TCR subunit specificity of CTLA-4-mediated signaling Eric Siu,* Beatriz M. Carreno,† and Joaquı´n Madrenas*,1 *The FOCIS Centre for Clinical Immunology and Immunotherapeutics, Robarts Research Institute, and The University of Western Ontario, London, Canada; and †Wyeth Research, Cambridge, Massachusetts
Abstract: Cytotoxic T-lymphocyte-associated antigen (CTLA)-4 is an activation-induced receptor that down-regulates T cell responses by antagonizing B7dependent costimulation and/or by transducing a negative signal. The mechanism of CTLA-4-mediated negative signaling is unknown. Recently, it has been postulated that CTLA-4 inhibits T cell activation by causing specific dephosphorylation of the T cell receptor (TCR)- chain of the antigen-receptor complex through an lck-dependent recruitment of the Src homology-2-containing tyrosine phosphatase-2. To test this hypothesis, we generated stably transfected T cell clones expressing doxycycline-inducible CTLA-4 with CD25:TCR- (CD25-) or CD25: CD3- (CD25-) fusion proteins. In these clones, ligation of CD25- or of CD25- with antibodies against CD25 induced full T cell activation, as illustrated by extracellular signal-regulated kinase (ERK) activation and interleukin (IL)-2 production. More importantly, coligation of CTLA-4 with CD25- or of CTLA-4 with CD25- in the respectively transfected clones inhibited ERK activation and IL-2 production, demonstrating that CTLA-4 does not specifically inhibit signals from TCR- but can also inhibit signals from CD3-. Our results suggest that the target specificity of CTLA-4 is determined by its coligation with any given transmembrane receptor rather than by its intracellular mediators. J. Leukoc. Biol. 74: 1102–1107; 2003. Key Words: SHP-2 䡠 ERK 䡠 interleukin-2 䡠 CTLA-4 䡠 costimulation 䡠 T cell activation
INTRODUCTION Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an activation-induced receptor on T cells that down-regulates immune responses by two different mechanisms [1]. One mechanism is direct antagonism of B7-dependent costimulation as a result of the higher affinity and avidity of CTLA-4 for B7 than CD28. This mechanism does not need the cytoplasmic tail of CTLA-4 but requires high levels of expression on the cell surface. The other mechanism involves delivery of a negative signal to the T cell. This requires at least the proximal region of the cytoplasmic tail of CTLA-4 and occurs at low levels of surface expression. A significant part of recent work on CTLA-4 has been focused on the signaling properties of this molecule in response 1102
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to coligation with the T cell receptor (TCR) complex. Specifically, the subcellular compartmentalization within lipid rafts and the polarization of CTLA-4 to the immunological synapse under conditions of T cell stimulation have been described [2– 4]. In addition, crystal structures for CTLA-4 binding to B7 have shown that the CTLA-4 dimer binds B7 molecules on different dimers (reviewed in ref. [5]), suggesting that oligomerization of the engaged CTLA-4 at the immunological synapse is a critical step to determine the nature of CTLA-4-mediated signaling. However, a key question that remains is how CTLA-4 signals. Recently, it has been reported that mouse CTLA-4 may associate with the phosphorylated TCR- chain within lipid rafts causing dephosphorylation of such a signaling unit of the TCR and subsequent cessation of TCR-induced activation [4, 6]. Reconstitution experiments in nonlymphoid cells have suggested that such dephosphorylation may be mediated by an lck-dependent recruitment of the Src homology-2-containing tyrosine phosphatase (SHP)-2 phosphatase. Implicit in this model is that CTLA-4 specifically inhibits the activating signals emanating from TCR-. If confirmed, this information may be critical to understand how CTLA-4 works and to design immunomodulatory drugs targeting this receptor. To test if the inactivation of T cells following CTLA-4 coengagement with the TCR is the result of specific inhibition of TCR--dependent signals, we took advantage of a welldefined system to analyze the contribution of different subunits of the TCR complex to T cell activation. Such a system uses Jurkat T cells transfected with cDNAs coding for fusion proteins of the extracellular and transmembrane domains of CD25 with the cytoplasmic domains of TCR- or CD3-ε [7, 8]. Jurkat T cells are particularly appropriate for these studies, as they do not express endogenous CD25 [9]. The resulting chimeric molecules (CD25- or CD25-ε) are expressed on the cell surface and can induce interleukin (IL)-2 production in vitro [8] and proliferation in vivo [7] upon ligation with monoclonal antibodies (mAb) against CD25. Therefore, we generated a panel of Jurkat T cell clones stably expressing CD25- or CD25-ε and doxycycline-inducible CTLA-4. Using these cells, we found that CTLA-4 inhibited T cell activation regardless of whether activation resulted from TCR- signals or from CD3-ε signals, and thus, we concluded that CTLA-4-mediated signaling does not have intrinsic specificity for the TCR- chain.
