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The Journal of Immunology, 2000, 164: 4533-4542.
Copyright © 2000 by The American Association of Immunologists

Crry/p65, a Membrane Complement Regulatory Protein, Has Costimulatory Properties on Mouse T Cells1

Elena Fernández-Centeno*, Gloria de Ojeda*, José M. Rojo{dagger} and Pilar Portolés2,*

* Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III; and {dagger} Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is known that certain type I membrane molecules (complement receptors type 1 and 2) belonging to the regulators of complement activation (RCA) family are involved in the regulation of B lymphocyte activation. In contrast, only GPI-anchored RCA molecules (CD55) have been described to be involved in T lymphocyte activation. In this study, we describe a novel function for the mouse RCA type I membrane protein Crry/p65 as a costimulatory molecule in CD4+ T cell activation. This is shown by increased anti-CD3-induced proliferation of CD4+ spleen T lymphocytes in the presence of the Crry/p65-specific mAb P3D2. Furthermore, Ab-induced coligation of Crry/p65 and CD3 favors IL-4 rather than IFN-{gamma} secretion in these cells. Crry/p65 signaling was also observed regardless of additional Ca2+, protein kinase C, or CD28-mediated costimuli. Analysis of intracellular intermediaries shows that Crry/p65-CD3 coligation enhances certain TCR/CD3-mediated signals, producing increased early tyrosine phosphorylation of many substrates and enhanced activation of the mitogen-activated protein kinase, extracellular signal-related kinase. These data fit well with the association of Crry/p65 with the tyrosine kinase Lck found in T cell lysates. The epitope recognized by the mAb P3D2 interferes with the protective role of Crry/p65 on C3 deposition. The relationship between protective function and costimulation by Crry/p65 is discussed. Our results support a multifunctional role for Crry/p65 in T cells and suggest new links between the natural and adaptive immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Complement is a system of plasma proteins that greatly enhances the ability of phagocytes to engulf and destroy certain microorganisms. In addition, complement activation is necessary to develop a normal adaptive immune response (1, 2, 3, 4). Mammals possess several mechanisms to protect bystander cells of the deleterious effects of autologous complement activation. These protective mechanisms involve serum and membrane molecules (complement regulatory proteins (CRP)3), which specifically recognize and inactivate components of the classical or alternative pathways of complement activation (5, 6). Many of these molecules, including complement receptors type 1 (CR1; CD35) and 2 (CR2; CD21), membrane cofactor protein (MCP; CD46), and decay-accelerating factor (DAF; CD55), belong to a family of structurally and genetically related proteins known as regulators of complement activation (RCA). Characteristically, these molecules contain homologous repetitive domains called "short consensus repeats," which are responsible for the protective function against autologous complement activation (5, 6). Proteins able to interact with membrane-bound C components possess one or both of two types of activity, known as "receptor" and "regulatory." The first one facilitates the recognition of complement factors deposited on surfaces or immune complexes, promoting their clearance. On the other hand, the main functions of membrane proteins showing complement regulatory activity are decreasing complement activation and deposition on the membrane in which they reside (reviewed in Ref. 5).

Several RCA proteins are expressed in B and T lymphocytes. In humans, membrane complement receptors CR1 and CR2, mainly recognizing C3b or C3d deposited on other surfaces, are expressed on B lymphocytes and certain T cells. Another two molecules of wide cellular distribution, DAF (CD55) and MCP (CD46), are expressed by lymphocytes to inactivate C3b- and C4b-containing C convertases deposited on their cell surface (reviewed in Ref. 5). CR2 in humans and CR1 and CR2 in mice are involved in the regulation of B cell Ag activation, amplifying Ab responses (1, 7, 8). In addition, expression of the CD28 ligands B7-1 and B7-2 in murine splenic B cells is increased by CR2/CR1/surface Ig co-cross-linking (9). Furthermore, experiments with knock-out mice suggest that certain T-dependent Ags are absolutely dependent on complement receptors for the development of normal immune responses (10, 11). Thus, activation of the complement cascade and ligation of complement C3 receptors in B cells represent an important bridge between innate and Ag-specific, acquired immunity. Apart from CR1 and CR2, mouse B lymphocytes also express Crry/p65 (Crry), a CRP of widespread distribution, which possesses both DAF and MCP activity (5, 12, 13). However, unlike CR1/CR2 ligation, binding of mAb to Crry does not modify B cell activation (9).

Expression of membrane CRPs is different in mouse and human T lymphocytes. Unlike human T cells, CR1, CR2, or MCP are not expressed in mouse T cells (14, 15, 16, 17). Thus, protection from autologous complement is mainly achieved by Crry and perhaps murine DAF (18). Data showing the costimulatory role of CRPs in T cells are limited so far to human DAF (CD55), a GPI-linked surface protein (19, 20). This makes it particularly interesting to analyze the possible influence of type I membrane proteins like Crry on mouse T cell activation.

In this paper, we describe a rat mAb (P3D2) that recognizes mouse Crry, which inhibits Crry-mediated protection of C3 deposition and has costimulatory properties on normal isolated CD4+ T cells and mouse T cell lines in vitro. Proliferation of purified CD4+ T cells induced by anti-CD3 is increased by Crry-mediated signaling, even in the presence of other costimulatory signals like anti-CD28. Analysis of secreted ILs shows that IL-4, rather than IFN-{gamma}, is favored, suggesting that Crry ligation may have implications in the development of Th1 and Th2 subpopulations as well as in the balance of immune responses. Furthermore, in vitro kinase assays show that the cytoplasmic Lck kinase coprecipitates with Crry. This association fits with the increase in phosphotyrosine (P-Tyr) substrates, including the mitogen-activated protein kinase (MAPK) extracellular signal-related kinase (ERK), found in lysates of CD4+ T cells stimulated by anti-CD3 plus anti-Crry.

To our knowledge, this is the first description of a bifunctional role of Crry in mouse T cells. The relationship between protective function and costimulation by Crry suggests new links between the natural and adaptive immune response.


