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The Journal of Immunology, 2003, 171: 106-114.
Copyright © 2003 by The American Association of Immunologists

Ephrin B2 Induces T Cell Costimulation1

Guang Yu*, Hongyu Luo*, Yulian Wu*,{ddagger} and Jiangping Wu2,*,{dagger}

* Laboratory of Immunology and {dagger} Nephrology Service, Notre Dame Hospital, Centre Hospitalier de l’Université de Montréal, Université de Montréal, Montreal, Canada; and {ddagger} Department of Surgery, Second Affiliated Hospital of Zhejiang Medical College, Zhejiang University, Hangzhou, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eph kinases form the largest family of receptor tyrosine kinases, and their ligands are ephrins (EFNs), which are cell surface proteins. Some Eph kinases and EFNs are expressed on T cells, B cells, and dendritic cells, but their functions in the immune system are largely unknown. In this study, we investigated the effect of EFNB2 on murine T cells. EFNB2 mRNA was expressed in the cortex of the thymus and white pulp of the spleen. At the protein level, it was expressed on T cells and monocytes/macrophages, but not on B cells. EFNB2Rs were expressed mainly on T cells. Solid-phase EFNB2 along with suboptimal anti-CD3 strongly stimulated T cell proliferation, with concomitant augmentation of IFN-{gamma} but not IL-2 or IL-4 secretion. The activity of cytotoxic T cells was also significantly enhanced in the presence of solid-phase EFNB2. These results indicate that EFNB2R cross-linking results in costimulation of T cells. EFNB2Rs were normally scattered on the T cell surface; after TCR cross-linking, they rapidly congregated to capped TCR complexes and then to patched rafts. This provides a morphological base for EFNB2Rs to participate in T cell costimulation. We also demonstrated that EFNB2R signaling led to augmented p38 and p44/42 mitogen-activated protein kinase activation. Our study shows that EFNB2 plays important roles in immune regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Eph family of receptor tyrosine kinases (RTK)3 is the largest among the RTK families, comprising ~25% of known RTKs (1). According to sequence homology, Eph family members are divided into EphAs (EphA1 to -9) and EphBs (EphB1 to -6) (1) (http://cbweb.med.harvard.edu/eph-nomenclature/cell_letter.html). Their ligands are cell surface molecules called ephrins (EFNs) (1). EFNs are classified into two subfamilies. There are six members in the EFNA subfamily (EFNA1 to -6), and they are GPI-anchored cell surface proteins (1, 2) (http://cbweb.med.harvard.edu/eph-nomenclature/cell_letter.html). The EFNB subfamily consists of three members (EFNB1 to -3), and they are transmembrane proteins (1, 2). Although they are ligands, EFNs, especially EFNB subfamily members, can reverse transduce signals into cells (2, 3). The interaction between Ephs and EFNs is not very strict: one Eph can bind to several different EFNs and vice versa. However, in general, EphAs only interact with EFNAs, and EphBs, with EFNBs (2, 3). Human genome sequences have revealed 14 Eph entries and 8 EFN entries (4). Therefore, most of the Ephs and their ligand EFNs probably have already been identified.

Because both Eph receptor kinases and their ligands are cell surface molecules, they can interact only with each other if expressed on adjacent cells. Not surprisingly, these receptors and ligands are known to control accurate spatial patterning and cell positioning. Many of these findings are derived from studies in the CNS, where most Eph kinases have high-level expression (3, 5, 6, 7, 8, 9). It has also been found that EFNB2 and its receptor EphB4 are involved in angiogenesis (10), and such a function is consistent with the known roles of Eph kinases in controlling spatial structure formation.

The expression of some Ephs and EFNs in immune cells has been documented. For example, EphA1 (11), EphA2 (12), EphA3, EphA4 (13), EphB2 (14), EphB4 (15), and EphB6 (16) are expressed in the thymus; EphB6 is expressed on mature T cells (17, 18); EphA3 is expressed in pre-B cell lines (11), and EphA4 and EphA7 are significantly expressed in B cells (19); EphA2 (19) and EphB1 (20) are expressed in certain types of dendritic cells; and some Ephs, such as EphA3 (21), EphB4 (22), and EphB6 (18, 23), are expressed in leukemia cells. As for EFNs, EFNA1 (24), EFNA3, EFNB1 (25), EFNA2, EFNA4, and EFNA5 (13) are expressed in the thymus; EFNA4 can be detected in peripheral T and B cells (19).

