Mitogenic signals through CD28 activate the protein kinase C
NF-
B pathway in primary peripheral T cells
Kevin M. Dennehy1,
Andreas Kerstan1,
Astrid Bischof1,
Jung-Hyun Park2,
Shin-Young Na1 and
Thomas Hünig1
1 Institute for Virology and Immunobiology, University of Würzburg, 97078 Würzburg, Germany 2 Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
Correspondence to: T. Hünig; E-mail: huenig{at}vim.uni-wuerzburg.de
Transmitting editor: D. R. Littman
 |
Abstract
|
---|
Mitogenic anti-CD28 antibody stimulates all peripheral T cells to proliferate in the absence of TCR ligation, providing an exception to the two-signal requirement of T cell responses. This antibody preferentially recognizes a mobilized signaling-competent form of CD28, normally induced following TCR ligation, thus providing a unique non-physiological tool to dissect CD28-specific signals leading to T cell proliferation. The protein kinase C (PKC)
NF-
B pathway has recently been shown to integrate TCR- and CD28-derived signals in co-stimulation. We now demonstrate that this pathway is activated by mitogenic anti-CD28 antibody stimulation. In contrast to conventional anti-CD28 antibody, mitogenic anti-CD28 antibody induced activation of phospholipase C
and Ca2+ flux in peripheral rat T cells despite no or low levels of inducible tyrosine phosphorylation of TCR
chain, TCR
-associated protein of 70 kDa (ZAP-70) or linker for activation of T cells (LAT)critical components of the TCR signaling machinery. Nevertheless, PKC
kinase activity in vitro was increased following mitogenic anti-CD28 antibody stimulation, as was membrane association of both PKC
and Bcl10. As downstream targets of PKC
activation, NF-
B components translocated to the nucleus at levels comparable to those after TCRCD28 co-stimulation. NF-
B translocation was diminished by PKC
inhibition, as was induction of the NF-
B/AP-1 responsive activation marker CD69. We propose that co-stimulation is a sequential process in which appropriate TCR engagement is required to mobilize CD28 into a signaling-competent form which then activates the PKC
NF-
B pathway necessary for IL-2 production and proliferation.
Keywords: CD28, co-stimulation, NF-
B, protein kinase C
 |
Introduction
|
---|
Induction of proliferation in primary resting T cells requires engagement of both the antigen-specific TCR and a co-stimulatory molecule, of which the most important on naive T cells is CD28 (1,2). How CD28 contributes to co-stimulation of T cells has remained elusive, partly because of the inability to distinguish between TCR- and CD28-specific signaling events. We have described a mitogenic anti-CD28 antibody which stimulates all resting rat T cells to proliferate in vitro and in vivo in the absence of TCR ligation (36). Interestingly, mitogenic anti-CD28 antibody binds poorly to unstimulated T cells, but more efficiently if the TCR is simultaneously ligated (6). The recognition of such an activation-induced epitope suggests that functionally distinct forms of CD28 exist, and that T cell proliferation can be induced by trapping and ligating a mobilized signaling-competent form of CD28 which in conventional co-stimulation is induced by antigen recognition. The comparison of mitogenic and conventional anti-CD28 antibody stimulation therefore provides a unique non-physiological tool to identify CD28-specific signaling events leading to IL-2 production and T cell proliferation in the absence of TCR-derived signals.
Recently, the novel protein kinase C (PKC) isoform PKC
was shown to integrate TCR- and CD28-specific signals leading to NF-
B transcription factor activation necessary for efficient IL-2 production and T cell proliferation (710). Since the relative contributions of TCR- and CD28-derived signals to the initiation of the PKC
NF-
B pathway remain unknown, we investigated whether mitogenic anti-CD28 antibody stimulation activates this pathway. Our results indicate that if CD28 mobilization and ligation are forced by mitogenic anti-CD28 antibody, downstream signaling via PKC
to NF-
B is indistinguishable from that induced by TCRCD28 co-stimulation.
 |
Methods
|
---|
Antibodies and reagents
Generation of antibodies to rat
ßTCR (R73, IgG1), and conventional (JJ319, IgG1) and mitogenic (JJ316, IgG1) antibodies to rat CD28 have been previously described (3,11). Mouse anti-rat CD3 (G4.18, IgG2a), mouse anti-phosphotyrosine (4G10, IgG2a), mouse anti-human PKC
(IgG2a) and streptavidinphycoerythrin were from Becton Dickinson (Mountain View, CA). Mouse anti-human TCR
-associated protein of 70 kDa (ZAP-70) (3.3.1, IgG2b) (12) and hamster anti-mouse TCR
(146968) (13) were kindly provided by Dr A. D. Beyers (University of Stellenbosch, South Africa) and Dr B. Malissen (Centre dImmunologie Marseille-Luminy, France) respectively. Rabbit anti-p56lck was provided by Dr J. Borst (The Netherlands Cancer Institute, Amsterdam). Mouse anti-human p95vav was from Upstate Biotechnology (Lake Placid, NY), and rabbit polyclonal antibodies to phospholipase C (PLC)
1, SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), Bcl10, NF-
B p65, NF-
B p50, USF-2, goat polyclonal anti-linker for activation of T cells (LAT) and donkey anti-goat IgG peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep anti-mouse IgG was from Boehringer Mannheim, and rat anti-mouse IgG, goat anti-mouse IgG peroxidase and goat anti-rabbit IgG peroxidase were from Dianova (Hamburg, Germany).
