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The Journal of Immunology, 2006, 177: 6833-6841.
Copyright © 2006 by The American Association of Immunologists, Inc.

TIRC7 Inhibits T Cell Proliferation by Modulation of CTLA-4 Expression

Grit-Carsta Bulwin*,{dagger}, Thomas Heinemann{ddagger}, Volker Bugge*, Michael Winter*, Anke Lohan{dagger}, Mirko Schlawinsky{dagger}, Anke Schulze*, Stephanie Wälter{dagger}, Robert Sabat*, Ralf Schülein§, Burkhard Wiesner§, Rüdiger W. Veh, Jürgen Löhler||, Richard S. Blumberg#, Hans-Dieter Volk* and Nalân Utku1,*,{dagger}

* Institut für Medizinische Immunologie, Campus Charité Mitte, Humboldt-Universität zu Berlin, Berlin, Germany; {dagger} GenPat77 Pharmacogenetics, Berlin, Germany; {ddagger} Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Bonn, Germany; § Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany; Anatomisches Institut, Campus Mitte, Humboldt-Universität zu Berlin, Berlin, Germany; || Molecular Pathology Group, Heinrich-Pette-Institute, Hamburg, Germany; and # Division of Gastroenterology, Brigham and Women’s Hospital, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ab targeting of TIRC7 has been shown previously to inhibit T cell proliferation and Th1 lymphocyte-associated cytokine production. In this study, we demonstrate that Ab targeting of TIRC7 induces early cell surface expression of CTLA-4. The majority of stimulated CD4+ and CD8+ human T cells coexpress CTLA-4 and TIRC7. Similar to CTLA-4, TIRC7 rapidly accumulates at the site of Ag adhesion upon T cell activation. TIRC7 seems to colocalize with CTLA-4 in human T cells, and both molecules are associated with clathrin-coated vesicles, indicating they share intracellular transport systems. Moreover, Ab targeting of TIRC7 results in an early activation of CTLA-4 transcription. The inhibition of cell proliferation mediated by TIRC7 is dependent on CTLA-4 expression because the TIRC7-mediated inhibitory effects on cell proliferation and cytokine expression are abolished by Ab blockade of CTLA-4. Splenocytes obtained from CTLA-4-deficient mice are not responsive to TIRC7 Ab targeting. Thus, TIRC7 acts as an upstream regulatory molecule of CTLA-4 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The activation of T lymphocytes involves a series of distinct events that are initiated by the interaction of an Ag-specific TCR with a peptide presented by an MHC molecule expressed on the surface of an APC (1, 2). Cooperating with the TCR-mediated signal is a complex network of receptor-ligand interactions viewed as costimulatory signals. Among those described, the function of the CD28 and CTLA-4 receptors on the T cells has been elucidated in great detail. Engagement of CD28 on T cells by B7 molecules (CD80, CD86) expressed on the surface of APCs provides an activating costimulatory signal that is counterbalanced by a negative signal provided by the subsequent cell surface expression of CTLA-4, which binds with higher affinity to B7 (3, 4, 5, 6, 7). The negative signaling mediated by CTLA-4 was demonstrated by inhibiting the function of CTLA-4 with anti-CTLA-4-specific mAbs capable of blocking the binding of ligands to CTLA-4. Blocking of CTLA-4 resulted in an increased expression of the Th1 lymphocyte-related cytokines IL-2 and IFN-{gamma} and an enhanced T cell proliferation in vitro after T cell stimulation (8) as well as a significant acceleration of the acute rejection of cardiac allograft in mice and rats in vivo (9, 10). However, the molecules specifically involved in regulating CTLA-4 expression are largely unknown.

We recently identified TIRC7 as an activation-induced membrane protein on human T lymphocytes that plays a central role in T cell activation (11). In particular, targeting TIRC7 with anti-TIRC7-specific Abs causes inhibition of Th1, but not Th2 lymphocyte-related cytokine expression in association with a prolonged state of T cell unresponsiveness in alloantigen- and mitogen-activated human PBL in vitro. Consistent with this, administration of anti-TIRC7 Ab prevents the rejection of kidney allograft in rats in vivo (11). We also demonstrated that in an acute cardiac allograft rejection model in mice the targeting of TIRC7 with an anti-TIRC7 mAb diminishes lymphocyte infiltration into the grafts, resulting in prolongation of allograft survival. This effect was associated with hyporesponsiveness of the lymphocytes and an enhanced up-regulation of CTLA-4 expression on the cell surface (12). The latter observation of CTLA-4 up-regulation is in agreement with a recent finding that in lymphocytes obtained from TIRC7-deficient mice, the intracellular and cell surface expression of CTLA-4 is markedly reduced compared with wild-type lymphocytes before and after activation, whereas no differences are observed for either CD28 or CD40L expression (13).

Based on these results and the similarities that exist between the effects caused by Ab targeting of TIRC7 and those mediated by CTLA-4, we were interested in further investigating the functional relationship between CTLA-4 and TIRC7 expression. In the present study, we demonstrate that both TIRC7 and CTLA-4 basically share the same intracellular trafficking system via clathrin-coated vesicles and that TIRC7 is a constituent of the T cell Ag binding site, as was shown for CTLA-4 by others (14, 15, 16). CTLA-4 and TIRC7 are coexpressed in activated human T cell subpopulations. Moreover, Ab binding to TIRC7 induces an early expression of CTLA-4 on the T cell surface that is thus responsible for the inhibitory effect of anti-TIRC7 mAb on T cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and isolation

PBL were isolated from healthy human volunteers using standard Ficoll centrifugation methods. For each experiment, 106 cells/ml were resuspended in RPMI 1640 (Biochrom) supplemented with 10% FCS (Biochrom), 2 mM L-glutamine, and penicillin/streptomycin (Invitrogen Life Technologies). Cells were stimulated with rIL-2 (100 U/ml; R&D Systems), PHA (1 µg/ml; Sigma-Aldrich), or alloantigen in a two-way MLR. Informed written consent was obtained from each volunteer.