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

Male or female mice of the C3H/He or BALB/c strains maintained in the facilities of the Centro de Investigaciones Biológicas or the Instituto de Salud Carlos III were used. Sex-matched mice aged 8–12 wk were used in every single experiment. Lewis rats were used for immunization and generation of rat x mouse hybridomas.

Cell lines

SR.D10 is a subclone (21) of the murine CD4+ Th2 cell line D10.G4.1 (22). It was maintained in Click’s EHAA medium supplemented with 10% heat-inactivated FCS (culture medium) by stimulation at 5 x 104/ml every 2 wk, with mitomycin C-treated H-2k spleen cells of C3H/He mice (2.5 x 105/ml) as APCs plus 100 µg/ml conalbumin (Sigma, St. Louis, MO). Cells were rested at least 10 days before using in vitro experiments. AE103 is an I-Ak-specific, Th1 CD4+ mouse T cell line (23) and was grown in culture medium supplemented with mouse IL-2 (10 U/ml).

Human K562 cells transfected with sense or antisense Crry/p65 constructs (12), were kindly provided by Dr. V. Michael Holers (Health Sciences Center, University of Colorado, Denver, CO) and were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml gentamicin, and 2 mM L-glutamine.

Generation of the P3D2 hybridoma

Lewis rats were immunized by four i.p. injections of 4 x 107 SR.D10 cells. Spleen cells from immunized rats were used to obtain hybridomas by fusion with Sp2/0 cells by standard fusion and selection methods (24). Wells containing Abs recognizing surface proteins expressed at higher levels in SR.D10 cells than in normal T lymphocytes were initially selected, cloned by limiting dilution, and analyzed for their effect on the activation of CD4+ T cells and T cell lines. One of these clones, termed P3D2, which produced a rat IgG2a, was chosen for further analysis and is described here.

Abs, lymphokines, and other reagents

The following mAbs were used: YCD3-1 (anti-mouse CD3{epsilon}, rat IgG2b) (25); GK1.5 (anti-mouse CD4, rat IgG2b) (26); 53-6.72 (TIB 105, anti-mouse CD8, rat IgG2a) (27); M1/70 (anti-CD11b, rat IgG2b) (28); 37.51 (anti-CD28, hamster IgG2) (29); M1/9.3.4HL.2 (anti-Pan CD45, rat IgG2a) (30); C363.16A (anti-CD45RB, rat IgG2a) (31); 11B11 (anti-mouse IL-4, rat IgG1) (32); and XMG1.2 (anti-mouse IFN-{gamma}, rat IgG1) (33). P2G9 (anti-mouse class I MHC Kk {alpha}3 domain, rat IgG2b) and P3E3 (anti-mouse class I MHC Kk {alpha}1–{alpha}2 domain, rat IgG2b) were obtained as described for P3D2. The Abs were purified from ammonium sulfate precipitates of culture supernatants by chromatography on protein G-Sepharose columns (Amersham Pharmacia Biotech, Little Chalfont, U.K.), except YCD3-1 and 37.51, which were purified on protein A-Sepharose columns (Amersham Pharmacia Biotech). 5D5 (anti-Crry, rat IgG1) (14) was kindly given by Dr. V. M. Holers. Purified XMG1.2 was biotinylated by standard procedures. Rat mAb specific for mouse IFN-{gamma} R4-6A2 (rat IgG1) was purchased from ImmunoKontact (Bioggio, Switzerland). Biotinylated anti-mouse IL-4 BVD6-24G2 (rat IgG1) was purchased from PharMingen (San Diego, CA). HRP-conjugated anti-P-Tyr Ab (PY-20-HRP) was obtained from Amersham Pharmacia Biotech. Affinity-purified rabbit polyclonal anti-active MAPK Ab (Promega, Madison, WI) was used to detect dually phosphorylated (Thr183 and Tyr185) p44 or p42 MAPK active forms (ERK1 and ERK2, respectively). Polyclonal rabbit anti-mouse Ig and anti-rat Ig antisera were obtained by immunization with protein A-purified normal mouse and rat Ig, respectively. Rabbit antisera to human Fyn (residues 35–51) and human Lck (residues 22–51) cross-reactive with murine Fyn and Lck, respectively, were purchased from Upstate Biotechnology (Lake Placid, NY). A rabbit antiserum to mouse CD4 (no. 19) was obtained by repeated immunization of a synthetic peptide (residues 49–62 of the mouse CD4 sequence) coupled to OVA with glutaraldehyde. HRP-conjugated goat anti-rabbit Ig Ab from Sigma-Aldrich (St. Louis, MO) and FITC-conjugated anti-mouse C3 Ab from ICN Cappel (Costa Mesa, CA) were used. Goat anti-rat IgG-FITC Ab was purchased from Calbiochem Novabiochem (San Diego, CA). Mouse recombinant IL-4 and IFN-{gamma} were from ImmunoKontact or from Genzyme (Boston, MA). As a source of mouse recombinant IL-2, culture supernatants from transfected cell line X63Ag8-653 BMGNeo mIL-2 (34) kindly provided by Drs. F. Melchers and C. A. Janeway, Jr., were used. Ionomycin (Iono) from Calbiochem Novabiochem and phorbol 12,13-dibutyrate (PDB) from Sigma were used.

Flow cytometry

A total of 5 x 105 cells were incubated for 30 min at 4°C with saturating amounts of Abs in 100 µl of staining buffer (0.1% NaN3 and 5% FCSi in PBS). The cells were then washed three times in the same buffer and incubated for 30 min with FITC-conjugated goat anti-rat Ab in the same conditions. Both incubations were performed in the presence of 10% heat-inactivated normal mouse serum to avoid nonspecific binding of Abs. After washing with PBS containing 0.1% NaN3, the cells were fixed in 1% paraformaldehyde and analyzed on a FACScan (Coulter Electronics, Miami, FL).