Generation of anti-rat CD69 mAb
The extracellular domain of rat CD69 was amplified by RT-PCR from whole RNA of concanavalin A-activated rat splenocytes using oligonucleotide primers with flanking BglII and HindIII sites (5'-tatagatcttacaattgcccaggcttgtac-3' and 5'-tataagctttcatctggagggcttgctgc-3') designed based on the mouse CD69 cDNA sequence. The nucleotide sequence was determined using an ABI Prism 377 DNA sequencer (Perkin Elmer, Foster City, CA) and deposited in GenBank (accession no. AF440759). The cloned rat CD69 extracellular region was expressed in the prokaryotic expression vectors pMAL-c2 (New England Biolabs, Beverly, MA) and pQE-16 (Qiagen, Hilden, Germany), and fusion proteins purified using standard procedures (14) were used for immunization of BALB/c mice, from which B cell hybridoma-producing antibodies were generated as described elsewhere (15). A clone was identified that produced an antibody termed Yuggu-F6 (IgG1
) which showed specific interaction with recombinant rat CD69, but not with other control fusion proteins in an ELISA (results not shown). Yuggu-F6 anti-CD69 antibody precipitated two bands of Mr 2729 and 3133 kDa from cell-surface biotinylated concanavalin A-activated rat lymph node T cells, a close approximate to the two differentially glycosylated forms of human and mouse CD69 (16,17). Consistent with previous observations in the mouse system (18), Yuggu-F6 anti-CD69 antibody labeled only TCRhi thymocytes, but all CD25+ thymocytes after concanavalin A + IL-2 stimulation and all thymocytes after phorbol myristate acetate (PMA) + ionomycin stimulation (results not shown).
Stimulation and FACS analysis
Lymph nodes were taken from 8- to 12-week-old Lewis rats and T cells were purified to
95% purity by nylon wool passage. For TCR and TCRCD28 stimulation, cells (7.5 x 105/ml in supplemented RPMI medium) were incubated on plastic dishes (Greiner, Frickenhausen, Germany) precoated with sheep anti-mouse IgG and then with 2 µg/ml anti-TCR antibody, without or with 0.5 µg/ml soluble conventional anti-CD28 antibody. For stimulation with anti-CD28 antibodies alone, cells were cultured on sheep anti-mouse IgG-coated plates with 5 µg/ml soluble conventional or mitogenic anti-CD28 antibody. For analysis of cell division, cells (1 x 107/ml in PBS) were labeled for 5 min with 2 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; MoBiTec, Göttingen, Germany), washed and stimulated for 2 days, after which dilution of CFSE was determined using a FACScan flow cytometer (Becton Dickinson). Cells stimulated for 16 h in the absence or presence of 10 µM Rottlerin, 0.5 µM Gö6976 or 0.5 µM Gö6850 (Calbiochem, San Diego, CA) were stained with Yuggu-F6 anti-rat CD69 antibody (30 min, 4°C), biotinylated using sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) according to the manufacturers instructions, followed by streptavidinphycoerythrin (30 min, 4°C). Flow cytometric data were collected in a logarithmic mode on light scatter-gated live cells using CellQuest software (Becton Dickinson).
Preparation of whole-cell lysates, immunoprecipitation and Western blotting
For biochemical analyses, T cells (25 x 107/ml in HBSS) were incubated for 3 h at 4°C with 10 µg/ml conventional or mitogenic anti-CD28 antibodies, a mixture of 3.3 µg/ml anti-TCR and 3.3 µg/ml anti-CD3 antibodies, or a mixture of 3.3 µg/ml anti-TCR, 3.3 µg/ml anti-CD3 and 3.3 µg/ml conventional anti-CD28 antibody. Cells were washed, incubated for 30 min at 4°C with 10 µg/ml sheep or rat anti-mouse IgG washed and incubated for the indicated times at 37°C before addition of NP-40 lysis buffer (12% NP-40, 25 mM Tris, pH 7.5, 140 mM NaCl, 2 mM EDTA, 1 mM Pefabloc, 5 mM iodoacetamide, 1 mM Na3VO4 and 1 mM NaF). For preparation of whole-cell lysates, 50 µl 2% NP-40 lysis buffer was added to 50 µl cell suspension containing 2 x 107 cells, and for immunoprecipitation, 1 ml 1% NP-40 lysis buffer was added to 100 µl cell suspension containing 5 x 107 cells. Lysates were centrifuged (14,000 r.p.m., 10 min, 4°C) and the supernatant was either directly resolved by SDS10% PAGE (2 x 106 cell equivalents/well) or added to Protein GSepharose precoated with 25 µg precipitating antibody. Beads were incubated with rotation for 2 h at 4°C and washed 4 times with lysis buffer before addition of 50 µl SDSPAGE sample buffer and electrophoresis (2 x 107 cell equivalents/well). Proteins were transferred to nitrocellulose membranes (100 V, 3 h, 4°C) which were blocked with 10% milk powder in TBS, probed with the indicated primary antibodies followed by the appropriate secondary antibodyperoxidase conjugate and developed using ECL (Amersham, Little Chalfont, UK).
Preparation of membrane and nuclear fractions
Membrane fractions were prepared using a procedure modified from Villalba et al. (19). Cells (3 x 107) stimulated for 16 h at 37°C on sheep anti-mouse IgG-coated plates were resuspended in 100 µl HBSS and lysed by addition of 1 ml cold hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 5 mM iodoacetamide and 1 mM Pefabloc) and vortexing. After centrifugation (14,000 r.p.m., 30 min, 4°C) the pellet was washed twice with hypotonic buffer, resuspended in 30 µl 1% NP-40 lysis buffer and shaken for 1 h at 4°C. The supernatant after centrifugation (14,000 r.p.m., 30 min, 4°C) was resolved by SDS10% PAGE (1 x 107 cell equivalents per well) and Western blotted. Autoradio graphs were analyzed using NIH Image software.
