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*
Transplantationslabor, Klinik für Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover,
Abteilung für Molekulare Pathologie, Universität Würzburg, Würzburg; and
Hoechst-Marion-Roussel, Wiesbaden, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, NF-AT, and proteins binding to the CD28
response element of the IL-2 promoter. On the other hand, stimulation
of T cells with mAb 9.3 increased the level of intracellular
Ca2+ and triggered the activation of
p56lck and c-Raf-1, but was unable to
induce the binding of transcription factors to the IL-2 promoter. In
contrast to the differential signaling of BW 828 and 9.3 in resting T
cells, the two mAbs exhibited a similar pattern of early signaling
events in activated T cells and Jurkat cells
(p56lck activation, association of
phosphatidylinositol 3-kinase with CD28), indicating that the signaling
capacity of CD28 changes with activation. These data support the view
that stimulation through CD28 can induce some effector functions in T
cells and suggest that this capacity is associated with a particular
pattern of early signaling events. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although stimulation of the CD28 molecule alone usually does not induce
effector functions in resting human T cells, several biochemical
signaling events induced by CD28 can be observed. Activation of the
tyrosine kinase p56lck and its association
with CD28 (4), activation of the kinase c-Raf-1 (5), formation of
reactive oxygen intermediates (6), and activation of an acidic
sphingomyelinase resulting in the generation of ceramide (7) have been
described. In addition, strong cross-linking of CD28 in resting T cells
can trigger an increase in the level of free cytoplasmic calcium ions
[Ca2+]i3
(8). Furthermore, in Jurkat cells, cross-linking of CD28 causes the
activation of phospholipase C
1 and subsequent phosphatidylinositol
hydrolysis (9). The role of these signals in the costimulatory function
of CD28 is not well defined. Since transcription factors binding to the
NF-AT and NF-B sites or the CD28 response element (CD28RE) of the
IL-2 promoter are not activated by sole CD28 stimulation of resting T
cells (6, 10, 11), it has been suggested that CD28 pathways do not
directly control gene transcription but have to converge with TCR
signals to fully activate transcription factors and thereby induce
cytokine synthesis. Current models assume that both pathways are
integrated on the level of the protein kinases JNK1 and JNK2, since
these kinases need both TCR and CD28 signals for full activation (12).
We have recently shown that stimulation of resting human T cells with the CD28-specific mAb BW 828 can trigger proliferation and IL-2 production without further requirement for TCR engagement (13). This agrees with the activity of a mAb specific for rat CD28 that triggers T cell proliferation in vitro and in vivo (14). Two very recent observations suggest that in addition to CD28-specific mAbs, binding of CD80 or CD86 to CD28 can induce T cell functions without TCR involvement. Firstly, in vitro interaction of CD80 with CD28 on resting memory cells has been described to provide sufficient stimulus for the generation of effector T cells in the absence of specific Ag (15). Secondly, in the absence of TCR signaling, binding of CD86/B7.2 to CD28 might deliver a death signal for B cells, since CD86/B7.2-transgenic mice exhibit reduced numbers of B cells (16). Taken together, these data support the view that isolated CD28 signaling is not a neutral event for T cells but can induce T cell functions independent of the TCR. Since binding of mAb BW 828 seems to mimic this particular function of CD28, the analysis of the signaling events initiated by this mAb could provide insights into the activation pathways leading to the induction of T cell functions by sole CD28 triggering. We now report that this type of activation is associated with a pattern of signaling events different from that of isolated CD3 or typical CD28 costimulation and is able to activate several transcription factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD28-specific mAbs 9.3 (IgG2a) (17) and BW 828 (IgG2a) (18)
were used for the induction of CD28-mediated signaling events. mAb 9.3
was obtained from Bristol Myers Squibb (Seattle, WA). TCR/CD3-mediated
signaling was induced by the CD3
mAb OKT3 (IgG2a), which was
provided by Cilag (Alsbach Hahnlein, Germany). In some experiments T
cells were partially activated by culture with soluble mAb BMA 031
(IgG2b), which detects a monomorphic determinant on the 
ß-TCR
(18). mAb 9.3 has been clustered to CD28 in leukocyte typing workshops.
A characterization of BW 828 has been described previously (13).