Inhibition by mAb of Crry regulatory activity on the classical pathway

The effect of mAb on Crry-mediated protection of C3 deposition was determined as described by Li et al. (14), with minor modifications. A total of 106 SR.D10 Th2 cells expressing Crry were incubated with the mAb to be studied (P3D2 or isotype-matched rat mAb) in Ca2+/Mg2+-containing medium (gelatin Veronal-buffered saline) for 30 min at 4°C. Then, 5 µl of fresh BALB/c mouse serum was added and kept for 30 min at 37°C to allow complement activation. Cells were washed three times at 300 x g with PBS containing 1% BSA and 0.1% sodium azide and were stained with FITC-conjugated goat anti-mouse C3 (ICN Cappel) as described above. Preliminary experiments were performed with K562 cell lines transfected with sense or antisense constructs of Crry cDNA (12) to determine the amount of mouse serum to be used; the amount of fresh serum used (5 µl) allows the binding of C3 in the linear range of the experiment producing less than maximal C3 deposition on cells expressing antisense constructs. In these conditions the cells were not lysed during the experiments.

Isolation of CD4+ T lymphocytes

CD4+ T cells were isolated by Ig/anti-Ig columns (35). Spleen cell suspensions depleted of erythrocytes were washed and incubated for 30 min at 4°C in culture medium containing anti-CD8 and anti-CD11b Abs (1 µg/106 cells). Then, cells were washed four times, resuspended in the same medium, and passed through a mouse Ig column previously incubated with rabbit anti-mouse Ig serum, which cross-reacts with rat Ig. The Ig-anti-Ig column was exhaustively washed before use. Cells in the flow-through were routinely >95% viable and flow cytometry analysis revealed a >95% CD3+, >90% CD4+, and <2% CD8+ population. These cells did not proliferate to soluble anti-CD3 Abs in the absence of a cross-linking Ab bound to a solid phase unless exogenous APCs were added, indicating negligible accessory cell contamination.

Stimulation of lymphocytes

In vitro primary stimulation was conducted in flat-bottom 96-well culture plates (catalog no. 3598; Costar, Cambridge, MA) coated with the Abs at concentrations and conditions indicated in the figure legends. Coating was performed by overnight incubation of 50 µl of affinity-purified Abs in PBS at 4°C. The plates were then thoroughly washed with PBS, and 105 purified CD4+ T cells were added in 200 µl of culture medium. The plates were centrifuged at 400 x g for 1 min and incubated for 3 days at 37°C in 5% CO2. Then, 0.1 ml of culture supernatants was taken for lymphokine analysis, and cell proliferation was determined by adding MTT as described (36). Initial experiments showed equivalent results using the MTT colorimetric assay or [3H]thymidine incorporation in this system. Only experiments with SE <= 10% of the mean of triplicate cultures are depicted. Statistical differences between groups within an experiment were calculated using the Statworks software and Student’s t test. A p value less than 0.05 was considered significant.

Secondary stimulation was performed using cells previously activated in vitro for 6 days with fixed Abs as described above, and then they were resuspended and washed in culture medium. Activated cells (6 x 104/well) were then cultured in a final volume of 200 µl in the presence of 10 µg/ml anti-CD3 Abs plus 5 x 105 irradiated spleen cells as feeders. After 3 days of incubation at 37°C in 5% CO2, 100 µl of culture supernatant were taken for lymphokine analysis, and cell proliferation was determined using the MTT colorimetric assay as described above.

Lymphokine assays

IL-4 and IFN-{gamma} were measured in serial dilutions of culture supernatants by specific capture ELISA as recommended by PharMingen (San Diego, CA). Reference standard curves were set up in each assay with purified recombinant lymphokines. For IL-4, 11B11 was used for capture, and biotinylated BVD6-24G2 was used for detection. For IFN-{gamma}, R4-6A2 was used for capture, and purified biotinylated XMG1.2 was used for detection. Development was performed using streptavidin-HRP and o-phenylenediamine (Sigma). Results show the mean of lymphokine concentration inferred from the titration of each supernatant. Only experiments with SE <= 15% of the mean are shown.

Immunoprecipitation

For each immunoprecipitation, 107 cells were biotinylated with 100 µg of Sulfo (N-hydroxysuccinimide)biotin (Pierce Europe) as described in Ref. 37 and lysed for 15 min on ice with 1 ml of lysis buffer (1% Triton X-100 in 50 mM Tris (pH 7.6), 300 mM NaCl, 1 mM sodium orthovanadate, 1 mM PMSF, and 10 µg/ml aprotinin). Postnuclear supernatants were precleared twice by end-over-end rotation with 20 µl of normal rat Ig covalently coupled to Sepharose 4B beads (Amersham Pharmacia Biotech). Immunoprecipitations were performed for 1 h at 4°C with 15 µl of specific Ab covalently coupled to Sepharose 4B or mAb adsorbed to goat anti-rat IgG Agarose (Sigma-Aldrich). The precipitates were washed five times with 0.2% SDS, 0.2% sodium deoxycholate, and 0.1% Triton X-100 in 50 mM Tris/HCl (pH 7.6) and 300 mM NaCl and were extracted with double- strength reducing SDS Laemmli sample buffer. Samples were separated in 10% acrylamide gels, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA). Biotinylated proteins were detected with streptavidin-HRP and the Enhanced Chemiluminescence system (Amersham Pharmacia Biotech) as suggested by the manufacturer.

In vitro kinase assays

Cells (107/determination) were lysed as above, except that 10 mM 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Pierce Europe, Oud-Beijerland, Holland) instead of Triton X-100 was used. Immunoprecipitates were processed as described above and washed four times with 2 mM CHAPS in 50 mM Tris (pH 7.6), 300 mM NaCl, 1 mM sodium orthovanadate, 1 mM PMSF, and 10 µg/ml aprotinin (washing buffer).

Kinase assays were performed by addition to washed immunoprecipitates of 50 µl of kinase assay buffer (2 mM CHAPS in 25 mM HEPES (pH 7.2), 10 mM MgCl2, 3 mM MnCl2, and 1 mM sodium orthovanadate) containing 10 µCi [{gamma}-32P]ATP (30 Ci/mmol). After incubation for 20 min at room temperature with frequent mixing, the reaction was stopped by addition of 1 ml of ice-cold washing buffer, centrifugation, and extraction with 60 µl of double-strength reducing SDS-PAGE sample buffer. The samples were boiled for 5 min, and the phosphorylated products were separated on 10% acrylamide gels and visualized by autoradiography.

