6,
A and B). The control cultures containing RtIg
did not induce any significant change in the growth of the cells.
|
Induction of the Secretion of IgG1 and IgG2a Isotypes by Anti-B7-2
mAb-stimulated WEHI-279 Cells--
Because anti-B7-1 and -B7-2 mAbs
could regulate the proliferative response in B cells and WEHI-279
lymphoma and the differentiation events in B cells, we were therefore
curious to know whether these antibodies could exert any impact on the
secretion of IgG1 and IgG2a by WEHI-279 cells. Accordingly, we
monitored the secretion of IgG1 and IgG2a isotypes, in the cultures of
WEHI-279, stimulated with antibodies to CD80 (B7-1), CD86 (B7-2), and
CD40 molecules. WEHI-279 cells cultured with medium alone or with LPS
failed to show detectable level of IgG-isotypes. Most surprisingly,
anti-B7-2 mAb could significantly induce WEHI-279 cells to secrete IgG1 and IgG2a (Fig. 7). However, anti-B7-1
and CD40 mAbs completely failed to stimulate WEHI-279 cells to produce
IgG isotypes. Similar effects were also observed in the case of A20
lymphoma (data not shown). The control
cultures comprising WEHI-279 cells and RtIg could not induce any
change.
|
|
Cross-linking of CD80 and CD40 but Not CD86 Induces the Expression of Fas and FasL on WEHI-279
Because anti-B7-1 and CD40 mAbs exerted decline in the growth of
WEHI-279, it became essential for us to monitor the cause of this
response. We therefore did flow cytometry to explore the possibility of
the expression of Fas and FasL on WEHI-279 cells incubated with
anti-B7-1, B7-2, and CD40 Abs (Fig. 8).
Importantly, anti-B7-1 and CD40 mAbs but not anti-B7-2 mAb could
successfully augment the expression of Fas and FasL on WEHI-279 cells
(Fig. 8, a and b). The control cultures involving
RtIg did not display any change in Fas and FasL expression.
|
Cross-linking of CD80 Induces WEHI-279 Cells to Undergo Apoptosis
We finally analyzed the functional activity of Fas and FasL
induced on the surface of WEHI-279 cells by propidium iodide staining. Interestingly, WEHI-279 cells incubated with anti-Fas mAb in the absence of CD80 cross-linking showed a MFI of 5.80 (Fig.
9A). However, cross-linking
of CD80 into the cultures exhibited 8.00-fold increase (MFI of 46.40)
in the number of cells undergoing apoptosis (Fig. 9B). CD86
cross-linking could induce only marginal change (Fig. 9C).
We also observed the DNA-ladder formation when CD80 was cross-linked
(data not shown).
|
Cross-linking of CD80 Enhances the Expression of Pro-apoptotic Molecules, whereas CD86 Up-regulates the Level of Anti-apoptotic Molecules
Finally, we tested the involvement of the apoptosis-regulatory
molecules representative from several classes of proteins, namely
caspase-3, caspase-8, Fas, FasL, Fas-associated death domain, Fas-associated phosphatase, Fas-associated factor, TNF-related apoptosis-inducing ligand (Fas2L), tumor necrosis factor (TNF) receptor
(p55), TNF receptor-associated death domain, receptor inhibitory
protein, Bak, Bax, Bad, Bcl-w, Bfl-1, Bcl-xL, and Bcl-2 (Figs. 10-12). The expression of the
molecules was analyzed by flow cytometry (Fig. 8), ribonuclease
protection assay (RPA) (Figs. 10 and
11), and Western blotting (Fig.