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Correspondence: Robarts Research Institute, P.O. Box 5015, 100 Perth Drive, London, Ontario, Canada N6A 5K8. E-mail:
[email protected] Received May 5, 2003; revised July 16, 2003; accepted August 12, 2003; doi: 10.1189/jlb.0503198.
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MATERIALS AND METHODS
Statistical analysis of results
Cells and plasmids
All the groups were examined in triplicate. Statistical significance was determined by ANOVA and Bonferroni’s multiple comparison tests. A difference was considered statistically significant when P ⬍ 0.05.
The stable Jurkat E6.1 T cell transfectants for doxycycline-inducible, wildtype CTLA-4 used for these studies have been described previously [10]. Dr. Arthur Weiss (University of California–San Francisco) kindly provided plasmids containing the cDNAs coding from fusion proteins of the extracellular and transmembrane domains of human CD25 with the cytoplasmic domains of TCR- (CD25-) or CD3-ε (CD25-ε). The nature of the insert was verified by sequencing. These plasmids were introduced into the inducible CTLA-4Jurkat transfectants by electroporation. Three types of stably transfected clones were used in these studies: those expressing CD25- and inducible CTLA-4 (abbreviated as CTLA-4 CD25-), those expressing CD25-ε and inducible CTLA-4 (abbreviated as CTLA-4 CD25-ε), and those expressing inducible CTLA-4 but no CD25 (abbreviated as CTLA-4). T cell clones were maintained at 37°C in 5% humidified CO2 in RPMI 1640 (Gibco-Life Technologies, Burlington, ON) supplemented with 10% fetal bovine serum (Gibco-Life Technologies). Hygromycin B (Roche Diagnostics, Laval, PQ; 0.2 mg/ml) was used for CTLA-4-transfectant selection, and geneticin (G418; Roche Diagnostics; 0.8 mg/ml) was used for CD25-/ε selection.
Antibodies The following mAb, all from BD PharMingen (Mississauga, ON), were used for flow cytometry: R-phycoerythrin (PE)-conjugated anti-human CD25 mAb, RPE-conjugated anti-human CTLA-4 mAb, fluorescein isothiocyanate (FITC)conjugated anti-human CD3 mAb, and FITC-conjugated anti-human CD28 mAb. The following mAb were used for functional studies: UCHT1, a mAb against human CD3-ε (BD PharMingen); anti-human CD25 mAb (M-A251; BD PharMingen); anti-human CD28 mAb (28.2; eBioscience, San Diego, CA); CTLA-4-20A mAb (Wyeth Research, Cambridge, MA); and P3, a mouse immunoglobulin G (IgG)1 Ig isotype-control mAb (eBioscience). The following antibodies were used for biochemical studies: a goat polyclonal antiserum against CD3ε (Santa Cruz Biotechnology, Santa Cruz, CA); an anti-TCR- mAb (Zymed Laboratories, San Francisco, CA); CTLA-4-24 mAb (for immunoprecipitation), CTLA-4-11 mAb (for immunoblotting; Wyeth Research); phosphoextracellular signal-regulated kinase (ERK)-1/2 mAb (Cell Signaling Technologies, Mississauga, ON); a rabbit polyclonal antibody against ERK-1/2 (Stressgen Biotechnologies, Victoria, BC); a rabbit polyclonal antiserum against human CD25 (Santa Cruz Biotechnology); sheep horseradish peroxidase (HRP)-conjugated anti-mouse polyclonal Ig (Amersham Pharmacia, Baie d’Urfe´ , PQ), goat HRP-conjugated anti-rabbit polyclonal IgG (Bio-Rad Laboratories, Mississauga, ON), and donkey HRP-conjugated anti-goat polyclonal IgG (Santa Cruz Biotechnology).