For reprecipitation of phosphorylated proteins, the kinase reaction was stopped by addition of 1 ml of ice-cold washing buffer and centrifugation. To disrupt weak, noncovalent bonds between phosphorylated polypeptides, the washed immunoprecipitates were then treated for 30 min with 0.5 ml of 1% SDS, 0.2% sodium deoxycholate, and 0.1% Triton X-100 in 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 1 mM sodium orthovanadate. After centrifugation, the supernatant was taken and diluted with 50 mM Tris-HCl (pH 7.6) and 300 mM NaCl to bring the SDS to 0.1%. Aliquots of the diluted supernatant were reprecipitated with anti-Fyn or anti-Lck antisera coupled to protein A-Sepharose. After rotation for 16 h at 4°C, the beads were washed twice with "washing buffer," and the proteins were extracted and analyzed by SDS-PAGE electrophoresis as described above.

Immunoblot for P-Tyr and active MAPK

Total P-Tyr and P-Tyr185/phosphothreonine183-MAPK were detected in lysates from 3.3 x 105 CD4+ T cells/well activated by plate-adsorbed Abs as described above, except that serum-free culture medium was used. Cells were briefly centrifuged, incubated at 37°C for the times indicated, and the plates were "flicked" and blotted on filter paper. Then, 0.1 ml of reducing SDS-PAGE Laemli sample buffer containing 1 mM sodium orthovanadate was added to each well. Contents from triplicate wells were pooled, sheared by passage through needles, and boiled for 5 min. A total of 60 µl of lysate (equivalent to 2 x 105 cells) was separated by SDS-PAGE in 10% acrylamide gels and transferred to PVDF membranes (Immobilon-P; Millipore). HRP-conjugated PY-20 anti-P-Tyr Ab was used to detect total P-Tyr. Blots were washed and developed using the Enhanced Chemiluminescence system (Amersham Pharmacia Biotech). "Stripping" of PVDF membranes was performed as suggested in the supplier’s protocol. The same membranes used for P-Tyr detection were sequentially probed to detect active MAPK or CD4 (as a control for loading and blotting of the membranes). Densitometric analysis of the bands was performed in a Fuji Bas Equipment (Fuji, Tokyo, Japan) with a PC Bas 2.09 computer program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mAb P3D2 binds to Crry

To identify new cell surface molecules involved in mouse T lymphocyte activation, Lewis rats injected with cells of the SR.D10 mouse Th2 clone (21) were used to obtain mouse x rat hybridomas. The P3D2 hybridoma secreted a rat IgG2a mAb that was initially selected because it recognized a surface protein expressed at higher levels in the SR.D10 clone than in normal T lymphocytes. This molecule was expressed by all hemopoietic cells analyzed, including cells from the spleen, thymus, lymph nodes, bone marrow, and erythrocytes (not shown).

Immunoprecipitation of biotinylated cell surface molecules from SR.D10 cells indicated that P3D2 recognizes a molecule of 56–60 kDa in this cell line. Its cellular distribution and its molecular mass characteristics suggested the C regulatory protein Crry as one possible candidate to be the Ag recognized by P3D2. In fact, the molecules immunoprecipitated by P3D2 and 5D5, a previously described rat anti-mouse Crry mAb (14), are similar (Fig. 1GoA). Preclearing of SR.D10 lysates with P3D2 or 5D5 and immunoprecipitation with P3D2 (Fig. 1GoA, right panel) indicated that both mAbs depleted the lysates of the molecule recognized by P3D2.



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  		FIGURE 1. P3D2 recognizes Crry, a murine complement regulatory molecule. A, Immunoprecipitation of biotinylated surface proteins from SR.D10 cells with P3D2 or the anti-Crry Ab, 5D5 (left panel). Anti-CD8 Ab (53-6.72, TIB 105) was used as control. Lysates immunodepleted with TIB 105, P3D2, or 5D5 were reprecipitated with P3D2 Ab (right panel). B, Flow cytometry analysis of P3D2 (filled histograms) binding to sense (right panel) or antisense (left panel) rCrry transfectants of human K562 cells. Anti-CD8 Ab was used as control (thin lines).

 
The specificity of P3D2 for Crry was confirmed by flow cytometry using human K562 cells transfected with Crry cDNA in sense or antisense orientation. Fig. 1GoB shows that Crry is specifically recognized: P3D2 binds K562 cells transfected with sense Crry cDNA (right panel) but not those transfected with antisense rCrry cDNA (left panel).

P3D2 inhibits the protective function of Crry

We analyzed whether the binding site of P3D2 was involved in Crry-mediated protection from C deposition on cell membranes. Fig. 2Go (top panel) shows that no C3 deposition was observed in the absence of added fresh mouse serum (broken line). Basal C3 deposition was determined by adding fresh mouse serum and 53-6.72, an isotype-matched rat mAb unable to bind to the cells (Fig. 2Go, top panel, thin line). When cells were incubated with anti-Crry Ab P3D2, C3 deposition increased 10-fold (Fig. 2Go, top panel, filled histogram). In contrast, incubation with C363.16A, a rat IgG2a Ab specific for CD45RB, did not increase C3 deposition (Fig. 2Go, top panel, thick line), despite the fact that the expression levels of Crry and CD45RB in SR.D10 cells are similar (Fig. 2Go, bottom panel). These results show that P3D2 inhibits the protective function of Crry against C3 deposition on Th2 cells. Similar results were obtained with the human cell line K562 transfected with sense or antisense murine Crry (data not shown).



