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The Journal of Immunology, 2004, 172: 5948-5956.
Copyright © 2004 by The American Association of Immunologists

Conversion of CTLA-4 from Inhibitor to Activator of T Cells with a Bispecific Tandem Single-Chain Fv Ligand1

Joaquín Madrenas2,*, Luan A. Chau*, Wendy A. Teft*, Paul W. Wu{dagger}, Jason Jussif{dagger}, Marion Kasaian{dagger}, Beatriz M. Carreno{dagger} and Vincent Ling3,{dagger}

* Federation of Clinical Immunology Societies Centre for Clinical Immunology and Immunotherapeutics, Robarts Research Institute, and Departments of Microbiology and Immunology, and Medicine, University of Western Ontario, London, Ontario, Canada; and {dagger} Wyeth Research, Cambridge, MA 02140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abs or their recombinant fragments against surface receptors of the Ig superfamily can induce or block the receptors’ native function depending on whether they induce or prevent the assembly of signalosomes on their cytoplasmic tails. In this study, we introduce a novel paradigm based on the observation that a bispecific tandem single-chain variable region fragment ligand of CTLA-4 by itself converts this inhibitory receptor into an activating receptor for primary human T lymphocytes. This reversal of function results from increased recruitment of the serine/threonine phosphatase 2A to the cytoplasmic tail of CTLA-4, consistent with a role of this phosphatase in the regulation of CTLA-4 function, and assembly of a distinct signalosome that activates an lck-dependent signaling cascade and induces IL-2 production. Our data demonstrate that the cytoplasmic domain of CTLA-4 has an inherent plasticity for signaling that can be exploited therapeutically with recombinant ligands for this receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CTLA-4 is an activation-induced, transmembrane glycoprotein in T lymphocytes that inhibits the responses of these cells when coligated with their clonotypic Ag receptors (TCR) (1, 2). Current evidence indicates that T cell inactivation by CTLA-4 involves two distinct mechanisms (3). One is antagonism of CD28-dependent costimulation because CTLA-4 and CD28 share ligands (B7.1/CD80 and B7.2/CD86), but CTLA-4 interacts with them with 10–100 times higher affinity and avidity. The other mechanism involves negative signaling through its cytoplasmic tail by a still uncharacterized mechanism (4, 5). Through either or both mechanisms, CTLA-4 maintains T cell homeostasis and tolerance. As such, CTLA-4 has emerged as a very attractive target for immunomodulatory drugs that block its inhibitory function and enhance T cell-mediated immunity (6), or alternatively, enhance its inhibitory function and suppress unwanted immune responses (4, 7).

Learning about the structural nature of CTLA-4 signaling is key to exploiting the full potential of CTLA-4 as a therapeutic target. The current paradigm of receptor-mediated signal transduction suggests two explanations (8, 9). One is that CTLA-4 signaling results from ligand-induced oligomerization that facilitates transactivation of CTLA-4-associated molecules. However, the fact that CTLA-4 is already expressed as a dimer under conditions in which no signaling from this molecule is apparent argues against this possibility because one would predict that, under these same conditions, kinases and/or phosphatases associated to its cytoplasmic tail may transactivate each other and randomly initiate signaling (10). Also, Ab-induced cross-linking of CTLA-4 fails to induce any detectable response, making a conventional oligomerization model unlikely. The other possibility is that CTLA-4 signaling is initiated by a ligand-induced conformational/architectural change that is transmitted to the cytoplasmic domain of CTLA-4 allowing for the recruitment of signaling molecules. This second possibility has not been considered likely because recent crystal structures for nonengaged and B7-engaged CTLA-4 did not reveal any obvious conformational change in CTLA-4 (11, 12).

The capacity to engineer mAbs against cell surface receptors has renewed the interest in the development of these molecules as biopharmaceuticals (13). One of the manipulations on Abs involves the deletion of the Fc portion and the combination of two single-chain variable region fragments (ScFv)4 joined by a polypeptide linker. The resulting dimeric molecules have better pharmacokinetics than whole Abs while lacking the Fc portion-dependent side effects, and can be generated with two different specificities to increase the avidity of the interaction. The biological effect of these recombinant Ab fragments has consistently been either enhancement or blockade of the function of the target molecule (14, 15, 16, 17). In this study, we introduce a novel paradigm by which a bispecific tandem ScFv ligand of CTLA-4 converts this inhibitory receptor on T cells into an activating receptor. Such a reversal of function is due to the assembly of a distinct signalosome on the cytoplasmic tail of CTLA-4, and thus reveals an unexpected plasticity of signaling through this surface receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

PBMC were isolated from heparinized blood on Ficoll-Hypaque gradients (Amersham Pharmacia Biotech, Uppsala, Sweden). T cell blasts were generated by culturing PBMC (7 x 106 cells/group) with 100 ng/ml PMA (Sigma-Aldrich, Oakville, Ontario, Canada) and 1 µg/ml ionomycin (Sigma-Aldrich) for 72 h at 37°C. Cells were rested for 15 h in fresh medium before restimulation. Jurkat E6.1, JRT3, and JCaM1.6 cells were obtained from American Type Culture Collection (Manassas, VA).