Cell preparations and induction of T cell activation
PBMC were isolated from heparinized peripheral human blood by Ficoll gradient centrifugation. Small resting T cells were then prepared by E-rosetting. The E+ T cell population was incubated overnight at 37°C to remove residual adherent mononuclear cells by plastic adherence. Usually >95% of the remaining cells were CD3+, and 98 to 99% reacted with CD2 mAb. This cell population did not contain monocytes as detected by flow cytometry (<0.3% CD14+ cells) and did not proliferate in response to mitogenic concentrations of CD3 mAb. To induce CD3- or CD28-mediated activation, T cells (2 x 106/ml) were incubated with OKT3 (3 µg/ml) alone, the CD28 mAbs alone (9.3; final ascites dilution of 1/3200; BW 828, 10 µg/ml), or combinations of CD3 and CD28 mAbs for 30 min on ice. Ab-loaded cells were washed, and cell-bound mAbs were cross-linked either by the addition of soluble goat anti-mouse Ig (gam Ig; 3–10 µg/ml) or by gam Ig (3 µg/ml) immobilized on the plastic surface of microtiter plates.
RNA extraction and PCR-assisted analysis of cytokine mRNA
T cells loaded with CD3, CD28, or CD3 plus CD28 mAbs as
described above were cultured in flat-bottom multidish plates (2.5
x 106 cells/2 ml/well) precoated with 3 µg/ml gam Ig.
After 4 h, total RNA was isolated from 5 x 106
cells as described by Chomczynsky and Sacchi (19) and transcribed into
cDNA using reverse transcriptase from Moloney murine leukemia virus
(Stratagene, La Jolla, CA). Amplification of cDNA was conducted with
2.5 U Taq polymerase (Stratagene) in a Gene Amp PCR System 9600
(Perkin-Elmer/Cetus, Norwalk, CT) by 30 three-temperature cycles
consisting of denaturation at 94°C (55 s), annealing at 55°C (60
s), and elongation at 72°C (60 s). The following primers were used:
IL-2: sense primer, 5'-GAATGGAATTAATAATTACAAGAATCCC-3'; antisense
primer, 5'-TGTTTCAGATCCCTTTAGTTCCAG-3'; IL-4: sense primer,
5'-CTTCCCCCTCTGTTCTTCCT-3'; antisense primer,
5'-TTCCTGTCGAGCCGTTTCAG-3'; IFN-: sense primer,
5'-AGTTATATCTTGGCTTTTCA-3'; antisense primer,
5'-ACCGAATAATTAGTCAGCTT-3'; and ß-actin: sense primer,
5'-GAAACTACCTTCAACTCCATC-3'; antisense primer,
5'-CTAGAAGCATTTGCGGTGGAC-3'. PCR products were separated in 2%
agarose gels and visualized by staining with ethidium bromide.
Western blot analysis
T cells (2 x 106/ml) were loaded with CD3 or CD28 mAbs and resuspended in culture medium without FCS. Cross-linking was performed with 10 µg/ml soluble gam Ig for 15 min. After stimulation, cells were spun and resuspended in lysis buffer containing 1% Nonidet P-40 in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, and 1 µg/ml each of leupeptin and pepstatin. Following 30- to 45-min incubation on ice, lysates were centrifuged (8000 x g, 10 min), and the supernatants were mixed with 5x SDS sample buffer (310 mM Tris-HCl (pH 6.8), 50% glycerol, 10% SDS, 25% 2-ME, and 0.5 mg/ml bromophenol blue). The samples were boiled for 5 min and electrophoresed through 8% SDS-polyacrylamide (PAA) gels. Proteins were transferred to nitrocellulose by semidry blotting. Phosphotyrosine-containing proteins were detected by the mAb 4G10 purchased from Biomol (Hamburg, Germany), p56lck was detected by a polyclonal rabbit anti-human lck kinase Ab (Biomol), and c-Raf-1 was detected after incubation of the membrane with a polyclonal rabbit anti-c-Raf-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA). Detection of bound Abs was performed using a standard alkaline phosphatase method (Dianova, Hamburg, Germany) with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate or CSPD (Tropix Luminescence, Serva, Heidelberg, Germany) as substrates.