For preparation of nuclear extracts, the protocol of Schreiber et al. (20) was followed. Cells (3 x 106/ml) were stimulated for 6 h at 37°C on sheep anti-mouse IgG-coated plates in the absence or presence of 10 µM Rottlerin, 20 µM PP1, 0.5 µM Gö6976 or 0.5 µM Gö6850. Harvested cells (1 x 107) were washed with cold PBS, resuspended and incubated for 2 min in 100 µl cold hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 1 mM Pefabloc, 2 µg/ml aprotinin and 2 µg/ml leupeptin) before addition of 5 µl 10% NP-40 and vortexing. The nuclei were collected by centrifugation (14,000 r.p.m., 3 min, 4°C), washed twice with hypotonic buffer, resuspended in 30 µl hypertonic buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM Pefabloc, 2 µg/ml aprotinin and 2 µg/ml leupeptin) and rocked on a shaking platform for 2 h at 4°C. After centrifugation (14,000 r.p.m., 10 min, 4°C), the protein concentration in the supernatant was measured using the Bradford assay (Bio-Rad, Hercules, CA) and 7.5 µg protein/lane was resolved by SDS10% PAGE and Western blotted. Autoradiographs were analyzed using NIH Image software.
Measurement of cytoplasmic Ca2+ levels
The protocol of Tedford et al. (21) was followed. Cells (1 x 107/ml) were loaded with 4.5 µM Indo-1 (Molecular Probes, Eugene, OR) in RPMI/1% FCS/0.003% pluronic F-127 for 30 min at 37°C, washed, incubated with 5 µg/ml of the indicated stimulating antibodies for 3 h at 4°C and washed again. Then, 100 µl aliquots containing 1 x 106 cells were diluted with 800 µl RPMI/1% FCS prewarmed to 37°C, and a baseline cytoplasmic Ca2+ level was visualized as the ratio of bound (Fl5) to unbound (Fl4) Indo-1 using a FACS Vantage flow cytometer and CellQuest software (Becton Dickinson). Rat anti-mouse IgG was added at a final concentration of 10 µg/ml and cytoplasmic Ca2+ levels were followed over time. Dot colors indicate the percentage of cells that fell within the range of Ca2+ values demarcated by the colors at a given time point (yellow: 50% of measured cells fall within the range of Ca2+ values marked by the yellow dots at any one time point; dark blue: 25%; orange: 12%; light blue: 6%; pink: 3%; green: 1%).
PKC
kinase assay in vitro
T cells (5 x 107) preincubated for 23 h at 4°C with stimulating antibodies were incubated for 5 min at 37°C in 200 µl of 50 µg/ml rat anti-mouse IgG before addition of 1% NP-40 lysis buffer and immunoprecipitation of PKC
. Immunoprecipitates were washed twice with lysis buffer, twice with 20 mM MOPS, pH 7.5 and incubated for 20 min at 30°C in 40 µl assay buffer [20 mM Tris, pH 7.5, 10 mM MgCl2, 0.5 mM CaCl2, 25 µM PKC pseudosubstrate peptide substrate (from Calbiochem), 1.5 µM ATP and 5 µCi [
-32P]ATP] before addition of 5 µl 50% trichloracetic acid. Precipitated proteins were removed by centrifugation (14,000 r.p.m., 5 min), ATP was removed from neutralized samples by affinity ultrafiltration (Calbiochem) and peptide substrate phosphorylation was assessed by Cerenkov counting. Results were statistically analyzed using Students t-test.
 |
Results
|
---|
Mitogenic anti-CD28 antibody stimulation does not induce tyrosine phosphorylation of TCR
chain or ZAP-70
The earliest events following stimulation through the TCR include tyrosine phosphorylation of the TCRCD3 immuno receptor tyrosine-based activation motif (ITAM) by Src-like kinases, recruitment of the TCR
-associated protein ZAP-70 to phosphorylated ITAM, and subsequent tyrosine phosphorylation and activation of ZAP-70 (22). We have previously shown at the population level that stimulation of T cells with mitogenic anti-CD28 antibody leads to T cell proliferation in the absence of a detectable increase in ZAP-70 tyrosine phosphorylation (6). Using a single-cell assay we show here that mitogenic anti-CD28 antibody stimulation efficiently induces proliferation of all T cells (Fig. 1). Since tyrosine phosphorylation of ZAP-70 requires its binding to phosphorylated ITAM of the TCRCD3 complex, we investigated whether the TCR
chain itself is tyrosine phosphorylated (Fig. 2). Purified peripheral T cells were stimulated with antibodies to the TCR complex or CD28 as indicated, lysed, and both ZAP-70 and TCR
were immunoprecipitated. Anti-phosphotyrosine immunoblotting demonstrated that there was no detectable increase in either TCR
or ZAP-70 tyrosine phosphorylation following stimulation with conventional or mitogenic anti-CD28 antibodies, whereas, as expected, increased phosphorylation was observed following stimulation with anti-TCR antibody (Fig. 2). No increased TCR
or ZAP-70 phosphorylation was observed after mitogenic anti-CD28 antibody stimulation at various time points between 30 s and 20 min (results not shown). Similarly, ZAP-70 did not co-precipitate with TCR
after stimulation with mitogenic anti-CD28 antibody, but, as expected, co-precipitation was observed after TCR stimulation (results not shown).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1. Purified peripheral T cells proliferate after stimulation with mitogenic, but not conventional, anti-CD28 antibody. Purified peripheral rat T cells (7.5 x 105/ml) were labeled with CFSE and incubated on sheep anti-mouse IgG-coated plates with the indicated stimulating antibodies for 2 days. Cell division as indicated by dilution of CFSE was assessed by FACS analysis.
