CD80 and CD86 Are Not Equivalent in Their Ability to Induce the
Tyrosine Phosphorylation of CD28*
Jacqueline M.
Slavik
,
Jill E.
Hutchcroft
§, and
Barbara E.
Bierer
¶
From the
Department of Pediatric Oncology,
Dana-Farber Cancer Institute, Boston, the ¶ Department of
Medicine, Harvard Medical School, Boston, Massachusetts 02115, and
the § Department of Biochemistry, Purdue University,
West Lafayette, Indiana 47907
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ABSTRACT |
Ligation of either CD80 (B7-1) or CD86 (B7-2),
two principal ligands for CD28, is thought to skew the immune response
toward Th1 or Th2 differentiation. We have examined early signal
transduction pathways recruited following T cell stimulation with
either CD80 or CD86. Purified human peripheral T cells or Jurkat T
cells were stimulated with Chinese hamster ovary (CHO) cells expressing
either human CD80 (CHO-CD80) or human CD86 (CHO-CD86) or with anti-CD28 monoclonal antibody (mAb). In the presence of phorbol 12-myristate 13-acetate, both CHO-CD80 and CHO-CD86, like anti-CD28 mAb, were capable of stimulating cytokine production from both human peripheral T
cells and Jurkat T cells. Both CHO-CD80 and CHO-CD86, in the presence
of anti-CD3 mAb, costimulated NFAT-dependent
transcriptional activation. Several intracellular signaling proteins,
such as CBL and VAV, were phosphorylated on tyrosine in response to
CD80, CD86, and anti-CD28 mAb. Surprisingly, although stimulation of Jurkat T cells with either CHO-CD80 or anti-CD28 mAb resulted in robust
tyrosine phosphorylation of CD28 itself, ligation with CHO-CD86 was
unable to induce detectable CD28 tyrosyl phosphorylation over a range
of stimulation conditions. In addition, the association of
phosphoinositide 3-kinase with CD28 and enhanced tyrosine
phosphorylation of phospholipase C
were seen after anti-CD28 mAb and
CHO-CD80 stimulation but to a much lesser extent after CHO-CD86
stimulation. Thus, ligation of CD28 with either CD80 or CD86 leads to
shared early signal transduction events such as the tyrosine
phosphorylation of CBL and VAV, to NFAT-mediated transcriptional
activation, and to the costimulation of interleukin-2 and
granulocyte-macrophage colony-stimulating factor production. However,
CD80 and CD86 also induce distinct signal transduction pathways
including the tyrosine phosphorylation of CD28 and phospholipase C
1
and the SH2-dependent association of phosphoinositide
3-kinase with CD28. These quantitative, if not qualitative, differences
between signaling initiated by these two ligands for CD28 may
contribute to functional differences (e.g. Th1 or Th2
differentiation) in T cell responses.
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INTRODUCTION |
Activation and maturation of resting T lymphocytes can be achieved
by antigen-specific interactions of the TcR-CD3 complex in concert with
a second, antigen-nonspecific signal. This second, costimulatory signal
has been shown to prevent the induction of T cell anergy and to enhance
cytokine production, notably
IL-21 (1-3). Found on more
than 95% of human CD4+ T cells and on about 50% of human
CD8+ T cells, the cell-surface molecule CD28 is a major T
cell costimulatory receptor. Engagement of the CD28 receptor with
anti-CD28 mAb or by ligand prevents the induction of T cell anergy and
supports IL-2 production and T cell proliferation.
The B7 family members CD80 (B7-1) and CD86 (B7-2) are two principal
ligands for CD28 and for CTLA-4 (CD152), a second CD28 family member.
Whereas only 25% homologous by amino acid sequence, CD80 and CD86 bind
CD28 with similar low affinities and bind CTLA-4 with similar high
affinities (4). However, CD86 has faster dissociation kinetics than
CD80 (5), and independent mutational analyses have confirmed that CD80
and CD86 bind to overlapping but not identical sites on CD28 (6-9).
The cell-surface expression of CD80 and CD86 differs both
quantitatively and qualitatively on antigen presenting cells. CD86 is
expressed constitutively on resting monocytes and can be rapidly
induced on activated B cells (10). CD80 is not expressed on resting
monocytes and only minimally on dendritic cells and, although
expression can be induced on activated macrophages, B cells, and NK
cells, the time course of CD80 induction of expression is slower than
of that of CD86 (11),
A growing body of evidence suggests that there are different functional
consequences of CD28 engagement by CD80 and CD86 (12-17). For example,
blocking the interaction between CD28 and CD80 with anti-CD80 mAb has
been shown to increase IL-4 production in mice (13). Conversely,
blocking the interaction of CD86 with CD28 in vitro was
shown to increase the production of interferon-
(13). These data
from murine systems support a model whereby CD80 costimulation promotes
the development of Th1 cells, whereas CD86 costimulation drives
differentiation toward Th2 cells. Costimulation of resting
CD4+ human T cells with CD80 or CD86 resulted in equivalent
IL-2 and interferon-
production (18). However, costimulation with
CD80 resulted in more GM-CSF production than with CD86, whereas
costimulation with CD86 gave more efficient production of IL-4 and
TNF-
than with CD80 (18). Thus, CD86 costimulation appeared to
direct the immune response toward Th2 development, whereas CD80
provided a less directive (and therefore Th1-like) signal. These data
are consistent with the observation that human CD80 but not CD86 was able to generate human cytolytic effector cells (15). In addition, CD86
is less effective than CD80 at down-regulating CD28 expression after
ligation (17). Taken together, these data provide evidence that the
functional outcome of CD80 and CD86 costimulation may differ.
A limited number of studies have directly compared the signals
generated following CD80 or CD86 engagement of CD28 (19-22), but no
differences have been observed that correlate with the differences in
structure and function between CD80 and CD86. In this report, we have
compared the intracellular signals generated by engagement of human
CD28 with either anti-CD28 mAb or with CHO cells transfected with human
CD80 or human CD86. We have used both purified human peripheral blood T
cells and CD4+ T cells isolated by negative selection.