Flow cytometry Expression of CD3, CD28, CD25, and CTLA-4 was assessed on Jurkat T cell clones (1⫻106) by direct immunofluorescence. FITC- or PE-conjugated, isotype-matched, irrelevant antibodies were used as negative controls. Cells were analyzed in a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), and statistical analysis was performed with CELLQuest computer software (BD Immunocytometry Systems, San Jose, CA).
T cell functional assays Magnetic beads (Dynal, Lake Success, NY) coated with anti-CD3 and antiIgG1 antibodies, with anti-CD3 and anti-CTLA-4-20A antibodies, with antiCD25 and anti-IgG1 antibodies, or with anti-CD25 and anti-CTLA-4-20A antibodies were prepared at a ratio of 1:4 (w:w, first:second antibody) as described previously [11]. These beads were used for T cell stimulation at a 1:1 bead-to-cell ratio. Soluble anti-CD28 mAb (20 g/ml) was added to all cultures. CTLA-4 expression on Jurkat T cell transfectants was induced with doxycycline (1 g/ml) for 24 h before the addition of stimulating reagents and maintained for the duration of the experiment. Forty-eight-hour culture supernatants were collected, and enzyme-linked immunosorbent assay (ELISA) measured IL-2 levels (BD PharMingen).
Protein biochemistry Whole-cell lysates were prepared, used for immunoprecipitation where indicated, and Western blotted as described previously [12]. For immunoprecipitation experiments, 10 ⫻ 106 Jurkat T cells per group were used.
RESULTS Phenotypic characterization of Jurkat T cell transfectants expressing CTLA-4 and CD25-/- To test if CTLA-4-mediated signaling specifically inhibits TCR--dependent signals, we took advantage of a well-defined system using Jurkat T cells transfected with cDNAs coding for fusion proteins of the extracellular and transmembrane domains of CD25 with the cytoplasmic domains of TCR- or CD3-ε [7, 8]. As shown in Figure 1, Jurkat T cells do not express endogenous CTLA-4 or CD25 [9]. Upon CTLA-4 transfection, significant levels of CTLA-4 on the cell surface were detected only after doxycycline induction (Fig. 1A). When these clones were also transfected for CD25- or CD25-ε, the resulting Jurkat T cell clones expressed significant levels of CD25 (Fig. 1A). All the clones studied expressed comparable levels of CD3-ε and CD28 (Fig. 1A). Biochemical analysis confirmed the presence of CTLA-4 on doxycycline induction (Fig. 1B) and of the appropriate CD25 chimeras, which run, as expected, as differentially glycosylated bands (Fig. 1C).