The doxycycline-inducible CTLA-4, stably transfected Jurkat T cell panel used for these studies has been previously described (3, 18, 19, 20). LG2, a B lymphoblastoid cell line expressing high levels of HLA-DR1 and B7, was provided by Dr. E. Long (National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD). All cell lines were maintained in culture in RPMI 1640 plus 10% FCS.

Abs and reagents

The mouse mAbs 11, 24, and 26 against human CTLA-4, and F(ab')2 of the 24 Ab were generated at Wyeth Research (Cambridge, MA) and have been previously reported (3, 18, 19, 20). The mouse IgG2b mAb against phosphotyrosine, 4G10, was kindly provided by Dr. B. Druker (Oregon Heath Sciences University, Portland, OR). A rabbit antiserum against {zeta}-associated protein of 70 kDa (ZAP-70) was kindly provided by Dr. J. M. Rojo (Centro de Investigaciones Biologicas, Madrid, Spain). The following commercially available Abs were used in these studies: an anti-dually phosphorylated extracellular signal-regulated kinase (ERK)-1/-2 mAb (Cell Signaling Technology, Beverly, MA), a rabbit antiserum against ERK-1/-2 (StressGen Biotechnologies, Victoria, British Columbia, Canada), a mouse IgG1 mAb against phospholipase C{gamma}1 (PLC-{gamma}1; Upstate Biotechnology, Lake Placid, NY), a goat polyclonal antiserum against serine/threonine phosphatase 2A (PP2A)A{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA), and a mAb anti-CD28 (CD28.2; BD Biosciences, Mississauga, Ontario, Canada). FITC-labeled anti-CD3, PE-labeled anti-CTLA-4, and FITC-TCR V{beta}8 were purchased from BD Biosciences. Okadaic acid was purchased from Sigma-Aldrich. A ScFv from PV-1 (PV-1A), a hamster Ab against mouse CD28, was used for the B7-binding ELISA (21).

Generation of 24:26

DNA sequences of Ig variable domains for the Abs against human CTLA-4 24 and 26 were obtained from the hybridomas A3.5H3.F2 and 6CTLA4.1.1.1, respectively (Wyeth Research). H and L chain sequences for 24 were derived from hybridoma cDNA library screening using radiolabeled oligonucleotides from mouse IgG2 C regions and {kappa} L chain. Twenty-six sequences were derived by RT-PCR using degenerate nucleotides from the mouse ScFv module (catalog no. 27-94000F; Pharmacia, Peapack, NJ). Sequences were assembled into His-tagged bispecific ScFv format by overlap PCR (22).

24:26 was produced in methotrexate-resistant Chinese hamster ovary cells. Chinese hamster ovary cells were cultured in serum-free R5CD1-PVA in roller bottle format. The culture supernatant was clarified with Pall Profile II disks (VWR International, West Chester, PA) and concentrated and diafiltered with 10-kDa Millipore prepscale TFF cartridges (Bedford, MA). Protein was purified using NiCl IMAC and Chelating Sepharose Fast Flow column (Pharmacia). Concentrated, diafiltered, conditioned medium from four harvests was loaded onto the column. The column was washed with 20 mM sodium phosphate/1 M NaCl pH 7.5, and washed again with 20 mM sodium phosphate/1 M NaCl/10 mM imidazole pH 7.5. Protein was eluted via a 10 CV linear gradient from 10 to 500 mM imidazole. Fractions containing 24:26 were formulated into PBS with 5 mM EDTA pH 7.2 using an Amicon-stirred cell equipped with a 10-kDa YM110 membrane (Beverly, MA). The formulated protein was filtered through a Millipore 0.22-µM syringe filter, and used.

ELISA for B7 binding

Plates were coated overnight with human CTLA-4/human IgG1 (10 µg/ml) followed by blocking with 1% BSA/PBS for 1 h. After washing, ScFv Abs were added from an initial concentration of 10 µg/ml and diluted down 1:3 in titrated series and incubated at room temperature for 1 h. Plates were washed and human B7-1/human IgG1 biotin was added at 5 µg/ml in dialyzed 10% FBS and incubated at room temperature for 1 h. Plates were washed and B7-binding activity was detected using Neutravidin HRP detection agent.

T cell stimulation, immunoprecipitation, and Western blotting

Doxycycline-induced Jurkat T cells were stimulated with 24:26 or with 24 or 26 Abs at the concentrations indicated for 10 min at 37°C. Cell lysates and immunoprecipitates were prepared and used for Western blotting as described (23). Image acquisition, analysis, and densitometry were done with the Fluorchem 8000 Advanced Imaging System (Alpha Innotech, San Leandro, CA) and Phoretix 1D software (NonLinear Dynamics, Durham, NC).

T cell functional assays

PBMC blasts, generated after 72 h incubation with PMA and ionomycin, were washed four times with 1x PBS, rested at 37°C for 15 h in complete fresh medium before stimulation (0.1 x 106/group) with 24:26, a mixture of 24 ScFv and 26 ScFv, 24 F(ab')2, or whole Abs, or Staphylococcus enterotoxin E (SEE) at the concentrations indicated in 96-well plates for 72 h at 37°C. Supernatants were harvested at 72 h and IL-2 measured by ELISA (BD Biosciences).