Immunoprecipitation of CD28-associated proteins
Jurkat cells (2 x 107; 4 x 106/ml) were left untreated or stimulated for 15 min with CD28 mAbs 9.3 (final dilution of ascites, 1/3200) or BW 828 (5 µg/ml). Cells were then lysed in buffer containing 1% Brij 96 in 50 mM Tris-HCl (pH 7.4), 50 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, and 1 µg/ml of each leupeptin and pepstatin for 30 min and spun, and the cleared cell lysates were subjected to CD28 immunoprecipitation by the addition of 50 µl protein G-Sepharose (Pharmacia, Freiburg, Germany). The same amount of CD28 mAbs used for stimulation was added to the cell lysates of untreated cells. After incubation for 2 h at 4°C, the Sepharose beads were washed three times with lysis buffer containing 0.5% Brij 96. The immunoprecipitated proteins were eluted by boiling the samples for 5 min with 25 µl of nonreducing SDS sample buffer (62 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and 0.5 mg/ml bromophenol blue). After electrophoresis through 8% SDS-PAA gels, the proteins were transferred to nitrocellulose. Coimmunoprecipitated PI 3-kinase was detected with a polyclonal rabbit Ab directed to the p85 subunit of PI 3-kinase (Biomol) and a standard alkaline phosphatase method as described above.
Measurement of [Ca2+]i levels
The concentration of cytoplasmic calcium was determined using the fluo-3 method and flow cytometry (20). T cells (5 x 106/ml) were loaded with 4 µM fluo-3/AM (Molecular Probes, Eugene, OR) in culture medium without FCS at 37°C in the dark. After 20 min, the cell concentration was diluted to 1 x 106/ml, and the incubation time was prolonged for 40 min. Cells were loaded on ice with CD3 and CD28 mAbs as described above. After washing, cells were suspended in Ca2+ buffer (25 mM HEPES (pH 7.2), 140 mM NaCl, 1.8 mM CaCl2·2H2O, 1 mM MgCl2·6H2O, 3 mM KCl, and 10 mM D-glucose) and stored at room temperature in the dark. At a final dilution of 2 x 105/ml, cells were analyzed on a FACScan flow cytometer using Chronys software (Becton Dickinson, San Jose, CA). For cross-linking of cell-bound Abs, 3 µg/ml gam Ig was added. Fluo-3 fluorescence was monitored continuously for 3 min at an excitation wavelength of 488 nm.
DNA probes and electrophoretic mobility shift assays (EMSAs)
For detection of DNA binding activities, T cells were induced
with CD3, CD28, or CD3 plus CD28 mAbs and cultured for 16 h in
flat-bottom multidish plates (2.5 x 106 cells/2
ml/well) precoated with 3 µg/ml gam Ig. Cells were then washed, and
nuclear extracts were prepared essentially as previously described
(21). Equal amounts of the extracts (3 µg of crude protein) were
incubated with the 32P end-labeled oligonucleotides.
Binding reactions were performed in the presence of 0.5 µg
poly(dI-dC) in buffer consisting of 60 mM HEPES (pH 7.9), 150 mM KCl,
12% Ficoll, 3 mM DTT, and 3 mM EDTA. After 10-min incubation on ice,
protein-DNA complexes were separated from free probe on a 5% PAA gel.
In supershift assays 0.5 µl of Ab was added to the reaction mixture
simultaneously with the protein, and the mixture was incubated as
described above. In the competition experiments a 25- to 50-fold excess
of unlabeled double-stranded oligonucleotide probe was added to the
incubation mixture 5 min before addition of the labeled probe. The
sequences of the oligonucleotides were as follows: NF-AT (distal NF-AT
binding site of the mouse IL-2 promoter), 5'-CCAAAGAGGAAAATTTGTTTCA-3';
NF-B (distal T cell element of the IL-2 promoter),
5'-GAGGGATTTCACCTAAA-3'; AP-1 (distal T cell element of the IL-2
promoter), 5'-AGAGTCATCAGAA-3'; and CD28RE (proximal TPA response
element; positions -166/-154 of the IL-2 promoter),
5'-AAAGAAATTCCAG-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The two CD28-specific Abs, BW 828 and 9.3, are directed to
different epitopes on the CD28 molecule. In combination with
TCR/CD3 signaling, both Abs induce comparable proliferative
responses in human T cells. In the absence of TCR/CD3 coengagement,
however, only mAb BW 828 is able to trigger a proliferative response
and IL-2 synthesis (13). Further analysis of this type of T cell
activation revealed a BW 828-mediated stimulation of IL-2, IL-4, and
IFN- gene transcriptions (Fig. 1
).