12). Anti-CD80 Ab mediated signaling
significantly and reproducibly up-regulated the levels of caspase-3,
caspase-8, Fas, FasL, Bak, and Bax (Figs. 10-12). Maximum increase was
observed in the case of Bax (8.16-fold) followed by Fas (8.0-fold), Bak
(4.95-fold), FasL (4.6), and caspase-8 (4.16) (Figs. 10 (a
and b) and 11 (a and b)). Further,
stimulation through CD80 molecule could also decrease the expression of
anti-apoptotic molecules Bcl-x(L) (4.95). The expression of other
pro-apoptotic (namely Fas-associated death domain, Fas-associated
phosphatase, Fas-associated factor, TNF-related apoptosis-inducing
ligand, TNF receptor, TNF receptor-associated death domain, receptor
inhibitory protein, and Bad) and anti-apoptotic (Bcl-w, Bfl-1, Bcl-2)
molecules remained unaltered. In contrast, CD86 mediated signaling
significantly enhanced the expression of anti-apoptotic molecules Bcl-w
(4.23-fold) and Bcl-xL (3.36-fold) (Fig. 11, a
and b). Potent decrease in the level of pro-apoptotic marker
caspase-8 (4.04-fold) and marginal down-regulation of Bak was also
noticed.
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
An array of costimulatory molecules, e.g. ICAM-1, LFA-1, VCAM-1, HSA, CD40, and B7 (1-5), are expressed on the surface of B cells. Their role has gained considerable significance in the activation of T cells, but very little is known in connection with the activation of B cells (7, 8). The exact mechanism of sequence of signals provided by different costimulatory molecules in the stimulation and inhibition of B cells is largely unknown.
In the present study, we have attempted to analyze the distinct role of CD80 and CD86 costimulatory molecules in the activation and differentiation of B cells. The following five major findings have emerged from this study. 1) Cross-linking by anti-B7-1 mAb (16-10A1) inhibited the proliferation and differentiation of B cells; 2) anti-B7-2 mAb (GL1) promoted the growth and differentiation of B cells; 3) anti-B7-1 and CD40 mAbs acted synergistically and declined the growth of B cell lymphomas; 4) induction of secretion of IgG isotypes by WEHI-279 cells was triggered with anti-B7-2 mAb; 5) anti-B7-1 mAbs induced apoptosis in B cell lymphoma by enhancing the expression of caspase-3, caspase-8, Fas, FasL, Bak, and Bax and decreasing the expression of anti-apoptotic molecules Bcl-x(L).
Our findings suggest that CD80 and CD86 costimulatory molecules are responsible for delivering the signals essential for the inhibition and expansion of B cells, respectively. Hence it may be inferred that anti-B7-1 mAb provides a type of inhibitory signals to B cells similar to those delivered by CTLA-4 to T cells (28). Different studies have suggested a role of CD28 and CTLA-4 in the regulation of Ig synthesis. Defective production of switched Ig isotypes is seen both in mice treated with soluble CTLA-4 and in mice transgenic for CTLA-4 (16, 29, 30). CD28-deficient mice have reduced basal levels of IgG1 and IgG2a and diminished immunoglobulin class switching (15). Further, in vivo blocking of CD86 with anti-B7-2 mAb has shown the arrest of the germinal center formation and resulted in the reduction in primary antibody production (31). These results are in line with our observations about the negative signal transduction through CD80. To best of our knowledge, this is the first report regarding the role of CD80 and CD86 molecules in influencing the B cell activity. In the present study, we used a T-independent system and preferred to ligate CD80 and CD86 with their respective mAbs to rule out the possibility of interference of other accessory signals when B cell interacts with T cell. As compared with CD28, CTLA-4 molecule binds to CD80 with 20-fold higher avidity. It is also known that signaling through CTLA-4 can function to suppress the production of cytokines produced by T-helper cells that helps the differentiation of B cells (32). CD86 is also known to bind to CTLA-4 but with low affinity as compared with CD80. Similarly, interaction of CD86 with CD28 has high affinity, and delivers positive signals for T cell activation. Further, the property of CD80 in delivering inhibitory signals may also be related to its slower off-rate for CTLA-4 binding, thereby providing ample time to deliver signaling events necessary for restraining B cells from being activated (33).