Doxycline-induced Jurkat E6.1 T cell transfectants (0.1 x 106/group) were stimulated with 24:26, or mixture of 24 and 26 ScFvs, or 24 Ab alone, or 26 Ab alone, with and without cross-linking with secondary Ab or with beads, in 96-well plates for 48 h at 37°C, and IL-2 in the supernatants measured by ELISA. Stimulation with anti-CD3/anti-MHC class I or anti-CD3/anti-CTLA-4 Ab-coated beads was performed as previously described (3, 18, 19, 20) in the presence of soluble anti-CD28 mAbs (5 µg/ml).

Flow cytometry

Jurkat T cells (1 x 106/group) were washed twice with PBS and stained with FITC-anti-CD3, FITC-anti-CD28, or PE-anti-CTLA-4 for 30 min at 4°C. Samples were then washed in PBS and analyzed by flow cytometry (FACScan and CellQuest software; BD Biosciences).

Calcium mobilization

Cells were washed into Ca2+Mg2+-free PBS containing 1% BSA, and loaded with the calcium chelator dye FLUO-3 (Molecular Probes, Eugene, OR), resuspended in Pluronic F-127 detergent (Molecular Probes), and used at a final concentration of 4 µg/ml. Cells were loaded for 30 min at 30°C, then washed with ice cold PACM buffer (25 mM PIPES, pH 7.2, containing 110 mM NaCl, 5 mM KCl, 5 mM CaCl2, 2 mM MgCl2, and 0.05% BSA). Cells were prewarmed to 37°C for 3 min before initiation of time-based data acquisition on a FACScan equipped with 37°C sample chamber. Once a baseline fluorescence level was established, acquisition was interrupted for addition of 25 µg/ml anti-CTLA-4 Abs or 24:26 or 0.5 µg/ml anti-CD3, then resumed for an additional 200–250 s/sample. Mean fluorescence intensity was plotted as a function of time.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
24:26, a bispecific tandem ScFv ligand of CTLA-4

While developing novel CTLA-4 ligands with immunomodulatory activity, we generated a bispecific tandem ScFv ligand of CTLA-4 that combines two ScFv against different epitopes of human CTLA-4, linked by a (KEA)n semirigid spacer (Fig. 1a). The epitopes recognized by this bispecific tandem ScFv ligand, from now on referred as 24:26, are located in a loop adjacent to the loop containing the B7-binding MYPPPY sequence, but facing the cell membrane (Fig. 1b). The 24 ScFv is directed against the M10 CTLA-4 epitope (58ELT60). The 26 ScFv recognizes the M11 CTLA-4 epitope (65SICT68). Epitope mapping was confirmed by loss of Ab binding upon site-directed mutagenesis (Fig. 1b). 24:26 blocks binding of CTLA-4 to B7.1(Fig. 1c), consistent with the fact that both 24 and 26 Abs block the interaction between B7 and CTLA-4. However, the inhibition of T cell activation by CTLA-4 does not require intact M10 or M11 epitopes although the efficiency of inhibition was decreased when both epitopes were mutated (Table I).



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  		FIGURE 1. 24:26: a bispecific tandem ScFv ligand of CTLA-4. a, Domain structure of 24:26. The coding sequences for the VL and VH regions of Abs 24 and 26 united by a coding unit for a trimer of GGGGS were linked with a DNA portion coding for a helical linker. Expression of the transcript is driven by the leader sequence for Ab 24. The 3' end of the cDNA has a region coding for a His tag. b, Epitope map for Abs 24 and 26. The B7-binding loop containing the MYPPPY sequence is shown in pink. Epitope mapping was performed by mutation of the three amino acids from the human to the mouse sequence and loss of Ab binding. c, 24:26 (24–26ScFv), but not the anti-CD28 ScFv PV-1A, blocks the binding of B7.1 to CTLA-4, as measured by ELISA using recombinant molecules.

 

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  		Table I. Mutations of the M10 or M11 epitopes do not abolish the inhibitory function of CTLA-4

 
24:26, by itself, activates primary human T lymphocytes through CTLA-4

Unexpectedly, when the biological effect of 24:26 was tested, we found that this reagent by itself activated CTLA-4-expressing, primary human T lymphocytes (Fig. 2). In these experiments, CTLA-4 expression on primary T cells was induced by stimulation with mitogens for 72 h (Fig. 2a). Upon a short period of rest, these cells (92% T cells based on CD3 expression) were stimulated with increasing concentrations of 24:26 for 72 h. As controls, cells were stimulated with 24 ScFv and 26 ScFv. In addition, to control for dimerization of CTLA-4 and because monospecific tandem ScFv were not available, we used 24 F(ab')2. Only when stimulated with 24:26 by itself did primary T cells produce significant amounts of IL-2 in a dose-dependent manner (Fig. 2b). Such an effect correlated with the ability of 24:26 to induce T cell proliferation in a directly proportional way to the amount of IL-2 production (data not shown). In contrast, the addition of 24 and 26 ScFvs or dimeric 24 F(ab')2 failed to induce IL-2 production and T cell proliferation.