|
|
Mitogenic anti-CD28 antibody stimulation induces tyrosine phosphorylation of PLC
, SLP-76 and p95vav, and activates PLC
without prominent LAT tyrosine phosphorylation
A major substrate for ZAP-70, and a critical component of TCR-mediated signaling, is the transmembrane adaptor protein LAT. Tyrosine phosphorylated LAT binds directly to PLC
1, thereby contributing to activation of this enzyme, and recruits SLP-76 and p95vav by means of the adaptor molecule Gads (22). Consistent with the lack of ZAP-70 tyrosine phosphorylation, LAT was tyrosine phosphorylated only at low levels after stimulation with conventional anti-CD28 or mitogenic anti-CD28 antibodies (Fig. 3). However, mitogenic anti-CD28 antibody stimulation induced prominent and prolonged phosphorylation of p95vav and SLP-76, whereas conventional anti-CD28 antibody stimulation induced more transient phosphorylation of these molecules. Similarly, mitogenic anti-CD28 antibody stimulation induced tyrosine phosphorylation of PLC
1, whereas conventional anti-CD28 antibody stimulation did not induce detectable PLC
1 tyrosine phosphorylation (Fig. 3). Since tyrosine phosphorylation of both PLC
1 and p95vav contributes to their enzymatic activation (23,24), these results suggest that these enzymes may be activated independently of LAT tyrosine phosphorylation. To verify that PLC
is activated by mitogenic anti-CD28 antibody stimulation we measured cytoplasmic Ca2+ levels (Fig. 4). TCR stimulation induced a rapid and transient increase in cytoplasmic Ca2+ levels. In contrast, mitogenic anti-CD28 stimulation induced a delayed and prolonged increase in cytoplasmic Ca2+ levels, and conventional anti-CD28 antibody stimulation did not induce increased cytoplasmic Ca2+ levels (Fig. 4).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 4. Mitogenic, but not conventional, anti-CD28 antibody stimulation induces Ca2+ flux. Purified peripheral rat T cells (1 x 107/ml) were loaded with Indo-1 for 30 min at 37°C and incubated with the indicated stimulating antibodies for 3 h at 4°C. Aliquots were diluted with RPMI/1% FCS prewarmed to 37°C and a baseline cytoplasmic Ca2+ level was visualized by FACS analysis as the ratio of bound (Fl5) to unbound (Fl4) Indo-1. Rat anti-mouse IgG was added at a final concentration of 10 µg/ml and cytoplasmic Ca2+ levels were measured over time.
|
|
PKC
is activated by mitogenic anti-CD28 antibody stimulation
Increased cytoplasmic Ca2+ levels and diacylglycerol generation as a result of PLC
activation contribute to activation of a number of PKC isoforms (25). However, p95vav is required for membrane recruitment of the novel PKC isoform PKC
(19), which in turn activates NF-
B factors required for IL-2 production and proliferation (710,2629). Given the prominent tyrosine phosphorylation of p95vav induced by mitogenic anti-CD28 antibody, we measured membrane recruitment of PKC
(Fig. 5A). In unstimulated cells there was a low level of membrane-associated PKC
, which was not increased by stimulation with conventional anti-CD28 antibody. TCRCD28 co-stimulation or stimulation with mitogenic anti-CD28 antibody (Fig. 5A) or anti-TCR antibody (results not shown) induced enhanced membrane recruitment of PKC
.
A direct or downstream substrate of PKC
is Bcl10. During co-stimulation the scaffold molecule CARMA1 functionally bridges PKC
and Bcl10, resulting in Bcl10 membrane recruitment and phosphorylation (30,31). Defective NF-
B activation in Bcl10-deficient T cells after stimulation with CD3 and CD28 antibodies, but not tumor necrosis factor-
or IL-1, indicates that Bcl10 specifically transduces co-stimulatory signals downstream to NF-
B (32). As with PKC
, co-stimulation or stimulation with mitogenic anti-CD28 antibody induced enhanced membrane association of Bcl10 (Fig. 5A). Such stimulation also induced a lower mobility band, which is phosphorylated Bcl10 (30,31). In contrast, the low levels of membrane-associated Bcl10 were not increased after stimulation with conventional anti-CD28 antibody (Fig. 5A).
In order to directly show PKC
kinase activation, we utilized a kinase assay in vitro (Fig. 5B). As previously described (7,33), PKC
was activated by TCRCD28 co-stimulation, but not by stimulation with either conventional anti-CD28 antibody or anti-TCR antibody. However, stimulation with mitogenic anti-CD28 antibody induced a significant level of PKC
activation (Fig. 5B).
Activation of NF-
B by mitogenic anti-CD28 antibody requires novel PKC isoform activity
Previously we have shown using electromobility shift assays that there is comparable nuclear translocation of NF-
B components after TCRCD28 co-stimulation or mitogenic anti-CD28 antibody stimulation (6). Western blotting of nuclear extracts for the NF-
B components p50 and p65 again demonstrated comparable nuclear translocation of these components after TCRCD28 co-stimulation and mitogenic anti-CD28 stimulation (Fig. 6A). In contrast, stimulation with conventional anti-CD28 antibody did not induce NF-
B and anti-TCR stimulation induced only a low level of NF-
B. Treatment of cells with Rottlerin, a preferential inhibitor of the novel isoforms PKC
and PKC
(7), inhibited nuclear translocation of NF-
B p50 and p65 (Fig. 6A). As Rottlerin may inhibit PKC isoforms indirectly (34), we also compared the effects of inhibition of conventional and both conventional and novel PKC isoforms (Fig. 6B). Inhibition of conventional PKC isoforms with Gö6976 did not strongly diminish NF-
B p50 and p65 nuclear translocation. However, inhibition of both novel and conventional PKC isoforms with Gö6850 strongly diminished NF-
B nuclear translocation. Previously we have shown that activation of the c-Jun N-terminal kinase requires Src-like kinase activity (6) and we show here that NF-
B nuclear translocation is diminished by Src-like kinase inhibition with PP1 (Fig. 6B).