The kinetics of rapid and high level of expression of CD86 in early immune responses and the delayed expression of CD80 on antigen presenting cells can be viewed as necessary activation and inhibitory signals, respectively, delivered by these molecules during the immune response (34).
Another important conclusion that can be drawn from our data is that
the IL-4- and IFN--induced secretion of IgG1 and IgG2a, respectively, could be inhibited by anti-B7-1 Ab. IL-4 and IFN- acted synergistically with anti-B7-2 Ab and augmented the production of
IgG1 and IgG2a, respectively. Interestingly, IL-4 could inhibit the
secretion of IgG2a induced by anti-B7-2 Ab but IFN- failed to
obstruct the secretion of IgG1, even though the latter has antagonistic
property for IL-4. At present, this activity of IL-4 and IFN- is
difficult to explain. However, it has been reported that IL-4 induces
the secretion of IgG1 by LPS-activated B cells but inhibits the
formation of IgG2a. In contrast, IFN- promotes the yield of IgG2a
and down-regulated the production of IgG1 (17).
In the present study, B7-1 and B7-2 served as counter-receptors that transduced distinct signals to B cells upon engagement with their respective antibodies. The differential signaling effect may also be because of the dissimilarity in the structure and, in particular, the nature of their cytoplasmic tail (18). Especially B7-2 has a significant cytoplasmic tail, which has a markedly different cytosolic domain from that of B7-1 and contains a tyrosine residue. Further, unlike B7-1, the cytoplasmic domain of B7-2 contains three potential sites for phosphorylation by protein kinase C (35-37). The structure of B7-1 has a short cytoplasmic tail and does not predict intrinsic tyrosine kinase activity; therefore, if B7-1 must transduce signals, specific tyrosine kinases must associate with the receptor (38, 39). Earlier studies have suggested the transmembrane signaling through B7/BB1 expressed on T lymphocytes (40). Finally, it has been observed recently that the two regions in CD80 cytoplasmic tail regulate T cell costimulation and CD80 redistribution. A 30-kDa phosphoprotein has been found to be associated with the cytoplasmic tail of CD80 after cell activation, suggesting it may play a role in CD80 function (41). In line with these observations, it has been demonstrated that CD80 and CD86 can mediate differential signal transduction, and such signaling could distinctly regulate the B cell's function as an effector cell (7, 8, 19, 20).
Our experiments also demonstrate that the augmentation or inhibition of B cell activity is a concerted effect of the signaling by B7 molecules and cytokines. It was still of concern that this activity may be because of survival/death status only, and all changes in levels of Ig production and costimulation simply lead to further enhancement or decrease in B cell survival. To address the issue, the experiments were carried to study the role of anti-B7 Abs in affecting B cell viability and trans-membrane signaling. The triggering of B cells by anti-B7-1 or anti-B7-2 mAbs could not cast any impact on the viability of normal B cells, as evidenced by propidium iodide staining. Further, CD80 cross-linking could not induce Fas or FasL expression on the surface of B cells. Furthermore, we established categorically that this event of B cell activity is because of signaling, as evidenced by rapid alteration in phosphorylation of tyrosine residues, and not because of overproliferation/death by CD86 cross-linking (data not shown). This was further substantiated by differential regulation of intracellular expression of IgG1 and IgG2a by CD80 and CD86 ligation. These experiments very clearly demonstrate that isotype production is not a mere function of proliferation/survival of B cell, rather it is because of delivery of stimulatory or inhibitory signals through CD80 and CD86 costimulatory molecules, respectively.