Additionally, we have used a Jurkat T cell line, which expressed CD28
but did not express CTLA-4, to eliminate any contribution of CTLA-4
ligation by ligand to signaling differences observed. We have
demonstrated that CHO cells transfected with either human CD80
(CHO-CD80) or human CD86 (CHO-CD86) were capable of costimulating IL-2
production both from purified human peripheral T cells and from Jurkat
T cells in the presence of a second signal provided by phorbol
myristate acetate (PMA), as well as costimulating GM-CSF production
from purified human peripheral T cells. Both CHO-CD80 and CHO-CD86, in
the presence of anti-CD3, costimulated NFAT-mediated transcriptional activation. Similar but not identical patterns of phosphoproteins were
revealed following stimulation of either Jurkat T cells or human
purified T cells with anti-CD28 mAb, CHO-CD80, or CHO-CD86. We
identified two of the higher molecular weight phosphoproteins as CBL
and VAV. In striking contrast, however, stimulation of Jurkat T cells
with CHO-CD80, but not with CHO-CD86, resulted in robust tyrosine
phosphorylation of CD28 itself. Furthermore, considerably less PI3-K
was associated with CD28 after CD86 stimulation compared with CD80 or
anti-CD28 mAb stimulation. Correlating with the difference in CD28
phosphorylation and PI3-K association was the induction of PLC-
1
phosphorylation after stimulation with CHO-CD80 but not with CHO-CD86.
Thus, the signal transmission pathways recruited by CD80 and CD86
ligation of CD28 appear to differ quantitatively, if not qualitatively.
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EXPERIMENTAL PROCEDURES |
Cells and Cell Culture--
The human T leukemia cell line
Jurkat (clone J77) was the generous gift of Kendall Smith (Cornell
University, New York). Human peripheral blood T lymphocytes were
isolated from the whole blood of normal donors by centrifugation
through Ficoll-Hypaque (SG 1.007, Organon Teknika, Durham, NC), plastic
adherence, and nylon wool filtration. Contaminating erythrocytes were
lysed with Tris-buffered ammonium chloride (Sigma). Where indicated,
CD8+ T cells were depleted following serial incubation with
5 µg/ml OKT8 and anti-mouse Ig microbeads using the MACs magnetic
cell separation system (Miltenyi Biotech, Inc., Sunnyvale, CA).
Purified T cells or purified CD4+ T cells were rested
overnight at 37 °C prior to use. Lymphocytes were cultured at
37 °C with 5% CO2 in RPMI 1640 (Mediatech, Herndon, VA)
supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma),
100 units/ml penicillin (Life Technologies, Inc.), 100 µg/ml
streptomycin (Life Technologies, Inc.), 10 mM Hepes, pH 7.2 (M.A. Bioproducts, Bethesda, MD), 2 mM glutamine (Life
Technologies, Inc.), and 50 µM 2-mercaptoethanol (Sigma)
(termed 10% RPMI). Chinese hamster ovary (CHO) cells transfected with
human CD80 or human CD86 were kindly provided by G. Freeman
(Dana-Farber Cancer Institute, Boston). These cells were grown in
Dulbecco's modified Eagle's/F12 medium (Cellgro, Fisher) containing
10% heat-inactivated FCS, 100 units/ml penicillin, 100 µg/ml
streptomycin, 10 mM Hepes, pH 7.2, 2 mM
glutamine, 15 µg/ml gentamicin (Life Technologies, Inc.) plus 400 µg/ml geneticin (G418) (Life Technologies, Inc.). CHO cells were
detached from 10-mm2 Petri dishes with 1:5000 Versene (Life
Technologies, Inc.) for 5 min at 37 °C and washed before use. CHO
transfectants were monitored periodically to verify comparable and
invariant cell-surface expression of B7 family proteins. All cell lines
were mycoplasma negative, as verified by routine screening with the
mycoplasma set primer (Stratagene, La Jolla, CA).
Antibodies--
The anti-human CD28 mAb 9.3 (Squibb), the murine
anti-human CD3 mAb OKT3, and the anti-human mAb OKT8D (American Type
Culture Collection, Rockville, MD) were used as purified ascites fluid. The horseradish peroxidase coupled anti-phosphotyrosine mAb RC20 was
purchased from Transduction Laboratories (Lexington, KY). The
agarose-conjugated anti-phosphotyrosine mAb, the polyclonal anti-VAV,
the polyclonal anti-CBL, and the polyclonal anti-PLC
1 were all
purchased from Santa Cruz Biotechnology, Inc. Polyclonal antisera
against the p85 subunit of PI3-K was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-human CD80 mAb 133 was
the gift of G. Freeman (Dana-Farber Cancer Institute, Boston). The
anti-human CD86 mAb was purchased from PharMingen (San Diego, CA).
Indirect Immunofluorescent Flow Cytometry--
Cells were
incubated with the indicated primary mAb for 30 min at 4 °C and then
washed twice with PBS supplemented with 2% FCS and 0.02%
NaN3. Cells were then incubated with 20 µg/ml fluorescein isothiocyanate-conjugated goat F(ab')2 anti-mouse antibody
(Tago, Inc., Burlingame, CA) for 30 min at 4 °C, washed twice with
PBS containing 2% FCS and 0.02% NaN3, resuspended in
propidium iodide (5-10 µg/ml; Sigma), and analyzed on a FACScan
(Becton Dickinson, Mountain View, CA). Dead cells were excluded by
propidium iodide uptake, and data analysis was performed using LYSYS or
Cellquest software.
Measurement of IL-2 and GM-CSF Production--
Human purified T
cells or Jurkat T cells were plated in 96-well flat-bottomed plates at
2 × 105/well in 10% RPMI with the anti-CD28 mAb 9.3 (100 ng/ml), the anti-CD3 mAb OKT3 (5 ng/ml), PMA (1.25 ng/ml, Sigma),
parental CHO cells or CHO cells transfected with either CD80 or CD86
(2 × 104/well), as indicated, in 200-µl total
volume. Following a 24 h incubation at 37 °C, cell culture
supernatants were collected, frozen, thawed, and assayed for the
presence of IL-2 or GM-CSF by their ability to support the
proliferation of an IL-2-dependent T-cell line CTLL-20 (23)
or BaF/3 cells stably transfected with the human GM-CSF receptor (24),
respectively. Proliferation of either the CTLL-20 cells or the
transfected BaF/3 cells was assessed by the incorporation of
[3H]thymidine (NEN Life Science Products) after a 6-h
pulse following an 18-h incubation. Results are expressed as units/ml
IL-2 or GM-CSF and were determined by extrapolation to standard
dilution curves using human recombinant IL-2 (Hoffmann-La Roche) or
recombinant GM-CSF (Genetics Institute, Boston), respectively.
Transient Transfection and Luciferase Assay--
Jurkat cells
(1 × 107) were incubated with 10 µg of a reporter
plasmid p3xNFAT-luc (25), carrying the luciferase gene driven by three
tandem repeats of the distal NFAT sequences derived from the IL-2
promoter, for 15 min at room temperature. Cells were then
electroporated at 250 V, 800 microfarads (Life Technologies, Inc.).