In order to follow the effect of PKC inhibition to an early inducible gene regulated by NF-
B, we employed a newly developed antibody to rat CD69 (Fig. 7). Mitogenic anti-CD28 antibody stimulation and TCRCD3 co-stimulation induced a comparable level of CD69 expression, whereas stimulation with conventional anti-CD28 antibody did not induce CD69. In line with the observed inhibition of NF-
B nuclear translocation, treatment of cells with Rottlerin strongly diminished the induction of CD69. Similarly, inhibition of both conventional and novel PKC isoforms prevented CD69 up-regulation, whereas inhibition of conventional PKC isoforms alone did not effect CD69 up-regulation (Fig. 7). Taken together these results suggest that PKC
activity is required for nuclear translocation and subsequent transcriptional activity of NF-
B components induced by stimulation with mitogenic anti-CD28 antibody.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7. Expression of the activation marker CD69 after mitogenic anti-CD28 antibody stimulation is diminished by inhibition of novel PKC isoforms. Purified peripheral rat T cells (7.5 x 105/ml) were incubated for 16 h at 37°C on sheep anti-mouse IgG-coated plates with the indicated stimulating antibodies in the absence or presence of 10 µM Rottlerin, 0.5 µM Gö6976 or 0.5 µM Gö6850. CD69 expression was assessed by FACS analysis.
|
|
 |
Discussion
|
---|
The most widely accepted hypotheses of how CD28 contributes to T cell co-stimulation are that CD28 stimulation enhances TCR signaling (35,36) or that integration of initially independent TCR- and CD28-specific signals is required for T cell responsiveness (7,26,37). More recently, Lee et al. (38) demonstrated that TCR signaling during T cellantigen-presenting cell (APC) conjugate formation lasts for less than the minimum 2 h signaling required for T cell proliferation. However, PKC
remains in the immunological synapse for at least 4 h (39), a time sufficient for full commitment to cell division. These latter authors suggest that a function of the immunological synapse may be to allow the late co-stimulatory signals required for IL-2 synthesis to be generated, a process in which CD28 and PKC
are critically involved. Here we provide the first evidence that mitogenic signals through CD28 are sufficient to activate the PKC
NF-
B pathway.
We have previously proposed that TCR signaling enables a CD28-mediated pathway leading to full T cell activation during physiological co-stimulation (6). This hypothesis was based on two key findings: the full activation of all subsets of peripheral T cells by a mitogenic anti-CD28 antibody in the absence of TCR triggering as determined by a lack of ZAP-70 phosphorylation and the slow on-rate of mitogenic anti-CD28 antibody when binding to resting T cells, which was dramatically enhanced by simultaneous TCR engagement. Accordingly, we suggested that mitogenic anti-CD28 antibody selectively recognizes a mobilized, signaling-competent form of CD28 and that in TCRCD28 co-stimulation, this mobilization is dependent on cognate antigen recognition by the TCR. In this scenario, mitogenic anti-CD28 antibody both mobilizes and cross-links CD28, while in co-stimulation, TCR signaling mobilizes CD28 which is then cross-linked by conventional anti-CD28 antibody or CD80/86. This hypothesis is not mutually exclusive with the boosting of TCR signaling by co-stimulation proposed by others (35,36). Rather, we anticipate that the recruitment of CD28 to the immunological synapse as a result of cognate antigen recognition by the TCR (40) provides a common physiological basis for both a CD28-mediated enhancement of TCR signaling and the transition of CD28 into a signaling-competent form.
In the present report, we further investigated the possible participation of the TCR signaling machinery in mitogenic anti-CD28 antibody stimulation and then asked whether the PKC
NF-
B pathway can be initiated by CD28-mediated signaling alone. This pathway was analyzed because it is of central importance to T cell activation and is highly co-stimulation dependent (7,10,33). Since, like CD28, PKC
translocates to the immunological synapse in cognate T cellAPC interactions (41,42), we reasoned that its activation by mobilized CD28 could provide an attractive mechanism for a TCR-controlled, CD28-mediated signaling pathway leading to NF-
B activation. We show here that stimulation with mitogenic anti-CD28 antibody does indeed activate the PKC
NF-
B pathway. Membrane recruitment and activation of PKC
was observed after either TCRCD28 co-stimulation or mitogenic anti-CD28 antibody stimulation (Fig. 5), and equally efficient NF-
B activation was observed in response to both stimuli (Fig. 6A). In contrast, stimulation with anti-TCR or conventional anti-CD28 antibodies alone did not result in significant activation of the PKC
NF-
B pathway. A link between PKC
and NF-
B activation in both mitogenic anti-CD28 antibody and TCRCD28 stimulation was established by the inhibitory effect of novel PKC isoform inhibitors on NF-
B nuclear translocation and on the induction of the NF-
B-dependent early activation marker CD69 (Figs 6 and 7).