Flow cytometry

Flow cytometry was performed, as described by Waldrop et al. (17). Directly labeled anti-TIRC7 mAb (12), anti-CD3 mAb, anti-CD28 mAb, anti-CD4 mAb, anti-CD8 FITC mAb (BD Biosciences), anti-CTLA-4 PE mAb, and control IgG PE (BD Biosciences), respectively, were used for the staining of lymphocytes.

Confocal fluorescence microscopy

Fluorescence microscopy was performed, as described (18), using anti-CTLA-4 mAb (BD Biosciences), anti-CD5 mAb (Dianova), and anti-TIRC7 polyclonal Abs (11) and mAbs (12) (GenPat77 Pharmacogenetics), respectively, which were labeled indirectly with Cy2-conjugated goat anti-mouse IgG Ab and Cy3-conjugated goat anti-rabbit IgG Ab (Dianova).

For analysis of colocalization of TIRC7 and clathrin-coated vesicles, cells were coincubated with anti-clathrin H chain mAb (BD Transduction Laboratories) and anti-TIRC7 polyclonal Ab (GenPat77 Pharmacogenetics), and labeled with Cy2-conjugated goat anti-mouse IgG Ab and Cy3-conjugated goat anti-rabbit IgG Ab (Dianova). Microscopic analysis was performed with a Pascal 5 confocal laser-scanning microscope (Zeiss).

Electron microscopy

Electron microscopy was performed, as described (19). Immobilization of the nonadherent PBL was achieved by preincubating culture plates with anti-CD3 mAb overnight at 4°C. The anti-TIRC7 (GenPat77 Pharmacogenetics) and goat anti-rabbit Ab coupled to 1 nm of gold were used for tagging.

Isolation of clathrin-coated vesicles from PBL

The purification of clathrin-coated vesicles from PBL was performed as described previously by Lindner (20).

Immunoblot

Proteins were separated by SDS-PAGE and blotted onto nitrocellulose filters (Schleicher & Schuell Microscience). Filters were incubated with anti-clathrin H chain mAb (BD Transduction Laboratories) and anti-TIRC7 polyclonal Ab (Genpat77 Pharmacogenetics) as primary Abs, and with alkaline phosphatase-conjugated anti-mouse and anti-rabbit Ab (Dianova) as secondary Abs. For staining, filters were incubated with 5-bromo-4-chloro-3-indolyl phosphate (0.56 mM)/NBT (0.48 mM) (Sigma-Aldrich) in 10 mM Tris-HCl.

Quantitative expression analysis by TaqMan

PBL from healthy human volunteers (1 x 106 cells/ml) were resuspended in RPMI 1640 (Biochrom) supplemented with 10% FCS (Biochrom), 2 mM L-glutamine, and penicillin-streptomycin (Invitrogen Life Technologies). PBL were activated with PHA (Sigma-Aldrich) (1 µg/ml) and incubated with and without anti-TIRC7 polyclonal Ab for 0, 3, 5, 16, and 48 h. The following Abs were used in the experiments: anti-TIRC7 polyclonal Ab 79 (11); mAb against TIRC7 (anti-TIRC7 mAb) (12); preimmune sera obtained before immunization of rabbits as control Abs (control Ab or control Ab1); and polyclonal Ab against intracellular epitopes of TIRC7 protein (control Ab2) (11).

After mRNA isolation using standard methods, the amount of CTLA-4 mRNA was analyzed by TaqMan (BD Biosciences).

Proliferation assay

Isolated and stimulated human PBL or mouse splenocytes were plated in separate wells of a 96-well microtiter plate in a final volume of 200 µl in the absence and presence of investigated Abs, and cultures were pulsed with 0.5 µCi of [3H]thymidine (ICN Biochemicals).

ELISA for determination of cytokines

IL-2, IL-4, and IFN-{gamma} were assayed in cell culture supernatants by commercial ELISA kits for IL-2 (Laboserv), IL-4 (ultrasensitive), and IFN-{gamma} (Laboserv).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TIRC7 is shifted from intracellular compartments to the cell surface upon T cell activation

To investigate the functional relationship between TIRC7 and CTLA-4, we first analyzed the expression kinetics of TIRC7 to constitute a basis for comparing it with the well-known expression kinetic of the CTLA-4 molecule. CTLA-4 represents a membrane receptor protein that is mainly expressed in intracellular vesicles and cycles between the cell surface and intracellular stores (15). After T cell stimulation, the cell surface expression of CTLA-4 is up-regulated within 24–48 h (5).