This cytokine pattern was also observed after simultaneous stimulation
of T cells with the CD3 mAb OKT3 plus BW 828 or OKT3 plus 9.3.
Stimulation with mAb 9.3 alone did not trigger any cytokines, whereas
OKT3 alone induced IFN- but neither IL-2 nor IL-4 mRNA.
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The stimulatory capacity of mAb BW 828 prompted us to compare the
early signaling events induced by this mAb with signals triggered by
mAb 9.3 or OKT3. Since CD28 and CD3 stimulation has been shown to
activate cellular protein tyrosine kinases, we first analyzed the
tyrosine phosphorylation pattern in CD3- and CD28-stimulated T cells.
As shown in Figure 2A, CD3
stimulation triggered tyrosine phosphorylation of several substrates.
In some experiments weak phosphorylation of proteins at about 120 kDa
was found after stimulation of T cells with mAb 9.3. In cells
stimulated by BW 828, however, no significant tyrosine phosphorylation
was observed. An analysis of the effect of CD28 triggering on the
kinases p56lck and c-Raf-1 revealed that
9.3 caused a shift in the electrophoretic mobility of lck
from 56 to 60 kDa as previously described (22) and induced
hyperphosphorylation of c-Raf-1 (5) (Fig. 2, B andC). Similar effects were found after OKT3 stimulation, whereas mAb
BW 828 triggered neither the appearance of
p60lck nor hyperphosphorylation of c-Raf-1.
The lack of p56lck modification and c-Raf-1
activation in BW 828-stimulated cells was not due to delayed kinetics
of signaling by this mAb, since prolonged cross-linking of BW 828 up to
60 min did not trigger these signals (data not shown).
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). A similar response was observed in
OKT3- and 9.3-stimulated T cells. Without cross-linking there was a
small increase in OKT3-stimulated cells but not in 9.3- or BW
828-stimulated cells (data not shown). This pattern confirms earlier
data on the signaling capacity of CD28, which showed that CD28
stimulation by soluble mAb does not induce an increase in
[Ca2+]i, whereas calcium-dependent pathways
are activated by strong cross-linking of the CD28 molecule (8). The
capacity of mAb BW 828 to induce Ca2+ mobilization also
shows that its failure to activate p56lck
and c-Raf-1 is not due to ineffective binding in these short term
assays. It is therefore most likely that the differential effects of BW
828 and 9.3 reflect the induction of alternative signaling pathways by
binding of the two mAbs to the CD28 molecule.
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Some CD28-mediated signals can be detected in activated cells and
the Jurkat cell line but not in resting T cells (3, 23). We therefore
asked whether the signaling events that were absent in BW
828-stimulated resting T cells can be found in activated T cells. In
PHA-induced lymphoblasts BW 828 was as effective as 9.3 at triggering
the appearance of the p60 form of p56lck
(Fig. 4A). The
p56lck shift mediated by BW 828 stimulation
was also found in Jurkat cells (data not shown). In addition, the
association of the p85 subunit of PI 3-kinase with CD28, which was
not observed in resting T cells (data not shown), could be readily
detected in Jurkat cells stimulated either by 9.3 or BW 828 (Fig. 4B).
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). However, partially activated T cells
proliferated in response to both 9.3- and BW 828-mediated stimulation.
Together these data support the assumption that the signaling capacity
of CD28 changes with activation (25, 26).
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The CsA sensitivity of BW 828-mediated T cell proliferation (13)
and the Ca2+ flux (Fig. 3
) induced by this mAb strongly
suggested the involvement of Ca2+-dependent activation
pathways. Since Ca2+-dependent signaling pathways play an
important role in the nuclear translocation of the transcription factor
NF-AT (27), we assumed that BW 828 stimulation might induce these
factors to bind to the NF-AT sites of the IL-2 promoter. To address
this question, nuclear extracts from CD28-, CD3-, or CD28- plus
CD3-stimulated T cells were analyzed for their binding to the
32P-labeled distal NF-AT site probe in EMSAs. In
unstimulated T cells little binding activity was detected, and it was
not enhanced by stimulation with the CD28 mAb 9.3 or by sole CD3
stimulation using mAb OKT3 (Fig. 5A). However, a moderate but
clear-cut increase was detected in BW 828-stimulated T cells, albeit
the protein/DNA complexes were less prominent, as after stimulation
with OKT3 plus 9.3 or OKT3 plus BW 828. This is in line with a
diminished IL-2 synthesis in BW 828-stimulated cells compared with that
in cells stimulated by TCR/CD3 plus CD28 mAbs (13). Binding of proteins
to the NF-AT oligonucleotide was partially inhibited by AP-1 competitor
DNA, and anti-NF-ATp Abs induced a distinct supershift,
indicating binding of NF-ATp and AP-1 to the distal NF-AT
site after BW 828 stimulation (Fig. 5B).
