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Proc Natl Acad Sci U S A. Sep 6, 2005; 102(36): 12861–12866.
Published online Aug 26, 2005. doi:  10.1073/pnas.0505802102
PMCID: PMC1192824
Immunology

CTLA-4 up-regulation of lymphocyte function-associated antigen 1 adhesion and clustering as an alternate basis for coreceptor function

Abstract

Although cytotoxic T lymphocyte antigen-4 (CTLA-4) negatively regulates T cell activation, the full range of functions mediated by this coreceptor has yet to be established. In this study, we report the surprising finding that CTLA-4 engagement by soluble antibody or CD80 potently up-regulates lymphocyte function-associated antigen 1 (LFA-1) adhesion to intercellular adhesion molecule-1 (ICAM-1) and receptor clustering concurrent with IL-2 inhibition. This effect was also observed with CTLA-4 ligation and not with other coreceptors. T cell antigen receptor (TcR)-induced lymphocyte function-associated antigen 1 function was also dependent on CTLA-4 expression as observed with reduced adhesion/clustering on CTLA-4-/- primary T cells. CTLA-4 up-regulated adhesion was mediated by regulator for cell adhesion and polarization type 1 (Rap-1) as shown by anti-CTLA-4-induced Rap-1 activation as well as Rap-1-N17 blockade and Rap-1-V12 mimicry of adhesion/clustering. Our findings identify a potent role for CTLA-4 in directing integrin adhesion and provide an alternate mechanism to account for aspects of CTLA-4 function in T cell immunity.

Keywords: integrin adhesion, regulator for cell adhesion and polarization type 1

Cytotoxic T lymphocyte antigen-4 (CTLA-4) negatively modulates T cell proliferation and cytokine production (1). CTLA-4-deficient (CTLA-4-/-) mice show an autoimmune phenotype with organ destruction (2, 3). However, the reported mechanisms include ectodomain competition for CD28 binding to CD80/86 (4), disruption of CD28 localization at the immunological synapse (5), modulation of phosphatases protein phosphatase 2A (PP2A) and Src homology 2 (SHP-2) (6-10), and interference with lipid raft expression (11-14). CTLA-4 engagement of CD80/86 on dendritic cells can also induce the release of indoleamine 2,3-dioxygenase (IDO) (15, 16), whereas CD4+CD25+ regulatory T cells modulate disease in CTLA-4-/- mice (17-20).

Of the multiple integrins on T cells, CD11a/CD18 or lymphocyte function-associated antigen 1 (LFA-1) binds to intercellular adhesion molecule-1 (ICAM-1) and -2 (21, 22). LFA-1 becomes activated by two mechanisms: an increase in affinity due to conformational changes and an increase in avidity due to clustering (23). Studies have focused on antigen-receptor (TcR/CD3 complex) and chemokines in the generation of inside-out signals needed for LFA-1 activation. Active LFA-1 is needed for the antigen presentation by antigen-presenting cells (APCs) and the migration of T cells to intravascular regions, to peripheral lymph nodes, and to inflammatory sites (24). In this regard, it is intriguing that a prominent feature of the CTLA-4-/- mouse is extensive lymphoadenopathy and T cell infiltration of tissues (2, 3).

The past few years have seen the identification of adaptor proteins and GTPases that account for inside-out signaling (25, 26). These mediators include VAV, ADAP (adhesion- and degranulation-promoting adaptor protein) (previously Fyb or SLAP), SKAP55 (src kinase-associated protein, 55 kDa), and the GTP-binding protein regulator for cell adhesion and polarization type 1 (Rap-1). Rap-1 is an allosteric regulatory element, switching between inactive GDP-bound and active GTP-bound conformations (27, 28). GTP-bound Rap-1 up-regulates LFA-1 clustering and T cell/APC conjugate formation (29-33). A dominant negative form of its ligand RapL (Rap enriched in lymphoid tissues) can block LFA-1 clustering (34). Although anti-CD3 can partially activate Rap-1, anti-CD3/CD28 coligation has been reported to inhibit this activation (35). Active Rap-1 may inhibit p21ras-extracellular signal-regulated kinase (ERK) signaling by competing for the effector mitogen-activated protein (MAP) kinase kinase kinase Raf-1 (27, 28, 36). It is a subject of debate whether Rap-1 can inhibit ERK activation and cytokine production in T cells (29, 30, 37). It is active in anergic T cells (38), whereas the Rap-1 GTPase-activating protein (GAP) (SPA-1)-deficient mice show an increase in numbers of anergic T cells (39). By contrast, transgenic mice expressing active Rap-1 fail to show defects in proliferation (30).