After electroporation, the cells were transferred to 10% RPMI and
incubated at 37 °C for 12 h. Transfected cells were stimulated for 6 h with CHO-Mock, CHO-CD80, or CHO-CD86 in the presence of anti-CD3 mAb (200 ng/ml) or with PMA (10 ng/ml) plus ionomycin (2 µM). Cells were washed with PBS, and samples were
prepared using the Enhanced Luciferase Kit (Analytic Luminescent
Laboratory, San Diego, CA), according to the manufacturer's
instructions. The relative luciferase units are presented as the
percentage of the stimulation induced by PMA plus ionomycin.
Cell Stimulation and Immunoprecipitation--
Cells were washed
twice with cold Buffer A (RPMI 1640 supplemented with 10 mM
Hepes, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin). Human purified T cells or Jurkat T cells were
resuspended in Buffer A, incubated on ice for 15 min with parental or
transfected CHO cells, anti-CD28 mAb 9.3, or anti-CD3 mAb OKT3, and
then warmed to 37 °C for the times indicated. The cells were washed
twice with cold Buffer A containing 1 mM sodium
orthovanadate (Sigma) and lysed for 15 min on ice in 1 ml of cold lysis
buffer (1% Nonidet P-40, 150 mM NaCl, 25 mM
Hepes, pH 7.5, 1 mM EDTA, 1 mM sodium
orthovanadate, 100 µg/ml soybean trypsin inhibitor, 10 µg/ml
leupeptin, and 10 µg/ml aprotinin). Detergent extracts were clarified
by centrifugation at 14,000 × g for 10 min at 4 °C.
The resulting supernatants were harvested and used for immunoprecipitations.
For immunoprecipitation, cellular extracts were incubated with the
indicated antisera and 25 µl of protein A-agarose for at least 2 h at 4 °C after which the resin was washed three times with lysis
buffer. For experiments examining the association of CD28 with
phosphoinositide 3-kinase (PI3-K), the immunoprecipitates were further
washed twice with Buffer B (0.5 M LiCl, 100 mM
Hepes, pH 7.5, 2 mM sodium orthovanadate, 10 µg/ml
aprotinin, 10 µg/ml leupeptin) and twice with Buffer C (100 mM NaCl, 25 mM Hepes, pH 7.5, 2 mM
sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin). In
peptide competition studies, 5 µM of HSDYMNMTPRR or
HSD-phospho-YMNMTPRR peptides (pYMNM) (the kind gift of S. Shoelson,
Joslin Diabetes Center, Boston) was added to the cell lysates prior to
incubation with anti-CD28 mAb and protein A-Sepharose. Immunoprecipitated proteins were separated by electrophoresis through
SDS-polyacrylamide gels (SDS-PAGE) (Protogel, National Diagnostics,
Atlanta, GA) transferred to polyvinylidene difluoride (PVDF) membranes
(Millipore, Bedford, MA), immunoblotted with the antibody of interest,
and detected by enhanced chemiluminescence (ECL) (Amersham Pharmacia
Biotech) according to the manufacturer's instructions.
Deglycosylation and precipitation of the CD28 polypeptide were
performed essentially as described (26). In brief, the
immunoprecipitates were washed twice with RIPA buffer (1% Triton
X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl,
50 mM Tris-Cl, pH 8, 1 mM sodium orthovanadate,
10 µg/ml leupeptin, and 10 µg/ml aprotinin), twice with RIPA buffer
containing 1 M NaCl, twice more with RIPA buffer, and once
with Buffer D (0.1% Triton X-100, 150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 1 mM sodium orthovanadate,
100 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). The immunoprecipitated proteins were denatured by
boiling for 10 min in a solution of 0.5% SDS and 1%
-mercaptoethanol and then adjusted to 50 mM sodium
phosphate, pH 7.5, and 1% Nonidet P-40. The proteins were incubated
for 1 h at 37 °C with 1,000 units of
peptide:N-glycosidase F (New England Biolabs, Beverly, MA), an enzyme that hydrolyzes N-glycan chains from
proteins. Deglycosylated proteins were separated on 10.5% SDS-PAGE
gels and visualized by immunoblotting as described above.
 |
RESULTS |
Both CD80 and CD86 Costimulate T cell cytokine Production and
NFAT-mediated Transcriptional Activation--
We used CHO cells
transfected with either vector alone (CHO-Mock) or the cDNA
encoding either human CD80 (CHO-CD80) or human CD86 (CHO-CD86) to
compare the intracellular signals triggered by stimulation of T cells
with the natural ligands of CD28. The transfected CHO cells used in
these studies had expressed comparable surface CD80 and CD86 as
detected by indirect immunofluorescence using either anti-CD80 or
anti-CD86 mAb (Fig. 1). As expected, CHO-CD80 cells expressed CD80 but failed to express CD86, and CHO-CD86
cells expressed CD86 but did not express CD80; CHO-Mock cells,
transfected with the vector encoding neomycin resistance (G418r) alone, expressed neither B7 family member.

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Fig. 1.
CD80 and CD86 expression on transfected CHO
cells. CHO cells were stained with the murine anti-human CD80 mAb
133 (1:200 dilution), the murine anti-human CD86 mAb IT2.2 (1 µg/ml),
or diluent alone for 20 min. Samples were washed and then incubated
with fluorescein isothiocyanate-conjugated goat anti-mouse (20 µg/ml)
for an additional 20 min. After washing, cells were resuspended in
propidium iodide (5-10 µg/ml) and analyzed on a Becton Dickinson
FACscan. 2000 events were collected, and dead cells were excluded from
analysis by propidium iodide uptake.
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Both CD80 and CD86 are capable of costimulating a number of T cell
functions including cytokine production and proliferation (reviewed in
Ref. 27). Resting, human purified CD4+ peripheral blood T
cells and CD3+ Jurkat cells were assayed for their ability
to produce IL-2 and GM-CSF in response to CHO-Mock, CHO-CD80, or
CHO-CD86 cells or to anti-CD28 mAb, in the presence or absence of the
phorbol ester PMA. T cells and Jurkat cells both produced IL-2 (Table
I) and T cells produced GM-CSF (Table
II) in response to the combination of PMA
and either anti-CD28 mAb, CHO-CD80, or CHO-CD86 cells. No IL-2 was
produced after stimulation with PMA, anti-CD28 mAb, or mock or
transfected CHO cells alone. As expected, costimulation of human
peripheral blood T cells with CHO-CD80 elicited more IL-2 than did
costimulation with CHO-CD86 (Table I), whereas there were no
reproducible, quantitative differences in the amount of IL-2 (Table I)
produced by Jurkat cells or GM-CSF (Table II) produced by human
peripheral blood T cells after CHO-CD80 or CHO-CD86 stimulation.