To further provide weight to our findings of the regulatory capacity of anti-B7-1 and -B7-2 Abs in the activation of B cells, we utilized B cell lymphomas, WEHI-279, which express CD80, CD86, and CD40 molecules. More surprisingly, anti-CD80 and CD40 Abs could significantly arrest the proliferation of WEHI-279. Thus it may be concluded that the signaling via CD80 and CD40 molecules may be responsible for inhibiting the growth of B cell lymphomas. In contrast, costimulation through CD86 may help lymphomas to proliferate and secrete antibodies that may help lymphomas to evade immune surveillance. Further, triggering through CD80 and CD40 may help the regression of the lymphomas in the early stages. Although signaling through CD80 retards the growth of B cell lymphomas by inducing the expression of Fas and FasL and in turn induces apoptosis, it fails to induce Fas and FasL or apoptosis in normal B cells. At present, it is difficult to explain why ligation of B7-1 with anti-B7-1 has a differential effect on normal versus tumor B cells. This could be because of the involvement of the distinct domains of CD80 in normal B cells and B lymphomas. Moreover, this observation is in agreement with the fact that, in the case of CD40 molecule, antibodies to CD40 stimulate normal B-lymphocytes but inhibit proliferation of B cell lymphoma (27). Further, binding of CD40 ligand to CD40 on a murine B lymphoma induces B7-1 and inhibits cell growth (12, 14). The dual nature of signaling events has also been reported in the case of BCR triggering. It has been shown that BCR signaling induces apoptosis in germinal center B cells but not in peripheral B cells. Mature germinal center B cells can re-acquire sensitivity to BCR-induced cell death following CD40 ligation. In contrast, neither virgin nor memory B cells become susceptible to antigen receptor-triggered apoptosis upon CD40 stimulation (42).
Apart from demonstrating the expression of Fas and FasL by FACScan and their involvement in inducing apoptosis, we also monitored by ribonuclease protection assay the involvement of CD80 and CD86 molecules in the regulation of the activation of several pro- and anti-apoptotic molecules in WEHI-279 B cell lymphoma. We have observed that signaling through CD80 molecule augmented the levels of pro-apoptotic molecules, i.e. caspase-3, caspase-8, Fas, FasL, Bak, and Bax. This suggests that CD80 signaling induces apoptosis in WEHI-279 via mechanism involving pro-apoptotic molecules caspase-3, caspase-8, Fas, FasL, Bak, and Bax, thus rendering WEHI-279 cells more vulnerable to apoptosis and therefore restricting the progression of the lymphoma. The T cell co-stimulatory molecule CD28, which has a high and low binding affinity for CD86 and CD80, respectively, promotes T cell survival by up-regulating Bcl-x(L) and down-regulating FasL expression (43). Based on these results and our findings, it may be postulated that there may exist a probability that, when CD28 is ligated with CD86, it may deliver a protective signal necessary for the clonal expansion of B cells. In contrast to this, when CTLA-4 gets engaged with CD80, it may deliver a lethal signal responsible for controlling the clonal expansion of B cell and B cell lymphomas. Further, overexpression of caspases is sufficient to cause apoptosis and Bax have been correlated with disease progression and shorter survival of B cell chronic lymphocytic leukemia patients (44, 45). In contrast, signaling through CD86 increased the expression of anti-apoptotic molecules Bcl-w and Bcl-x(L). Thus, there may be a possibility that ligation of B7-2 on WEHI-279 may promote their survival by increasing the expression of anti-apoptotic proteins Bcl-w and Bcl-x(L). It has also been reported earlier that increased expression of Bcl-x(L) but not Bcl-2 could prevent the apoptosis in B cell lymphoma WEHI-231 (46). Further, it has been reported that signaling through CD40 up-regulated Bcl-x(L) and Bfl-1 and protected B cell lymphoma from apoptosis (47).
Recent studies have highlighted the lack of costimulatory molecules as a predominant reason for the inefficient tumor rejection (13, 14, 48). Moreover, if the lymphomas express CD80, signaling through this molecule may impede their growth by inducing the expression of pro-apoptotic and down-regulating the levels of anti-apoptotic molecules and thereby facilitating apoptosis (49, 50). It is important to mention here that CD80-mediated delivery of inhibitory signals may not only be responsible for apoptosis of CD80-bearing tumor cells, but may also be responsible in the regulation of immune responses and maintaining self-tolerance in B cells. Thus, in the case of tumors, ligation of B7-1 by anti-CD80 Ab may also deliver effective anti-tumor immunity.