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  		FIGURE 2. P3D2 binding inhibits Crry-mediated protection, increasing C3 deposition on cell membranes. C3 deposition on SR.D10 Th2 cells was determined by flow cytometry using FITC-labeled anti-C3 Ab (top panel) as described in Materials and Methods. No C3 was detected in the absence of added fresh normal mouse serum (broken lines). Basal C3 deposition was determined by adding fresh mouse serum (as a source of complement) and an isotype-matched rat mAb (anti-CD8 53-6.72, TIB 105) unable to bind to CD4+ Th cells (thin line). Incubation with fresh serum and C363.16A rat IgG2a anti-CD45RB (thick line) does not increase basal C3 deposition on these cells. Binding of anti-Crry P3D2 to SR.D10 cells in the former conditions clearly increases C3 deposition (filled histogram). Crry and CD45RB are expressed at similar levels on SR.D10 cell surface (bottom panel). Flow cytometry analysis of SR.D10 cells stained as described in Materials and Methods: anti-CD8 (53-6.72, TIB 105; thin line), anti-CD45RB (C363.16A; thick line), and anti-Crry P3D2 (filled histogram). All of these mAbs are rat IgG2a.

 
Effect of Crry ligation on the activation of spleen T lymphocytes

To elucidate whether Crry ligation can activate T cells or modify TCR activation, assays were set up in which normal spleen purified CD4+ T lymphocytes were induced to proliferate in vitro by means of anti-CD3 bound to culture plastic plates in the presence of a mAb recognizing the putative costimulatory molecule. Fig. 3GoA shows a clear costimulatory effect mediated by mAb P3D2 or 5D5 binding Crry or by the GK1.5 mAb specific for CD4. As a control, mAb recognizing membrane molecules expressed at levels similar to those of Crry (class I MHC or CD45) were included. The costimulatory effect of mAb specific for class I MHC depends on the epitope recognized, whereas mAb M1/9.3 did not costimulate proliferation in these in vitro assays, despite the high levels of CD45 expression in T cells. In every case, no proliferation was observed in the absence of plated anti-CD3 (open bars).



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  		FIGURE 3. Costimulation mediated by specific mAbs is dependent on the surface molecule recognized on mouse T cells (A). In vitro CD3-dependent proliferation of CD4+ T cells was conducted in plates coated with anti-CD3 (YCD3-1, 50 µg/ml; hatched bars) and a second mAb (at 10 µg/ml) recognizing another surface molecule as indicated in the figure. No proliferation was detected in the presence of a control Ab (open bars) instead of anti-CD3. Costimulation mediated by anti-Crry P3D2 mAb requires physical proximity to the TCR/CD3 complex (B). Assays were performed in the presence of anti-CD3 (50 µg/ml) and a second mAb (10 µg/ml), both bound to plastic plates (condition 2), or bound anti-CD3 plus a soluble second mAb (condition 3). Results in the absence of anti-CD3 but in presence of plastic-bound (condition 1) or soluble (condition 4) second mAb are also shown. Second Abs recognizing CD4 (GK1.5), Crry (P3D2), or isotype-irrelevant control (TIB 105, anti-CD8) were added as indicated in the graph. Anti-CD11b mAb was used as a control in the absence of anti-CD3 (conditions 1 and 4).

 
Because some surface molecules, like CD4, can modify cell proliferation in a positive or negative manner depending on their proximity to the TCR/CD3 complex (38), in vitro assays were set up to analyze this possibility in the case of Crry. Thus, anti-Crry mAb P3D2 was used either bound to plastic plates together with anti-CD3 (YCD3-1) or in solution. Fig. 3GoB shows that coligation of Crry and CD3 using Abs bound to the same solid surface strongly enhanced CD3-induced activation (Fig. 3GoB, condition 2), although P3D2 alone did not induce cell proliferation (Fig. 3GoB, condition 1). The effect is much lower when soluble P3D2 is added to cultures activated by plate-bound anti-CD3 (Fig. 3GoB, condition 3). Also, assays were set up in which T cells were induced to proliferate by plated hamster anti-mouse CD3{epsilon} mAb 2C11, in the presence of cross-linked soluble anti-Crry. In this case, Crry was not able to deliver costimulatory signals (data not shown). These results indicate that signals mediated by P3D2 binding to Crry enhance CD3-dependent proliferation of CD4+ T lymphocytes, provided the TCR/CD3 and Crry are in close proximity because the effect is low when CD3 and Crry are independently cross-linked (as when soluble P3D2 and plastic bound anti-CD3 are used). For comparison, the results obtained with mAb recognizing other costimulatory molecules like CD4 were as expected, namely, costimulation of CD3-dependent proliferation using plate-bound mAbs (Fig. 3GoB, condition 2) or inhibition when soluble anti-CD4 was used (Fig. 3GoB, condition 3).

Crry ligation enhances CD3-dependent activation of CD4+ cells in the presence of other costimuli

To analyze whether costimulatory signals from Crry are overridden by other costimuli, freshly isolated CD4+ cells were challenged to proliferate by plastic bound anti-CD3 and anti-Crry mAb P3D2 plus anti-CD28 mAb (37.51), PDB, or the calcium ionophore Iono. Results shown in Fig. 4Go confirm that signals from Crry need CD3 activation to be detected. PDB, Iono, both, or anti-CD28 did not induce proliferation in the presence of anti-Crry P3D2. On the other hand, anti-Crry mAb P3D2 strongly potentiated CD3 activation when costimulatory signals mediated by PDB or soluble anti-CD28 were present (p < 0.05 when cultures with or without P3D2 are compared). This is not the case for the Ca2+ ionophore Iono, because proliferation mediated by anti-CD3 plus P3D2 becomes only slightly enhanced by Iono.



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  		FIGURE 4. Crry ligation potentiates CD3-dependent proliferation of CD4+ cells in the presence of other costimuli. Proliferation assays of isolated purified CD4+ lymphocytes were performed in the presence of combinations of plate-bound mAbs recognizing CD3 (YCD3-1, 50 µg/ml), Crry (P3D2, 10 µg/ml), or control mAb, as indicated in the figure. Additional costimuli were PDB (2 ng/ml), Iono (200 ng/ml), PDB plus Iono, soluble anti-CD28 (1 µg/ml), or culture medium (None), as indicated in the figure.