However, we have very limited knowledge about the function of Eph and EFN in the immune system, and publications in this area are numbered. We have recently reported that EphB6, although it lacks intrinsic tyrosine kinase activity due to a mutation in its kinase domain (16), is able to transduce signals into T cells, probably via adaptor proteins such as Cbl, Grb2, and CrkL (23), and via EphB1 with which it associates (26). EphB6+ Jurkat cells cross-linked with anti-EphB6 mAb undergo Fas-mediated apoptosis (23). Further detailed study showed that mAb against EphB6 costimulates normal human T cells, in terms of p38 mitogen-activated protein kinase (MAPK) activation, lymphokine secretion, and proliferation (17). Initial examination of EphB6-/- mice shows no gross anomaly in the thymic structure and thymocyte populations (27), suggesting compensatory mechanisms at work. Munoz et al. (13) have reported that a few soluble EphAs and EFNAs interfere with T cell development in thymic organ culture.

In this study, we investigated the immune regulatory role of EFNB2, which is a ligand of several EphB kinases, including, but not restricted to, EphB6. The EphBs binding to EFNB2 are collectively termed EFNB2Rs in this study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In situ hybridization

A 656-bp cDNA fragment of mouse EFNB2 cDNA from positions 29 to 684 (accession no. NM010111) was fetched with PCR from a mouse embryonic tissue cDNA library and cloned into pGEM-4Z (Invitrogen, San Diego, CA). The resulting construct pGEM-4Z-mB2 was used to transcribe antisense probes with SP6 RNA polymerase or to transcribe sense probes with T7 RNA polymerase, using digoxigenin (DIG) RNA labeling kits (Roche Diagnostics, Laval, Quebec, Canada). Mouse thymus and spleen were embedded in optimal cutting temperature compound, sectioned at 7-µm thickness, and kept at -80°C until tested. The sections were thawed for 5 min at 55°C and fixed in 4% paraformaldehyde in PBS for 10 min at 4°C. After rinsing in PBS (pH 7.4) for 2 min, the sections were immersed in 0.2 N HCl for 10 min at room temperature, briefly washed three times in PBS, and digested with 1 µg/ml proteinase K in 50 mM EDTA and 100 mM Tris (pH 8) at 37°C for 30 min. Proteolysis was arrested by immersing the slides in 4% paraformaldehyde in PBS for 5 min at 4°C, followed by three 2-min washes in PBS. After the above pretreatments, the slides were incubated in a solution containing 50% deionized formamide and 2x SSC (1x SSC: 150 mM NaCl, 15 mM trisodium citrate (pH 7.0)) at 37°C for 30 min. The slides were exposed to 40–60 µl of hybridization mixture containing 1.5–3 ng/µl DIG-labeled antisense or sense RNA probes in hybridization buffer (50% deionized formamide, 2x SSC, 0.1% SDS), heated at 55°C for 5 min, and then incubated at 42°C for 16–18 h in a chamber humidified with 50% formamide in 2x SSC. Finally, the slides were washed twice in 4x SSC, once in 2x SSC, and once in 1x SSC for 15 min each. Signals were detected by alkaline phosphatase-conjugated anti-DIG Ab, with 5-bromo-4-chloro-3-indolyl phosphate as substrate and 4-nitroblue tetrazolium chloride as chromogen, according to instructions for the use of DIG detection kits (Roche Diagnostics). The sense probe was taken as a negative control. As additional negative controls, one slide for each experiment was treated with RNase A before hybridization to deplete mRNA, one slide was hybridized with omission of the probes, and one slide was detected with omission of the primary Ab during the DIG detection procedure. These additional negative controls revealed no signals, and photographs of them are not shown.

Generation of mouse EFNB2-Fc

The coding sequence of the extracellular domains of mouse EFNB2 from positions 29 to 684 was cloned in-frame upstream of the human IgG1-Fc coding sequence in an expression vector pCMVhFc. The constructs and pcDNA3 were then transfected into CHO/dhfr- cells with Lipofectamine (Invitrogen, Burlington, Ontario, Canada). The cells were cultured in selection medium ({alpha}MEM without ribonucleosides and deoxyribonucleosides containing 5% dialyzed FCS, 0.01 mM methotrexate, 0.8 mg/ml G418, and 0.1 mg/ml gentamicin). After 2 wk of culture, well-isolated clones were handpicked and expanded in the selection medium without G418. The culture supernatants were assayed by ELISA for human IgG-Fc-positive clones, which were then expanded. Fusion proteins were isolated from supernatants of the positive clone by protein A columns, then analyzed with 10% SDS-PAGE to confirm their molecular sizes, and verified by N-terminal peptide sequencing (Sheldon Biotechnology Center, McGill University, Montreal, Canada).