We recently described that TIRC7 is present in lymphocytes at the cell membrane and in intracellular compartments (11). We demonstrated that TIRC7 expression on the cell surface is induced within 30 min upon T cell activation and reaches maximum levels within 2 h. To understand the expression kinetics of TIRC7 in greater detail, permeabilized and nonpermeabilized human peripheral blood T lymphocytes were submitted to FACS analysis for TIRC7 levels in intracellular stores and on the cell membrane before and after activation with human rIL-2 for 8 h. As shown in Fig. 1, TIRC7 localization, expressed as medium fluorescence intensity, was ~100-fold higher in the intracellular compartments of lymphocytes compared with the cell surface. Upon activation, the protein reached maximum expression levels within 2 h on the cell surface (Fig. 1A), whereas the intracellular levels were reduced to a minimum at that time (Fig. 1B). The intracellular and extracellular distribution levels of TIRC7 were restored within 6 h. These results indicate that TIRC7 is present in intracellular compartments as a preformed protein and, upon T cell activation, is shifted within few hours to the cell surface.


Figure 1
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  		FIGURE 1. TIRC7 cycles from intracellular compartments to the cell surface. PBL were isolated from healthy human volunteers, as described in Materials and Methods. PBL were stimulated with 100 U/ml human rIL-2 and stained for the relevant markers. The kinetic of extracellular (A) and intracellular (B) TIRC7 was determined by flow cytometry in parallel in human CD28+/CD4+ cells in comparison with control. Shown is one example of four experiments.

 
TIRC7 cycles between intracellular stores and the surface of the cell

It was demonstrated that CTLA-4 cycles between the cell surface and intracellular stores (15). If this would be the case for TIRC7 as well, anti-TIRC7 Ab should accumulate intracellularly in lymphocytes expressing TIRC7. To test this possibility, we used indirect immunofluorescence microscopy to compare the accumulation of either the anti-TIRC7 Ab (Ab) or anti-CTLA-4 Ab as positive control, and rabbit IgG as negative control, in human PBL stimulated with PHA at 37°C for 3 h. As shown in Fig. 2C, when cells were incubated with rabbit IgG, very little accumulation was observed. In contrast, significant intracellular staining was observed in cells incubated with anti-TIRC7 Ab (Fig. 2A) as well as with anti-CTLA-4 Ab (Fig. 2B).


Figure 2
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  		FIGURE 2. TIRC7 is stored in intracellular compartments. A, Human PBL were cultured in medium containing anti-TIRC7 polyclonal Ab, anti-CTLA-4 mAb as positive control, and rabbit IgG as negative control, respectively, for 3 h at 37°C. After incubation, cells were washed, fixed, permeabilized, and stained by indirect immunofluorescence with the corresponding secondary Abs. Anti-TIRC7 Ab shows intracellular staining in red fluorescence according to accumulation of the Ab within the cell. B, Anti-CTLA-4 Ab shows intracellular staining in green fluorescence according to accumulation within the cell. C, Comparatively, only little intracellular immunostaining was observed after incubation with rabbit IgG. D, Human PBL were isolated, stimulated for 48 h with CD3/CD28 mAb, and stained with either anti-TIRC7 mAb FITC or anti-CD152 PE for flow cytometric analysis. TIRC7 is expressed in predominantly CTLA-4-positive cells, and all CTLA-4-positive CD4+CD8+ T cells express TIRC7. However, TIRC7 is also present in ~10% of CTLA-4-negative CD4+ and CD8+ T cells. Data represent one experiment of four.

 
To further analyze whether both proteins were coexpressed on lymphocytes, human PBL were isolated, stimulated for 48 h with anti-CD3/CD28 mAb, and stained with either anti-TIRC7 mAb FITC or anti-CD152 PE for flow cytometric analysis. As shown in Fig. 2C, TIRC7 is predominantly expressed in CTLA-4-positive cells, and all CTLA-4-positive, stimulated CD4+ and CD8+ cells expressed TIRC7. However, TIRC7 was also present in ~10% of CTLA-4-negative CD4- and CD8-positive T cells.

These results indicate that CTLA-4 and TIRC7 are coexpressed and colocalized to similar cellular areas during T cell activation. TIRC7, similar to CTLA-4, is redistributed from the cell surface into intracellular compartments and, in combination with the shift to the cell surface upon T cell activation, cycles between the cell membrane and cytoplasmic stores. In addition, the coexpression pattern of TIRC7 with CTLA-4 supports the hypothesis that TIRC7 might be involved in regulation of CTLA-4 via direct binding to CTLA-4.

TIRC7 accumulates at the Ag binding site upon T cell activation

Upon activation of T cells by Ag, a high local concentration of a number of molecules required for T cell activation congregates at the site of membrane adhesion between T cell and APC, which is referred to as the immunologic synapse (21, 22, 23, 24, 25). On the basis of the above results, we were interested in determining whether TIRC7 was associated with the APC binding site, which would support the putative role of TIRC7 in the activation process. It was demonstrated previously that immobilization of PBL on an activating anti-CD3 mAb, but not on nonactivating anti-CD18 mAb, induced orientation of CTLA-4 toward the Ag binding site (15). We therefore used a similar assay to examine TIRC7 orientation upon T cell activation. Nonpermeabilized PBL were immobilized on chambered coverslips coated with either anti-CD3 mAb or anti-CD18 mAb. Subsequently, immune staining of TIRC7, CTLA-4, and CD5 was performed using fluorescently labeled specific Ab. Confocal microscopy revealed that in cells stimulated with anti-CD3 Ab TIRC7 as well as CTLA-4 residing on the cell surface accumulated at the Ag binding site (anti-CD3), as shown in Fig. 3A (upper panel A and B). In contrast, uniform cell surface distribution was observed for CD5 (Fig. 3A, upper panel C). The polarized distribution of intracellular TIRC7 was similar to that of CTLA-4 toward the Ag binding site and was specific for the antigenic stimulus as TIRC7 and CTLA-4 did not show focal accumulation in lymphocytes immobilized on chamber slides coated with nonactivating anti-CD18 mAb (Fig. 3A, lower panel A and B). Likewise, the CD5 distribution pattern was not changed (Fig. 3A, lower panel C). These results demonstrate that TIRC7, like CTLA-4, is specifically concentrated at the APC binding site upon Ag activation.