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B binding proteins was already found in the
nuclei of unstimulated peripheral T cells (Fig. 5C).
Stimulation with mAb 9.3 did not enhance these levels, whereas
stimulation of cells with mAbs BW 828 or OKT3 resulted in a strong
increase in protein binding to the NF-B probe. A further increase
was found when T cells were stimulated with CD3 plus CD28 mAbs. The
NF-B complexes were retarded by anti-p50 and anti-p65 Abs
and marginally inhibited by anti-c-Rel (Fig. 5D),
indicating the binding of p50 and p65 to the B probe of the IL-2
promoter.
Binding of proteins to the CD28RE was analyzed using an oligonucleotide
spanning positions -154 to -166 of the IL-2 promoter. Triggering of T
cells with the nonstimulatory mAb 9.3 was without effect on protein
binding to the CD28RE (Fig. 6). In BW
828-stimulated cells, however, the generation of complexes consisting
of two proteins was observed. The binding of these proteins was also
induced in OKT3-treated cells and in cells stimulated with both CD28
plus OKT3. Generation of the lower complex could be inhibited by a
50-fold excess of a cold NF-B site and could be supershifted by
anti-p65 Abs. The upper complex was supershifted by an Ab to
NF-ATp, indicating the binding of RelA/p65 and
NF-ATp to the CD28RE after BW 828 and/or OKT3 induction of
T lymphocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Binding of the nonstimulatory Ab 9.3 to CD28 triggered a shift in
electrophoretic mobility of the tyrosine kinase
p56lck, activated the kinase c-Raf-1 (Fig. 2), and increased the level of [Ca2+]i (Fig. 3). This pattern of signaling events confirms earlier observations on
the stimulatory capacity of mAb 9.3 (4, 5, 8). The role of these
signals is not well defined. Since stimulation of T cells with 9.3 did
not induce the transcription factors NF-AT and NF-B (Figs. 5 and 6)
or cytokine gene transcription (Fig. 1), it is likely that the
activation of the kinases p56lck and
c-Raf-1 in concert with Ca2+ mobilization are insufficient
to induce transcriptional events. It has recently been shown that T
cells triggered by mAb 9.3 alone can become refractory to subsequent
CD28 stimulation (28). One might therefore speculate that signals
observed in 9.3-stimulated T cells not only are insufficient to trigger
cytokine gene transcription without TCR signaling but may be
inhibitory. Indeed, 9.3-stimulated cells tend to contain a lesser
amount of transcription factors compared with unstimulated cells (Figs. 5 and 6).
In contrast to the failure of mAb 9.3 to induce cytokine gene
transcription, IL-2, IL-4, and IFN- mRNA could readily be detected
in T cells stimulated with mAb BW 828 (Fig. 1). Since the two mAbs
induced a differential pattern of early signaling events, it is
unlikely that the different stimulatory capacities of 9.3 and BW 828
are due to quantitative differences in their potency to trigger
signaling events. The most likely explanation is therefore the
induction of alternative signaling pathways by the two mAbs. The early
signaling steps induced by BW 828 remain speculative. The absence of
p56lck modification could indicate that this
molecule is not involved in the signaling pathway. The existence of
p56lck-independent signaling mechanisms via
CD28 is suggested by the observation that the
p56lck-deficient Jurkat cell line J.CAM
does not produce IL-2 when stimulated by PMA/ionomycin but can be
triggered to produce some IL-2 when stimulated by PMA/ionomycin plus
immobilized CD28 mAb (29). On the basis of the assumption that
p56lck is not involved in CD28-mediated
triggering by BW 828, it is tempting to speculate that phosphorylation
of CD28, which is required for the recruitment of the adaptor molecules
PI 3-kinase (23, 30), GRB-2/SOS (31), and ITK/EMT (32) to CD28, is
mediated by a different kinase. Since neither ITK nor ZAP-70 seems to
phosphorylate the receptor (33), the kinase
p59fyn could be a good candidate.