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Table I
IL-2 production after costimulation with CHO cells expressing CD80 or
CD86
Human peripheral blood T cells or Jurkat T cells, 2 × 105
cells/well, were cultured with 2 × 104 CHO/well, 1.25 ng/ml PMA, 100 ng/ml anti-CD28 mAb, as indicated. Production of IL-2
was determined as described under "Experimental Procedures" and is
expressed as units/ml by comparison with a standard curve generated
with recombinant human IL-2. A representative assay from a total of
three assays performed with human peripheral blood T cells is shown.
The mean ± S.D. IL-2 production of three independent assays
performed using Jurkat T cells is shown.
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Table II
GM-CSF production after costimulation with CHO cells expressing CD80 or
CD86
Human peripheral blood T cells, 2 × 105 cells/well, were
cultured with 2 × 104 transfected CHO cells/well or 100 ng/ml anti-CD28 mAb with or without 1.25 ng/ml PMA, as indicated.
Production of GM-CSF (units/ml) was determined as described under
"Experimental Procedures" by comparison with a standard curve
generated using recombinant human GM-CSF. A representative assay from a
total of three assays performed is shown.
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Cytokine production is a somewhat distal readout of
CD28-dependent stimulation; to analyze a more proximal
readout of CD28-dependent stimulation, we compared the
relative abilities of the two ligands to costimulate
NFAT-dependent transcriptional activation. Jurkat T cells
were transiently transfected with a reporter plasmid carrying the
luciferase gene driven by three tandem repeats of the distal NFAT
sequences derived from the IL-2 promoter (25). The cells were then
stimulated with CHO-Mock, CHO-CD80, or CHO-CD86 in the presence of
anti-CD3 mAb or with PMA plus ionomycin (Fig.
2). NFAT-mediated transcriptional
activity was induced by CHO-CD80, and more so by CHO-CD86, whereas
CHO-Mock induced the same amount of activity as anti-CD3 mAb alone. The
addition of a blocking CD28 mAb to the stimulations abrogated both
CD80- and CD86-dependent NFAT-mediated transcriptional
activation, confirming that both CD80 and CD86 were signaling through
CD28 (data not shown).

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Fig. 2.
Costimulation with CD80 or CD86 induces
NFAT-mediated transcriptional activity in Jurkat T cells. Jurkat T
cells (1 × 106) transiently transfected with an
NFAT-luciferase reporter plasmid were stimulated for 6 h with
soluble anti-CD3 mAb alone (200 ng/ml) (dashed bar) or with
soluble anti-CD3 mAb plus CHO-Mock (shaded bar), CHO-CD80
(open bar), or CHO-CD86 (solid bar) or with PMA
(10 ng/ml) plus ionomycin (2 µM). Cells were washed with
PBS, and samples were prepared using the Enhanced Luciferase Kit
(Analytic Luminescent Laboratory, San Diego, CA), according to the
manufacturer's instructions. The relative luciferase units are
presented as the percentage of the activity induced by PMA plus
ionomycin and represent the mean plus S.D. of three separate
assays.
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Stimulation with CD80 or CD86 Induces the in Vivo Tyrosine
Phosphorylation of Several T Cell Proteins Including CBL and
VAV--
Having established, in this system, that the CHO-CD80 and
CHO-CD86 cells were both competent to costimulate cytokine production as well as NFAT-mediated transcriptional activation, we compared the
early signaling events induced by anti-CD28 mAb with those induced by
each of the two natural ligands. Jurkat cells were incubated with
either mock or transfected CHO cells or, for comparison, anti-CD28 or
anti-CD3 mAb for 5 min at 37 °C. Analysis of post-nuclear lysates by
phosphotyrosine immunoblotting yielded no significant differences
between CHO-Mock, CHO-CD80, and CHO-CD86 cell stimulations (data not
shown). To enhance the detection of tyrosine-phosphorylated proteins,
anti-phosphotyrosine immunoprecipitates were prepared from stimulated
Jurkat cell lysates, separated by SDS-PAGE, transferred to PVDF
membranes, and detected by anti-phosphotyrosine Western blot (Fig.
3A). As expected, a distinct
pattern of tyrosine phosphorylated proteins was detected after
stimulation with anti-CD3 mAb (Fig. 3A, lane 7). Increases
in tyrosyl-phosphorylated proteins were also seen in response to
stimulation with soluble (Fig. 3A, lane 5) and cross-linked
(Fig. 3A, lane 6) anti-CD28 mAb 9.3. There was an increase
in detection of both an ~95-kDa protein (indicated by the open
arrowhead) after anti-CD3 mAb treatment (Fig. 3A, lane
7) and an ~115-120-kDa protein (indicated by the solid
arrowhead) in both the anti-CD3 mAb and the anti-CD28
mAb-stimulated cells (Fig. 3A, lanes 5-7). The
phosphorylation of the ~95-kDa band was preferentially enhanced after
stimulation with CHO-CD80 (Fig. 3A, lane 3) when compared
with CHO-CD86- (Fig. 3A, lane 4) or CHO-Mock (Fig. 3A,
lane 2)-treated cells. The CHO cells alone, in the absence of
Jurkat cells, contained few tyrosine-phosphorylated proteins (Fig.
3A, lane l, and data not shown).

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Fig. 3.
Tyrosine phosphorylation in Jurkat
(A) or purified peripheral blood (B)
human T cells after stimulation with anti-CD3 mAb, anti-CD28 mAb,
CHO-Mock, CHO-CD80, and CHO-CD86. A,
anti-phosphotyrosine immunoprecipitates were prepared from detergent
lysates of CHO-CD80 cells alone (lane 1) or of Jurkat cells
(2 × 107) left untreated (lane 8) or
treated with CHO-Mock (lane 2), -CD80 (lane 3),
-CD86 (lane 4), CHO cells (1 × 107), 4 µg/ml anti-CD28 mAb in the absence (lane 5) or presence of
10 µg/ml rabbit anti-mouse (lane 6) or with 6 µg/ml
anti-CD3 mAb (lane 7). All cells were incubated for 30 min
on ice, followed by 5 min at 37 °C. Stimulations were stopped by the
addition of cold Buffer A containing 1 mM
Na3VO4; cells were washed once with Buffer
A/Na3VO4 and then lysed at 4 °C for 15 min
in 1 ml of 1% Nonidet P-40 lysis buffer. Lysates were clarified by
centrifugation and immunoprecipitated with anti-phosphotyrosine
antibodies precoupled to agarose for 2 h at 4 °C. Lysates were
separated by electrophoresis through a 6-15% SDS-PAGE and then
transferred to PVDF membranes. Tyrosine-phosphorylated proteins were
detected with anti-phosphotyrosine antibody (RC20 1:2500 dilution) for
2 h, and the proteins were visualized by ECL. The migration of
molecular mass markers are indicated. B,
anti-phosphotyrosine immunoprecipitates were prepared from detergent
lysates of 2 × 107 human peripheral blood T cells as
in A. Lysates were prepared from CHO-CD80 cells alone
(lane 1), from unstimulated T cells (lane 5), or
from T cells stimulated with CHO-Mock (lane 2), CHO-CD80
(lane 3), CHO-CD86 (lane 4) cells (1 × 107), anti-CD28 mAb (lane 7) plus rabbit
anti-mouse (lane 8) or anti-CD3 mAb (lane
6).