Figure 3
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  		FIGURE 3. TIRC7 accumulates at the Ag binding site upon T cell activation. A, PBL were immobilized on anti-CD3 mAb- or anti-CD18 mAb-coated coverslips for 48 h and fixed in situ. Various proteins involved in T cell activation, i.e., TIRC7, CTLA-4, and CD5, were stained with the respective Cy3- and Cy2-coupled Abs, and cellular localization was examined by confocal microscopy. A–C, Left upper panel shows fluorescence imaging in vertical optical sections of the cell; the dotted line represents the Ag binding site (anti-CD3) on coverslip (CS). Upon activation with anti-CD3 mAb, TIRC7 accumulates in the vicinity of the Ag recognition site on the coverslip (CS), resulting a cap-like image (upper panel B). The same pattern was observed for CTLA-4 upon anti-CD3 mAb activation (upper panel B). The corresponding horizontal sections of the cells, from which the vertical sections were taken, are shown below in a–c. The scheme at the right side of the upper panel depicts the frontal view of a T cell immobilized on an anti-CD3 mAb (pink dot)-coated coverslip (white layer). Along the vertical plane of the T cell (black plane), the fluorescing molecules that accumulate in a caplike structure (dark blue line) toward the coverslip corresponding to caplike structure of TIRC7 and CTLA-4 (A and B, left upper panel). In contrast, a uniform T cell membrane distribution pattern without any capping is displayed for CD5 in anti-CD3 mAb-activated cells (C, left upper panel). The same molecules immobilized on nonactivating anti-CD18 mAb exhibit a uniform ringlike distribution pattern (lower panel A–C and a–c). The scheme at the right side of the lower panel shows the frontal view of a T cell immobilized on an anti-CD18 mAb-coated (green dot) coverslip (white layer). Along the vertical plane of the T cell (black section plane), the fluorescing molecules (dark blue line) that do not aggregate toward the contact site is shown. In the lower panel, A–C shows vertical sections of cells, and the panel below (a–c) shows corresponding cells in horizontal sections. B, PBL were immobilized and activated on anti-CD3 mAb-coated coverslips for 24 h, tagged with TIRC7-coupled gold particles, and analyzed by electron microscopy. The orientation of intracellular and surface TIRC7 toward the Ag binding site is indicated by arrowheads. C, PBL were activated with PHA for 3 days, then immobilized on anti-CD3 mAb- or anti-CD18 mAb-coated coverslips for 6 h and fixed in situ. Cells were stained and analyzed, as described in A. Fluorescence images in vertical optical sections of the cell are shown in A–C, left upper panel. Upon activation with anti-CD3 mAb, TIRC7 (A) and CTLA-4 (B) are (Figure legend continues) localized around the Ag recognition site (anti-CD3) on the coverslip (CS), forming a caplike structure. CD5 mAb staining resulted in a uniform T cell membrane image without any capping in anti-CD3 mAb-activated cells (C of the upper panel). Left upper panel a–c, Shows the corresponding horizontal sections of the respective cells, from which the vertical sections were taken. Likewise, the same molecules immobilized on nonactivating anti-CD18 mAb show a uniform ringlike distribution. A–C left lower panel, Vertical sections; a–c, the corresponding cells in horizontal sections. The schemes on the right side are explained in the legend of A.

 
The shift of intracellular TIRC7 toward the Ag binding site was associated with a reorientation of labeled cell surface TIRC7 as visualized by electron microscopy (Fig. 3B). Lymphocytes immobilized and stimulated for 24 h on anti-CD3-coated culture plates showed directed accumulation of TIRC7 at the site of adhesion. The reorientation of cell surface TIRC7 toward the Ag binding site was further examined by performing the same set of experiments as shown in Fig. 3A, using human T cells previously incubated with PHA for 3 days. Because mitogen activation results in random distribution of molecules on the cell surface as was shown for CTLA-4 (26), reorientation of membrane molecules toward the Ag binding site should become visible. After 3 days of PHA activation, cells were collected and immobilized on anti-CD3 mAb-coated or anti-CD18 mAb-coated coverslips for 6 h. Confocal microscopy revealed accumulation of TIRC7 and CTLA-4, as described previously for CTLA-4 (15), toward the Ag binding site (Fig. 3C, upper panel A and B). The cell surface distribution of CD5, in contrast, remained uniform (Fig. 3C, upper panel A–C). Again, no reorientation toward the binding site of TIRC7, CTLA-4, and CD5 was induced by immobilization on an anti-CD18 mAb-coated coverslip (Fig. 3C, lower panel a–c). In summary, TIRC7 localized on the cell surface or in intracellular stores is shifted to the Ag binding site upon anti-CD3 activation.