 
Crry signals modify the pattern of IL secreted by CD4+ spleen T cells in vitro

As the development of Th1 and Th2 responses is influenced by the balance between IL-4 and IFN-{gamma} production by T cells, the effect of Crry ligation on the production of these murine lymphokines was determined in culture supernatants at different times. Fig. 5GoA shows the costimulatory effect of plate-bound P3D2 on anti-CD3-dependent proliferation in the absence of exogenously added ILs. As shown in this figure and in the former one, the effect of P3D2 is largely independent of costimulation induced by anti-CD28 Ab. IL-4 secretion is clearly enhanced by CD3-Crry coligation in the presence (Fig. 5GoB, right panel) or absence (Fig. 5GoB, left panel) of CD28-mediated signaling (p < 0.05). In fact, in this in vitro system, CD28 and Crry produce qualitatively distinct modifications of the IL secreted pattern: CD28 increases IFN-{gamma} secretion in anti-CD3 stimulated cells as shown in Fig. 5GoC (compare open symbols in left and right panels) but does not affect IL-4 secretion (compare open symbols in left and right panels in Fig. 5GoB). However, Crry-CD3 coligation promotes an increment in IL-4 secretion (compare open and filled symbols in Fig. 5GoB, left panel). IFN-{gamma} secretion was similar in cultures containing anti-CD3 plus anti-Crry or anti-CD3 and control Ab (compare filled and open symbols in Fig. 5GoC, left panel). These results indicate that Crry can deliver costimulatory signals to CD3 activation in CD4+ T lymphocytes. These signals can be observed as increased T cell proliferation and IL-4 production in these cells.



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  		FIGURE 5. Crry signals modify the pattern of ILs secreted by spleen CD4+ T cells stimulated in vitro. Effect on mouse CD4+ lymphocyte proliferation (A) of CD3-Crry coligation by combinations of adsorbed mAbs recognizing CD3 (YCD3-1, 50 µg/ml) and Crry (P3D2, 10 µg/ml; filled bars) in the absence or presence of soluble anti-CD28 (CD28, 1 µg/ml), as indicated in the figure. Isotype-matched anti-CD8 Ab was used as a control (open symbols). IL-4 (B) and IFN-{gamma} (C) production was determined by ELISA in the same cultures activated with anti-CD3 plus P3D2 (filled symbols) or plus the anti-CD8 control Ab (open symbols) in the presence or absence of soluble anti-CD28 (CD28), as indicated. Lymphokines secreted in cultures lacking anti-CD3 were below the detection levels of the system and for simplicity are not shown.

 
As costimulation by P3D2 enhances IL-4 production, the effect of Crry-CD3 coligation on the functional development of CD4+ cells was followed in vitro. Primary cultures of CD4+ T lymphocytes were activated for 6 days with plate-bound anti-CD3 plus or minus P3D2 as described above. Then the cells were harvested, washed, and restimulated with anti-CD3 mAb. After 3 days, supernatants were taken to measure IL-4 and IFN-{gamma}, and proliferation was assessed by MTT assay. IL-4 production by CD4+ T cells that had been stimulated in a primary in vitro culture by anti-CD3 plus P3D2 was increased about 10-fold (Fig. 6GoB), whereas the levels of secreted IFN-{gamma} were similar compared with those in cellsfrom cultures activated by anti-CD3 alone (Fig. 6GoC). However, cells costimulated by P3D2 in a primary culture proliferated less upon secondary in vitro stimulation (Fig. 6GoA). These results indicate that Crry ligation by P3D2 can deliver costimulatory signals to CD4+ T cells, leading to increased proliferation of primary cells and IL-4 secretion. This increased IL-4 secretion is particularly clear in in vitro secondary cultures, suggesting a bias toward the development of a Th2 profile.



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  		FIGURE 6. Effect of Crry ligation on in vitro secondary responses of mouse CD4+ lymphocytes. Isolated CD4+ T cells were stimulated by coligated anti-CD3 plus anti-Crry P3D2 or by anti-CD3 plus control mAb, as indicated in the figure (1ary stimulation). Then, CD4+ T cells were restimulated (2nd stimulation) by 10 µg/ml of anti-CD3 (filled bars) or a control mAb (open bars), as indicated in the figure and under conditions described in Materials and Methods. No anti-Crry mAb was present in the secondary culture in any case. Proliferative responses (A), IL-4 secretion (B), and IFN-{gamma} secretion (C) in the same secondary cultures are shown.

 
In view of these results, we also examined whether Crry signals were exclusively linked to IL-4 production using established Th1 and Th2 CD4+ T cell lines. The results indicated that Crry-mediated costimulation increased IL-4 secretion by Th2 clones but also increased IFN-{gamma} secretion by Th1 cells (data not shown).

Crry ligation modifies early TCR/CD3 signaling

We searched for modifications induced in early TCR/CD3-mediated signaling, in purified CD4+ T cells, or in differentiated Th1 and Th2 cells. Fig. 7GoA shows the kinetics of tyrosine phosphorylation in freshly isolated CD4+ spleen T lymphocytes activated by plastic bound anti-CD3 in the presence or absence of anti-Crry P3D2 as a ligand. Tyrosine phosphorylation of polypeptides of 33, 36–38, 42–43, 45, 55–60, 70–80, and 116–120 kDa as well as of other high molecular mass polypeptides was increased and appeared earlier when T cells were activated by combined anti-CD3 plus P3D2. TCR/CD3-induced early tyrosine phosphorylation was also enhanced by P3D2 in Th2 (SR.D10) or Th1 (AE103) cells (results not shown).