CTLA-4 down-regulates IL-2 production in response to TCR- signaling and CD3- signaling Using these T cell clones, we first examined the functional responses to TCR ligation and to TCR:CTLA-4 coligation to exclude a loss of CTLA-4 function upon cotransfection with CD25- or CD25-ε. As shown in Figure 2A, ligation of the TCR complex with beads coated with antibodies against CD3 and CD28 induced significant IL-2 production in the CTLA4-transfected T cells as well as in the CTLA-4/CD25- and CTLA-4/CD25-ε transfectants. As previously reported [1, 3, 10, 11], coligation of the TCR with CTLA-4 using anti-CD3/ anti-CTLA-4-coated beads significantly down-regulated the production of IL-2 in these clones (Fig. 2A). From these experiments, we concluded that CTLA-4 was functional in the three different types of T cell transfectants under study. Next, we tested if CTLA-4 specifically inhibited TCR-induced IL-2 production or could also inhibit CD3-ε-induced IL-2 production. As expected, stimulation with anti-CD25coated beads did not induce IL-2 production in the absence of CD25-/ε transfection but induced significant IL-2 production in T cell clones transfected with CD25- or CD25-ε (Fig. 2B). The levels of IL-2 production in response to CD25 ligation were consistently higher in the CD25--expressing clones than in CD25-ε-expressing clones, reflecting the amount of CD25- or CD25-ε expressed on the surface of these clones (Fig. 1A) as well as the presence of three immune-receptor tyrosine-based motifs (ITAMs) in the -tail compared with only one ITAM in the ε-tail [13]. More importantly, upon coligation of CD25 with CTLA-4, we observed a significant inhibition of IL-2 production in T cell clones expressing CD25- and in T cell clones expressing CD25-ε (Fig. 2B). To assess if the efficiency of such Siu et al. CTLA signal specificity
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Fig. 1. Characterization of Jurkat T cells transfected for an inducible CTLA-4 or cotransfected for an inducible CTLA-4 and CD25- or CD25-ε. (A) Surface expression of CD3 (solid line in left-column panels), CD28 (dotted line in left-column panels), CD25, and CTLA-4 on these three types of T cell clones. One million T cells from each clone were used for flow cytometry analysis of each molecule. CTLA-4 expression was assessed in noninduced T cells (gray line) and after induction with doxycycline (1 g/ml) overnight (thick line). These profiles are representative of at least three independent analyses and several clones for each combination (five for CD25-ε and seven for CD25-). (B and C) Biochemical detection of CTLA-4 (B), CD25-ε, and CD25- (C) molecules. CTLA-4 immunoprecipitates (IP) from noninduced or doxycycline (Doxy)-induced transfectants were immunoblotted (IB) with an anti-CTLA-4 antibody. CD3-ε immunoprecipitates and TCR- immunoprecipitates from CTLA-4 transfectants or from CTLA-4:CD25- transfectants or CTLA-4:CD25-ε transfectants were immunoblotted with an antibody against the N terminus of human CD25.
inhibition was similar for ε-mediated and -mediated signaling, we titrated the effect of CTLA-4 inhibition and expressed it as percentage of the maximal IL-2 response for each clone. We observed that the level of inhibition was comparable for both clones (Fig. 2C). These results demonstrate that the CTLA-4-mediated inhibition of IL-2 production following T cell activation is not specific for signals emanating from the TCR- chain.
Inhibition of ERK activation by CTLA-4 is not specific for signals from the cytoplasmic domain of TCR- or CD3- The inhibition of IL-2 production by CTLA-4 was apparent regardless of whether the activating signal came from the cytoplasmic domains of the TCR- chain or of the CD3-ε chain. To strengthen this conclusion, we examined whether an earlier event that has been consistently shown to be inhibited by CTLA-4, such as ERK activation, was also inhibited. To approach this issue, we first established the optimal conditions for activation of ERK-1/2 following the ligation of CD25- or CD25-ε with anti-CD25-coated beads. As shown in Figure 3A, ERK-1/2 activation, as reflected by dual phosphorylation of these kinases, peaked at 2 min after ligation of CD25- or of CD25-ε and slowly decreased for the next 30 min after ligation. This profile is similar to that reported following TCR ligation with peptide:major histocompatibility complexes or with mAb against CD3 [14]. 1104
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Having determined the optimal stimulation time for maximum activation of ERK-1/2, the next step was to examine ERK activation under conditions of CD25/CTLA-4 coligation. We found that coligation of CTLA-4 with CD25- or with CD3-ε in the respectively transfected T cell clones inhibits significantly the activation of ERK-1 and ERK-2 (Fig. 3B). Again, the magnitude of activation of ERK-1/2 was proportional to the levels of CD25 expression. As expected, T cell clones transfected with CTLA-4 only, but not with CD25- or CD25-ε, did not show any response to ligation of CD25 or to coligation with CD25 and CTLA-4. As control for activation of ERK-1/2, as detected by dual (tyrosine and threonine) phosphorylation of these kinases, we used pervanadate, a tyrosine phosphatase inhibitor that induces massive phosphorylation of intracellular proteins [15]. These results conclusively demonstrate that CTLA-4 can inhibit ERK activation, resulting from the cytoplasmic domains of the TCR- chain or the CD3-ε chain upon coligation with these TCR subunits.