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  		FIGURE 2. 24:26 by itself, but not 24 ScFv and 26 ScFv, induces IL-2 production in primary human T cells. PBMC were isolated from normal volunteers and stimulated with PMA and ionomycin for 72 h to induce CTLA-4 expression. At this time, cells were harvested, washed in fresh medium, and rested for 15 h. Expression of CTLA-4 in these cells was verified by flow cytometry (a) (thick line, anti-CTLA-4 stain; thin line, isotype-matched Ab stained), before being stimulated with increasing concentrations of 24:26 or 24 ScFv and 26 ScFv or 24 F(ab')2 against CTLA-4 (b) for 72 h. Supernatants were then collected, and used for measurement of IL-2 production by ELISA.

 
To establish the mechanism of T cell activation by 24:26, we used a well-characterized panel of Jurkat T cells stably transfected with doxycycline-inducible wild-type CTLA-4 or mutant forms of CTLA-4 that lack either the first cytoplasmic tyrosine residue (Y165F CTLA-4) or the cytoplasmic tail (Tailless CTLA-4) (Fig. 3) (3, 18, 19, 20). The Y165F CTLA-4 is expressed on the cell surface at much higher levels than the wild-type CTLA-4 because it cannot be internalized efficiently through AP-2 (24, 25) (Fig. 3a). The tailless CTLA-4 cannot be internalized either, and thus it is also expressed at very high levels on the cell surface (Fig. 3a).



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  		FIGURE 3. The IL-2 response induced by 24:26 is dependent on the level of CTLA-4 expression, and requires the cytoplasmic tail of CTLA-4. a, Expression of CTLA-4 on the surface of Jurkat T cells stably transfected with a doxycycline-inducible wild-type CTLA-4 (WT CTLA-4), the Y165F mutant CTLA-4 (Y165 CTLA-4), or tailless CTLA-4 (Tless CTLA-4) in the absence (thick line) or presence of doxycycline (1 µg/ml; thick, filled profile). Isotype-matched controls are shown in thin line profiles. b, CTLA-4 inhibits T cell activation when coligated with the TCR. IL-2 production of T cells expressing wild-type CTLA-4, Y165F CTLA-4, or tailless CTLA-4 in response to 24 h stimulation with anti-CD3/anti-MHC class I Ab-coated beads or anti-CD3/anti-CTLA-4 Ab-coated beads in the presence of anti-CD28 mAbs. IL-2 in the culture supernatants was measured by ELISA. c, 24:26 binding to CTLA-4 induces IL-2 production. IL-2 production in response to 24:26 in the presence ({blacktriangleup}) or absence of doxycycline ({diamondsuit}), or to cross-linked whole 24 and 26 Abs in the presence ({blacktriangledown}) or absence ({blacksquare}) of doxycycline for wild-type CTLA-4 (left panel), Y165F CTLA-4 (center panel), and tailless CTLA-4 (right panel). d, Production of IL-2 in response to 24:26 is directly proportional to levels of CTLA-4 expression. Y165F CTLA-4-expressing Jurkat T cells were stimulated with 24:26 ({blacksquare}) or 24 ScFv and 26 ScFv ( ) in the presence of increasing concentrations of doxycycline, and IL-2 production measured by ELISA. *, p < 0.05.

 
As expected from previous studies (3, 18, 26, 27), coligation of the TCR with wild-type CTLA-4 or with Y165F CTLA-4, but not with tailless CTLA-4, inhibited IL-2 production by negative signaling (Fig. 3b). In contrast, when the same T cells expressing wild-type CTLA-4 or Y165F CTLA-4 were stimulated with 24:26 by itself, in the absence of APC, they responded with significant IL-2 production (Fig. 3c). The response occurred only when CTLA-4 was expressed on the cell surface (Fig. 3d), ruling out the possibility that 24:26 was acting by binding to a receptor other than CTLA-4. Intact 24 and 26 Abs (soluble or cross-linked with secondary Abs or by beads) or 24 ScFv and 26 ScFv separately did not induce IL-2 production. The activating effect of 24:26 was dependent on the cytoplasmic tail of CTLA-4 because T cells expressing tailless CTLA-4 molecules did not respond to 24:26 (Fig. 3c). It also was directly proportional to the amount of CTLA-4 expressed on the cell surface as demonstrated by the more pronounced effect on the Y165F-CTLA-4 expressing T cells than on the wild-type CTLA-4-expressing T cells (Fig. 3, a and c), and by the titration with doxycycline, the inducer of CTLA-4 expression in these stably transfected Jurkat T cell clones (Fig. 3d). Activation following CTLA-4 engagement with 24:26 was independent of B7.1 and B7.2 as indicated by the fact that Jurkat T cells do not express these CTLA-4 ligands (J.M. and L.A.C., unpublished observations).

T cell activation following 24:26 binding to CTLA-4 requires the expression of functional TCR/CD3, and benefits from CD28 costimulation

The above-mentioned results demonstrate that 24:26 binding to CTLA-4, without concomitant TCR ligation, converts CTLA-4 from an inhibitory receptor into an activating receptor for T cells. Because the T cell response to 24:26 mimicked the response to TCR ligation, we tested whether the expression of a functional TCR/CD3 complex was required for the response to 24:26. To address this issue, we used the Jurkat T cell subclone JRT3 that lacks expression of the {beta}-chain of the TCR complex (28). This defect translates into a lack of expression of TCR heterodimer and of TCR-{zeta}- and CD3 chains on the cell surface (29). For an optimal window of response, the following experiments were performed using Y165F CTLA-4 transfectants. In Y165F CTLA-4-transfected JRT3 cells, we failed to see an IL-2 response to 24:26 (Fig. 4a) despite the fact that these cells expressed similar amounts of CTLA-4 on the cell surface and produced similar amounts of IL-2 in response to mitogens. Therefore, the biological activity of 24:26 requires not only expression of CTLA-4, but also expression of a functional TCR/CD3 complex on the surface of T cells.