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  		FIGURE 7. Crry ligation modifies early TCR/CD3 signaling. Tyrosine phosphorylation (A) or active MAPK ERK (p42) detection (B) in lysates of CD4+ T lymphocytes. Cells were activated for the times shown (in minutes) in culture wells containing adsorbed anti-CD3 (YCD3-1, 50 µg/ml) plus P3D2 anti-Crry or a control Ab (10 µg/ml) as indicated and were lysed as described in Materials and Methods. The results using cells stimulated with P3D2 or isotype-matched control mAb in the absence of anti-CD3 Abs are also shown. The same membrane in A was stripped and probed (B) sequentially with Abs against phosphorylated active forms of ERK (P-ERK) and anti-CD4 serum (CD4) as a control of load. Densitometric analysis of P-ERK bands normalized against CD4 immunoblot is shown in C ({blacktriangleup}, lysates of cultures stimulated with anti-CD3 plus P3D2 anti-Crry; {square}, lysates of cultures stimulated in the presence of anti-CD3 plus a control mAb).

 
Because one of the main targets of P3D2 costimulation is one polypeptide of apparent molecular mass of 42 kDa and TCR/CD3 activation involves the activation of the 42–44 ERK MAPK, we used an Ab specific for active, Thr- and Tyr-phosphorylated forms of ERK1 (p44) and ERK2 (p42) to determine the effect of P3D2 on ERK activation. Indeed, CD3 plus Crry ligation increased the amount of active, phosphorylated 42-kDa ERK (ERK2) in normal CD4+ lymphocytes between 1 and 10 min of activation (Fig. 7GoB). Densitometric analysis of phosphorylated active forms of ERK (P-ERK) bands is shown in Fig. 7GoC. After 30 min, the differences were smaller. No active MAPK was detected in cells that were not activated by anti-CD3, although in those treated with P3D2 a faint band at 42–44 kDa appeared.

In T cells, early tyrosine phosphorylation induced by TCR/CD3 ligation is mediated by the activity of protein-tyrosine kinases like p56lck, p59fyn, and ZAP-70. Because P3D2 binding alters early tyrosine phosphorylation, we analyzed the possibility that Crry might associate tyrosine kinase activity. Thus, in vitro kinase assays were performed in P3D2 immunoprecipitates (Fig. 8Go) from SR.D10 Th2 or AE103 Th1 cell lines. Fig. 8GoA shows that, in Th2 cells, Crry coprecipitates kinase activity, which phosphorylates polypeptides of 56, 60, and >116 kDa. Reprecipitation with anti-Lck and anti-Fyn specific antisera suggests that p56lck, rather that p59fyn, is the Crry-associated kinase in these cells. Kinase activity could be also coprecipitated with Crry from Th1 cells (Fig. 8GoB).



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  		FIGURE 8. In vitro kinase assay in Crry immunoprecipitates from lysates of SR.D10 Th2 (107/lane) (A) or AE-103 Th1 (B) using Sepharose-conjugated P3D2 mAb. Sepharose-conjugated anti-CD8 mAb (TIB 105) was used as a negative control. Reprecipitation of phosphorylated polypeptides in Crry precipitates from SR.D10 was performed with specific anti-Lck or anti-Fyn to identify the associated kinase. NRS, immunoprecipitation with normal rabbit serum.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The activation of CD4+ T cells depends on signals coming from the TCR complex (responsible for Ag recognition) and from other costimulatory molecules and coreceptors expressed in the cell membrane. The integration of these signals determines which activation pathways will be triggered, resulting in full activation, anergy, or apoptosis as well as the development toward Th1 or Th2 functional phenotypes.

We have found that coligation of the mouse RCA type I membrane protein Crry and the TCR/CD3 complex by means of mAbs strongly enhances CD3-mediated activation in CD4+ spleen T lymphocytes and CD4+ T cell lines. To our knowledge, this is the first time that Crry is described as a molecule involved not only in the control of complement damage to cell membranes but also in T cell activation. Several data suggest the relevance of Crry as an activation molecule: 1) Crry is the main, if not the only, surface molecule protecting mouse T cells from autologous complement attack (see below), 2) it can associate tyrosine kinases of the src family (Fig. 8Go) and can modify early T cell signaling (Fig. 7Go) as well as the pattern of lymphokine secretion in CD4+ T lymphocytes toward a Th2 profile (Figs. 5Go and 6Go), 3) because Crry is widely expressed in cells of hemopoietic origin, it is worth analyzing its role in the functional activation of different cells, making it a possible target to manipulate immune responses, and 4) recently, autoantibodies to Crry have been implicated in the development of pathologies in rats (39). Also, we have shown qualitative differences between Crry and CD28 costimulatory signals: mainly, in these in vitro systems, costimulation by CD28 promotes IFN-{gamma} secretion, whereas that by Crry favors IL-4 production. These findings would allow one to include Crry in the group of surface molecules that deliver signals altering the development of Th1/Th2 populations and participate in the fine balance of the immune response.

Other cell surface molecules of the RCA family are involved in T or B lymphocyte activation in the human and the mouse. These include costimulation of T cell activation by GPI-anchored forms of human DAF, a functional homologue of Crry (40), as well as enhanced Ag activation by human CR2 (CD21) (7) or mouse CR1 (CD35) and CR2 in B cells (9). On the other hand, ligation of CD46 (MCP), another functional homologue of Crry, inhibits certain functions in activated human monocytes (41). However, to our knowledge, the participation of Crry in T cell activation has not been described.

As mentioned above, current data suggest that this membrane molecule is the main, if not the only, C3/C4 regulatory protein expressed in mouse T lymphocytes. Mouse T cells do not express CR2 and, unlike some human T cells, they do not express CR1 either (15, 16, 42). The complement regulatory activity of Crry makes it a functional homologue of human MCP (CD46), DAF (CD55), and CR1 (CD35) (13, 43). Recently, mouse CD46 has been described, but it has a preferential expression in the testis and is barely expressed in other tissues (17). On the other hand, two forms of mouse DAF cDNA have been reported (44), but it is not clear whether these genes encode the same surface molecule recognized by Abs (18), and surface expression in different mouse cell populations needs additional studies. We have also observed that C3 deposition in the surface of Crry-deficient mutants from the mouse T cell line SR.D10 is very strong; in fact, it is stronger that C3 deposition induced by P3D2-mediated blockade of Crry function in SR.D10 cells (J. M. Rojo, E. Fernández-Centeno, and P. Portolés, unpublished observations). This suggests that P3D2 is blocking one of the two (MCP or DAF) Crry activities but also that Crry expression is the main control for C3 deposition in these cells.