Lymphocyte preparation and culture

Cells were flushed out from the BALB/c mouse spleen, and RBCs were lysed with 0.84% NH4Cl, as described elsewhere (28). The resulting cells were referred to as spleen cells. Splenic T cells were purified by deleting mouse IgG (H+L)-positive cells from spleen cells with T cell columns according to the manufacturer’s instructions (Cedarlane, Hornby, Ontario, Canada). In some experiments, the T cells were fractionated from spleen cells into CD4+ and CD8+ cells using magnetic beads (Miltenyi Biotec, Auburn, CA). The cells were cultured in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, and penicillin-streptomycin. Solid-phase EFNB2 and anti-CD3 were prepared by coating 96-well Costar 3595 plates (Costar, Cambridge, MA) overnight with anti-mouse CD3 (clone 2C11) in PBS at 4°C, followed by incubating EFNB2-Fc or normal human IgG (NHIgG; as a control; Southern Biotechnology, Birmingham, AL) of different concentrations at 37°C for 2 h. The plates were finally incubated on ice for 1–2 additional hours before use.

Flow cytometry

Flow cytometry was used for measurement of EFNB2R expression as well as EFNB2 expression in different cell populations. BALB/c spleen cells were stained with EFNB2-Fc/goat anti-human IgG-PE or with goat anti-EFNB2 (R&D Systems, Minneapolis, MN)/donkey anti-goat IgG-PE (Cedarlane). For the second color, anti-Thy1.2-FITC (Caltag Laboratories, Burlingame, CA), anti-B220-FITC (clone RA3-6B2), and anti-F4/80-FITC (clone CI:A3-1) were used. To measure the expression of activation markers on T cells, splenic T cells were stained with anti-Thy1.2-PE in combination with anti-CD25-FITC (clone M-A251), anti-CD44-FITC (clone IM7), anti-CD54-FITC (clone HA58), or anti-CD69-FITC (clone FN50). All of these mAbs were from BD PharMingen (San Diego, CA), unless indicated otherwise.

[3H]Thymidine uptake assay

Splenic T cells were cultured in 96-well Costar plates coated with different mAb or recombinant proteins, and [3H]thymidine uptake was measured, as described previously (29).

Cytotoxic T cell assay

The assay was performed as detailed earlier (30). Briefly, C57BL/6 or transgenic 2C mouse spleen cells (H-2b with most of their T cells specific to Ld; 0.4 x 106 cells/well) were stimulated with an equal amount of mitomycin C-treated BALB/c mouse spleen cells (H-2d) in flat-bottom 96-well plates, which were precoated with goat anti-human IgG (5 µg/ml) followed by EFNB2-Fc or NHIgG coating (both at 10 µg/ml). The cells were cultured in the presence of 10 U/ml IL-2 for 6 days. On day 6, cells receiving the same treatment were pooled and counted, and their CTL activity was measured by a standard 4-h 51Cr release assay, using 51Cr-labeled P815 cells (H-2d) as targets at different E:T ratios. The lysis percentage of the test sample was calculated as follows: % lysis = (cpm of the test sample - cpm of spontaneous release)/(cpm of maximal release - cpm of spontaneous release).

Cytokine measurement

Culture supernatants of splenic T cells placed in anti-CD3- and/or EFNB2-Fc-coated wells were harvested 1–3 days after initiation of culture. IL-2, IL-4, and IFN-{gamma} in the supernatants were quantified by ELISA (R&D Systems) according to the manufacturer’s instructions.

Laser scanning confocal microscopy

Five million BALB/c splenic T cells were first blocked with 100 µl of PBS containing 2% BSA on ice for 30 min. Five micrograms of EFNB2-Fc and 1 µg of biotinylated anti-CD3 (clone 2C11; hamster mAb) were then added to the cell suspension, which was incubated for another 30 min on ice. After washing with cold PBS, the cells were reacted with goat anti-hamster IgG (5 µg/sample) for 30 min on ice. The cells were washed with cold PBS and transferred to 100 µl of warm PBS to start the cross-linking process at 37°C. The cells were then immediately fixed with 2 ml of 3.7% formalin at room temperature for 10 min. For TCR and EFNB2R staining, the cells were reacted with streptavidin-Alexa Fluor 594 (1 µg/sample) and goat anti-human IgG-Alexa Fluor 488 (1 µg/106 cells) on ice for 30 min. For raft and EFNB2R staining, the procedure was similar to that for TCR and EFNB2R staining, but cholera toxin-Alexa Fluor 594 (0.5 µg/sample) was used in place of streptavidin-Alexa Fluor 594. The stained cells were then washed with PBS and mounted on slides with Prolong antifade mounting medium (Molecular Probes, Eugene, OR). The slides were examined under a confocal microscope. Digital images were processed with Photoshop (Adobe, Seattle, WA).