TIRC7 protein is localized within clathrin-coated vesicles and colocalizes with CTLA-4 in activated human T cells

CTLA-4 has been demonstrated previously to be transported within clathrin-coated vesicles (14, 27, 28, 29). The functional association between TIRC7 and CTLA-4 led us to hypothesize that the same mode of cellular transport would be observed for TIRC7. To examine this possibility, PBL were immobilized on chambered coverslips, permeabilized, and costained with an anti-TIRC7 Ab and anti-clathrin H chain mAb, and the intracellular localization of TIRC7 was compared with that of clathrin by confocal microscopy. As shown in Fig. 4A, anti-TIRC7 Ab and anti-clathrin mAb staining as defined by red (Fig. 4Aa) and green (Fig. 4Ab) fluorescence, respectively, colocalized, resulting in a yellow fluorescence after overlay of both pictures (Fig. 4Ac). These results were confirmed by Western blot analysis of enriched fractions of clathrin-coated vesicles from human PBL using an anti-clathrin mAb (Fig. 4Ba) and anti-TIRC7 Ab (Fig. 4Bb).


Figure 4
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  		FIGURE 4. TIRC7 is associated with clathrin-coated vesicles and colocalizes with CTLA-4. A, The association of TIRC7 with clathrin-coated vesicles was demonstrated by confocal microscopy. PBL were immobilized on glass coverslips, fixed, permeabilized, and coincubated with anti-TIRC7 polyclonal Ab and an anti-clathrin mAb. The corresponding secondary Ab for the anti-TIRC7 Ab was conjugated with Cy3 (red fluorescence) (a), and the secondary Ab for the anti-clathrin mAb with Cy2 (green fluorescence) (b). Areas of red and green fluorescence overlay result in yellow fluorescence (c) (arrowheads), indicating partial colocalization. B, Colocalization of TIRC7 and clathrin-coated vesicles was also demonstrated by Western blot analysis. a, Clathrin-coated vesicles were isolated from PBL, and a vesicle-enriched fraction was obtained. Lysed PBL (PBL lysates), clathrin-coated vesicle fraction from bovine brain (CCV bovine brain), and clathrin-coated PBL vesicle fraction from PBL (CCV PBL) were incubated with anti-clathrin mAb and analyzed by Western blot. The 180-kDa band in all specimens depicts the enriched clathrin-coated vesicles (b). The same specimens were analyzed with anti-TIRC7 polyclonal Ab and show a distinct band that is specific for TIRC7 for the PBL fractions. C, The colocalization of TIRC7 and CTLA-4 was demonstrated by confocal microscopy. Human PBL were immobilized on chambered coverslips, fixed, permeabilized, and incubated with anti-TIRC7 mAb (a) and anti-CTLA-4 mAb (b). The corresponding secondary Ab for the anti-TIRC7 Ab was conjugated with Cy3 (red fluorescence) and for anti-CTLA-4 mAb with Cy2 (green fluorescence). Areas of red and green fluorescence overlay result in yellow fluorescence (c).

 
To examine whether TIRC7 and CTLA-4 were also colocalized within human T cell during activation, PBL were activated with PHA, immobilized on chambered coverslips, permeabilized, and costained with anti-TIRC7 Ab and anti-CTLA-4 mAb, and the localization of TIRC7 was compared with that of CTLA-4 by confocal microscopy. Again, as shown in Fig. 4C, anti-TIRC7 Ab and anti-CTLA-4 mAb staining, as defined by red (Fig. 4Ca) and green (Fig. 4Cb) fluorescence, respectively, colocalized, resulting in yellow fluorescence (Fig. 4Cc).

Ab targeting of TIRC7 up-regulates CTLA-4 mRNA expression and causes rapid induction of CTLA-4 protein expression in intracellular compartments and on the cell surface

To test whether the anti-TIRC7 Ab-mediated CTLA-4 expression on the cell surface (12) is only the consequence of a shift and subsequent recycling of preformed vesicle-bound CTLA-4 or whether anti-TIRC7 Ab causes de novo synthesis of CTLA-4 as well, CTLA-4 mRNA levels were determined in human PBL activated with PHA and incubated with and without anti-TIRC7 Ab. Cells were collected immediately after incubation and after 3, 5, 16, and 48 h, and mRNA was isolated and quantified using TaqMan analysis. As shown in Fig. 5A, anti-TIRC7 Ab treatment resulted in significantly higher levels of CTLA-4 encoding mRNA compared with cells cultured in the absence of anti-TIRC7 Ab. The maximal effect of anti-TIRC7 Ab-mediated mRNA induction was observed 3 h after incubation, followed by a slight reduction after 5 h (Fig. 5A). Baseline levels were reached after 16 and 48 h (data not shown). The results suggest that in activated cells, anti-TIRC7 Ab induces rapid CTLA-4 mRNA expression that substantially exceeds that induced by PHA activation alone. Based on these results, we next examined whether the time course of the cell surface expression of CTLA-4 was modulated by targeting TIRC7 with anti-TIRC7 Ab. Human PBL were activated with PHA in in vitro culture in the presence of anti-TIRC7 Ab, and cell surface expression of CTLA-4 was analyzed by FACS analysis. As shown in Fig. 5, B and C, the cell surface expression of CTLA-4 was significantly accelerated in anti-TIRC7 Ab-treated cells compared with untreated and control Ab-treated lymphocytes, reaching a maximum level of expression as early as 4 h after activation. A similar level of CTLA-4 expression was observed in control PBL not earlier than 24 h after PHA activation, consistent with the known course of CTLA-4 expression after anti-CD3 stimulation of PBL (3). When the ligation of the anti-TIRC7 Ab to TIRC7 protein was competed with a soluble peptide corresponding to the TIRC7 domain recognized by the Ab, the TIRC7-induced up-regulation of CTLA-4 expression was inhibited. These results indicate that TIRC7 stimulates rapid CTLA-4 transport to the cell surface in a pathway that is transcriptional.