In conclusion, we report here that selective inhibition of B cell
proliferation and differentiation via CD80 signaling may be viewed as a
novel strategy to restrict the undesired stimulation of B cell and
progression of B cell lymphomas. The triggering through B7-2 may play a
crucial role in the activation of B cells. This observation may
therefore explain why there is the existence of both the isomers on a
single cell. Taken together with the data from others, these
observations allow us to propose a sequential positive and negative
feedback model between B and T cells. The predominance of the signals
transduced through CD28 and CTLA-4 govern the outcome of a T cell
immune response. As the CD80 and CD86 have been found to have
differences in their ability to bind CD28 and CTLA-4, the predominant
engagement of CD86 with CD28 leads to proliferation of T cells, whereas
stronger avidity of CTLA-4 with CD80 delivers a down-regulatory signal
for T cell responses (29). On the other hand, our results show that
stimulation through CD80 and CD86 can modulate the humoral response by
transducing positive and negative signals in B cells and may control
the progression of B cell lymphomas.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Dr. S. Swain (Trudeau Institute, Saranac Lake, NY) and Dr. S. E. Hasnain (Center for DNA Fingerprinting and Diagnostics, Hyderabad, India) for providing facilities for RPA and Western blotting experiments, respectively, and to Dr. T. Randall (Trudeau Institute) for critically evaluating the manuscript. We are also grateful to N. Lepak for helping in the RPA assay and analysis, Dr. S. K. Basu and Dr. R. Anand (National Institute of Immunology, New Delhi, India) for providing animals, and Naresh Sharma for helping in the preparation of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported in part by the Council of Scientific and Industrial Research, Government of India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Council of Scientific and Industrial Research fellowship.
** To whom correspondence should be addressed. Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
Published, JBC Papers in Press, November 28, 2001, DOI 10.1074/jbc.M105902200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: LPS, lipopolysaccharide; Ab, antibody; SN, supernatant; MFI, mean fluorescence intensity; RPA, ribonuclease protection assay; TNF, tumor necrosis factor; mAb, monoclonal antibody; IL, interleukin; IFN, interferon; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; BCR, B cell receptor; SSC, side scatter; FSC, forward scatter.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Mueller, D. L., Jenkins, M. K., and Schwartz, R. H. (1989) Annu. Rev. Immunol. 7, 455-480 |
| 2. |
Agrewala, J. N.,
Susmit, S.,
Verma, R. K.,
and Mishra, G. C.
(1998)
J. Immunol.
160,
1067-1077 |
| 3. | Damle, N. K., Klussman, K., Linsley, P. S., and Aruffo, A. (1992) J. Immunol. 148, 1985-1992[Abstract] |
| 4. | Agrewala, J. N., Vinay, D. S., Joshi, A., and Mishra, G. C. (1994) Eur. J. Immunol. 24, 2092-2097[Medline] [Order article via Infotrieve] |
| 5. | Clark, E. A., and Ledbetter, J. A. (1994) Nature 367, 425-428[CrossRef][Medline] [Order article via Infotrieve] |
| 6. | Rathmell, J. C., Townsend, S. E., Xu, J. C., Flavell, R. A., and Goodnow, C. C. (1996) Cell 87, 319-325[CrossRef][Medline] [Order article via Infotrieve] |
| 7. |
Jeannin, P.,
Delneste, Y. S.,
Henchoz, L.,
Gauchat, J. F.,
Ellis, J.,
and Bonnefoy, J. Y.