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Comparable results were obtained in purified human peripheral T cells
(Fig. 3B). Characteristic increases in tyrosine
phosphorylation were observed after stimulation with anti-CD3 mAb and
with soluble or cross-linked anti-CD28 mAb (Fig. 3B, lane
6-8). Induction of tyrosine phosphorylation was also observed
following stimulation with CHO-CD80 (Fig. 3B, lane 3) or
CHO-CD86 (Fig. 3B, lane 4). These differences were subtle,
however, and therefore the ability of CD80 and CD86 to induce the
tyrosine phosphorylation of specific signaling proteins was examined.
VAV is a 95-kDa protein expressed exclusively in hematopoietic cells
that is rapidly phosphorylated on tyrosine after ligation of the
TcR-CD3 complex (28) or CD28 (29). This tyrosine phosphorylation leads
to the activation of VAV as an exchange factor for Rac-1 (30).
Stimulation of Jurkat T cells with either CHO-CD80 or CHO-CD86
induced tyrosine phosphorylation of VAV that was detectable by 30 s and appeared to decrease by 10 min of stimulation (Fig.
4). Comparable
activation-dependent changes in the tyrosine
phosphorylation of VAV after CD80 and CD86 stimulation were also
observed in peripheral human T cells (data not shown), although maximal
signals were not as high.

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Fig. 4.
Both CHO-CD80 and CHO-CD86 induce tyrosine
phosphorylation of VAV in Jurkat T cells. Anti-VAV
immunoprecipitates were prepared from detergent lysates of Jurkat cells
(2 × 107) not treated, stimulated with CHO-Mock,
CHO-CD80, or CHO-CD86 for 15 min on ice, and then warmed to 37 °C
for the indicated times. Stimulations were stopped and cells lysed as
in Fig. 3A. Lysates were clarified by centrifugation and
immunoprecipitated with anti-VAV and protein A-Sepharose for 2 h
at 4 °C. Lysates were separated by SDS-PAGE, transferred to PVDF,
and tyrosine-phosphorylated proteins detected by ECL (top).
The blot was stripped and reprobed with anti-VAV antibody to
demonstrate equivalent loading of proteins (bottom).
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CBL is an ~120-kDa protein that is rapidly tyrosine-phosphorylated in
response to TcR (31) and Fc
R stimulation (32). CBL is an
intracellular docking protein that associates with a number of SH2- and
SH3-containing proteins including p59Fyn, PI3-K, Zap-70,
and the adapter protein Grb2 (reviewed in Ref. 33). Stimulation of
Jurkat cells for 5 min with CD80, CD86, anti-CD28 mAb, and, as
expected, anti-CD3 mAb all induced the tyrosine phosphorylation of CBL
(Fig. 5). Thus, CD80 and CD86 share the
ability to induce the tyrosine phosphorylation of several intracellular
signaling proteins, including CBL and VAV.

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Fig. 5.
Tyrosine phosphorylation of CBL after
stimulation with anti-CD28 mAb, anti-CD3 mAb, CHO-CD80, and
CHO-CD86. Anti-CBL immunoprecipitates were prepared from detergent
lysates of CHO-CD80 cells alone or from Jurkat cells (2 × 107) left untreated or stimulated with CHO-Mock, CHO-CD80,
or CHO-CD86 cells (1 × 107), 6 µg/ml anti-CD3 mAb,
or 4 µg/ml anti-CD28 mAb for 15 min on ice and then 5 min at
37 °C. Stimulations were stopped and cells lysed as in Fig.
3A. Lysates were clarified by centrifugation and
immunoprecipitated with anti-CBL and protein A-Sepharose for 2 h
at 4 °C. Lysates were separated by electrophoresis through SDS-PAGE
and then transferred to PVDF membranes. Tyrosine-phosphorylated
proteins were detected with anti-phosphotyrosine antibody (RC20 1:2500
dilution) for 2 h, and the proteins were visualized by ECL
(top). The migration of molecular mass markers are
indicated. The blot was stripped and reprobed with anti-CBL to verify
equivalent protein levels (bottom).
|
|
CD80 and CD86 Differ in Their Ability to Induce Tyrosine
Phosphorylation of PLC-
1 and CD28--
The ability of CD80 and CD86
to induce the tyrosine phosphorylation of PLC-
1 differed.
Anti-phosphotyrosine immunoprecipitates of Jurkat T cells stimulated
with CHO-Mock (Fig. 6A, lane
2), CHO-CD80 (Fig. 6A, lane 3), CHO-CD86
(Fig. 6A, lane 4), mAb to CD3 (Fig. 6A, lane 6),
mAb to CD28 (Fig. 6A, lane 7), or left unstimulated (Fig.
6A, lane 5) for 5 min were probed by anti-PLC-
1 antisera.
Stimulation with CHO-CD80 induced a significant increase in PLC-
1
stimulation, whereas stimulation with CHO-CD86 induced, like CHO-Mock
treatment, a much smaller increase. Similar results were obtained using
CD4+ peripheral human T cells (Fig. 6B). Thus,
CD80 and CD86 differed in their ability to induce the tyrosine
phosphorylation of PLC-
1 in both Jurkat and CD4+ human
peripheral blood T cells.

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Fig. 6.
Phospholipase C 1
tyrosine phosphorylation is induced by CHO-CD80 but not by CHO-CD86.