The mechanism by which mobilized CD28 recruits and activates PKC
remains obscure. Importantly, we could extend our previous observations by showing that NF-
B activation requires Src-like kinase activity (Fig. 6B), but nevertheless occurs in the absence of inducible tyrosine phosphorylation of ZAP-70 and TCR
(Fig. 2). Since tyrosine phosphorylation of TCR
and ZAP-70 is diagnostic of TCR-mediated signaling, these results underscore that TCR ligation is not required for signal transduction induced by mitogenic anti-CD28 antibody. A previous study has shown that PKC
activation during TCRCD28 co-stimulation requires ZAP-70 (33). We are currently addressing whether and how constitutive TCR signals may contribute to signaling induced by mitogenic anti-CD28 antibody.
A key event during T cell activation is activation of PLC
. We show here that mitogenic, but not conventional, anti-CD28 antibody activates PLC
and induces intracellular Ca2+ flux in primary peripheral T cells (Figs 3 and 4). Thus, CD28-mediated signaling not only boosts TCR-induced intracellular Ca2+ flux during co-stimulation as shown previously (36), but upon appropriate triggering induces intracellular Ca2+ flux without TCR engagement or proximal TCR signaling. During TCR stimulation LAT is essential for PLC
activation and induction of intracellular Ca2+ flux (43). The low levels of LAT tyrosine phosphorylation observed during mitogenic anti-CD28 antibody stimulation (Fig. 3) suggest that LAT is not required for CD28-mediated activation of PLC
. Accordingly, we were not able to detect LAT associated with PLC
after mitogenic anti-CD28 antibody stimulation (result not shown). However, our qualitative biochemical assays may not be sufficiently sensitive to detect low levels of LAT associated with PLC
, and we are currently addressing whether LAT is required using a genetic approach of comparing LAT- deficient and -reconstituted cell lines.
Another adaptor required for both TCR-mediated activation of PLC
and TCRCD28 co-stimulation is SLP-76 (33,44). Here we demonstrate that both SLP-76 and p95vav, an exchange factor for Rho GTPases, are prominently tyrosine phosphorylated after mitogenic anti-CD28 antibody stimulation (Fig. 3). SLP-76 can be recruited to tyrosine phosphorylated CD28 by means of the adaptor protein Grid/Gads (45). More recently, Raab et al. (46) showed using overexpression systems that an association between tyrosine phosphorylated SLP-76 and p95vav is required for CD28-mediated cytokine transcription independent of TCR engagement. Such an association occurs after mitogenic anti-CD28 antibody stimulation and at a lower level after conventional anti-CD28 antibody stimulation (result not shown). Whether this association is necessary for mitogenic anti-CD28 antibody stimulation will be addressed using a genetic approach.
In summary, our present results provide further evidence that CD28 can fully activate primary resting T cells to proliferate without classical TCR signaling. We show that this involves the key CD28-dependent pathway activated in TCRCD28 co-stimulationactivation of PKC
which ultimately leads to nuclear translocation of NF-
B, and activation of multiple genes required for T cell proliferation and function. These results lend further support to the hypothesis that a mobilized signaling-competent form of CD28, normally induced by TCR ligation (6), is triggered by the non-physiological stimulation with mitogenic anti-CD28 antibody. We propose that upon recognition of appropriate antigen, the TCR alerts the cell to further mitogenic signals by inducing and recruiting mobilized signaling-competent CD28 molecules which stimulate the PKC
NF-
B pathway once ligated by CD80/86. This two-step mechanism would ensure the need for dual-receptor engagement by appropriate ligands for T cell responsiveness, and hence the proposed checkpoint between tolerance and immunity provided by the requirement for CD80/86+ cells for T cell activation. Current models of synapse formation provide an elegant structural basis for this concept (40). Since we believe that the non-physiological recruitment of mobilized CD28 by mitogenic antibody mimics an activation step normally induced in TCRCD28 co-stimulation, it may be relevant that mitogenic antibodies to both rat and human CD28 recognize the same epitope, which is distinct from the CD80-binding site (47). By contrast, the conventional antibody used in this study recognizes an epitope adjacent to the CD80-binding site. Such studies may contribute to an understanding of a molecular mechanism by which mitogenic antibodies activate T cells, as well as the physiological role of CD28 during co-stimulation.
 |
Acknowledgements
|
---|
We thank Dr A. D. Beyers and Dr B. Malissen for reagents, and Sonja Rotzoll and Peter Zigan for technical assistance. Supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 465 and Graduiertenkolleg Immunmodulation 520.
 |
Abbreviations
|
---|
APCantigen-presenting cell
CFSEcarboxyfluorescein diacetate succinimidyl ester
ITAMimmunoreceptor tyrosine-based activation motif
LATlinker for activation of T cells
PKCprotein kinase C
PLCphospholipase C
PMAphorbol myristate acetate
SLP-76SH2 domain-containing leukocyte protein of 76 kDa
ZAP-70TCR
-associated protein of 70 kDa
 |
References
|
---|
- Frauwirth, K. A. and Thompson, C. B. 2002. Activation and inhibition of lymphocytes by costimulation. J. Clin. Invest. 109:295.[Free Full Text]
- Shahinian, A., Pfeffer, K., Lee, K. P., Kundig, T. M., Kishihara, K., Wakeham, A., Kawai, K., Ohashi, P. S., Thompson, C. B. and Mak, T. W. 1993. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261:609.[ISI][Medline]
- Tacke, M., Hanke, G., Hanke, T. and Hünig, T. 1997. CD28-mediated induction of proliferation in resting T-cells in vitro and in vivo without engagement of the T-cell receptor: evidence for functionally distinct forms of CD28. Eur. J. Immunol. 27:239.[ISI][Medline]
- Rodriguez-Palmero, M., Kerkau, T. and Hünig, T. 1999. Rapid reconstitution of the peripheral T cell compartment in severely immunosuppressed rats with a mitogenic CD28-specific mAb. Immunobiology 200:590.
