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(Journal of Leukocyte Biology. 2003;73:178-182.)
© 2003 by Society for Leukocyte Biology

Short cytoplasmic SDYMNM segment of CD28 is sufficient to convert CTLA-4 to a positive signaling receptor

Li Yin*,{dagger}, Helga Schneider*,{dagger},{ddagger} and Christopher E. Rudd*,{ddagger},§

* Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Departments of
{dagger} Medicine and
§ Pathology, Harvard Medical School, Boston, Massachusetts; and
{ddagger} Department of Haematology, Imperial College of Science, Technology and Medicine, London, United Kingdom

Correspondence: Christopher E. Rudd, Imperial College of Science, Technology and Medicine, Department of Haematolgy, Division of Investigative Science, Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 ONN, UK. E-mail: c.rudd{at}ic.ac.uk


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ABSTRACT
 
CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 are key coreceptors on the surface of T cells that have opposing effects on T cell activation. Although CD28 enhances proliferation, CTLA-4 markedly inhibits the activation process. These opposing roles are particularly surprising given the structural similarity of the cytoplasmic residues of the two receptors. These include the related CD28SDYMNM and CTLA-4GVYVKM motifs. In this study, we have directly addressed whether these related motifs may play different roles in the activation process by swapping the CTLA-4GVYVKM motif with the CD28SDYMNM motif. Remarkably, stable transfectants of the T cell hybridoma DC27.10 showed that substitution of CTLA-4GVYVKM was sufficient to convert CTLA-4 from a negative signaling coreceptor to a positive CD28-like coreceptor. CD28SDYMNM is therefore sufficient to convey positive signals within CTLA-4. These results demonstrate that CD28SDYMNM and CTLA-4GVYVKM motifs contain sufficient information to distinguish positive versus negative coreceptor signaling in T cells.

Key Words: coreceptor • phosphatidylinositol 3-kinase


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INTRODUCTION
 
At least two signals from antigen-presenting cells are required for optimal activation of T cells as mediated by coreceptors CD28 and inducible costimulatory receptor (ICOS) [1 , 2 ]. CD28 is expressed on naive and activated T cells, and cytotoxic T-lymphocyte antigen (CTLA)-4 is expressed solely as a consequence of activation [3 ]. CD28 and CTLA-4 have opposing effects, with CD28 providing positive signals and CTLA-4 acting as a potent, negative regulator of the response [3 ]. CD28 augments proliferation and can also rescue cells from apoptosis [4 ]. Despite the importance of these cosignals, the signaling mechanisms responsible for these opposing effects are unclear. CD28 has a phosphotyrosine-based motif pYMNM, which can bind the Src homology (SH)2 domains of phoshatidylinositol 3-kinase (PI3-K), growth factor receptor-bound protein-2 (Grb-2)/Grb-2 related adaptor downstream of Shc (GADS), T cell-specific protein tyrosine kinase (PTK; IL-2 inducible kinase, ITK), and phosphatase PP2A [5 6 7 8 9 10 ]. PtdIns 3-kinase is a heterodimer, consisting of an adaptor subunit (p85) with two SH2 domains, coupled to a p110 catalytic subunit, and is important to signaling by PTK receptors. By contrast, CTLA-4 has a YVKM motif that can bind to PI3-K, phosphatases SHP-2, and PP2A, as well as adaptor complexes AP-1 and AP-2 [10 11 12 ]. Mutations that disrupt the YMNM motif, or selectively disrupt PI3-K binding, attenuate signaling [7 , 13 14 15 ], while the same mutants behave normally in Jurkat cells [16 , 17 ]. This discrepancy may now be explained by the finding that Jurkat cells are defective in phosphoinositide phosphatase (PTEN), an enzyme that removes the phosphate moiety on the third position of the inositol ring [18 ]. High constitutive PtdIns 3,4-P2 and PtdIns 3,4,5-P3 expression can potentially circumvent the need for additional PI3-K. Further, the reliability of the hybridoma system has been confirmed in in vivo models documenting the importance of the YMNM motif in CD28 function [19 20 21 ]. In vivo reconstitution studies of YMNM mutants in mice have confirmed the central importance of the motif in CD28-mediated graft-versus-host responses [19 , 20 ] and in the induction of BcL-XL [21 ]. The major downstream target of PI3-K, AKT, or protein kinase B has recently been implicated in CD28 regulation of interleukin (IL)-2 but not of T helper cell type 2 cytokines [22 ]. In addition to the regulation of IL-2 expression, CD28 associated PI3-K has been implicated in other functions such as Bcl-XL expression ß-1 integrin adhesion and CD28 receptor endocytosis [23 ]. The opposing roles of CD28 and CTLA-4 are particularly surprising considering the structural similarity of the cytoplasmically related CD28YMNM and CTLA-4YVKM motifs. Besides slight differences within the YxxM motif, the coreceptors have different, adjacent N-terminal residues as well (i.e., GVYVKM vs. SDYMNM). In this study, we have addressed whether these related motifs may be sufficient to account for the opposing functions of the two receptors by substituting the CTLA-4GVYVKM motif with the analogous SDYMNM motif of CD28. Remarkably, this substitution converted CTLA-4 from a negative to a positive cosignaling receptor. The results demonstrate that a restricted cytoplasmic region within the two receptors accounts for positive versus negative coreceptor signaling in T cells.