Immunoblotting

Twelve-well plates were coated overnight with anti-CD3 (0.8 µg/ml, 500 µl/well) at 4°C. After washing, the wells were incubated with EFNB2-Fc or NHIgG (both at 10 µg/ml, 500 µl/well) at 37°C for 1–2 h, and then at 0°C for another 2 h. BALB/c splenic T cells were seeded in the precoated plates at 5 x 106 cells/well, and the plates were centrifuged at 228 x g for 5 min to achieve rapid contact between the cells and the bottom of the culture wells. The cells were then cultured at 37°C for 2 h before being harvested. The remainder of the procedure has been detailed in our previous publication (28). Briefly, the harvested cells were washed and lysed in lysis buffer for 10 min; the cleared lysates were resolved in 10% SDS-PAGE with 50 µg of protein/lane and were then blotted onto polyvinylidene difluoride membranes. The membranes were sequentially hybridized with rabbit anti-phospho-p38 MAPK Ab followed by rabbit anti-p38 MAPK Ab, or with rabbit anti-phospho-p44/42 MAPK Ab followed by anti-p44/42 MAPK. All of the Abs used in immunoblotting was from New England Biolabs (Mississauga, Ontario, Canada). Signals were revealed by ECL.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of EFNB2 in the thymus and spleen according to in situ hybridization

The BALB/c mouse thymus and spleen were examined by in situ hybridization using DIG-labeled EFNB2 antisense and sense probes. As shown in Fig. 1, EFNB2 was prominently expressed in the thymic cortex and spleen white pulp. This expression pattern suggests that EFNB2 might have a function in lymphocytes cells, particularly T cells.



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  		FIGURE 1. EFNB2 expression in the thymus and spleen according to in situ hybridization. Thymus and spleen sections were hybridized with DIG-labeled antisense or sense EFNB2 probes. The original magnification was x100.

 
EFNB2 and EFNB2R expression in T cells, B cells, and monocytes/macrophages according to flow cytometry

We first examined EFNB2 expression (Fig. 2A). Splenic T cells were double-labeled with anti-CD3/anti-EFNB2 and analyzed by flow cytometry. Cells with EFNB2 intensity above the control goat IgG were referred to as EFNB2-positive cells. In freshly prepared cells, 7.1% T cells were EFNB2 positive. The T cells rapidly up-regulated their EFNB2, and EFNB2-positive T cells reached 23.2% after overnight culture in medium. The expression of EFNB2 on CD4 and CD8 cells was similar (Fig. 2C); after overnight culture, 26.1 and 28.8% of CD4 and CD8 cells, respectively, became EFNB2 positive. Activation of CD3+ T cells by anti-CD3 or anti-CD3 plus anti-CD28 (both on solid phase) for 24–48 h did not augment EFNB2 expression, as shown in Fig. 2D; actually, there was a moderated decrease of EFNB2+ cells at 24 h after the activation. EFNB2 was minimally expressed in freshly isolated or cultured B cells (B220+) (0.2 and 4.9%, respectively) (Fig. 2A). Monocytes/macrophages (F4/80+) were another cell population showing significant expression of EFNB2 after culture, with 5.2% positive in freshly isolated ones and 31.2% positive after culture (Fig. 2A).



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  		FIGURE 2. Flow cytometry analysis of EFNB2 and EFNB2R expression on immune cells. All experiments were performed more than twice and were reproducible. Representative results are shown. Two-color flow cytometry was used to assess EFNB2 or EFNB2R expression on T cells, B cells, and monocytes/macrophages. T cells were stained with anti-CD3; B cells were stained with anti-B220; and monocytes/macrophages were stained with F4/80. One-color flow cytometry was used to assess EFNB2 or EFNB2R expression on magnetic bead-purified CD4 or CD8 cells. A, EFNB2 expression on CD3+ T cells, B220+ B cells, or F4/80+ monocytes/macrophages. B, EFNB2R expression on CD3+ T cells, B220+ B cells, or F4/80+ monocytes/macrophages. C, EFNB2 and EFNB2R expression on magnetic bead-purified CD4 or CD8 cells. D, Kinetics of EFBB2 and EFNB2R expression on CD3+ T cells activated with solid-phase anti-CD3 (suboptimal) or anti-CD3 (suboptimal) plus anti-CD28. E, EFNB2 and EFNB2R expression on CD3+ T cells cultured in 100% untreated or heat-inactivated mouse serum. The percentages of positive cells are indicated.

