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*
Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, PA, 19104;
Department of Pediatrics, North Shore University Hospital-New York University School of Medicine, Manhasset, NY 11030; and
Department of Molecular Pathology, Institute of Pathology, University of Wuerzburg, Wuerzburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of activated TCR/CD3 complexes by interactions between the
SH2 domain of p56lck and several
TCR-associated proteins, including adaptor proteins and PTKs ZAP-70
and/or Syk, LAT (linker for activated T cells), SLP-76, and vav
(4, 5). The coordinated activity of these PTKs activates
downstream signal cascades, including phosphatidylinositol-3 (PI-3)
kinase, phospholipase C 
1, and
p21ras-involving pathways, which culminate in
the nucleus and result in changes in gene expression.
Signals transduced through CD4 molecules on T cells are distinct from
those transduced through the TCR/CD3 complex. Triggering of both
receptors activates p21ras, albeit in divergent
ways. TCR/CD3- and CD4-mediated signals lead to the tyrosine
phosphorylation of Shc on different sites. This seems to reflect
differences in the functional role of Shc in the signal transfer from
the TCR and CD4 receptors to p21ras(6). Ligation of the CD4 molecule also induces activation
of PI-3 kinases and NF-
B (7, 8, 9). Apart from
costimulatory signals, CD4 coreceptors can also transmit inhibitory
signals that are able to interfere with TCR/CD3- (10, 11)
or PMA + ionomycin- (12) mediated T cell activation to
induce a functional unresponsiveness of T cells. The molecular
mechanisms of the inhibitory effects of anti-CD4 Abs involve
inhibition of TCR-induced ZAP-70 phosphorylation, ras activation,
mitogen-activated protein kinases (MAPK), and, finally, inhibition of
IL-2 gene transcription (13). In vivo studies in mice and
humans have implicated that anti-CD4 mAb preferentially inhibit the
Th1 pathway and spare Th2 responses (14, 15). The
molecular nature of this response, however, is unclear.
In the two-signal model for T cell activation (16, 17, 18, 19), ligation of the TCR/CD3 complex delivers signal 1, which up-regulates activation molecules on T cells, e.g., CD40 ligand (L). Interaction of CD40L with CD40 leads to expression of B7 family proteins on APC, which, in turn, bind to the costimulatory molecule CD28 on T cells (20, 21). Signals transduced through CD28 comprise the signal 2 (17). Abrogation of the sequential cascade leading to signal 2, using mAbs to CD40L, B7-1/B7-2, or CTLA4Ig, has been demonstrated to lead to T cell unresponsiveness (22, 23). The biochemical nature of CD28-mediated signals has been shown to involve the activation of c-Jun N-terminal kinase (JNK), a critical event in the complete activation of T effector cell function (24).
In this study, we have investigated signals initiated by CD4-specific mAbs that are able to interfere with the CD3-mediated activation of CD4+ T cells. We show that triggering of CD4 molecules on T cells induces extracellular signal-related kinase (Erk) 2, but not JNK, activation. Pretreatment of T cells with anti-CD4 mAbs suppressed the CD3-mediated induction of Erk2 activity, as well as that of the IL-2 promoter factors NF-AT and AP-1. This suppression could be overcome by costimulation of T cells with agonistic anti-CD28 Abs, suggesting that CD4-mediated signals inhibit signal 1 transduced through the TCR/CD3 complex, but do not affect signal 2, mediated through CD28 molecules. The in vivo role of anti-CD4 mAbs was confirmed when mice treated with anti-CD4 mAb had markedly inhibited IL-2 secretion and proliferation in response to Ag-specific stimuli. These results indicate that a "partial activation" of T cells through CD4 coreceptors can lead to a functional unresponsiveness, similar but not identical to T cell anergy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs were used: mAb to murine CD4 (YTS177, rat
IgG2b; purified from a hybridoma provided by Drs. H. Waldmann and S.
Cobbold (University of Oxford, Oxford, U.K.) (43); murine anti-CD28
mAb (clone 37.51; PharMingen, San Diego, CA); murine anti-CD3
mAb (clone 2C11; PharMingen), anti-active MAPK (Erk2) (Promega,
Madison, WI; catalog no. V6671); anti-active JNK (Promega; catalog
no. V7931).