The CD28-specific mAb BW 828 was unable to trigger a shift in
electrophoretic mobility of the tyrosine kinase
p56lck and to activate the kinase c-Raf-1
in resting T cells, whereas both signals could be detected after
stimulation of PHA-activated T cells and Jurkat cells (Fig. 4). This
pattern strongly suggests that the signaling capacity of CD28 changes
with activation and is in line with data showing that several CD28
signaling events can only be observed in preactivated T cells and in
Jurkat cells (3). The molecular differences between CD28 on resting vs
activated T cells are not known exactly. It has been suggested that
previous TCR-mediated activation induces phosphorylation of CD28 and
binding of PI 3-kinase (3). In addition,
p56lck is associated with CD28 in
exponentially growing, but not in resting, Jurkat cells (32). It could
therefore be possible that previously activated or memory T cells
express a particular form of CD28 that is biased to mediate T cell
activation without requirements for TCR/CD3 coengagement. On the basis
of this assumption, TCR-stimulated T cells should express a stimulatory
CD28 molecule that can trigger activation when cross-linked by any CD28
mAb. The observation that T cells preactivated by TCR triggering
responded to both 9.3 and BW 828 stimulation (Table I) is in line with
this concept. In experiments using isolated T cell subsets we found
that naive CD4+CD45RA+ T cells did not
proliferate in response to sole CD28 stimulation, whereas previously
activated/memory CD4+CD45R0+ T cells
proliferated in response to stimulation with mAb BW 828 but not in
response to stimulation with mAb 9.3. (13). Thus, it is unlikely that
CD28 molecules expressed on previously activated T cells in general
have the capacity to induce T cell functions on their own. If
CD28-mediated activation occurs in vivo, it might be possible that
particular binding properties and/or a high concentration of CD28
ligand(s) are required to induce critical configurational changes in
CD28 resulting in the induction of stimulatory pathways.
Stimulation of the CD28 molecule without triggering of the TCR usually
does not induce binding of transcription factors to the IL-2 promoter.
Therefore, it has been assumed that CD28 signals have to converge with
TCR signals to activate transcription factors and induce cytokine gene
transcription (12, 34). This view of the signaling capacity of the CD28
molecule is in line with our observation, that T cell stimulation with
mAb 9.3 alone did not activate transcription factors, whereas
stimulation with 9.3 plus OKT3 induced binding of proteins to the
NF-AT, NF-B, and CD28RE sites of the IL-2 promoter (Figs. 5 and 6).
The CD28-specific mAb BW 828 activated these transcription factors
without requirement for TCR coengagement, strongly suggesting that CD28
can control T cell activation at the transcriptional level. The
individual steps of the signaling cascade leading to the activation of
transcription factors by sole CD28 stimulation are not known. Since BW
828 stimulation induced a particular pattern of early signaling events
(Figs. 2 and 3) the early steps of this putative pathway may differ
from TCR/CD3 signals and CD28-mediated costimulatory signals. An
important downstream event of the signaling cascades that regulate the
transcription factor NF-AT is the activation of the Jun kinases JNK1
and JNK2, which are regarded as requiring TCR- as well as CD28-mediated
signals for full activation (12). It remains to be established whether
the potency of BW 828 to induce NF-AT results from the capacity of this
mAb to fully activate JNK1 and JNK2 without further requirement for TCR
signaling.
The data presented in this paper and recent observations from other researchers (14, 15, 16) suggest that stimulation of CD28 with mAbs or with the ligands CD80 and CD86 is not a neutral event but can activate T cells without further requirement for TCR stimulation. Presently it is not clear whether this function of CD28 is operative in vivo. If so, it might be possible that this type of activation plays a role in the regulation of lymphocyte homeostasis. In T cells, CD28-mediated activation might control Ag-independent expansion of memory T cells (15). In the B cell compartment, elimination of irrelevant and/or potentially dangerous B cells could be regulated via T cell factors that are produced by CD28/CD86 interaction without TCR involvement (16). Further knowledge of the biochemical signaling events leading to T cell activation by sole CD28 stimulation could provide the basis for therapeutic inhibition or potentiation of this novel CD28 function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Reinhard Schwinzer, Transplantationslabor K25, Klinik für Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany. E-mail:
3 Abbreviations used in this paper: [Ca2+]i, intracellular Ca2+ concentration; CD28RE, CD28 response element; gam Ig, goat anti-mouse Ig; PAA, polyacrylamide; CsA, cyclosporin A; EMSA(s), electrophoretic mobility shift assay(s); AP-1, activator protein-1; PI 3-kinase, phosphatidylinositol 3-kinase.