A, anti-phosphotyrosine immunoprecipitates were prepared
from detergent lysates of CHO-CD80 cells alone (lane 1) or
from Jurkat cells (2 × 107) left untreated
(lane 5), or treated with CHO-Mock (lane 2),
CHO-CD80 (lane 3), or CHO-CD86 (lane 4) cells
(1 × 107), 4 µg/ml anti-CD28 mAb (lane
7) or 6 µg/ml anti-CD3 mAb (lane 6) for 30 min on
ice, followed by 5 min at 37 °C. Sample were processed as in Fig.
3A. Proteins were visualized by Western blotting with
anti-PLC 1 antibody. The migrations of molecular mass markers are
indicated. B, anti-phosphotyrosine immunoprecipitates were
prepared, processed, and proteins visualized as in A from
detergent lysates of CHO-CD80 alone or from CD4+ human
peripheral blood T cells (2 × 107) treated with
CHO-Mock, CHO-CD80, or CHO-CD86 cells (1 × 107).
|
|
We developed a deglycosylation procedure that allowed the detection and
identification of a distinct CD28 protein band, facilitating the
analysis of activation-dependent changes in the state of
phosphorylation of CD28 itself (26). We have reported that anti-CD28
mAb stimulation led to the Lck-dependent, in
vivo phosphorylation of CD28 on tyrosine as detected with
anti-phosphotyrosine antibodies (26). We used this technique to compare
the ability of CD80 and CD86 to induce the in vivo tyrosine
phosphorylation of CD28. Jurkat T cells were stimulated for 5 min with
anti-CD28 mAb or mock-, CD80-, or CD86-transfected CHO cells (Fig.
7A). Stringently washed
anti-CD28 immunoprecipitates were treated with
peptide:N-glycosidase F to remove N-linked sugars from CD28. Stimulation of Jurkat T cells with CHO-CD80 cells or with
anti-CD28 mAb induced the in vivo tyrosine phosphorylation of CD28 (Fig. 7A). Surprisingly, parallel treatments of
Jurkat cells with CHO-CD86 failed to elicit any detectable CD28
tyrosine phosphorylation. To eliminate the possibility that the
difference in CD28 phosphorylation after CD80 and CD86 stimulation
resulted from an artifact of the CHO-human B7 transfectants, we tested the ability of murine B7 family members to induce CD28 phosphorylation in murine cells and found that CD80 but not CD86 induced the tyrosine phosphorylation of murine CD28 (data not shown). Time course studies were performed to determine if the difference in the ability of CD80
and CD86 to induce CD28 tyrosine phosphorylation was a qualitative or a
kinetic difference (Fig. 7B). Robust CD28 tyrosine
phosphorylation was detected at 5 min and was still detectable at 30 min following ligation by the ligand CD80; in striking contrast, no
CD28 tyrosine phosphorylation was detected following CD86 ligation at
any time point analyzed (Fig. 7B).

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Fig. 7.
CHO-CD80 but not CHO-CD86 induces the robust
tyrosine phosphorylation of CD28. A, anti-CD28
immunoprecipitates were prepared from detergent lysates of 2.5 × 107 Jurkat cells left unstimulated or stimulated with
CHO-Mock, CHO-CD80, CHO-CD86 cells (1.25 × 107) or
with 4 µg/ml anti-CD28 mAb. Stimulations were stopped and cells lysed
by the addition of 2× detergent lysis buffer. Anti-CD28 mAb and
protein A-Sepharose were added to all samples post-lysis, and the
samples were incubated at 4 °C for 2 h. The samples were washed
as described under "Experimental Procedures" and then incubated
with 1000 units peptide:N-glycosidase F for 1 h before
separation on SDS-PAGE. The proteins were transferred to PVDF, blotted
with anti-phosphotyrosine antibody, and detected by ECL. B,
anti-CD28 immunoprecipitates were prepared and processed as in
A from 2.5 × 107 Jurkat cells stimulated
with 1.25 × 107 CHO-CD80 or with 1.25 × 107 CHO-CD86 for the times indicated.
|
|
CD80 and CD86 Differ in Their Ability to Induce the Association of
CD28 with PI3 Kinase--
Differential protein phosphorylation is one
mechanism regulating the local assembly of functional protein
complexes. Given the dramatic ligand-specific differences in the
tyrosine phosphorylation of CD28, we examined the effect of CD80 and
CD86 on the recruitment of intracellular signaling proteins to the CD28
receptor complex. Stimulation with anti-CD28 mAb has been shown to lead
to the SH2-dependent association of the heterodimeric
p85/p110 PI3-K with the -Tyr(P)193-Met-Asn-Met- sequence in
the cytoplasmic domain of CD28 (34-37). There is a concomitant
increase in PI3 lipid kinase activity that may be important in CD28
signaling pathways, particularly in those involved in the prolongation
of T cell survival (35, 36, 38). Given the dramatic difference in the
tyrosine phosphorylation of CD28 after stimulation with CD80
versus CD86, we examined the ability of CD28 to bind PI3-K
as an in vivo functional correlate of CD28 tyrosyl
phosphorylation. CD28 was immunoprecipitated from Jurkat cells
stimulated with CHO-Mock, CHO-CD80, CHO-CD86, or the anti-CD28 mAb;
coprecipitated PI3-K were detected using an antibody directed against
the p85 subunit of PI3-K. Stimulation with either CD80 or anti-CD28 mAb
induced the association of PI3-K with CD28; in contrast, the PI3-K/CD28
association was much less dramatic following CD86 stimulation (Fig.
8A). This association was
evident by 2 min after stimulation with CD80 and continued for at least
30 min after activation (data not shown). We then tested the
ligand-specific association of PI3-K with CD28 in purified CD4+ human T cells (Fig. 8C). Stimulation for 15 min with CD80 and mAb to CD28 induced the association of PI3-K with
CD28, whereas CD86 did so to a much lesser extent. The time course of
PI3-K association appeared to be slightly different in CD4+
T cells than in Jurkat cells, in that induction of PI3-K association at
15 min exceeded that at 5 min (data not shown). Therefore, as predicted
by the in vivo tyrosine phosphorylation of CD28, there was
much less PI3-K associated with CD28 in both Jurkat and
CD4+ human peripheral T cells after CD86 stimulation
compared with CD80 stimulation.

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Fig. 8.
PI3-K associates in an
SH2-dependent fashion with CD28 after stimulation of T
cells with anti-CD28 mAb, CHO-CD80, and CHO-CD86. A,
anti-CD28 immunoprecipitates were prepared from detergent lysates of
1 × 107 Jurkat cells treated as indicated with
CHO-Mock, CHO-CD80, CHO-CD86 cells (5 × 106) or 4 µg/ml mAb 9.3 for 15 min at 4 °C and then warmed to 37 °C for 5 min. Stimulations were stopped and cells lysed by the addition of 2×
detergent lysis buffer. Anti-CD28 mAb and protein A-Sepharose were
added to all samples post-lysis, and the samples were incubated at
4 °C for 2 h. Immunoprecipitates were washed as described under
"Experimental Procedures," and proteins were separated by
electrophoresis through a 6-15% SDS-PAGE and transferred to PVDF.