DISCUSSION The mechanism of CTLA-4-mediated signaling remains elusive. The complete identity of the cytoplasmic domain of CTLA-4 across species suggests that the signaling mechanism used by this molecule is fully conserved in evolution [16]. In its short cytoplasmic tail of 36 amino acids, CTLA-4 has several http://www.jleukbio.org
Fig. 2. Inhibition of IL-2 production by CTLA-4 is not specific for signals from TCR-. (A) CTLA-4 coligation with the TCR inhibits IL-2 production in CTLA-4 transfectants, CTLA4:CD25- transfectants, and CTLA-4:CD25-ε transfectants. (B) CTLA-4 inhibits IL-2 production upon coligation with CD25 in CTLA-4:CD25- transfectants and in CTLA-4:CD25-ε transfectants. T cells (5⫻105) were stimulated with beads coated with the appropriate antibodies at a 1:1 ratio. (C) CTLA-4 inhibits IL-2 production in CTLA-4:CD25- transfectants and CTLA-4: CD25-ε transfectants with similar proportional efficiencies. T cells (5⫻105) were stimulated with anti-CD25-coated beads (1:1 ratio) or anti-CD25- and anti-CTLA-4-coated beads at ratios of 1:1, 1:2, 1:4, and 1:8 for 2 min for 48 h in the presence of soluble antibodies against human CD28 (20 g/ml) and doxycycline (1 g/ml) to induce CTLA-4 expression. IL-2 concentrations were normalized against the maximum level of IL-2 production for each clone. ELISA measured IL-2 in culture supernatants. *, P ⱕ 0.05. At least three clones for each transfectant were used for these experiments.
regions reminiscent of signaling motifs. These include a lysinerich region that binds the regulatory subunit of the serine/ threonine phosphatase PP2A (PP2AA), two tyrosine residues susceptible to phosphorylation, a proline stretch in between these two tyrosine residues, and a YVKM motif that could be involved in binding to phosphatidylinositol-3 kinase, SHP-2, and the catalytic subunit of PP2A (PP2AC) [6, 17–20]. None of these motifs is essential for CTLA-4-mediated, negative signaling, although some of them, such as the two tyrosine residues, are clearly implicated in CTLA-4 export to the membrane and subsequent compartmentalization and internalization [21, 22]. Recently, Bluestone and colleagues [4, 6] have proposed a model to explain CTLA-4-mediated signaling based on the ability to detect association among mouse CTLA-4, SHP-2, and TCR-. This model suggests that TCR signaling would induce an lck-dependent interaction between CTLA-4 and TCR- through SHP-2. Such interaction would result in dephosphorylation of the TCR- chain, removal of this chain from lipid rafts, and abrogation of TCR-mediated signaling [4, 6]. Implicit in this model is that negative signaling through CTLA-4 is specific for TCR--initiated responses. However, it would be surprising that the SHP-2-mediated interaction between CTLA-4 and TCR- is specific, as SHP-2 has been found associated to costimulatory receptors such as CD28 [22] and as CTLA-4 is functional under conditions in which no association between CTLA-4 and SHP-2 or TCR- can be detected [10, 18, 23, 24]. Thus, we decided to address the ability of CTLA-4 to
inhibit TCR- signals specifically, to narrow the search for the mechanism of CTLA-4 signaling and to design CTLA-4 agonists. Here, we report that CTLA-4 inhibition of T cell activation is not restricted to signals and responses from TCR- but can also inhibit those signals from CD3-ε. A remarkable feature of CTLA-4-mediated inhibition of T cell activation is that it requires coligation of CTLA-4 with the TCR/CD3 complex in cis, i.e., on the same surface, in contrast to CD28 costimulation that can work in trans [1, 10, 23–25]. Such arrangement is consistent with recent reports on CTLA-4 compartmentalization during T cell stimulation, showing that it relocates to the immunological synapse, where it coclusters with the TCR and partitions within lipid rafts [2– 4]. These observations imply that the mechanistic basis of CTLA-4 function requires close proximity between the TCR complex and CTLA-4 to allow for their interaction