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  		FIGURE 4. T cell activation by 24:26 requires expression of a functional TCR complex on the cell membrane and Lck, and is enhanced by CD28-dependent costimulation. a, E6.1 parental Jurkat T cells or their subclones JRT3, which lack TCR-{beta} expression, and JCaM1.6, which lacks functional Lck kinase, were stably transfected for doxycycline-inducible Y165F CTLA-4. Single cell clones of these T cell lines, not expressing or expressing CTLA-4, were examined for their ability to produce IL-2 in response to either PMA and ionomycin or 24:26 (10 µg/ml) or single-chain Abs 24 and 26 after 24 h. Results are representative of three experiments testing three different clones. The levels of surface expression of CTLA-4 on these clones as measured by flow cytometry are shown (thin line, isotype control; thick line, CTLA-4 staining in the absence of doxycycline; black profile, CTLA-4 staining in the presence of doxycycline). The response of these clones to stimulation with PMA and ionomycin in different experiments was: Y165F E6.1 = 984.7 ± 27.2 pg/ml; Y165F JRT3 = 836.4 ± 22.1 pg/ml; and Y165F JCaM1.6 = 181.3 ± 24.8 pg/ml. b, Noninduced and doxycycline-induced Y165F CTLA-4 transfected T cells were stimulated with 24:26 at 1, 10, and 100 µg/ml in the presence of soluble anti-CD28 mAb (5 µg/ml) or with 24:26 at 100 µg/ml without anti-CD28, for 24 h. Supernatants were collected and tested for IL-2 by ELISA. *, p < 0.05. c, IL-2 production in response to anti-CD3 mAb (10 µg/ml) alone or in the presence of 24, 26, 24:26, or a combination of 24 ScFv and 26 ScFv. All the anti-CTLA-4 molecules were used at 20 µg/ml, final concentration.

 
Under physiological conditions, T cell activation and IL-2 production requires concomitant delivery of signals from the TCR/CD3 complex and from costimulatory molecules such as CD28. Thus, we hypothesized that ligation of CD28 would enhance the IL-2 response to 24:26 stimulation by providing costimulation. We observed that simultaneous treatment of CTLA-4-expressing T cells with 24:26 and with an Ab against CD28 decreased the threshold of activation of these cells: similar levels of IL-2 production were achieved with 10x less 24:26 when CD28 was concomitantly ligated (Fig. 4b). This finding shows that 24:26-induced CTLA-4 ligation triggers a TCR-dependent signaling pathway that can be synergized with CD28-dependent costimulation.

Next, we tested whether the signaling cascade initiated by 24:26 through CTLA-4 could be enhanced by simultaneous ligation of the TCR. We found that activation by 24:26 could be synergized by simultaneous ligation of the TCR, leading to enhanced IL-2 production (Fig. 4c). Such an effect was not seen when using 24 ScFv or 26 ScFv in isolation or in combination, nor when an isotype-matched control was used.

CTLA-4 binding to 24:26 triggers a TCR-like signaling pattern

Given the requirement for TCR expression and the effect of costimulation, we hypothesized that 24:26 was triggering a TCR-like signaling cascade. This was tested by examining the proximal events associated with activation of T cells through the TCR. These include tyrosine phosphorylation of the TCR-{zeta} immune receptor tyrosine-based activation motifs, recruitment and phosphorylation of the ZAP-70 kinase, phosphorylation of the transmembrane linker for activation of T cells (LAT) and of PLC-{gamma}1, activation of ERK-1/-2 kinases, and calcium mobilization (30). In CTLA-4-expressing T cells, we observed that 24:26 by itself induced a biochemical cascade of events that was qualitatively similar to that triggered by TCR ligation, including assembly of tyrosine phosphorylated TCR-{zeta}, ZAP-70, and LAT complexes (Fig. 5, a and b), tyrosine phosphorylation of PLC-{gamma}1 (Fig. 5c), net calcium mobilization (Fig. 5d), and activation of ERK-1/-2 (Fig. 5e). Furthermore, these events were not secondary to CTLA-4 aggregation because cross-linking 24 and 26 Abs against CTLA-4 did not induce phosphorylation of TCR-{zeta} and its association with ZAP-70 (Fig. 5b). These results explain the requirement for TCR expression for 24:26-induced activation because expression of TCR-{beta} is critical for TCR-{zeta} expression on the T cell surface (29). Therefore, we concluded that binding of 24:26 to CTLA-4 triggers a TCR-dependent signaling pathway that is qualitatively similar to that seen following TCR ligation with agonist ligands.