Cells and cell culture
BALB/c mouse splenocytes purified by Ficoll-Hypaque density
gradient centrifugation were used for the studies on cytokine secretion
and proliferation. Purified CD4+ T cells were
obtained by removal of adherent cells and subsequent depletion of
B220+ and CD8+ cells on
magnetic beads (Miltenyi Biotec, Auburn, CA). The purity of the CD4 T
cells was 95–97%, as determined by flow cytometry. EL4 T cells were
cultured in RPMI 1640 media supplemented with 10% FCS. To trigger the
T cells through CD4 receptors, cells were treated with YTS177 mAb for
murine T cells (1 µg/ml) for 2 h and washed once with PBS. Cells
were resuspended in PBS with 2% FCS and goat anti-rat (GAR) IgG
(10 µg/ml) for the time intervals indicated. For proliferation and
cytokine secretion assays, cells were stimulated with plate-bound
anti-CD3 mAb (clone 2C11, 1 µg/ml) in the absence or presence
of soluble anti-CD28 mAb (clone 37.51; agonistic Ab; 1 µg/ml) or
with a combination of 12-O-tetradecanoylphorbol-12-acetate
(TPA; 10 ng/ml) and ionomycin (1 µM).
DNAs and DNA transfections
Chloramphenicol acetyltransferase (CAT) reporter gene constructs
bearing multiples of the Pu-bA (NF-AT, IL-4)
(25), Pu-bd (NF-AT, IL-2)
(26), TREcoll (AP-1) (27), and a B site
from the MHC class II invariant chain promoter (NF-B)
(28) were used for transient transfections into EL4 cells
using a conventional DEAE dextran sulfate transfection protocol, as
described previously (26). Then, 20 h after
transfection, the cells were divided and either left untreated or
stimulated with YTS177 for 2 h, washed with PBS, followed by
stimulation with plate-bound anti-CD3 mAb in the absence or
presence of soluble anti-CD28 mAb or with a combination of TPA and
ionomycin. The cells were incubated for another 20 h, harvested,
sonicated, and used for CAT assays. For the CAT assay, 100 µg of cell
extracts were incubated with 14C-chloramphenicol
and acetyl CoA. Acetylated CoA was extracted in ethyl acetate and
separated by TLC. Percent acetylation was calculated by PhosphoImaging
(Molecular Dynamics, Sunnyvale, CA). Transient transfection experiments
were done only in EL4 T cells, due to the poor transfection efficiency
of primary murine T cells.
Western blot analyses
To detect activated Erk2 and JNK in cell extracts, EL4 cells were stimulated with anti-CD4, anti-CD3, and anti-CD28 mAbs under various conditions, as described above. A total of 50 µg of proteins were electrophoresed on a 10% SDS-PAGE and transferred to nitrocellulose membrane (Hybond-ECL; Amersham, Arlington Heights, IL). Blots were blocked in 5% milk solution for 2 h. The presence of active Erk2 and JNK was detected by probing membranes with Abs to active MAPK and JNK. The reactivity of the Abs was measured by incubating with peroxidase-conjugated anti-rabbit IgG Ab. The reaction was detected by the enhanced chemiluminescence kit (Amersham) and quantitated by densitometry (Bio-Rad, Hercules, CA). The density of each band was calculated over background density of the autoradiogram (5 arbitrary units).
Animal experiments
BALB/c mice (6–8 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME). For Ab treatment, animals were injected i.p. with a 100 µg/dose of YTS177 mAb or control rat IgG2b on days -1 and 1. Mice were immunized s.c. with 100 µg of OVA emulsified in CFA on day 1. Anti-CD28 Ab (clone 35.1) was administered i.p. on days 1 and 3 at concentrations of 100, 10, and 1 µg/dose/mouse. Three mice were used in each group. Splenocytes were harvested on day 7 and analyzed for OVA-induced proliferative responses, as well as IL-2 and IL-4 secretion.