(1997)
J. Biol. Chem.
272,
15613-15619 |
| 8. | Hirokawa, M., Kuroki, J., Kitabayahi, A., and Miura, A. B. (1996) Immunol. Lett. 50, 95-98[CrossRef][Medline] [Order article via Infotrieve] |
| 9. | Freeman, G. J., Boussiotis, V. A., Anumanthan, A., Bernstein, G. M., Ke, X. Y., Rennet, P. D., Gray, G. S., Gribben, J. G., and Nadler, L. M. (1995) Immunity 2, 523-532[CrossRef][Medline] [Order article via Infotrieve] |
| 10. | Sethna, M. P., Van Parijs, L., Sharpe, A. H., Abbas, A. K., and Freeman, G. J. (1994) Immunity 1, 415-421[CrossRef][Medline] [Order article via Infotrieve] |
| 11. | Gordon, J., Millsum, M. J., Guy, G. R., and Ledbetter, J. A. (1988) J. Immunol. 140, 1425-1430[Abstract] |
| 12. | Goldstein, M. D., and Watts, T. H. (1996) J. Immunol. 157, 2837-2843[Abstract] |
| 13. |
Nakajima, A.,
Kodama, T.,
Morimoto, S.,
Azuma, M.,
Takeda, K.,
Oshima, H.,
Yoshino, S.,
Yagita, H.,
and Okumura, K.
(1998)
J. Immunol.
161,
1901-1907 |
| 14. | Bergamo, A., Bataille, R., and Pellat-Deceunynck, C. (1997) Br. J. Haematol. 97, 652-655[CrossRef][Medline] [Order article via Infotrieve] |
| 15. |
Shahinian, A.,
Pfeffer, K.,
Lee, K. P.,
Kundig, T. M.,
Kishihara, K.,
Wakeham,
Kawai, A. K.,
Ohashi, P. S.,
Thompson, C. B.,
and Mak, T. W.
(1993)
Science
261,
609-612 |
| 16. |
Ronchese, F.,
Hausmann, B.,
Hubele, S.,
and Lane, P.
(1994)
J. Exp. Med.
179,
809-817 |
| 17. |
Snapper, C. M.,
and Paul, W. E.
(1987)
Science
236,
944-947 |
| 18. | Ikemizu, S., Gilbert, R. J., Fennelly, J. A., Collins, A. V., Harlos, K., Jones, E. Y., Stuart, D. I., and Davis, S. J. (2000) Immunity 12, 51-60[CrossRef][Medline] [Order article via Infotrieve] |
| 19. | Bajorath, J., Peach, R. J., and Linsley, P. S. (1994) Protein Sci. 3, 2148-2150[Abstract] |
| 20. | Heath, A. W., Chang, R., Harada, N., Argumedo, L. S., Gordon, J., Hannum, C., Campell, D., Shanafelt, A. B., Clark, E. A., Torres, R., and Howard, M. (1993) Cell. Immunol. 152, 468-480[CrossRef][Medline] [Order article via Infotrieve] |
| 21. | Borriello, F., Sethna, M. P., Boyd, S. D., Schweitzer, A. N., Tivol, E. A., Jacoby, D., Strom, T. B., Simpson, E. M., Freeman, G. J., and Sharpe, A. H. (1997) Immunity 6, 303-313[CrossRef][Medline] [Order article via Infotrieve] |
| 22. |
Murphy, E.,
Shibuya, K.,
Hosken, N.,
Openshaw, P.,
Maino, V.,
Davis, K.,
Murphy, K.,
and Garra, A. O.
(1996)
J. Exp. Med.
183,
901-903 |
| 23. | Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. J. (1991) J. Immunol. Methods 139, 271-279[CrossRef][Medline] [Order article via Infotrieve] |
| 24. | PharMingen (1998) PharMingen Instruction Manual: Apoptosis, Applied Reagents and Technologies, 2nd Ed., pp. 77-89, San Diego |
| 25. |
Sah, N. K.,
Taneja, T. K.,
Pathak, N.,
Begum, R.,
Athar, M.,
and Hasnain, S. E.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4838-4843 |
| 26. | Hasnain, S. E., Taneja, T. K., Sah, N. K., Mohan, M., Pathak, N., Sahdev, S., Athar, M., and Begum, R. (1999) J. Biosci. 24, 101-107 |
| 27. |
Funakoshi, S.,
Longo, D. L.,
Beckwith, M.,
Conley, D. K.,
Tsarfary, G.,
Tsarfaty, L.,
Armitage, R. J.,
Fanslow, W. C.,
Spriggs, M. K.,
and Murphy, W. J.