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  		FIGURE 5. 24:26 engagement with CTLA-4 triggers a TCR-like signaling cascade. a, Y165F CTLA-4 expressing T cells were stimulated with OKT3 or 24:26 (10 µg/ml) for 10 min. Whole cell lysates were prepared and used for immunoprecipitation with Abs against ZAP-70 and immunoblotting for phosphotyrosine. The identity of the bands was confirmed by direct re-blotting for indicated molecules. Chemiluminescence signal was quantified using Phoretix 1D software (NonLinear Dynamics). b, ZAP-70 immunoprecipitates from Y165F CTLA-4-expressing Jurkat T cells left unstimulated or stimulated for 10 min with either OKT3, 24:26, or with 24 and 26 Abs cross-linked with goat-anti-mouse Abs (X-24+26), were immunoblotted with an anti-phosphotyrosine Ab to assess association with phospho-TCR-{zeta}. Identity of this band was confirmed by TCR-{zeta} blotting. Similar levels of ZAP-70 were loaded in each lane as confirmed by ZAP-70 blotting. c, PLC-{gamma}1 immunoprecipitates from Y165F CTLA-4-expressing and nonexpressing T cell transfectants stimulated with increasing concentrations of 24:26 and immunoblotted for phosphotyrosine. Equal protein loading was confirmed by subsequent immunoblotting for PLC-{gamma}1. d, Time course of calcium mobilization in Y165F CTLA-4 T cell transfectants in response to OKT3 (•), 24:26 ({blacksquare}), 24 and 26 ({diamondsuit}), 24 ScFv ({blacktriangleup}), and nonstimulated cells ({circ}), as measured by FLUO-3 mean fluorescence intensity. e, Whole cell lysates from Y165F CTLA-4-expressing and -nonexpressing T cell transfectants stimulated with increasing concentrations of 24:26 were immunoblotted for dually phosphorylated, active ERK-1/-2 (pERKs), and for total ERK.

 
T cell activation by 24:26 follows increased association of PP2A to CTLA-4, and activation of Lck

How can 24:26 binding to CTLA-4 induce TCR-like signaling and T cell activation? We and others (19, 31) have recently reported that the function of CTLA-4 and CD28 might be regulated by PP2A. Specifically, we have shown that the regulatory subunit of PP2A (PP2AA) binds to the juxtamembrane lysine-rich (152KMLKKR157) sequence in the cytoplasmic domain of CTLA-4 (19), and the catalytic subunit (PP2AC) binds to the 165YVKM168 region of that tail (31). Together, they block the inhibitory function of CTLA-4. The interaction between PP2AA or PP2AC and CTLA-4 reflects association of the whole enzyme to CTLA-4 because individual regulatory units of PP2A are not found by themselves but always associated with the catalytic subunit of PP2A (32, 33). Coligation of the TCR with CTLA-4 correlates with dissociation of PP2A from CTLA-4 and down-regulation of T cell responses (19). Because PP2A regulates cell activation and proliferation through many different signaling pathways (32), and has also been implicated in the induction of IL-2 gene expression in activated T cells (34, 35), we hypothesized that 24:26 activates T cells by increasing the association of PP2A to CTLA-4. To test this hypothesis, we immunoprecipitated PP2AA from T cells expressing wild-type CTLA-4 or Y165F CTLA-4 undergoing TCR-CTLA-4 coligation or only CTLA-4 ligation with 24:26, respectively, and immunoblotted these precipitates for CTLA-4. As previously reported, we found that TCR coligation with wild-type CTLA-4 induced dissociation of CTLA-4 from PP2A. In Y165F CTLA-4 expressing T cells, the decreased association upon TCR-CTLA-4 coligation was not as apparent, likely due to the very high levels of expression of this form of CTLA-4 (data not shown). However, in these cells, 24:26 induced an increase in the association of PP2A to CTLA-4, apparent after 10 min of stimulation and further increasing by 60 min (Fig. 6a). Such an association was not seen when CTLA-4 lacked the cytoplasmic tail (data not shown).



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  		FIGURE 6. The induction of T cell activation and IL-2 production by 24:26 correlates with increased association of PP2A to CTLA-4 and activation of CTLA-4-associated Lck, and is blocked by okadaic acid. a, Whole cell lysates from Jurkat T cells stably transfected for wild-type CTLA-4 and stimulated with SEE and APC, or Y165F CTLA-4 and stimulated with 24:26 for the indicated times were used for immunoprecipitation of PP2AA and sequential immunoblotting for CTLA-4, PP2AA, and PP2AC. b, CTLA-4 molecules that cannot bind PP2A (K-less) do not respond to 24:26. Y165F CTLA-4-expressing Jurkat T cells or K-less CTLA-4-expressing T cell transfectants were stimulated with 24:26 for 24 h, and IL-2 accumulation in culture supernatants was measured by ELISA. c, Y165F CTLA-4-expressing Jurkat T cells were stimulated with 24:26 and increasing concentrations of okadaic acid (a selective inhibitor of PP2A) or with wortmannin (an inhibitor of phosphatidylinositol 3-OH kinase) for 24 h, and IL-2 production measured by ELISA. In addition, noninduced and doxycycline-induced Y165F CTLA-4 T cell transfectants were stimulated with APC and SEE in the presence or absence of okadaic acid (10–2 µM) for 24 h and IL-2 production measured by ELISA. d, CTLA-4-associated Lck is activated by 24:26. CTLA-4 immunoprecipitates from nonstimulated or 24:26-stimulated (10 min) CTLA-4-expressing T cells were immunoblotted for Lck and re-blotted for CTLA-4.