- Rodriguez-Palmero, M., Hara, T., Thumbs, A. and Hunig, T. 1999. Triggering of T cell proliferation through CD28 induces GATA-3 and promotes T helper type 2 differentiation in vitro and in vivo. Eur. J. Immunol. 29:3914.[CrossRef][ISI][Medline]
- Bischof, A., Hara, T., Lin, C.-H., Beyers, A. and Hünig, T. 2000. Autonomous induction of proliferation, JNK and NF
B activation in primary resting T-cells by mobilized CD28. Eur. J. Immunol. 30:876.[CrossRef][ISI][Medline]
- Coudronniere, N., Villalba, M., Englund, N. and Altman, A. 2000. NF-
B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-
. Proc. Natl Acad. Sci. USA 97:3394.[Abstract/Free Full Text]
- Lin, X., OMahony, A., Mu, Y., Geleziunas, R. and Greene, W. C. 2000. Protein kinase C-
participates in NF-
B activation induced by CD3CD28 costimulation through selective activation of I
B kinase ß. Mol. Cell. Biol. 20:2933.[Abstract/Free Full Text]
- Khoshnan, A., Bae, D., Tindell, C. A. and Nel, A. E. 2000. The physical association of protein kinase C
with a lipid raft-associated inhibitor of
B factor kinase (IKK) complex plays a role in the activation of the NF-
B cascade by TCR and CD28. J. Immunol. 165:6933.[Abstract/Free Full Text]
- Sun, Z., Arendt, C. W., Ellmeier, W., Schaeffer, E. M., Sunshine, M. J., Gandhi, L., Annes, J., Petrzilka, D., Kupfer, A., Schwartzberg, P. L. and Littman, D. R. 2000. PKC-
is required for TCR-induced NF-
B activation in mature but not immature T lymphocytes. Nature 404:402.[CrossRef][ISI][Medline]
- Hünig, T., Wallny, H.-J., Hartley, J. K., Lawetzky, A. and Tiefenthaler, G. 1989. A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation. J. Exp. Med. 169:73.[Abstract]
- Dennehy, K. M., Broszeit, R., Garnett, D., Durrheim, G. A., Spruyt, L. L. and Beyers, A. D. 1997. Thymocyte activation induces the association of phosphatidylinositol 3-kinase and pp120 with CD5. Eur. J. Immunol. 27:679.[ISI][Medline]
- Rozdzial, M. M., Kubo, R. T., Turner, S. L. and Finkel, T. H. 1994. Developmental regulation of the TCR
-chain. Differential expression and tyrosine phosphorylation of the TCR
-chain in resting immature and mature T lymphocytes. J. Immunol. 153:1563.[Abstract/Free Full Text]
- Park, J. H., Choi, E. A., Cho, E. W., Hahm, K. S. and Kim, K. L. 1998. Maltose binding protein (MBP) fusion proteins with low or no affinity to amylose resins can be single-step purified using a novel anti-MBP monoclonal antibody. Mol. Cells 8:709.[ISI][Medline]
- Park, J. H., Na, S. Y., Lee, H. H., Lee, Y. J. and Kim, K. L. 2001. Detection of pET-vector encoded, recombinant S-tagged proteins using the monoclonal antibody ATOM-2. Hybridoma 20:17.[CrossRef][ISI][Medline]
- Yokoyama, W. M., Koning, F., Kehn, P. J., Pereira, G. M., Stingl, G., Coligan, J. E. and Shevach, E. M. 1988. Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation. J. Immunol. 141:369.[Abstract/Free Full Text]
- Hamann, J., Fiebig, H. and Strauss, M. 1993. Expression cloning of the early activation antigen CD69, a type II integral membrane protein with a C-type lectin domain. J. Immunol. 150:4920.[Abstract/Free Full Text]
- Hare, K. J., Jenkinson, E. J. and Anderson, G. 1999. CD69 expression discriminates MHC-dependent and -independent stages of thymocyte positive selection. J. Immunol. 162:3978.[Abstract/Free Full Text]
- Villalba, M., Bi, K., Hu, J., Altman, Y., Bushway, P., Reits, E., Neefjes, J., Baier, G., Abraham, R. T. and Altman, A. 2002. Translocation of PKC
in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C. J. Cell Biol. 157:253.[Abstract/Free Full Text]
- Schreiber, E., Matthias, P., Müller, M. M. and Schaffner, W. 1989. Rapid detection of octamer binding proteins with mini-extracts prepared from a small number of cells. Nucleic Acids Res. 17:6419.[ISI][Medline]
- Tedford, K., Nitschke, L., Girkontaite, I., Charlesworth, A., Chan, G., Sakk, V., Barbacid, M. and Fischer, K. D. 2001. Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling. Nat. Immunol. 2:548.[CrossRef][ISI][Medline]
- Leo, A., Wienands, J., Baier, G., Horejsi, V. and Schraven, B. 2002. Adapters in lymphocyte signaling. J. Clin. Invest. 109:301.[Free Full Text]
- Liu, K. Q., Bunnell, S. C., Gurniak, C. B. and Berg, L. J. 1998. T cell receptor-initiated calcium release is uncoupled from capacitative calcium entry in Itk-deficient T cells. J. Exp. Med. 187:1721.[Abstract/Free Full Text]
- Aghazadeh, B., Lowry, W. E., Huang, X. Y. and Rosen, M. K. 2000. Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 102:625.[ISI][Medline]
- Shirai, Y. and Saito, N. 2002. Activation mechanisms of protein kinase C: maturation, catalytic activation, and targeting. J. Biochem. 132:663.[Abstract]