Membranes were incubated with antibody to the p85 subunit of PI3-K for
2 h at room temperature, washed, incubated with secondary antibody
coupled to horseradish peroxidase, and proteins were visualized by ECL.
B, jurkat T cells were stimulated as described in
A. In addition to anti-CD28 mAb and protein A-Sepharose, 5 µM -Tyr-Met-Asn-Met- or -Tyr(P)-Met-Asn-Met-containing
peptide was added to samples post-lysis. C, anti-CD28
immunoprecipitates were prepared as described in A from
2 × 107 CD4+ T cells left unstimulated,
stimulated with CHO-Mock, -CD80, -CD86 CHO cells (1 × 107) or with 4 µg/ml anti-CD28 mAb or 15 min at 4 °C,
and then warmed to 37 °C for 15 min. Immunoprecipitates were treated
exactly as in A. D, CD4+ human
peripheral T cells were stimulated with CHO-Mock, CHO-CD80, or CHO-CD86
as described under "Experimental Procedures." In addition to
anti-CD28 mAb and protein A-Sepharose, 5 µM
-Tyr-Met-Asn-Met- or -Tyr(P)-Met-Asn-Met-containing peptide was added
to samples post-lysis. Immunoprecipitates were washed as described
under "Experimental Procedures" and treated as in
A.
|
|
Peptide competition studies were performed to ensure that the
CD86-dependent CD28-PI3-K complex formed in the absence of
detectable levels of CD28 tyrosine phosphorylation remained
SH2-dependent. Incubation of CD28 immunoprecipitates
prepared from Jurkat cell lysates (Fig. 8B) or
CD4+ human peripheral blood cells (Fig. 8D) with
the -Tyr(P)-Met-Asn-Met-containing peptide, but not with its
unphosphorylated -Tyr-Met-Asn-Met- counterpart, inhibited the
association of PI3-K with CD28 after ligation with either CD80 and
CD86. Thus, CD86 induced minimal increases in the in vivo
phosphorylation of Tyr173 below the limits of detection
using the deglycosylation procedure described above. The minimal
CD28 phosphorylation is consistent with the relatively poor ability of
CD86 to induce the binding of PI3-K to CD28 when compared directly with
anti-CD28 mAb or CD80 stimulation.
 |
DISCUSSION |
We have found quantitative differences between the
CD28-dependent signaling responses elicited by the two B7
family members, CD80 and CD86. In our studies, CD80 and anti-CD28 mAb,
but not CD86, induced the robust and sustained tyrosine phosphorylation of CD28 itself. CD86 stimulation was unable to induce detectable in vivo tyrosine phosphorylation of CD28 at any time point
examined. This dramatic difference in the ability of CD80 and CD86 to
stimulate the tyrosine phosphorylation of CD28 was reflected in the
differential ability of the two ligands to stimulate the
SH2-dependent association of PI3-K with CD28.
The data reported here and other studies (21, 22) have shown that both
CD80 and CD86 are able to induce the association of PI3-K with CD28 and
that this association is dependent upon the phosphorylated YMNM motif
of the CD28 cytoplasmic domain. Our conclusions differ, however, from
those of Rudd and colleagues (22) who reported no difference in the
ability of CD80 and CD86 to induce the SH2-dependent
association of PI3-K and CD28. These investigators studied a murine
CD28+ hybridoma transfected with human CD28 that was
stimulated with plate-bound CHO cells expressing murine CD80 and CD86
proteins; both CD80 and CD86 induced the association of PI3-K with
CD28. It remains possible that endogenous murine CD28 with the murine ligands contributed to the responses observed in their system or, more
likely, that the interspecies interaction may have masked the
intraspecies specificity, i.e. that the interaction of human CD28 with the murine ligands did not faithfully reflect
species-specific signaling events. Alternatively, the lack of
quantitative differences observed may relate to differences between our
specific experimental culture conditions. Using purified human T cells,
Olive and colleagues (39) also demonstrated that both CD80 and CD86
induced the SH2-dependent association of CD28 with PI3-K.
However, they further demonstrated that wortmannin, a specific
inhibitor of lipid kinase activity of PI3-K via binding to the p110
subunit, inhibited CD80 costimulation of IL-2 production more
efficiently than CD86 costimulation (IC50 25 and 110 nM, respectively) (39). This suggested that the dependence on PI3 lipid kinase activity of CD80 and CD86 stimulation differed, an
observation consistent with our results. PI3-K has been shown to be
upstream of Akt/PKB in other systems (40, 41) as well as upstream of
p70 S6 kinase (42, 43), both of which may impact on cell survival and
proliferation and therefore on the functional outcome of CD80
versus CD86 stimulation.
In contrast to CD80, CD86 failed to induce the detectable tyrosine
phosphorylation of CD28. We appreciate that PI3-K binding to the CD28
receptor is a more sensitive assay for CD28 tyrosyl phosphorylation
than immunoblotting of precipitated, deglycosylated CD28 polypeptide.
Nevertheless, dramatic quantitative differences between the ability of
the two ligands to induce CD28 tyrosine phosphorylation were observed,
and a number of potential mechanisms to explain this observation should
be entertained. The more rapid dissociation of CD86 from CD28 (5) may
not permit the robust tyrosine phosphorylation of CD28 that the
relatively more prolonged binding of CD80 induced. Simply increasing
the expression of CD86 did not overcome or compensate for the observed
difference (data not shown). Not only do kinetic differences in the
association/dissociation of CD80/CD28 and CD86/CD28 exist but
structural differences in these interactions have been documented (6,
7). Mutation of the LDN residues of the
M99YPPPYLDN107 region of CD28 reduced both CD80
and CD86 binding (6), whereas mutation of Tyr100 and
Tyr104 selectively reduced CD86 binding without impacting
upon CD80 binding (6, 7). Thus, the discrepancy in the conformation of
CD28 upon CD80 binding from that resulting from the CD86 binding may
have resulted in divergent downstream signaling cascades. Finally, CD86
may selectively and rapidly recruit and/or activate a tyrosine
phosphatase that dephosphorylated the -Tyr(P)-Met-Asn-Met- motif of the
CD28 cytoplasmic tail, thereby resulting in lower levels of CD28
phosphorylation and diminished PI3-K binding. It is important to note,
however, that despite the absence of detectable CD28 phosphorylation,
CD86 costimulated cytokine production and NFAT mediated transcriptional
activation. The NFAT-mediated transcriptional activation was inhibited
by the addition of blocking CD28 mAb (data not shown), confirming that
CD86 was signaling through CD28. It remains possible that the mechanism
of CD86-dependent cytokine production and transcriptional
activation may differ from CD80-dependent activation of the
same events. CD86 stimulation may recruit another protein (cytoplasmic
or transmembrane) to CD28, resulting in activation of a slightly
different cascade of signaling events that nonetheless culminate in
cytokine gene transcription. Regardless, either the minimal level of
CD28 phosphorylation achieved by CD86/CD28 interactions is sufficient
for cytokine production or CD28 phosphorylation is itself not required
for cytokine production.