T cell assays
Proliferative responses of murine T cells were measured by
standard [3H]thymidine incorporation followed by liquid
scintillation. Briefly, 2 x 105 purified
CD4 T cells were cultured in the presence of various stimuli. For
OVA-stimulated cultures, adherent cells from naïve C57BL/6 mice
were treated with OVA, irradiated, and used as APC. Cell cultures were
harvested on day 3 for CD3, CD3 + CD28, or OVA-stimulated cultures. In
some cases, cells were cultured with a combination of TPA + ionomycin.
Lymphoproliferation was assessed by addition of 1 µCi
[3H]thymidine 16 h before harvesting and
radioactivity determined as cpm on a liquid scintillation counter
(Wallach, Gaithersburg, MD). Cytokine secretion was measured in culture
supernatants of murine T cells stimulated under various conditions for
48 h. IL-2, IL-4, and TNF-
were measured using commercial ELISA
kits (BioSource International, Camarillo, CA) using the manufacturers’
protocol. Optical densities were read at 405 nm on a MRX Dynatech
(Chantilly, VA) microplate reader.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the influence of CD4 coreceptors on T cell
proliferation and secretion of IL-2 and TNF-, splenic
CD4+ T cells were pretreated with the
CD4-specific mAb YTS177. Fig. 1
shows
that YTS177 treatment of purified CD4+ splenic T
cells induced the secretion of TNF-, but did not stimulate IL-2
secretion and T cell proliferation. Pretreatment of the same cells with
YTS177 inhibited the CD3-mediated secretion of IL-2 and T cell
proliferation, while it did not affect the CD3-induced TNF-
secretion. Pretreatment of T cells with control mouse IgG2b Ab had no
effect on anti-CD3-induced proliferation and cytokine secretion
(data not shown).
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). These results suggest that CD4
activation could interfere with the TCR/CD3, but not CD28-mediated
signals.
At the transcriptional level, the expression of the IL-2 gene is
controlled by several inducible transcription factors
(29). In transient transfection assays using CAT reporter
gene constructs bearing multiples of factor binding sites, we have
investigated the effect of anti-CD4 treatment on the activation of
three prominent inducible transcription factors involved in IL-2
promoter activity, namely NF-AT, AP-1, and NF-B. Fig. 2 shows that YTS177 treatment of
CD4+ EL4 T cells induced NF-B, but not NF-AT
or AP-1, activity. Importantly, pretreatment of cells with YTS177
inhibited the anti-CD3 mAb-induced activation of NF-AT and AP-1,
whereas the activation of NF-B was not inhibited by YTS177
pretreatment.
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, YTS177 pretreatment inhibited
both the anti-CD3 mAb-induced activation of the IL-2 (48% to
5.9%; Pu-bd) and IL-4 NF-AT sites (29.9% to
6.9%, Pu-bA), indicating that NF-AT factors are
direct targets of anti-CD4 mAb pretreatment. Costimulation of T
cells with anti-CD3 + CD28 Abs overcame the inhibitory effect of
anti-CD4 mAbs on NF-AT and AP-1 factors (Fig. 2). The inhibitory
effects of CD4 signals are proximal to the cell surface, since
anti-CD4 mAb did not inhibit TPA + ionomycin-induced NF-AT and AP-1
activation, which bypass the requirement of receptor-associated
kinases. CD4 signals modulate MAPK activation
To delineate the signals transduced through CD4 molecules to
downstream targets, we investigated the activation of MAPK by YTS177.
Fig. 3 shows that stimulation of
CD4+ T cells with YTS177 gave rise to a transient
activation of Erk2. Maximal activity of Erk2 was observed after
stimulation for 30 min, declining to the basal level within 2 h.
In striking contrast, no JNK activation was detected under identical
induction conditions, whereas TPA + ionomycin treatment induced both
Erk2 and JNK activities.
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). Anti-CD3 alone did not induce JNK
activation (Fig. 4, lanes 2–5). Costimulation with
anti-CD3 + CD28 Abs could not overcome the inhibitory effect of
YTS177 mAb on Erk2 activation completely (Fig. 4, lanes
7–10). Fig. 4 shows that pretreatment of
CD4+ EL4 cells with YTS177 for 2 h resulted
in an inhibition of anti-CD3 + CD28 mAbs-induced Erk2 activation in
a dose-dependent manner (Fig. 4, lanes 2–5). In contrast,
the CD3 + CD28 Abs-mediated activation of JNK remained resistant to the
anti-CD4 mAb pretreatment (Fig. 4, lanes 7–10). These
findings show that anti-CD4 mAbs inhibit TCR/CD3-mediated Erk2
activation, but do not affect CD3 + CD28-induced JNK activation.