Given the extensive lymphocytic infiltration of tissues in CTLA-4-/- mice (2, 3), we reexamined CTLA-4 function in the context of LFA-1 integrin adhesion. Here, we report that CTLA-4 is an integrin activator, generating inside-out signals that potently up-regulate LFA-1/ICAM-1 adhesion and clustering. Its importance is underscored by the observation that TcRζ/CD3-induced LFA-1 adhesion/clustering was markedly dependent on CTLA-4 expression as observed in CTLA-4+/+ vs. CTLA-4-/- T cells. These effects were mediated by Rap-1 where anti-CTLA-4 activated the G protein, inactive Rap-1-N17 reversed adhesion and active Rap-1-V12 could substitute for the coreceptor. The role of CTLA-4 in LFA-1 clustering/adhesion provides a novel pathway by which the coreceptor can modulate T cell immunity.

Materials and Methods

Cells and Antibodies. CTLA-4 and CD28 expressing DC27.10 hybridoma T cells (DC27.10-CTLA-4 and DC27.10-CD28, respectively) were cultured in RPMI medium 1640 supplemented with FCS (5%) (40, 41). CD4+ T cells from CTLA-4+/+ and CTLA-4-/- mice were purified and cultured as described (42). Peripheral blood lymphocytes were isolated from buffy-coats by lymphocyte separation medium density gradient centrifugation. Anti-murine CD3 (145-2C11) and human CD3 (OKT3) were obtained from American Type Culture Collection, anti-human CTLA-4 (BNI3) and anti-human CD28 (9.3) mAbs were from B. Bröker (Ernst-Moritz-Arndt University, Greifswald, Germany), and Bristol-Myers Squibb, respectively. Anti-mouse CTLA-4 (UC10-4F10-11), anti-mouse CD28 (37.51), anti-mouse CD2, anti-mouse CD11a, and anti-mouse FITC-labeled CD18 mAbs were purchased from Pharmingen. HA-tagged pRK5-Rap-1 WT, Rap-1-N17, and Rap-1-V12 were a kind gift from J. de Gunzburg (Institut Curie, Paris). Recombinant ICAM-1-Fc (human and mouse) was purchased from R & D Systems. Ral GDS-RBD agarose beads and anti-Rap-1 mAb were bought from Upstate Biotechnology (Cambridge, U.K.).

IL-2 Detection by Intracellular Staining. DC27.10-CTLA-4 cells were either left unstimulated or activated with plate-bound anti-CD3 (3 μg/ml), anti-CTLA-4 (30 μg/ml), and anti-CD3/CTLA-4 mAbs for 10 h. Brefeldin (10 μg/ml) was added 6 h before fixation with 4% paraformaldehyde. After permeabilization with 0.3% saponin, the cells were stained with allophycocyanin-labeled anti-IL-2 and analyzed by FACS. Allophycocyanin-labeled rat IgG2a served as an isotype control.