- Kalli, K., Huntoon, C., Bell, M. and McKean, D. J. 1998. Mechanism responsible for T-cell antigen receptor- and CD28- or interleukin 1 (IL-1) receptor-initiated regulation of IL-2 gene expression by NF-
B. Mol. Cell. Biol. 18:3140.[Abstract/Free Full Text]
- Shapiro, V. S., Truitt, K. E., Imboden, J. B. and Weiss, A. 1997. CD28 mediates transcriptional upregulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites. Mol. Cell. Biol. 17:4051.[Abstract]
- McGuire, K. L. and Iacobelli, M. 1997. Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells. J. Immunol. 159:1319.[Abstract]
- Lai, J. H., Horvath, G., Subleski, J., Bruder, J., Ghosh, P. and Tan, T. H. 1995. RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter. Mol. Cell. Biol. 15:4260.[Abstract]
- Wang, D., You, Y., Case, S. M., McAllister-Lucas, L. M., Wang, L., DiStefano, P. S., Nunez, G., Bertin, J. and Lin, X. 2002. A requirement for CARMA1 in TCR-induced NF-
B activation. Nat. Immunol. 3:830.[CrossRef][ISI][Medline]
- Gaide, O., Favier, B., Legler, D. F., Bonnet, D., Brissoni, B., Valitutti, S., Bron, C., Tschopp, J. and Thome, M. 2002. CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-
B activation. Nat. Immunol. 3:836.[CrossRef][ISI][Medline]
- Ruland, J., Duncan, G. S., Elia, A., del Barco Barrantes, I., Nguyen, L., Plyte, S., Millar, D. G., Bouchard, D., Wakeham, A., Ohashi, P. S. and Mak, T. W. 2001. Bcl10 is a positive regulator of antigen receptor-induced activation of NF-
B and neural tube closure. Cell 104:33.[ISI][Medline]
- Herndon, T. M., Shan, X. C., Tsokos, G. C. and Wange, R. L. 2001. ZAP-70 and SLP-76 regulate protein kinase C-
and NF-
B activation in response to engagement of CD3 and CD28. J. Immunol. 166:5654.[Abstract/Free Full Text]
- Soltoff, S. P. 2001. Rottlerin is a mitochondrial uncoupler that decreases cellular ATP levels and indirectly blocks protein kinase C
tyrosine phosphorylation. J. Biol. Chem. 276:37986.[Abstract/Free Full Text]
- Viola, A., Schroeder, S., Sakakibara, Y. and Lanzavecchia, A. 1999. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283:680.[Abstract/Free Full Text]
- Michel, F., Attal-Bonnefoy, G., Mangino, G., Mise-Omata, S. and Acuto, O. 2001. CD28 as a molecular amplifier extending TCR ligation and signaling capabilities. Immunity 15:935.[ISI][Medline]
- Avraham, A., Jung, S., Samuels, Y., Seger, R. and Ben-Neriah, Y. 1998. Co-stimulation-dependent activation of a JNK-kinase in T lymphocytes. Eur. J. Immunol. 28:2320.[CrossRef][ISI][Medline]
- Lee, K. H., Holdorf, A. D., Dustin, M. L., Chan, A. C., Allen, P. M. and Shaw, A. S. 2002. T cell receptor signaling precedes immunological synapse formation. Science 295:1539.[Abstract/Free Full Text]
- Huang, J., Lo, P. F., Zal, T., Gascoigne, N. R., Smith, B. A., Levin, S. D. and Grey, H. M. 2002. CD28 plays a critical role in the segregation of PKC
within the immunologic synapse. Proc. Natl Acad. Sci. USA 99:9369.[Abstract/Free Full Text]
- Bromley, S. K., Iaboni, A., Davis, S. J., Whitty, A., Green, J. M., Shaw, A. S., Weiss, A. and Dustin, M. L. 2001. The immunological synapse and CD28CD80 interactions. Nat. Immunol. 2:1159.[CrossRef][ISI][Medline]
- Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N. and Kupfer, A. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395:82.[CrossRef][ISI][Medline]
- Bi, K., Tanaka, Y., Coudronniere, N., Sugie, K., Hong, S., van Stipdonk, M. J. and Altman, A. 2001. Antigen-induced translocation of PKC-
to membrane rafts is required for T cell activation. Nat. Immunol. 2:556.[CrossRef][ISI][Medline]
- Zhang, W., Irvin, B. J., Trible, R. P., Abraham, R. T. and Samelson, L. E. 1999. Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11:943.[Abstract/Free Full Text]
- Yablonski, D., Kadlecek, T. and Weiss, A. 2001. Identification of a phospholipase C-
1 (PLC-
1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-
1 and NFAT. Mol. Cell. Biol. 21:4208.[Abstract/Free Full Text]
- Ellis, J. H., Ashman, C., Burden, M. N., Kilpatrick, K. E., Morse, M. A. and Hamblin, P. A. 2000. GRID: a novel Grb-2-related adapter protein that interacts with the activated T cell costimulatory receptor CD28. J. Immunol. 164:5805.[Abstract/Free Full Text]
- Raab, M., Pfister, S. and Rudd, C. E. 2001. CD28 signaling via VAV/SLP-76 adaptors: regulation of cytokine transcription independent of TCR ligation. Immunity 15:921.[ISI][Medline]
- Lühder, F., Huang, Y., Dennehy, K. M., Guntermann, C., Müller, I., Winkler, E., Kerkau, T., Ikemizu, S., Davis, S. J., Hanke, T. and Hünig, T. 2003. Topological requirements and signaling properties of T-cell activating, anti-CD28 antibody super agonists. J. Exp. Med., in press.