Figure 5
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  		FIGURE 5. Ab targeting of TIRC7 up-regulates CTLA-4 mRNA and causes rapid cell surface expression of CTLA-4 protein. A, Human PBL were activated with PHA and incubated with and without anti-TIRC7 Ab. Cells were harvested immediately and at 0, 3, and 5 h after incubation, and mRNA was isolated. Incubation of PBL with anti-TIRC7 Ab (TIRC7 Ab) results in maximal induction of CTLA-4 mRNA expression after 3 h, exceeding the induction caused by PHA stimulation alone, followed by a slight reduction after 5 h. No induction of CTLA-4 mRNA was observed in the presence of control Ab (control Ab 1 and control Ab 2). B, PBL were activated with PHA and incubated with anti-TIRC7 Ab (TIRC7 Ab), in the presence of the corresponding soluble TIRC7 peptide (TIRC7 Ab + P), control Ab (Control Ab1), and PHA alone (PHA), respectively. Cells were double stained with FITC-labeled anti-CD3 mAb and PE-labeled anti-CTLA-4 mAb 4F10, and CTLA-4 cell surface expression was determined by flow cytometry. Shown is the mean fluorescence intensity (MFI) of CTLA-4 expression. Cell surface expression of CTLA-4 was increased in anti-TIRC7 Ab-treated cells, reaching a maximum level of expression as early as 4 h after activation. No increase was obtained when the ligation of the anti-TIRC7 Ab to the TIRC7 protein was competed with the corresponding peptide, as well as in control Ab-treated and PHA-stimulated lymphocytes, respectively. Data represent means and SD of seven independent experiments (*, p < 0.01 vs controls). C, Histograms of a representative flow cytometric analysis of human PBL stained with anti-CD152 PE (CTLA-4) Ab (4F10) (a–c) and control IgG PE (d). CTLA-4 surface expression was measured 4 h after incubation with PHA in the presence of anti-TIRC7 Ab (a), anti-TIRC7 Ab plus the corresponding soluble peptide (b), in the presence of an unrelated control Ab (c), and staining control IgG PE (d), respectively. CTLA-4 surface expression was increased in cells treated with anti-TIRC7 Ab, whereas no enhancement was obtained in cells after coincubation with the anti-TIRC7 Ab plus the corresponding soluble peptide or in lymphocytes incubated with control Ab. Data represent one example of seven independent experiments. D, Proliferation was determined by DNA incorporation of [3H]thymidine in PBL activated for 48 h with PHA alone (PHA), in the presence of anti-TIRC7 Ab (TIRC7 Ab), coincubation of anti-TIRC7 Ab and the nonlabeled blocking anti-CTLA-4 mAb 4F10 (TIRC7 Ab + CTLA-4 Ab), coincubation of anti-TIRC7 Ab and the corresponding soluble TIRC7 peptide (TIRC7 Ab + P), anti-CTLA-4 mAb 4F10 (CTLA-4 Ab), nonfunctional anti-TIRC7 Ab (Control Ab1), and isotypic control mouse IgG Ab (Control Ab2), respectively. Incubation of PBL with anti-TIRC7 Ab inhibited the PHA-induced proliferation. Control Abs Ab1 and Ab2 had no effect. The inhibition of proliferation was prevented by coincubation of anti-TIRC7 Ab with its soluble peptide, and coincubation of anti-TIRC7 Ab and anti-CTLA-4 mAb 4F10 showed a similar effect. Anti-CTLA-4 mAb 4F10 alone and TIRC7 Ab in combination with CTLA-4 mAb caused an increased proliferation compared with control cells. The results represent the means and SD in cpm of seven independent experiments (*, p < 0.01 vs controls). E, The same set of experiments was performed, as described in D, and the levels of the cytokines IL-2, IFN-{gamma}, and IL-4 in the culture supernatants were determined 48 h after PHA activation of PBL. Treatment of PHA-activated cells with anti-TIRC7 Ab resulted in an inhibition of IL-2 and IFN-{gamma} secretion. The inhibitory effect of the anti-TIRC7 Ab was competed with the corresponding soluble peptide. A similar effect on the inhibition of secretion of IL-2 and IFN-{gamma} was obtained by coincubation of anti-TIRC7 Ab with anti-CTLA-4 mAb 4F10. No Ab exhibited a significant effect on the secretion of the Th2-related cytokine IL-4.

 
Ab blockade of CTLA-4 abolishes the anti-TIRC7 Ab-mediated inhibition of T cell activation

The results obtained so far led us to examine whether the antiproliferative effect associated with Ab targeting of TIRC7 was dependent on CTLA-4 expression. Specifically, we investigated whether Ab blockade of CTLA-4 abolished the antiproliferative effect mediated by TIRC7. For this purpose, the anti-CTLA-4 mAb (mAb) 4F10 was used that has been demonstrated previously to prevent ligand binding of CTLA-4 to B7 and to inhibit the negative costimulatory effect of CTLA-4 on lymphocyte proliferation in a dose-dependent manner (3). PHA-activated human PBL were coincubated with anti-TIRC7 Ab in either the presence or absence of anti-CTLA-4 mAb 4F10, and T cell proliferation was assessed by DNA incorporation of [3H]thymidine. As shown in Fig. 5D, the anti-TIRC7 Ab alone strongly inhibited the PHA-induced proliferation of PBL, as predicted by previous studies (11). When anti-CTLA-4 mAb 4F10 was added to the culture, however, the inhibitory effect of the anti-TIRC7 Ab was completely reversed. A similar inhibition of proliferation was prevented by coincubation of anti-TIRC7 Ab with its soluble peptide corresponding to the TIRC7 domain recognized by anti-TIRC7 Ab. When mAb 4F10 was administered alone, an increase of [3H]thymidine incorporation was observed in comparison with control cells, as has been described previously by others (3). The results indicate that the inhibition of proliferation associated with anti-TIRC7 Ab targeting is, at least to a substantial part, mediated by CTLA-4.