LFA-1 Adhesion and Clustering. For ICAM-1-binding assays, 96-well flat-bottom plates were coated with recombinant ICAM-1-Fc (mouse or human; 12.5 μg/ml) in PBS overnight at 4°C as described (43-45). Cells (including CD4+ age-matched CTLA-4+/+ and CTLA-4-/- T cells) were left either unstimulated or were stimulated for 30 min with soluble anti-CD3 (1 μg/ml), anti-CD28 (10 μg/ml), anti-CTLA-4 (10-25 μg/ml), anti-CD2 (10 μg/ml), anti-CD8 (10 μg/ml) alone and in combination with anti-CD3. Secondary rabbit anti-mouse/hamster (5 μg/ml) was included for crosslinking. DC27.10-CTLA-4 cells or peripheral blood lymphocytes (PBLs) were transiently transfected with Rap-1-N17 or Rap-1-V12 by electroporation (41) or by the Amaxa (Cologne, Germany) Nucleofector Kit, respectively. For clustering assays, DC27.10-CTLA-4, DC27.10-DC28, CTLA-4+/+ and CTLA-4-/- T cells were stimulated with the indicated mAbs for 30 min as described above, and then stained with FITC-labeled LFA-1 mAb or anti-LFA-1 mAb and Alexa Fluor 568-conjugated goat anti-rat antibody for 1 h. Anti-LFA-1 capping was defined by the presence of a discrete polarized cap as described (43).

Rap-1 Pull-Down Assay. DC27.10-CTLA-4 cells were stimulated with anti-CD3 (1 μg/ml), anti-CD3/CTLA-4 (1 μg/ml/10 μg/ml), and anti-CTLA-4 (10 μg/ml and 20 μg/ml) antibodies plus secondary rabbit anti-mouse/hamster (5 μg/ml) for the times indicated in Fig. 4. Activated Rap-1 was isolated from cell lysates by using the GST-RalGDS fusion protein as described (35).

Fig. 4.
CTLA-4 up-regulates LFA-1 clustering on primary T cells. (A) Preactivated primary CD4+ T cells from CTLA-4 WT and CTLA-4 knockout (KO) mice stimulated with anti-CD3, anti-CTLA-4, and anti-CD3/CTLA-4 were stained with FITC-labeled anti-CD18 and analyzed ...

Results

CTLA-4 Increases LFA-1 Adhesion and Clustering. To assess CTLA-4 regulation of LFA-1-mediated adhesion, CTLA-4-expressing DC27.10 hybridoma T cells (DC27.10-CTLA-4) and activated CTLA-4-positive primary T cells were crosslinked with various soluble antibodies and/or CD80-Ig for 30 min and assessed for binding to immobilized ICAM-1 (43). DC27.10-CTLA-4 cells were compared with DC27.10 cells expressing the same level of CD28 (DC27.10-CD28) (40, 41). Consistent with TcR/CD3 mediated up-regulation of LFA-1 adhesion (46), soluble anti-CD3 increased the percentage of cells to bind to ICAM-1 from 5-7% to 16-18% in DC27.10-CTLA-4 and DC27.10-CD28 cells (Fig. 1A Upper). Anti-CD28 failed to further increase the enhancement of adhesion induced by the TcR (Fig. 1A Upper). At no point was CTLA-4 expressed on the DC27.10-CD28 cells. The lower panel shows the light microscopic images of adhered cells (Fig. 1 Ab vs. Aa). By contrast, soluble anti-CTLA-4 markedly increased ICAM-1 binding from 16-18% to 33% on DC27.10-CTLA-4 cells (Fig. 1A Upper). Anti-CTLA-4 alone increased binding to the same degree as anti-CD3 alone (Fig. 1A Upper and Lower Ac and Ad vs. Aa and Ab). These results were observed in five independent experiments. Further, soluble natural ligand CD80-Ig plus anti-CD3 increased adhesion from 5% to 37-38% of DC27.10-CTLA-4 cells, but not with DC27.10-CD28 cells (Fig. 1B). CD80-Ig alone also had an effect on DC27.10-CTLA-4 cells. As a control, anti-LFA-1 effectively blocked adhesion (Fig. 1C). These observations indicate that CTLA-4 generates inside-out signals that potently up-regulate LFA-1 binding to ICAM-1.

Fig. 1.
CTLA-4 ligation markedly increases LFA-1 binding to ICAM-1. (A) DC27.10-CD28 and DC27.10-CTLA-4 cells were activated with anti-CD3, anti-CTLA-4, anti-CD28, anti-CD3/CD28, or anti-CD3/CTLA-4 antibodies. (Upper) Histogram showing the percentage of cells ...