Current models for TCR-dependent activation (for reviews, see Refs. 45, 46, 47) hold that TCR ligation triggers at least three signaling cascades critical to IL-2 transcription. The first one is mediated by phospholipase C-{gamma} and inositol 3,4,5-triphosphate and increases intracellular calcium to activate calmodulin and calcineurin to induce NF-AT dephosphorylation and translocation to the nucleus. The second cascade is the classical MAPK pathway induced by Grb-2/mSOS recruiting, which activates p21ras. The third one (the stress-activated MAPK pathway, c-Jun N-terminal kinase 1/2) involves Rac activation through Grb-2/Vav and is also the main target for CD28 costimulation. Several experiments were performed to elucidate whether Crry ligation increases TCR/CD3-induced signals or activates a different signaling pathway. Our results suggest that Crry basically enhances the signals triggered by TCR/CD3 activation. First, Crry ligation alone does not induce lymphokine secretion or cell proliferation even in the presence of PDB, Iono, or anti-CD28 ( Figs. 3–5GoGoGo). Second, Crry ligation by P3D2 clearly enhances TCR/CD3 activation ( Figs. 3–5GoGoGo). Moreover, coligation of Crry and TCR/CD3 produced a faster and stronger early tyrosine phosphorylation of a number of cell substrates including polypeptides of 33, 36–38, 42–44, 45, 55–60, 70–80, and 116–120 kDa (Fig. 7GoA). One of these substrates could be identified as the MAPK ERK, which has been previously described to be involved in TCR/CD3-mediated signals (reviewed in Refs. 46, 48). Previous data (reviewed in Ref. 49) indicate that the duration of ERK activation is critical for cell signaling. Whether differences in ERK kinetics are directly related to preferential induction of IL-4 by Crry signals needs further studies. The nature of other, as yet unidentified, substrates might give some clues on additional pathways involved in the costimulatory mechanisms mediated by Crry.

Enhanced early tyrosine phosphorylation by Crry/CD3 coligation suggests that tyrosine kinases of the src or Syk families are involved in Crry costimulation. Indeed, Crry from Th1 and Th2 T cell lines coprecipitates kinase activity, which is at least partially due to p56lck (Fig. 8Go). It is remarkable that in vitro kinase assays performed with Th1 and Th2 Crry precipitates reveal the association of different substrates in both types of differentiated T cell lines. Its possible implication in signaling needs to be ascertained. The nature of the phosphorylated band of 60–64 kDa (Fig. 8GoA) is currently under study because it could be Crry itself. It should be noted that Crry has a short cytoplasmic domain that has no consensus immunoreceptor tyrosine-based activation motif sequences but contains three Tyr residues (50) which, upon phosphorylation, could mediate association to kinases or adapter proteins by interaction with Src homology 2 domains. In this regard, it has been recently observed that in activated macrophages, kinase activity is associated with another RCA molecule, namely human MCP (CD46) (51). Interestingly, in activated human monocytes, CD46 ligands like measles virus, C3b dimers, or anti-CD46 Abs suppress the secretion of certain lymphokines including IL-12 (41). This is yet another example of how CRPs serve as bridges between innate and acquired immunity.

The question remains of the nature of ligands to mediate the costimulatory function described here under physiological or pathological conditions. Our assays performed with P3D2 mAb indicate that the same epitope or a conformational change induced by a ligand may be implicated in both functions: protection against autologous complement and costimulation. All the possible ligands should comply with the requirement of keeping Crry close to TCR/CD3 for the costimulation to occur (as demonstrated by results in Fig. 3Go and data not shown). Because membrane Crry did not show receptor ability to bind complement molecules in other cell surfaces (12), C3b or C4b acting as putative costimulatory ligands should be deposited on the T cell surface. Another possibility is raised by the fact that some pathogens bind to complement regulators, e.g., this is the case of MCP which is the receptor for measles virus in humans (52) and also binds to protein M from Streptococcus pyogenes (53) or CR2, which is the receptor for EBV (54). In this case, Crry might bind an unknown microorganism. Because many bacteria and viruses are complement activators and some of their molecules have superantigen properties, it is tempting to speculate that these superantigens or microorganism particles covered with complement factors could cross-link the TCR and Crry together. Additional experiments will help in elucidating the implication of Crry in the biology of T lymphocytes.


   Acknowledgments
 
We thank Dr. S. Rodriguez-de Córdoba for helpful comments and revising the manuscript, Dr. V. M. Holers for providing 5D5 mAb and Crry-transfected K562 cell lines, and Drs. C. A. Janeway, F. Melchers, J. Allison, and other colleagues for many Abs and cell lines. The technical assistance of I. Ramos, B. Dorado, M. L. del Pozo, and A. del Pozo is gratefully acknowledged.


   Footnotes
 
1 This research was supported by grants from Fondo de Investigaciones Sanitarias (FIS/950381 and FIS/980037) and from Comisión Interministerial de Ciencia y Tecnología (Spain) (SAF94-0035). P.P. is a tenured scientist of the Consejo Superior de Investigaciones Cientificas at the Centro Nacional de Biologia Fundamental, Instituto de Salud Carlos III. Back

2 Address correspondence and reprint requests to Dr. Pilar Portolés, Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo km. 2, E-28220 Madrid, Spain. Back

3 Abbreviations used in this paper: CRP, complement regulatory protein; CR1, complement receptor type 1; MCP, membrane cofactor protein; DAF, decay-accelerating factor; RCA, regulators of complement activation; Crry, Crry/p65; P-Tyr, phosphotyrosine; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; Iono, ionomycin; PDB, phorbol 12,13-dibutyrate; PVDF, polyvinylidene difluoride; CHAPS, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate; P-ERK, phosphorylated active forms of ERK. Back

Received for publication May 10, 1999. Accepted for publication February 18, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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