CTLA-4 blockade abolishes TIRC7 Ab-mediated cytokine inhibition

The functional association between Ab targeting of TIRC7 and CTLA-4 expression was also examined at the level of cytokine production. In vitro and in vivo studies by others have demonstrated that blockade of CTLA-4 with mAb 4F10 resulted in an increased Th1-associated cytokine production after T cell activation (8, 9). To examine whether Ab blockade of CTLA-4 altered the decrease of Th1 cytokine production associated with Ab targeting of TIRC7 (11), PHA-activated PBL were coincubated with anti-TIRC7 Ab in either the presence or absence of anti-CTLA-4 mAb 4F10, and the production of IL-2, IFN-{gamma}, and IL-4 was examined 48 h after stimulation. As shown in Fig. 5E, coincubation of PHA-activated cells with anti-TIRC7 Ab alone resulted in a significant down-regulation of both IL-2 and IFN-{gamma} production. As expected, the inhibitory effect of the anti-TIRC7 Ab was competed successfully with soluble TIRC7 peptide, confirming the specificity of cytokine down-regulation by anti-TIRC7 Ab. A similar rescue of cytokine production was achieved when the cell culture was coincubated with anti-CTLA-4 mAb 4F10 in the presence of anti-TIRC7 Ab. In contrast, neither Ab had a significant effect on production of the Th2-related cytokine IL-4 (Fig. 5E). Thus, the results further indicate that the TIRC7-mediated modulation of the immune response, as defined by down-regulation of Th1-associated cytokine production, relies on signals provided by CTLA-4.

Anti-TIRC7 Ab exhibits antiproliferative effects only in the presence of CTLA-4

To differentiate between the CTLA-4-mediated antiproliferative effects and a possible inhibition of proliferation mediated by TIRC7 blockade or other TIRC7-triggered pathways, the effect of anti-TIRC7 Ab was examined in lymphocytes obtained from CTLA-4-deficient mice. Lymphocytes isolated from these mice were previously demonstrated to exhibit hyperreactivity with evidence of spontaneous activation (30, 31, 32). If the antiproliferative effect of anti-TIRC7 Ab was mediated by mechanisms other than CTLA-4, at least an attenuation of the hyperreactivity of CTLA-4-deficient lymphocytes would have been expected. Lymphocyte activation was examined by [3H]thymidine incorporation in the presence and absence of anti-TIRC7 mAb. As shown in Fig. 6, no alteration in the proliferation of CTLA-4-deficient lymphocytes was obtained by treatment with anti-TIRC7 Ab. In contrast, a strong inhibitory effect of this Ab was observed in lymphocytes isolated from control BALB/c mice. Also, lymphocytes isolated from IL-2-deficient mice showed substantial inhibition of proliferation when incubated with anti-TIRC7 Ab, indicating that the lack of IL-2 was not sufficient to abolish the effect of the anti-TIRC7 Ab. These results confirm that the antiproliferative function of TIRC7 blockade is based, at least to a substantial part, on the up-regulation of CTLA-4 expression on T lymphocytes.


Figure 6
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  		FIGURE 6. The antiproliferative effect of anti-TIRC7 Ab is abolished in the absence of CTLA-4. Lymphocytes from CTLA-4-deficient mice (CTLA-4–/–) and the corresponding wild type (CTLA-4+/+) from BALB/c mice (BALB c), IL-2-deficient mice (IL-2–/–), and the corresponding wild type (IL-2+/+), respectively, were isolated and cultured either in the presence of anti-TIRC7 mAb (TIRC7 Ab) or isotypic control mouse IgG1 Ab (Control Ab), and proliferation was examined by determination of [3H]thymidine incorporation. The proliferation of CTLA-4-deficient lymphocytes was not affected by incubation with anti-TIRC7 Ab, whereas a strong inhibitory effect of this Ab was observed in lymphocytes isolated from control BALB/c mice. Also, lymphocytes isolated from IL-2-deficient mice showed inhibition of proliferation when incubated with anti-TIRC7 mAb. Data represent one experiment of three.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although the biochemical mechanisms of CTLA-4 signaling remain unclear to date and the specific pathways are not well defined, CTLA-4-derived signals have been described as being crucial for the regulation of peripheral tolerance in several studies (10, 33, 34, 35). We have demonstrated recently that TIRC7 is involved in the modulation of the immune response in vitro and in vivo, exhibiting similar effects as described for CTLA-4 (11). We therefore aimed to determine whether CTLA-4 is involved in TIRC7-mediated signaling. Previous observations from our laboratory are in support of this hypothesis. Lymphocytes from TIRC7-deficient mice before and after activation exhibit a markedly reduced intracellular and cell surface expression of CTLA-4 compared with wild-type lymphocytes, indicating a disturbed regulation of CTLA-4 expression in the absence of TIRC7 (13). In addition, in mice treated with cardiac transplantation with a full mismatch barrier, the administration of anti-TIRC7 Ab was associated not only with hyporesponsiveness of the lymphocytes and significantly prolonged allograft survival, but also with an increase of the CTLA-4 expression on the cell surface (12).