directly or indirectly through a third receptor such as CD28 [26]. This claim is consistent with recent DNA microarray analysis of T cell activation through TCR and CD28 or the inducible costimulator ICOS, showing that CTLA-4-mediated inactivation of T cells correlates with the down-regulation of expression of those genes reflecting the effects of CD28 on TCR signaling [26]. Our results demonstrate that the inhibition of T cell activation by CTLA-4 is not specific for TCR--dependent signaling but can also be seen for signaling emanating from the CD3-εchain. In vitro evidence indicates that TCR- and CD3-ε chains may have differential signaling effects as a result of the heterogeneity of the primary sequence of their ITAMs, which Siu et al. CTLA signal specificity
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Fig. 3. -Induced and ε-induced ERK activation can be inhibited by CTLA-4. (A) Kinetics of ERK-1/2 activation in response to CD25 ligation in CD25-- and in CD25-ε-transfected T cell clones. CTLA-4:CD25- and CTLA-4:CD25-ε T cell transfectants (1⫻106/group) were stimulated with anti-human CD25 mAb-coated beads for 0, 1, 2, 5, 15, and 30 min at a 1:1 cell-to-bead ratio. Whole-cell lysates, equalized for total protein content, were resolved in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and were immunoblotted for dually phosphorylated (active) ERK-1/2 (pERK1/2) and for total ERK. (B) CTLA-4 inhibits ERK activation resulting from CD25- and CD25-ε signaling. T cell transfectants (1⫻106 cells/group) were stimulated with anti-CD25-coated or anti-CD25- and anti-CTLA-4-coated beads (1:1 bead-to-cell ratio) for 2 min or pervanadate in the presence of doxycycline (1 g/ml). Cell lysates were resolved in a 10% SDS-PAGE gel and were analyzed for active and total ERK. At least two clones for each transfectant were used for these experiments.
translates into differential molecular associations with SH2 domain-containing molecules. However, in vivo experiments favor the concept that the differences in signaling may result from quantitative differences as a result of the presence of three ITAMs in TCR- versus one ITAM in CD3-ε, respectively [27, 28]. In addition, there may be intrinsic differences in the regulation of expression for these chimeric molecules, as we and others [13] have shown that chimeric molecules with the ε-tail are always expressed at lower levels than chimeric molecules with the -tail, despite multiple transfection attempts. As we failed to see complexes between the CD25 chimeras and endogenous TCR signaling units (Fig. 1C), it is fair to conclude that in our experimental system, each chain (TCR- or CD3-ε) can contribute on its own to the activation of ERK and the induction of IL-2 gene expression. This validated the claim that CTLA-4-mediated inhibition was effective for signaling emanating from either chain. Our findings may have therapeutic implications for the generation of immunomodulatory drugs using CTLA-4 as target. It is tempting to speculate that CTLA-4 may inhibit signaling from surface receptors other than the TCR inasmuch as coligation of these receptors with CTLA-4 occurs. While waiting for the characterization of signaling steps linking CTLA-4 with the inhibition of TCR-dependent activation, one can envision the generation of bispecific antibodies against CTLA-4 and other receptors as a way to down-regulate undesirable cellular responses. 1106
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ACKNOWLEDGMENTS This work was supported by grants from the Canadian Institutes of Health Research, the Kidney Foundation of Canada, and the Ontario Research and Development Challenge Fund. E. S. held a Natural Sciences and Engineering Research Council scholarship, and J. M. holds a Canada Research Chair in Transplantation and Immunobiology. We thank the members of the Madrenas laboratory for helpful comments and criticisms regarding this work.
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