(1994)
Blood
83,
2787-2794 |
| 28. | Kearney, E. R., Walunas, T. L., Karr, R. W., Morton, P. A., Loh, D. Y., Bluestone, J. A., and Jenkins, M. K. (1995) J. Immunol. 155, 1032-1036[Abstract] |
| 29. | Greenfield, E. A., Nguyen, K. A., and Kuchroo, V. K. (1998) Crit. Rev. Immunol. 18, 389-418[Medline] [Order article via Infotrieve] |
| 30. |
Linsley, P. S.,
Wallace, P. M.,
Johnson, J.,
Gibson, M. G.,
Greene, J. L.,
Ledbetter, J. A.,
Singh, C.,
and Tepper, M. A.
(1992)
Science
257,
792-795 |
| 31. | Han, S., Hathcock, K., Zheng, B., Kepler, T. B., Hodes, R., and Kelsoe, G. (1995) J. Immunol. 155, 556-567[Abstract] |
| 32. | Linsley, P. S., Brady, W., Grosmaire, L., Arutto, A., Damle, N. K., and Ledbetter, J. A. (1991) J. Exp. Mol. 174, 561-569 |
| 33. | Linsley, P. S., Greene, J. L., Brady, W., Bajorath, J., Ledbetter, J. A., and Peach, R. (1994) Immunity 1, 793-801[CrossRef][Medline] [Order article via Infotrieve] |
| 34. | June, C. H., Bluestone, J. A., Nadler, L. M., and Thompson, C. B. (1994) Immunol. Today 15, 321-331[CrossRef][Medline] [Order article via Infotrieve] |
| 35. |
Freeman, G. J.,
Gribben, J. G.,
Boussiotis, V. A., Ng, J. W.,
Restivo, V. A., Jr.,
Lombard, L. A.,
Gray, G. S.,
and Nadler, L. M.
(1993)
Science
262,
909-911 |
| 36. | Allison, J. P. (1994) Curr. Opin. Immunol. 6, 414-419[CrossRef][Medline] [Order article via Infotrieve] |
| 37. | Azuma, M., Ito, D., Yagita, H., Okumura, K., Phillips, J. H., Lanier, L. L., and Somoza, C. (1993) Nature 366, 76-79[CrossRef][Medline] [Order article via Infotrieve] |
| 38. |
Freeman, G. J.,
Borriello, F.,
Hodes, R. J.,
Reiser, H.,
Gribben, J. G., Ng, J. W.,
Kim, J.,
Goldberg, J. M.,
Hathcock, K.,
and Laszlo, G.
(1993)
J. Exp. Med.
178,
2185-2192 |
| 39. | Doty, R. T., and Clark, E. A. (1996) J. Immunol. 157, 3270-3279[Abstract] |
| 40. | Hirokawa, M., Kitabayashi, A., Kuroki, J., and Miura, A. B. (1995) Immunol. 86, 155-161[Medline] [Order article via Infotrieve] |
| 41. |
Doty, R. T.,
and Clark, E. A.
(1998)
J. Immunol.
161,
2700-2707 |
| 42. | Billian, G., Mondiere, P., Berard, M., Bella, C., and Defrance, T. (1997) Eur. J. Immunol. 27, 405-414[Medline] [Order article via Infotrieve] |
| 43. | Budd, C. R. (2001) Curr. Opin. Immunol. 13, 356-362[CrossRef][Medline] [Order ar |