 
To test whether the increase in PP2A association to CTLA-4 was functionally relevant, we first examined whether T cells expressing mutant CTLA-4 molecules that cannot interact with PP2A responded to 24:26. We have previously reported that PP2A interaction with CTLA-4 requires the three lysine residues (152, 154, and 155) on the juxtamembrane portion of the cytoplasmic tail of CTLA-4 (19). When we used T cells expressing CTLA-4 molecules with these three lysine residues mutated to alanine residues (K-less CTLA-4), we failed to see any response to 24:26 (Fig. 6b). Next, we studied whether the activity of PP2A was required for T cell activation by 24:26, using okadaic acid, a PP2A-selective inhibitor (36). As a negative control, we used wortmannin, an inhibitor of phosphatidylinositol 3-OH kinase, another signaling molecule that interacts with the cytoplasmic tail of CTLA-4. We found that okadaic acid inhibited the IL-2 response induced by 24:26 (90% at 10–3 µM) while wortmannin had no effect (Fig. 6c). In addition, okadaic acid at 10x higher concentration (10–2 µM) had no effect on the inhibition of IL-2 production upon TCR-CTLA-4 coligation (Fig. 6c). These findings indicate that T cell activation triggered by CTLA-4 ligation with 24:26 is dependent on the phosphatase activity of PP2A.

PP2A can interact with several protein tyrosine kinases, including Lck, in an interplay causing bidirectional positive and negative regulation of each other’s enzymatic activity (37, 38, 39, 40, 41). Specifically, Lck can tyrosine-phosphorylate and inactivate the catalytic subunit of PP2A (42), and this is dramatically enhanced by okadaic acid, implying that PP2A itself may act as a tyrosine phosphatase on itself and perhaps on other substrates. In addition, tyrosine phosphorylation of the catalytic subunit of PP2A has been detected in a small PP2A fraction from activated T cells (33). Because CTLA-4 and Lck can interact with each other, at least in in vitro cotransfection epithelial cell systems (43), and because the signaling events induced by 24:26 upon binding to CTLA-4 mimicked the lck-dependent events triggered by TCR signaling (44), we hypothesized that the increased association of PP2A with CTLA-4 induced by 24:26 might result in lck activation.

To test this hypothesis, we first examined the status of CTLA-4-associated Lck under basal conditions and upon binding to 24:26. As shown in Fig. 6d, we documented a low but consistent level of Lck association to CTLA-4. More importantly, we observed that 24:26 induced the appearance of significant amounts of p59lck, a serine-phosphorylated form of Lck that reflects its activation (45). To further support the involvement of Lck on 24:26-induced T cell activation, we examined the effect of 24:26 in CTLA-4-expressing, Lck-deficient Jurkat T cells (JCaM1.6 cells). In these cells, the level of Y165F CTLA-4 expression upon doxycycline induction was similar to that seen in CTLA-4-expressing parental Jurkat T cells (Fig. 4a). However, 24:26 failed to induce any detectable IL-2 production by these cells, demonstrating that Lck is required for the biological effect of 24:26 (Fig. 4a).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that bispecific tandem ScFv against human CTLA-4 can have an unexpected biological effect by revealing the plasticity for signaling through the cytoplasmic domain of this surface receptor. On one hand, when CTLA-4 binds B7.1 or B7.2 in the context of coligation with the TCR, it transduces a signal that inhibits T cell activation (reviewed in Refs. 1 , 2 , and 4, 5, 6). On the other hand, we show in this study that binding of a bispecific tandem ScFv ligand of CTLA-4 causes the same cytoplasmic domain to transduce a signal that, by itself, activates the T cell without the need for coligation of the TCR. Although triggered by different ligands that bind to different sites on CTLA-4, both responses involve PP2A. The inhibitory function of CTLA-4 correlates with a decrease in its association with PP2A (19), while the activating function of CTLA-4 correlates with an increase in its association with PP2A.

Activation of T cells by 24:26 is not the result of functional blockade of the B7-CTLA-4 inhibitory interaction for two reasons. One is that such a mechanism does not induce T cell activation on its own, but requires concomitant TCR ligation (6). The other reason is that the effect of 24:26 occurs in APC-free cultures of T cells that do not express B7. It is also unlikely that 24:26-induced T cell activation results from nonspecific aggregation of lipid rafts and transphosphorylation of Src kinases (46, 47, 48), as one would expect to see a similar outcome upon cross-linking of CTLA-4 with B7 or whole Abs given that surface CTLA-4 is mostly within lipid rafts (20, 49). But this is not the case (Fig. 5b). Furthermore, preliminary evidence indicates that GPI-anchored CTLA-4, which is expressed almost exclusively within lipid rafts at high levels, does not induce T cell activation to the same degree (data not shown). Therefore, we concluded that 24:26-induced T cell activation is the result of differential CTLA-4-mediated signaling.

The biological effects of 24:26 on CTLA-4 reveal the capacity of this receptor to assemble two different signalosomes on its cytoplasmic tail, one inhibitory and the other activating, in response to two different types of ligands. This plasticity is inherent to the cytoplasmic tail of CTLA-4 rather than artificially induced by cytoplasmic domain swapping with activating receptors such as CD28 (50). PP2A is the key regulator of such plasticity either by direct activation of the CTLA-4-associated pool of Lck or by sequestration of an inhibitor of Lck. Whether the role of PP2A is linked to a "tonic off-signal" on the T cell is currently unknown. However, preliminary data indicate that such an off-signal, if it exists, is not dependent on TCR-mediated recognition of peptide:MHC complexes because Jurkat T cells do not express class II MHC molecules and blockade of TCR:class I MHC interactions did not prevent the activation by 24:26 (data not shown).