Similar results were observed using human anti-CD4 Ab and human
Jurkat CD4+ T cells (data not shown).
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To confirm these observations in vivo, BALB/c
H-2d mice were injected i.p. with either control
rat IgG2b or a nondepleting anti-CD4 mAb, YTS177, followed by s.c.
immunization with OVA in CFA, with or without anti-CD28 mAb (see
Materials and Methods). CD4 T cells isolated 7 days later
were analyzed for OVA-induced proliferative responses, IL-2, and IL-4
secretion. Fig. 5 shows that CD4 T cells
from animals treated with YTS177 mAb were inhibited in their ability to
proliferate and secrete IL-2 in response to OVA stimulation, compared
with those treated with control IgG2b. In vivo administration with
anti-CD28 mAb overcame the inhibition of T cell functional
responses (Ag-specific proliferation and IL-2 secretion).
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Taken together, these findings demonstrate that signals transduced
through the CD4 molecule (e.g., Erk2 and NF-B) inhibit TCR-mediated
IL-2 secretion, but not those induced through the costimulatory
molecule CD28. Furthermore, ligation of CD4 molecules with anti-CD4
mAb potentiates Ag + CD28-induced IL-4 secretion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
induction. In our view, this observation might be of relevance for the
induction of a transient unresponsiveness of peripheral T cells and,
possibly, thymocytes in vivo. Costimulation through the CD28 Ab
overcame this inhibitory effect of anti-CD4, by inducing JNK
activity. Moreover, the anti-CD28 Ab-mediated switch of immune
responses to a Th2 phenotype in vivo was substantially potentiated by
anti-CD4 treatment. Naive T cells require two signals for complete activation, resulting in maximal IL-2 secretion and effector cell functions (18, 23). Signal 1 is delivered by recognition of Ag peptide-MHC class II on APC by TCR/CD3 on T cells. This signal initiates a cascade of intermolecular interactions involving CD40L (on T cells), CD40 (on APC) followed by B7 (on APC), and CD28 (on T cells). Signal 2 comprises of biochemical events triggered through CD28. The ability of anti-CD4 mAb to inhibit TCR/CD3-induced signal 1 leads to disruption of intermolecular signal cascades required for complete T cell activation. Thus, lack of provision of signal 2 in vivo results in Ag-specific T cell unresponsiveness. In this study, we have demonstrated that anti-CD4 mAb inhibited TCR-mediated signal 1. Restoring the cascade by providing exogenous anti-CD28 mAb overcame the inhibitory effect of anti-CD4 mAb in vitro and in vivo.
Engagement of the CD4 molecule on T cells with mAb before TCR
stimulation has been shown to trigger negative signals leading to T
cell unresponsiveness (reviewed in Ref. 13). Several
signals, including calcium flux and the activation of
p21ras, PI-3, and Raf-1 protein kinases, have
been shown to be enhanced following CD4 ligation. We have confirmed
previous reports (31) that triggering of
CD4+ T cells with anti-CD4 mAb led to partial
activation of T cells, including activation of Erk2. However, CD4
ligation failed to induce activation of JNK. In addition, analyses of
transcription factors involved in the IL-2 promoter induction showed
that YTS177 treatment resulted in consistent activation of NF-B
(7, 9), but not that of NF-AT or AP-1. It is presently
unclear how activation leads to the induction of one but not other
signaling pathways and transcription factors involved in T cell
activation. The published data on the signals transduced through the
CD4 molecule are quite controversial (13). While the
differences may be due to various culture systems and Abs used for CD4
stimulation, all these studies demonstrated that CD4 ligation leads to
delivery of "partial" and transient signals to T cells. However,
the physiological role of these signals remains elusive. Recent studies
have implicated that CD4 signals may contribute to regulation of T
helper cell differentiation (32, 33, 44).