The conversion of LFA-1 from a low to intermediate/higher avidity form involves clustering (22). To assess this, anti-CD3 and/or CTLA-4 was used to ligate cells followed by staining for clustering with anti-LFA-1/Alexa Fluor 568 (43). Although anti-CD3 increased LFA-1 clustering from 2% to 6-13% of DC27.10-CD28 and DC27.10-CTLA-4 cells, CTLA-4 coligation increased this further to 22-24% (Fig. 2A Upper). Anti-CD28 coligation had no additional effect. Fig. 2A Lower shows cells stained with anti-LFA-1/Alexa-Fluor 568 as viewed by immunofluorescent microscopy. Caps were seen on both single and aggregated cells. This result indicates that CTLA-4 up-regulates binding to ICAM-1 by virtue of enhanced LFA-1 clustering on the surface of cells.

Fig. 2.
CTLA-4 ligation markedly increases LFA-1 capping. DC27.10-CD28 and DC27.10-CTLA-4 cells stimulated with anti-CD3, anti-CD3/CD28, and anti-CD3/CTLA-4 antibodies were stained with anti-CD11a and Alexa Fluor 568-conjugated goat anti-rat antibody and assessed ...

Significantly, anti-CD3/CTLA-4 coligation also inhibited IL-2 production, as detected by intracellular staining (Fig. 2B). This inhibition was observed with plate-bound, or soluble antibodies crosslinked with secondary antibody (47, 48) (data not shown). Anti-CTLA-4 can therefore up-regulate LFA-1 adhesion by promoting clustering (i.e., positive signal) under the same conditions that lead to an inhibition of IL-2 production (i.e., negative signal).

Human and mouse primary T cells were also activated for 48 h to induce CTLA-4 expression followed by coligation with anti-CD3 or anti-CD3/CTLA-4 (Fig. 3). With human peripheral T cells, anti-CD3 increased adhesion from 4% to 9% of cells, whereas CTLA-4 coligation increased this further to 14% (Fig. 3A Left). Anti-CTLA-4 alone increased adhesion to ICAM-1 to the same level as observed for anti-CD3 alone. This increase occurred with CTLA-4 expression on 17% of cells (Fig. 3A Right). No enhancing effect was observed with anti-CD3/CD28 coligation. Similar results were obtained in a comparison of activated age-matched CTLA-4+/+ versus CTLA-4-/- primary mouse T cells (Fig. 3B). Although anti-CD3 increased ICAM-1 binding from 5% to 11% of CTLA-4+/+ T cells, CTLA-4 coligation increased this binding further to 14-15% (Fig. 3B). This finding occurred with CTLA-4 expression on 20% of cells (data not shown). No increase in binding was evident with CTLA-4-/- T cells, although they were hyperproliferative, as reported (2, 3) (Fig. 3B Right). A similar difference between CTLA-4+/+ and CTLA-4-/- T cells was observed for LFA-1 clustering (Fig. 4A). Anti-CD3 increased clustering from 2% to 13% of cells, whereas CTLA-4 coligation augmented this further to 28%. Anti-CTLA-4 alone increased clustering to levels seen with anti-CD3 alone. As expected, no effect was evident with CTLA-4-/- T cells. Fig. 4 Right shows immunofluorescence images of LFA-1 distribution. As a control, neither anti-CD28, -CD2, or -CD8 coligation was able to increase adhesion under the short-term incubation conditions of the study (Fig. 4B). Our findings confirm that CTLA-4 acts as a selective integrin activator of LFA-1 on primary human and mouse T cells by increasing receptor clustering.

Fig. 3.
CTLA-4 up-regulates LFA-1-mediated adhesion in primary T cells, whereas anti-CD3 up-regulation of LFA-1 clustering requires CTLA-4 expression. (A Left) Preactivated human peripheral T cells were stimulated with anti-CD3, anti-CD3/CTLA-4, anti-CTLA-4, ...