Received for publication August 11, 1997. Accepted for publication April 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation through CD28 requires reactive oxygen production by 5-lipoxygenase. EMBO J. 14:3731.[Medline]
1. Adv. Exp. Med. Biol. 323:23.[Medline]
B-like response element. J. Biol. Chem. 266:14179./ß-TCR and anti-CD3 monoclonal antibodies. Transplant. Proc. 23:272.[Medline]
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A. Skapenko, P. E. Lipsky, H.-G. Kraetsch, J. R. Kalden, and H. Schulze-Koops Antigen-Independent Th2 Cell Differentiation by Stimulation of CD28: Regulation Via IL-4 Gene Expression and Mitogen-Activated Protein Kinase Activation J. Immunol., April 1, 2001; 166(7): 4283 - 4292. [Abstract] [Full Text] [PDF]
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 C. S. Heinly, G. D. Sempowski, D. M. Lee, D. D. Patel, P. M. McDermott, R. M. Scearce, C. B. Thompson, and B. F. Haynes Comparison of thymocyte development and cytokine production in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice Int. Immunol., February 1, 2001; 13(2): 157 - 166. [Abstract] [Full Text] [PDF]
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C.-T. Yu, H.-m. Shih, and M.-Z. Lai Multiple Signals Required for Cyclic AMP-Responsive Element Binding Protein (CREB) Binding Protein Interaction Induced by CD3/CD28 Costimulation J. Immunol., January 1, 2001; 166(1): 284 - 292. [Abstract] [Full Text] [PDF]
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B. Luettig, L. Pape, U. Bode, E. B. Bell, S. M. Sparshott, S. Wagner, and J. Westermann Naive and Memory T Lymphocytes Migrate in Comparable Numbers Through Normal Rat Liver: Activated T Cells Accumulate in the Periportal Field J. Immunol., October 15, 1999; 163(8): 4300 - 4307. [Abstract] [Full Text] [PDF]
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G. Woerly, N. Roger, S. Loiseau, D. Dombrowicz, A. Capron, and M. Capron Expression of Cd28 and Cd86 by Human Eosinophils and Role in the Secretion of Type 1 Cytokines (Interleukin 2 and Interferon {gamma}): Inhibition by Immunoglobulin a Complexes J. Exp. Med., August 16, 1999; 190(4): 487 - 496. [Abstract] [Full Text] [PDF]
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B. L. Leung, L. Haughn, A. Veillette, R. G. Hawley, R. Rottapel, and M. Julius TCR{alpha}{beta}-Independent CD28 Signaling and Costimulation Require Non-CD4-Associated Lck J. Immunol., August 1, 1999; 163(3): 1334 - 1341. [Abstract] [Full Text] [PDF]
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A. Skapenko, J. Wendler, P. E. Lipsky, J. R. Kalden, and H. Schulze-Koops Altered Memory T Cell Differentiation in Patients with Early Rheumatoid Arthritis J. Immunol., July 1, 1999; 163(1): 491 - 499. [Abstract] [Full Text] [PDF]
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M. Bonnard, L. Haughn, and M. Julius CD4-Mediated Inhibiton of IL-2 Production in Activated T Cells J. Immunol., February 1, 1999; 162(3): 1252 - 1260. [Abstract] [Full Text] [PDF]
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 S. Bounou, N. Dumais, and M. J. Tremblay Attachment of Human Immunodeficiency Virus-1 (HIV-1) Particles Bearing Host-encoded B7-2 Proteins Leads to Nuclear Factor-kappa B- and Nuclear Factor of Activated T Cells-dependent Activation of HIV-1 Long Terminal Repeat Transcription J. Biol. Chem., February 23, 2001; 276(9): 6359 - 6369. [Abstract] [Full Text] [PDF]
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