There are precedents for two different ligands of a single receptor to
signal differently through that receptor even when contacting amino
acids overlap. A compelling example is T cell receptor stimulation by
native versus altered peptide ligand; here, peptides that
differ at specific amino acid residues interact with a clonogenic TcR
to modify early intracellular signaling events (e.g.
phosphorylation of Zap-70 and calcium mobilization) (44). In addition,
nonmitogenic antibodies to CD3 have been shown to result in ineffective
TcR
and Zap-70 phosphorylation, minimal PLC-
1 phosphorylation,
and diminished calcium mobilization (45), compared with mitogenic
antibodies binding to different sites on the same CD3 molecule.
Although the TcR is a multichain complex and CD28 exists as a
homodimer, oligomerization of CD28 may be one mechanism permitting
analogous regulation of CD28 signaling pathways (5). In a completely
different system, estrogen receptor binding by two different estrogenic
entities (estrogen and raloxifene) has been shown to regulate two
different DNA response elements (46). Indeed, the biochemical
differences following CD80- and CD86-dependent signaling we
report here might have been predicted by earlier work (47)
demonstrating that stimulation with different anti-CD28 mAbs had
different outcomes with regard to calcium flux and IL-2 production.
To a first approximation, the activation of PLC-
1 has been shown to
generate diacylglycerol and inositol triphosphates that in turn result
in the activation of protein kinase C and the release of calcium from
intracellular stores, respectively. CD80 stimulates the rapid tyrosine
phosphorylation of PLC-
1; there is no discernible phosphorylation
following stimulation with CD86 (Fig. 6). The ability of CD80 to induce
the tyrosine phosphorylation of PLC-
1 correlated with its ability to
induce the in vivo tyrosine phosphorylation of CD28 itself
and the enhanced association of PI3-K with CD28. Buhl et al.
(48) recently demonstrated that the absence of CD19 resulted in
decreased antigen-induced PI3-K activity, as well as decreased
phosphoinositide hydrolysis and calcium mobilization. Furthermore, they
showed that this phenotype could be reproduced either by treatment with
the PI3-K inhibitor wortmannin or by mutation of the tyrosine residues
in the PI3-K-binding motif of CD19. Our results are consistent with a
correlation between PI3-K recruitment and PLC-
1 activation. Whether
the correlation is explained by the recent observation that binding of
phosphatidylinositol 3,4,5-triphosphate to the pleckstrin homology
domain of PLC-
1 results in targeting of PLC-
1 to the membrane
remains to be shown (49).
We and others (19-22) also found several shared biochemical events
triggered by stimulation with either CD80 or CD86. Both ligands were
capable of inducing the rapid tyrosine phosphorylation of signaling
proteins involved in the earliest stages of T cell activation,
including VAV and CBL. VAV has been shown to bind the tyrosine kinase
Zap-70 and Slp-76 and may bind to adapter proteins such as Grb2 and Shc
as well as the nuclear proteins Ku-70 and human ribonucleoprotein-K
(reviewed in Ref. 50). More recent data indicate that phosphorylated
VAV is an exchange factor for Rac-1 and that VAV-GEF is enhanced by
incubation with the Src family kinase Lck (30). Our findings confirmed
the prior demonstration that both CD80 and CD86 induce the tyrosine
phosphorylation of VAV (19). We have also demonstrated that both CD80
and CD86 are able to induce the phosphorylation of CBL, a protein able to bind constitutively with PI3-K and, upon activation, with
p59Fyn (51-53). Thus, the phosphorylation of both VAV and
CBL after stimulation with either CD80 or CD86 may be to provide a
means to assemble a macromolecular signaling complex, common features
of which may initiate signaling transmission cascades leading to their
shared functional properties.
We have shown differing biochemical outcomes between CD80 and CD86
ligation; although these differences appear to be quantitative and not
absolute, quantitative signaling differences have been shown to result
in qualitatively different outcomes ("all or none response") (54).
However, these biochemical differences do not exclude other operational
means by which B7 ligands may contribute in differing outcomes in T
cell responses and differentiation. Clearly, if CD80 and CD86 are able
to direct T cell differentiation and functional program, differential
expression of B7 family members represent another mechanism by which
CD28 and CTLA-4-dependent costimulatory responses are
regulated. The affinities of both CD80 and CD86 for CD28 are low, and
thus the outcome of an in vivo immune response will depend
upon the level of CD80 and CD86 expression on local antigen-presenting
cells. Our data provide further evidence that even in relatively
similar environments, wherein the surface expression of both ligands is
equivalent and other potentially costimulatory molecules are
comparable, and wherein the responding T cell(s) is/are identical, CD80
and CD86 differ in their capacity to stimulate the tyrosine
phosphorylation of CD28, the recruitment of PI3-K to CD28, and the
tyrosine phosphorylation of PLC-
1. These differences demonstrate the
potential for ligand-specific regulation of CD28-dependent
intracellular signaling pathways. Taken together, both the context and
environment of T cell stimulation and ligand-specific signaling
cascades are able to contribute to regulation of T cell differentiation.
 |
FOOTNOTES |
*
This work was supported by the National Institutes of
Health.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.
To whom correspondence and reprint requests should be
addressed: NHLBI, Bldg. 10, Rm. 5D49, 10 Center Dr., Bethesda, MD
20892. Tel.: 301-402-6786; Fax: 301-480-1792; E-mail:
BiererB{at}nih.gov.
The abbreviations used are:
IL, interleukin; CHO, Chinese hamster ovary; PLC, phospholipase C; mAb, monoclonal
antibody; GM-CSF, granulocyte-macrophage colony-stimulating factor; PBS, phosphate-buffered saline; FCS, fetal calf serum; PI3-K, phosphoinositide 3-kinase; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; TCR, T cell receptor.
 |
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