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MATERIALS AND METHODS
 
Cells, reagents, and antibodies
The murine T cell hybridoma DC27.10 (a kind gift of Dr. Rose Zamoyska, MRC, London) was cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS; Sigma Chemical Co., St. Louis, MO) and 50 µM 2-mercaptoethanol, L-glutamine, and penicillin/streptomycin. DC 27.10 cells were stably transfected with pSR{alpha} vector, wild-type human CD28, wild-type human CTLA-4, and human CTLA-4 chimera (SDYMNM). Chinese hamster ovary (CHO) cells stably transfected with FcR or FcR-CD80 were grown in Dulbecco’s modified Eagle’s medium with 5% FBS. G418 was purchased from Life Technologies (Grand Island, NY). Anti-human CD28 monoclonal antibody (mAb; 4B10) was from Coulter (Hialeah, FL), and anti-human CTLA-4 mAb was from PharMingen (San Diego, CA). Anti-murine CD3 (145–2C11) was purchased from American Type Culture Collection (Manassas, VA). Anti-p85 Ab was kindly provided by Dr. Morris White (Joslin Diabetes Center, Boston, MA).

Generation of CTLA-4 chimeric transfectants
Human CTLA-4 chimera was achieved by Altered Sites II in vitro mutagenesis system of Promega (Madison, WI). The CTLA-4 cytoplasmic motif GVYVKM was replaced by the human CD28 cytoplasmic motif SDYMNM. The CTLA-4 chimera was generated by using oligonucleotides carrying the CD28 motif SDYMNM. The product was verified by dideoxynucleotide sequencing.

The CTLA-4 chimera was resubcloned into the pSR{alpha} expression vector [24 ]. Stable transfections were generated using DC27.10 cells transfected with CTLA-4 chimera plasmid together with pSVneo containing a neomycin-resistance gene. Electroporation was conducted at 260 V and 960 µF. Cells were selected with 1.5 mg/ml G418 for 2 weeks, and cells from different populations were assayed for human CD28 or human CTLA-4 expression by fluorescence-activated cell sorter (FACS).

Flow cytometry
Cytometric analysis was performed as described [6 ]. Briefly, 5 x 105 cells were stained with anti-CD28 or anti-CTLA-4 mAb for 1 h on ice, washed twice, and then incubated with secondary fluorescein isothiocyanate (FITC)-labeled antibodies under the same conditions. Cytometric analysis was conducted using FACScan (Becton Dickinson, Mountain View, CA).

IL-2 assay
CHO cells expressing FcR or FcR-CH0-CD80 were plated at 5 x 103/well in 96-well plates and cultured overnight. Cells were washed twice and incubated with DC27.10 cells stably transfected with vector, wild-type human CD28, wild-type CTLA-4, and human CTLA-4 chimera at 1 x 105/well together with anti-CD3 mAb (2C11) at indicated concentrations. The final volume was 200 µl/well. Each condition was assayed as a triplicate. Cells were then cultured for 24 h before the supernatants were collected for IL-2 measurement.

CTLL-2 cells grown in the presence of recombinant human IL-2 were washed and plated at 2500 cells/well in a 96-well plate. A series of twofold-diluted supernatants was added to CTLL-2 cells. Cells were cultured for 24 h followed by an 18-h pulsing with 1 µCi [3H]thymidine before harvesting and counting. The amount of IL-2 in these supernatants was determined by comparing with the standard curve established with IL-2.

Immunoprecipitation and immunoblotting
For immunoprecipitation, 80 x 106 cells were lysed in ice-cold lysis buffer containing 1% Triton X-100 in 20 mM Tris-HCl, pH 8.3, 150 mM NaCl. The lysis buffer contained protease and phosphatase inhibitors. Postnuclear lysates were incubated for 1 h with the indicated antibody. Protein A-sepharose beads (30 µl; Pharmacia, Upspsala, Sweden) were added and incubated for 1 h at 4°C. The eluted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose for immunoblotting. The membranes were blocked with 5% milk in Tris-buffered saline (10 mM Tris-HCl, pH 7.6, 150 mM NaCl) and incubated with the indicated antibody. Bound antibody was revealed with the appropriate secondary antibody, and protein was visualized by enhanced chemiluminescence (Amersham, Little Chalfont, UK).