Based on the data presented in this study, we propose that, on the T cell surface, nonengaged CTLA-4 exists in two different forms with distinct signaling properties. One of these has its cytoplasmic tail not accessible to PP2A, likely because of folding of the cytoplasmic tail on the inner leaflet of the cell membrane as suggested for TCR-{zeta} (51). The other has its cytoplasmic tail accessible to PP2A. In both states, CTLA-4 is inactive. When CTLA-4 is coligated with the TCR, the CTLA-4-bound PP2A pool is phosphorylated and dissociates from CTLA-4, making this molecule an inhibitory receptor (19, 31). In contrast, binding of 24:26 to CTLA-4 increases the accessibility and binding of PP2A to CTLA-4, and the initiation of an Lck-dependent signaling cascade similar to that triggered by TCR ligation.

The precise effects of 24:26 on the structure of CTLA-4 are unknown. We think that activation by 24:26 requires simultaneous binding to both the M10 and M11 epitopes on CTLA-4 because dimeric 24 F(ab')2 did not induce a similar effect. This is further suggested by preliminary data showing that very high concentrations of unlinked 24 ScFv and 26 ScFv are able to induce low and transient calcium mobilization and low amounts of IL-2 production (data not shown). We cannot rule out that 24:26 induces the formation of an architecturally unique aggregate of CTLA-4 dimers (e.g., tetramers) that favors recruitment of PP2A and activation of Lck. The inability of the combination of whole 24 and 26 Abs to induce T cell activation may then be the result of steric hindrance. A similar model may explain the capacity of some Abs against CD28 to induce T cell activation on their own (52), although in that case the response to ligand binding is the same as that triggered by the natural ligand. Of interest, these superagonist Abs also target a loop of CD28 that is adjacent to the B7-binding site, and also need expression of TCR to induce T cell activation (53).

Our data point toward a novel paradigm by which two different ligands that bind to the same receptor trigger the formation of different signalosomes that cause different downstream responses. This paradigm is clearly different from the well-established "one ligand-two receptors" scenario (e.g., TNF-{alpha} acting on TNFR type I or TNFR type II to induce cell death or proliferation, respectively), but is reminiscent of the agonist trafficking of receptor signal model recently proposed to explain the capacity of G protein-coupled receptors to undergo a conformational change in response to different ligands and assemble different signaling subunits of the trimeric G protein (54, 55, 56). We are currently characterizing the profile of response to 24:26, including the effect on production of cytokines other than IL-2.

Our results raise the question about the existence of an endogenous ligand for CTLA-4 that mimics 24:26 and that can activate T cells. Initial in vivo data using Abs against CTLA-4 had suggested that CTLA-4 ligation played a costimulatory role on T cell activation (57, 58). Although blockade of CTLA-4 function may have been operational in these studies, there is no conclusive evidence ruling out other mechanisms. The data reported in this study renew the interest for the search of novel B7 family members with potential capacity to costimulate T cells through CTLA-4.

The biological response induced by 24:26 may be of clinical interest in conditions requiring a boost in immunity. For example, some of the difficulties encountered in effective vaccine development for disease prevention (59, 60, 61) or cancer immunotherapy (62, 63, 64) have been linked to CTLA-4-mediated regulation of T cell responsiveness. By targeting activated T cells that express CTLA-4, 24:26 may enhance the responsiveness to the priming Ag, and thus increase the efficacy of vaccine preparations.


   Acknowledgments
 
We thank Drs. Maria Luisa Alegre, Jeff Bluestone, Stephen Ferguson, David Margulies, and Ron Wange for critical reading of the manuscript and helpful suggestions, Dr. Dave Erbe for the generation of Ab epitope mutants, and the members of the Madrenas laboratory for helpful comments and criticisms.


   Footnotes
 
1 This work was supported by grants from the Canadian Institutes of Health Research and the Kidney Foundation of Canada. J.M. holds a Canada Research Chair in Transplantation and Immunobiology. Back

2 Address correspondence and reprint requests to Dr. Joaquín Madrenas, Robarts Research Institute, P.O. Box 5015, 100 Perth Drive, London ON N6A 5K8, Canada. E-mail address: madrenas{at}robarts.ca Back

3 Current address: Compound Therapeutics, 1365 Main Street, Waltham, MA 02451. Back

4 Abbreviations used in this paper: ScFv, single-chain variable region fragment; ERK, extracellular signal-regulated kinase; LAT, linker for activation of T cells; PP2A, serine/threonine phosphatase 2A; PP2AA, regulatory subunit of PP2A; PP2AC, catalytic subunit of PP2A; PLC-{gamma}1, phospholipase C{gamma}1; SEE, Staphylococcus enterotoxin E; ZAP-70, {zeta}-associated protein of 70 kDa. Back

Received for publication November 11, 2003. Accepted for publication March 10, 2004.


   References
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 Results
 Discussion
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