The biochemical mechanism involved in T cell unresponsiveness induced by anti-CD4 mAb resembles induction of anergy by incomplete activation of T cells (34, 35, 36). CD4 cross-linking has been shown to induce tyrosine phosphorylation of several substrates through activation of p56lck (reviewed in Ref. 37). Pretreatment of CD4+ T cells with CD4 Abs or expression of truncated CD4, both associated with reduced tyrosine phosphorylation of several substrated, suggests that p56lck plays an important role in membrane-proximal events following CD4 ligation (38, 39, 40). In this study, we have examined MAPK downstream of p56lck activation. In the case of anti-CD3, a partial activation of T cells through CD3 or CD4 molecules interfere with MAPK pathways. However, pretreatment of T cells with anti-CD4 mAbs suppressed the following: CD3-induced Erk2 but not JNK kinase activation, whereas pretreatment with anti-CD3 mAbs led to the inhibition of Erk as well as JNK activation by anti-CD3 and anti-CD28 mAbs. Moreover, costimulation of the CD28 pathway overcame the inhibitory effect of anti-CD4 mAbs, whereas CD28 activation is unable to overcome CD3-mediated T cell anergy. These findings indicate that anti-CD4 mediated inhibition differs from the "classical" pathway of T cell anergy.
Previous studies have shown that anti-CD4 mAb treatment induces a shift to Th2 phenotype (14, 15). Although, in our experiments, the inhibitory effects of anti-CD4 mAb on TCR-mediated activation in vitro or in vivo did not lead to such a shift, we observed a modulation of OVA-specific IL-4 secretion induced by anti-CD28 mAb treatment in vivo. Thus, mice treated with anti-CD4 mAb showed an enhanced Th2 type response after activation of CD28 signaling pathway. One interpretation of this effect is the contribution of CD4-mediated stimuli in the modulation of signaling cascades and transcription factors involved in Th2 cell differentiation. The different strengths of anti-CD4 mAb used in earlier studies and our work may explain the need for CD28 costimulation to observe the stimulatory effects of CD4 Ab on IL-4 secretion (32, 33, 41).
In summary, our results have demonstrated the role of CD4-mediated signals in regulation of T cell functions in vivo. Our studies warrant immune suppressive regimens with anti-CD4 mAb to be combined with inhibitors of signal 2 (e.g., CTLA-4Ig (42)). These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Narendra Chirmule, Institute for Human Gene Therapy, University of Pennsylvania, 304 SCL, 422 Currie Boulevard, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: PTK, protein tyrosine kinase; Erk, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PI-3, phosphatidylinositol-3; MAPK, mitogen-activated protein kinase; L, ligand; CAT, chloramphenicol acetyltransferase; TPA, 12-O-tetradecanoylphorbol-12 acetate.
Received for publication February 25, 1999. Accepted for publication April 28, 1999.
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complex. J. Exp. Med. 181:1997.B. Biochem. Biophys. Res. Commun. 203:498.[Medline]
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regulates expression of the major histocompatibility complex II associated invariant chain by binding of an NF-B like factor to a promoter element. Mol. Cell. Biol. 10:4146.This article has been cited by other articles:
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S. E. Kerry, J. Buslepp, L. A. Cramer, R. Maile, L. L. Hensley, A. I. Nielsen, P. Kavathas, B. J. Vilen, E. J. Collins, and J. A. Frelinger Interplay between TCR Affinity and Necessity of Coreceptor Ligation: High-Affinity Peptide-MHC/TCR Interaction Overcomes Lack of CD8 Engagement J. Immunol., November 1, 2003; 171(9): 4493 - 4503. [Abstract] [Full Text] [PDF]
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S. M. L. Tamma, V. S. Kalyanaraman, S. Pahwa, P. Dominguez, and R. R. Modesto The lectin jacalin induces phosphorylation of ERK and JNK in CD4+ T cells J. Leukoc. Biol., May 1, 2003; 73(5): 682 - 688. [Abstract] [Full Text] [PDF]
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S. Harding, P. Lipp, and D. R. Alexander A Therapeutic CD4 Monoclonal Antibody Inhibits TCR-{zeta} Chain Phosphorylation, {zeta}-Associated Protein of 70-kDa Tyr319 Phosphorylation, and TCR Internalization in Primary Human T Cells J. Immunol., July 1, 2002; 169(1): 230 - 238. [Abstract] [Full Text] [PDF]
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