Defective Anti-CD3-Induced LFA-1 Clustering and Adhesion on CTLA-4-/- T Cells. In addition to anti-CTLA-4 effects on LFA-1 adhesion, our findings uncovered another surprising observation, namely that CTLA-4 expression is required for the optimal ability of antigen receptor to increase LFA-1 adhesion (Figs. (Figs.3B3B and and4A).4A). This finding was observed in a comparison of CTLA-4+/+ and CTLA-4-/- primary cells. Anti-CD3 induced a much lower increase in ICAM-1 binding with CTLA-4-/- T cells when compared with CTLA-4+/+ T cells (i.e., from 5% on unstimulated cells to 11% on CTLA-4+/+ T cells versus 7% of CTLA-4-/- T cells). Similarly, anti-CD3-induced LFA-1 clustering was impaired in CTLA-4-/- T cells [i.e., from 2% of unstimulated cells to 13% on CTLA-4+/+ T cells relative to 6% for anti-CD3-ligated CTLA-4-/- T cells (Fig. 4A)]. These findings underscore the importance of CTLA-4 in adhesion whereby the loss of CTLA-4 partially decouples the TcR/CD3 complex from the up-regulation of LFA-1 clustering/adhesion.

CTLA-4-Mediated Adhesion Is Rap-1-Dependent. Given the ability of anti-CTLA-4 to induce inside-out signaling, a question concerned the nature of the pathway. The GTPase Rap-1 has been reported to increase LFA-1 adhesion (27-34). This finding suggested a possible link between our observed effects of CTLA-4 and Rap-1. To assess this observation, DC27.10-CTLA-4 T cells were ligated with anti-CD3, anti-CD3/CTLA-4, or anti-CTLA-4 and followed by precipitation with GST-RalGDS and blotting with anti-Rap-1 (27, 28). RalGDS binds only to the active GTP-bound form of Rap-1 (49). Anti-CD3 induced a 2-fold increase in the precipitation of GTP-bound Rap-1 (Fig. 5A, lane 2 vs. 1) as described (37). Significantly, CTLA-4 coligation increased this activation by an additional 2.5-fold (Fig. 5A, lane 3 vs. 2). Further, anti-CTLA-4 ligation alone also induced Rap-1 activation (Fig. 5A, lanes 4 and 5). The presence of Rap-1 was confirmed by anti-Rap-1 immunoblotting (Fig. 5A Lower). A time course of Rap-1 activation by anti-CD3/CTLA-4 showed peak activation by 5-10 min followed by a decrease at 15 min (Fig. 5B). Recently, Stork and coworkers reported a similar result (50). Our combined findings indicate that CTLA-4 ligation activates Rap-1 beyond that observed with TcR/CD3 ligation alone.

Fig. 5.
CTLA-4 up-regulation of adhesion is mediated via Rap-1. (A) CTLA-4 ligation activates Rap-1. DC27.10-CTLA-4 cells were activated with combinations of anti-CD3, anti-CD3/CTLA-4, and/or anti-CTLA-4 antibodies for 7 min followed by precipitation with GST-RalGDS ...

To test for a link between CTLA-4 activation of Rap-1 and increased LFA-1/ICAM-1 binding, DC17.10-CTLA-4 and activated CTLA-4-positive human T cells were transfected with inactive Rap-1-N17 and assessed for adhesion to ICAM-1 (Fig. 5C Upper Left and Right, respectively). Anti-CD3, CD3/CTLA-4, and CTLA-4-induced adhesion to ICAM-1 was markedly impaired by Rap-1-N17. Expression of HA-Rap-1-N17 was confirmed by anti-HA immunoblotting (Fig. 5C Inset). Significantly, expression of the constitutively active form Rap-1-V12 substituted for CTLA-4 in promoting adhesion (Fig. 5C Lower). Anti-CD3 stimulation of Rap-1-V12-transfected cells increased ICAM-1 binding to levels comparable with anti-CD3/CTLA-4 coligation. These combined observations showing that inactive Rap-1-N17 can block CTLA-4 increased adhesion and that Rap-1-V12 can mimic CTLA-4 in its cooperation with anti-CD3 argue strongly that CTLA-4 increases LFA-1-mediated adhesion via the Rap-1 pathway.