The results of our present study confirm the hypothesis that the hyporesponsiveness of T lymphocytes induced by Ab targeting of TIRC7 is mediated, at least to a substantial part, by CTLA-4. Although other signaling pathways, like pathways in B lymphocytes (13), may also be affected by TIRC7 and contribute to the TIRC7-mediated inhibition of proliferation, the complete reversal of both the effects of anti-TIRC7 Ab-induced T cell hyporesponsiveness and the inhibition of Th1-specific cytokine production by Ab blockade of CTLA-4 strongly suggests that the inhibitory effect of TIRC7 on T cells is executed mainly via CTLA-4. The up-regulation of CTLA-4 mRNA in human T cells upon TIRC7 Ab targeting and the early and sustained up-regulation of CTLA-4 expression on the cell surface within only few hours indicate a close regulatory link between both molecules, and strongly suggest that TIRC7 functions as an upstream molecule in a negative signaling cascade involving CTLA-4. The induction of CTLA-4 mRNA synthesis and CTLA-4 expression upon TIRC7 cross-linking may be due to enhancement of CTLA-4 translocation of preformed intracellular CTLA-4 to the cell membrane and/or increased CTLA-4 synthesis via specific signaling through TIRC7. These findings gain significance in the light of recent studies that suggest that the level of constitutively expressed CTLA-4 on lymphocytes may play an important role in regulating autoimmune responses (35), and that establishing tolerance in peripheral lymphocytes is to a great extent dependent on CTLA-4 induction (36). The results of our study indicate that anti-TIRC7 Ab delivers signals through a novel TIRC7-mediated mechanism that on the one hand shifts the time course of CTLA-4 up-regulation, which normally occurs ~48 h after induction of T cell activation, to an earlier time point, and in contrast causes a sustained induction of CTLA-4. Both types of modulation may override the proliferative signaling pathways provided by CD28 and other molecules and may lead to an early and prolonged state of T cell anergy. Thus, the results suggest that TIRC7 represents an important modulating molecule for CTLA-4 expression.

The idea of TIRC7 being involved in early signaling pathways is further substantiated by the finding that, upon alloactivation, TIRC7 is directionally transported toward the site of Ag contact, which is also referred to as the immunologic synapse (21, 25). Upon T cell activation, a great number of receptor molecules that are known to be involved in the costimulation of T lymphocytes have been demonstrated to form a cluster in the cell membrane area next to the attached Ag, including the TCR, CD4, ICAM-1 (37), LFA-1 (22), as well as CTLA-4 (15) and CD28 (16). The orientation of TIRC7 toward the Ag adhesion site was found to be induced shortly after Ag recognition. This is in accordance with the results of previous studies demonstrating the early cell membrane expression of TIRC7 upon T cell activation (11), and suggests that TIRC7 is involved in early events in the T cell activation process.

In addition, the present results suggest that TIRC7 is transported toward and from the cell membrane via clathrin-coated vesicles, as was also described for several other costimulatory molecules (15, 38, 39, 40). Moreover, the early appearance at the Ag adhesion site is also a result of reorientation of membrane-bound TIRC7 toward the immunologic synapse. The finding of colocalization of TIRC7 and CTLA-4 in clathrin-coated vesicles raises the question as to whether the effect of TIRC7 on CTLA-4 expression may be due to a TIRC7-triggered regulation of vesicle trafficking. Although the exact function of TIRC7 remains to be elucidated, the sequence homologies of TIRC7 with subunits of a variety of vacuolar proton pump/H+-ATPases, including the clathrin-coated vesicle-bound rat vacuolar H+-ATPase subunit (11), may indeed suggest a role of this protein in the regulation of cellular and vesicle ion gradients. However, if TIRC7 would be acting by effects on vesicle trafficking, the vesicle transport of molecules other than CTLA-4 would be most likely affected as well. As demonstrated in an earlier study, in lymphocytes isolated from TIRC7-deficient mice, the membrane expression of molecules known to be transported via clathrin-coated vesicles such as CD71 (38) was found not to be altered, whereas CTLA-4 surface expression was virtually absent (13). These results strongly suggest that the selective effect of TIRC7 on CTLA-4 expression is induced by mechanisms other than a general function of TIRC7 in the clathrin-coated vesicle system.

The overall results of this study indicate a close link between TIRC7 signaling and functional expression of CTLA-4, and provide strong evidence for the conclusion that TIRC7 acts as an upstream regulating molecule of CTLA-4 expression and function. Also, the study demonstrates a regulatory effect of TIRC7 on CTLA-4 in that TIRC7 alters the expression and time course of CTLA-4 expression upon T cell activation. Given the importance of CTLA-4-mediated negative costimulation for the function of lymphocytes (5, 41, 42) and the use of the CTLA-4 pathway in the treatment of autoimmune disease in current clinical studies (43), the selective modulation of the T cell-mediated immune response by anti-TIRC7 Ab binding might provide a novel avenue for therapeutic interventions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
N. Utku is Director and Scientific Founder of GenPat 77 Pharmacogenetics AG, and also owns stock in the company.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Nalân Utku, Humboldt University of Berlin, Schumannstrasse 20/21, D-10115 Berlin, Germany. E-mail address: nalan.utku{at}charite.de Back

Received for publication February 22, 2006. Accepted for publication August 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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