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RESULTS
 
CD28 and CTLA-4 share an ability to bind PI3-K in T cells [6 7 ]. They possess related SDYMNM and GVYVKM motifs, which can bind the SH2 domains of the p85 subunit [5 , 6 ]. These motifs share a methionine in the plus-3 position but differ in several adjacent residues. A major question is whether these motifs determine the opposing signals sent by the two receptors. To address this, a chimeric form of CTLA-4 was generated in which the CTLA-4GVYVKM cytoplasmic motif was substituted with its CD28 counterpart (i.e., CD28SDYMNM; Fig. 1A ). Stable transfectants were then generated that express wild-type human CD28, wild-type human CTLA-4, or the human CTLA-4SDYMNM chimera (Fig. 1B) . Clones were selected that express equivalent levels of CD28, CTLA-4, or CTLA-4 chimeric receptor on the surface of cells as determined by FACS (Fig. 1B , left and middle panels). As an additional control, the level of expression of endogenous mouse CD3 was found to be similar for each clone (Fig. 1B , right panel). Wild-type CTLA-4 and CTLA-4 chimera were negative for human CD28, and wild-type CD28 was negative for human CTLA-4 (i.e., identical pattern to negative control; data not shown).



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  		Figure 1. Depiction of the CD28/CTLA-4 wild-type and CTLA-4 chimeric construct. (A) CD28 WT, CTLA-4 WT, and CTLA-4 chimera are illustrated. Human CTLA-4 with the cytoplasmic GVYVKM motif exchanged for the CD28SDYMNM motif (CTLA-4SDYMNM chimera). (B, left and middle panel) FACS profile of the expression of the various constructs. The T cell hybridoma DC27.10 was transfected with vector, wild-type human CD28 WT, human CTLA-4 WT, and CTLA-4 chimera as outlined in Materials and Methods. Vector, CD28 WT, CTLA-4 WT, and CTLA-4 chimera (clone #5)-transfected cells are shown. The negative control shows the profile of secondary antibody alone against vector-transfected cells. (B, right panel) FACS profile of the TcR/CD3 expression on the various transfectants. Expression levels of endogenous CD3 were analyzed by flow cytometry using mAb 2C11. The control corresponds to cell staining with secondary Ab alone (FITC-conjugated goat anti-hamster mAb).

CD28 and CTLA-4 bind to PI3-K [5 6 7 , 11 ]. To assess whether the exchange of the GVYVKM/SDYMNM motifs altered PtdIns 3-kinase binding, the CTLA-4 chimera (clone #5) was precipitated with anti-CTLA-4 and assessed for the presence of the p85 subunit by immunoblotting. As controls, CD28 and wild-type CTLA-4 were also assessed for the presence of coprecipitated p85. Under these conditions, the CTLA-4 chimera was found to precipitate p85 (Fig. 2 , lane 5) at a level comparable with wild-type CTLA-4 (lane 4). As an additional positive control, anti-CD28 was also found to precipitate p85 (lane 2). Over several experiments, no consistent difference was noted in the level of p85 coprecipitated by CD28WT, CTLA-4WT, and the CTLA-4 chimera (data not shown). These findings show that the CTLA-4SDYMNM chimera retained the ability to bind to PI3-K in T cells.



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  		Figure 2. CTLA-4 chimera associates with PI3-K. Equal amount of lysates from DC27.10 cells stably transfected with vector, human CD28 WT, human CTLA-4 WT, and human CTLA-4 chimera were lysed and immunoprecipitated with anti-CD28 (lanes 1 and 2) or anti-CTLA-4 (lanes 3–5) mAbs. The precipitates were subjected to immunoblotting with anti-p85 antiserum. Whole-cell lysates served as a positive control (lane 6). Lane 1, Vector; lane 2, CD28 WT; lane 3, vector; lane 4, CTLA-4 WT; lane 5, CTLA-4 chimera; lane 6, cell lysate.