Discussion

Integrins play key roles in regulating the migration and localization of T cells (21, 51). This fact combined with the observation that CTLA-4-/- mice show extensive lymphocytic infiltration suggested to us that CTLA-4 might regulate integrin-mediated adhesion on T cells. In support of this notion, we have shown that CTLA-4 can generate inside-out signals that potently up-regulate LFA-1 adhesion by means of receptor clustering. Adhesion was induced by engagement of coreceptor alone or with TcR/CD3, and not by other coreceptors such as CD28, CD2, and CD8. TcR/CD3-CTLA-4 coligation induced an increase in LFA-1 adhesion that was accompanied by an inhibition of IL-2 production. The importance of CTLA-4 was underscored by the fact that CTLA-4-/- T cells also showed a marked impairment of TcR/CD3-induced LFA-1 adhesion and clustering. This finding indicates that CTLA-4 expression is required for the optimal coupling of the TcR complex with LFA-1 adhesion. These events were mediated by Rap-1 because CTLA-4 activated the GTP-binding protein, and inactive Rap-1-N17 abrogated coreceptor-induced adhesion. In addition, active Rap-1-V12 cooperated with anti-CD3 to increase adhesion to levels comparable with anti-CD3/CTLA-4. Our findings require a revision of the view that CTLA-4 operates solely as a negative signaling receptor. Instead, the coreceptor plays an unusual role in positively up-regulating an event that is a central T cell function. Other stimulatory events linked to CTLA-4 include the binding of phosphatidylinositol 3-kinase (PI3-K) (52) and the differential activation of c-jun kinases (JNKs) (42). This unexpectedly potent role in LFA-1-mediated adhesion provides an alternate route to account for CTLA-4 function in T cell immunity.

Previous studies have focused on the antigen receptor (TcR/CD3) in the generation of inside-out signals that modulate LFA-1 adhesion (46). CTLA-4 must now be considered to be an alternate integrin activator on T cells. Antibody against each receptor induced similar levels of adhesion and together had an additive effect on adhesion. Whether the two receptors play equal roles in antigen presentation remains to be determined. The strength of the TcR/CD3 signal is expected to vary with the avidity of agonist, whereas CD80/CD86 binds to CTLA-4 with a constant avidity. The CD80 effect was similar to that observed with anti-CTLA-4 (Fig. 1 A and B). We therefore predict that CTLA-4 up-regulation of LFA-1 adhesion will dominate in responses to many agonists, especially with low-affinity peptide (i.e., self-antigen). This is especially the case given that CTLA-4 expression is required for the optimal coupling of the TcR complex with LFA-1 adhesion. In one scenario, increased adhesion provided by CTLA-4 may induce greater adhesion of T cells to APCs or target cells. However, hyperactivation of adhesion is often accompanied by increased T cell motility and migration. Increased motility could actually lead to a reduction in the residency period between T cell and APC, which in turn may limit the number of engaged TcRs. This reduced contact could serve to increase the threshold of TcR signaling and to limit the response to self-antigen (i.e., prevent autoimmunity). In this model, autoimmunity could result from altered adhesion and not vice versa. Altered adhesion could also contribute to the increased migration and lymphocytic infiltration of organs and/or lymphoadenopathy as observed in CTLA-4-deficient mice (2, 3).

Significantly, CTLA-4 up-regulation of adhesion occurred under different ligation conditions, both in the context of TcR/CD3 coligation, and with ligation of the coreceptor alone (i.e., with antibody or CD80-Ig) (Figs. (Figs.1,1, ,2,2, ,3,3, ,4).4). These different ligation conditions have been reported to elicit different functional outcomes. As observed in this and previous studies (47, 48), TcR/CD3-CTLA-4 coligation inhibits IL-2 production (Fig. 1). We now show that the same condition induces a maximal increase in LFA-1-mediated adhesion concurrent with an inhibition of cytokine production. This seemingly paradoxical situation (i.e., where one function is up-regulated and another inhibited) has been described in other biological systems such as embryogenesis. It is related to the prioritization of one function relative to another function that is no longer required. In this context, CTLA-4 is expressed as an activation antigen on effector T cells involved primarily in secondary responses (1). For example, cytolytic T cells (CTLs) rely on enhanced adhesion for the optimal killing of targets, and may be less reliant on additional cytokine production. It is interesting that CD80, the preferred ligand for the coreceptor, is a more efficient costimulator of anti-tumor responses (53).

By contrast to coligation, the administration of soluble anti-CTLA-4 alone in in vivo models can augment antitumor responses (1, 54, 55). Past studies have attributed this observation to “blocking effects” and the modulation or clearance of regulatory T cells (TRegs) (54, 55). Our findings that anti-CTLA-4 can increase adhesion and activate Rap-1 adds a new perspective to this issue. Increased LFA-1 adhesion may facilitate increased cell-cell contact and/or the frequency of interaction with target cells. The coreceptor will also alter T cell motility, intravascular migration, and migration to peripheral organs induced by chemokines. The altered localization of CTLA-4-bearing cells will in turn affect the micro-environment with different surrounding cells, possibly affecting activation and cytokine production. This autonomous function of CTLA-4 may be similar to CD28 where, once phosphorylated, the coreceptor can independently modulate cytokine production (56, 57).

Lastly, our findings show that CTLA-4 modulation of LFA-1 adhesion and clustering is mediated by the GTPase Rap-1 (Fig. 5). This observation combined with the demonstration that Rap1-N17 can block CTLA-4-induced adhesion and Rap1-V12 can substitute for CTLA-4 implicates Rap-1 in the regulation of CTLA-4-induced adhesion. CTLA-4 ligation activated Rap-1 by 10-fold relative to unstimulated cells, a finding that is supported by a recent report (50). The increase was observed by using soluble crosslinked antibody or immobilized antibody. In our hands, anti-CD3 induced only moderate levels of Rap-1 activation that was augmented by anti-CTLA-4 (Fig. 5). This reduced contact is likely to occur at even lower levels in response to low-intermediate avidity agonist. In this way, TcR/CD3 may increase adhesion without exerting a possible inhibitory effect on the ERK pathway and IL-2 production. Although Rap-1 can inhibit ERK activation in some systems (27, 28), it is uncertain whether it operates in the same fashion in T cells (29, 30, 35, 37). Transgenic mice expressing active Rap-1 fail to show defects in proliferation (30). If under certain conditions T cell responses can be inhibited, it would potentially provide a model whereby Rap-1 hyperactivation by CTLA-4 would have the dual effect of inhibiting IL-2 production (i.e., preventing hyperactivation) and increasing T cell adhesion and motility (i.e., affecting tissue infiltration). Future studies will be needed to resolve these outstanding issues.

Acknowledgments

We thank Drs. Ana Izcue and Fiona Powrie (Oxford University, Oxford) for providing a few of the CTLA-4-/- mice used in this study. This work was supported by a grant from the Wellcome Trust, London (C.E.R. is the recipient of a Principal Research Fellow Award) and by the Biotechnology and Biological Sciences Research Council (H.S.).

Notes

Author contributions: H.S., E.V., S.d.R.D., and C.E.R. designed research; H.S., E.V., S.d.R.D., B.W., and C.E.R. performed research; H.S. and C.E.R. analyzed data; and H.S. and C.E.R. wrote the paper.

Abbreviations: CTLA-4, cytotoxic T lymphocyte antigen-4; LFA-1, lymphocyte function-associated antigen 1; ICAM-1, intercellular adhesion molecule-1; TcR, T cell antigen receptor; APC, antigen-presenting cell; Rap-1, regulator for cell adhesion and polarization type 1; ERK, extracellular signal-regulated kinase.

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