CD28 is well established in its ability to up-regulate IL-2 production, and CTLA-4 inhibits this response [3 , 25 ]. Mutation of tyrosine or methionine residues in the pYMNM motif can attenuate anti-CD28-induced IL-2 production [6 , 7 , 13 ]. These findings have been confirmed in in vivo reconstitution studies [19 , 20 ]. However, none of the above studies addressed whether the SDYMNM motif is itself sufficient to distinguish between the positive and negative function mediated by CD28 and CTLA-4, respectively. Therefore, we next assessed whether the CTLA-4 chimera could support positive or negative responses to anti-CD3 plus CHO-FcR-CD80. Initially, a titration of anti-CD3 was conducted from 0 to 1.65 to 16.5 ng/ml (Fig. 3A ). Although 16.5 ng/ml anti-CD3 was able to induce IL-2 secretion, this was not observed at 1.65 ng/ml. However, the combination of anti-CD3 plus CHO-CD80 induced a dose-response shift so that IL-2 production was now observed at 1.65 ng/ml. By contrast, the combination of anti-CD3 plus anti-CTLA-4 failed to induce IL-2 production and instead, inhibited anti-CD3-induced IL-2 at 16.5 ng, as described by others [3]. Significantly, by contrast with wild-type CTLA-4, the CTLA-4SDYMNM chimera no longer inhibited anti-CD3-induced IL-2 production and instead, potentiated the anti-CD3 response fivefold (Fig. 3A) . The potentiation of IL-2 production by the CTLA-4SDYMNM chimera was actually comparable with that achieved with coligation of wild-type CD28. This effect was observed in at least five experiments. To control for clonal variability, we also included results from two other independently derived CTLA-4 transfectants (clones #8 and #10; Fig. 3B ). As observed with clones #5 and #9, the CTLA-4SDYMNM chimera induced positive costimulation in response to anti-CD3 plus CHO-FcR-CD80 cells. Clones 5, 8, 9, and 10 were chosen based on their comparable CTLA-4 expression levels and binding to PI3-K. Collectively, these results therefore make the important point that substitution of the CTLA-4GVYVKM motif with the CD28SDYMNM motif converts CTLA-4 from a negative to a positive signaling receptor, comparable with CD28. The YxxM motifs in both receptors therefore play central roles in defining negative and positive signaling by the two coreceptors.



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  		Figure 3. The YMNM motif of CD28 cytoplasmic tail is sufficient for IL-2 production. IL-2 production in DC.27.10 cells, which had been transfected with vector, human CD28 WT, human CTLA-4 WT, or human CTLA-4 chimera, was analyzed by incubation with anti-CD3 or anti-CD3 plus CHO-FcR-CD80 cells. Cells were cultured for 24 h in the presence of anti-CD3 mAb (2C11) alone or together with CHO cells stably transfected with FcR or FcR-CD80. Supernatants were harvested and analyzed for IL-2 in triplicates. (A) Clones #5 and # 9; (B) clones #8 and #10.


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DISCUSSION
 
In summary, our findings demonstrate for the first time that the exchange of the CTLA-4GVYVKM motif for the CD28SDYMNM motif is sufficient to convert CTLA-4 to a positive, costimulatory antigen. Hence, a surprisingly limited number of amino acid residues in CD28 and CTLA-4 are sufficient to determine positive versus negative signaling. The effect was so dramatic that the CD28SDYMNM motif converted CTLA-4 to a receptor that signals with the same potency as its wild-type CD28 counterpart (Fig. 3) . Further, the loss of the CTLA-4GVYVKM motif therefore eliminated the ability of CTLA-4 to generate negative signals. The reliability of our system has been previously documented in in vivo models that confirmed the importance of the YMNM motif in CD28 function [19 20 21 ]. An important remaining question concerns the nature of the signaling component(s) that can account for the function linked to this motif. PI3-K is likely to be required for positive signaling by producing D-3 lipids for pleckstrin homology domain recruitment to the cell surface, as previously reported [7 , 13 14 15 , 19 20 21 ]. However, in comparing different clones, we were not able to establish a clear relationship between level of PI3-K binding to different clones (i.e., with CD28 or the CTLA-4 chimera) and function. The only clear rule was that the presence of PI3-K with the cytoplasmic tail was correlated with the ability of CD28 or the CTLA-4 chimera to generate positive signals. One explanation is that there may be a threshold where a certain level of PI3-K is needed, and additional levels are redundant. The loss of negative signaling with the substitution of the CD28 motif indicates that negative signaling also maps to this region [7, 13]. Although the GVYVKM and SDYMNM motifs possess conserved tyrosine and methionine residues (in the plus-3 position) needed for PtdIns 3-kinase binding, they differ in the adjacent N-terminal GV and SD residues, as well as in the VK and MN residues in the plus-1 and -2 positions. Significantly, we previously showed that the CTLA-4SDYMNM chimera can no longer bind to the AP-1 and AP-2 tetrameric adaptor proteins [26 ]. AP-2 plays a central role in modulating the rate of CTLA-4 down-regulation from the cell surface and as such, may alter the surface topography of surface receptors needed for efficient signaling [27 ]. Indeed, the CTLA-4 chimera showed rates of receptor down-modulation similar to that observed for CD28 (data not shown). Residues other than the tyrosine within the GVYVKM-to-SDYMNM motifs could therefore contribute to these differences. Further studies will be needed to determine which of these individual residues are needed for the conversion from negative to positive signaling.

Received July 22, 2002; revised September 10, 2002; accepted September 24, 2002.


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