 
Next, we examined EFNB2R expression on these cells (Fig. 2B). EFNB2Rs were predominantly expressed on T cells, with 8.6% positivity in freshly isolated T cells and 42.9% positivity in cultured ones. Both CD4 and CD8 cells expressed EFNB2R, but more CD4 cells expressed EFNB2R than CD8 cells after overnight culture (47.9 vs 28.9%) (Fig. 2C). The expression of EFNB2R was not significantly affected by the status of T cell activation; T cells activated by solid-phase anti-CD3 or anti-CD3 plus anti-CD28 for 24 or 48 h had no increase in EFNB2R expression, but a moderate decrease of the expression could be consistently observed at 24 h (Fig. 2D). B cells had minimal receptor expression either before (data not shown) or after culture (Fig. 2B). Some monocytes/macrophages (16.9%) become EFNB2R positive after culture (Fig. 2B).

The low expression of EFNB2 and EFNB2R in freshly isolated T cells and their subsequent up-regulation after culture was intriguing. We suspected that some serum factors were responsible for this phenomenon. To test this possibility, we cultured the T cells overnight in 100% fresh untreated mouse serum as well as in 100% heat-inactivated mouse serum. As shown in Fig. 2E, T cells (CD3+) cultured in the former but not the latter failed to up-regulate both EFNB2 and EFNB2R. It is probable that heat-sensitive serum proteases rapidly cleave EFNB2 and EFNB2R from the cell surface. Such cleavage is not unusual for many cell surface molecules, including several TNF family members. Alternatively, a heat-sensitive factor(s) in serum might be suppressive of EFNB2 and EFNB2R expression.

These results revealed the expression pattern of both EFNB2 and EFNB2R in T cells, B cells, and monocytes/macrophages, and suggest that EFNB2 might have an important effect on T cells in the form of either T cell-T cell collaboration or T cell-monocyte/macrophage interaction during Ag presentation.

EFNB2 enhances T cell proliferation and modulates activation marker expression

The predominant expression of EFNB2R on T cells led us to investigate whether EFNB2 could modulate T cell function. For this purpose, both anti-CD3 mAb and EFNB2-Fc were put on solid phase. Anti-CD3 (at a suboptimal concentration) or EFNB2-Fc (at an optimal concentration) alone caused negligible T cell proliferation (Figs. 3, B and C), but EFNB2-Fc dose-dependently induced their proliferation in the presence of anti-CD3 (A). Next, T cells were cultured in wells coated with an optimal amount of EFNB2-Fc and various amounts of anti-CD3. As shown in Fig. 3B, EFNB2-Fc but not NHIgG (a negative control for EFNB2-Fc) augmented T cell proliferation when anti-CD3 was used at different concentrations. This result suggests that EFNB2R cross-linking reduces the T cell response threshold, and that EFNB2-expressing cells might be able to costimulate T cells. We also compared the costimulation by EFNB2-Fc with that by anti-CD28 mAb (Fig. 3C). The magnitude of costimulation mediated by EFNB2R was lower than the classical costimulating molecule CD28. CD4+ cells and CD8+ cells were both sensitive to EFNB2 costimulation, because their proliferation was similarly augmented by solid-phase EFNB2-Fc in the presence of suboptimal anti-CD3 (Fig. 3D).



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  		FIGURE 3. EFNB2R cross-linking enhances the T cell response to TCR stimulation. All experiments were conducted more than three times and were reproducible. Representative results are shown. The prefix "anti-" used in conjunction with mAbs is simplified as "{alpha}" in this and all of the other figures of this article. All of the concentrations indicated in the figure legends represent those used during the coating procedure. NHIgG served as a control for EFNB2-Fc. In some cases, the wells were only coated with anti-CD3, followed by PBS incubation (PBS), and were used as additional blank controls. BALB/c T cells were cultured in wells coated with a suboptimal amount of anti-CD3 (0.8 µg/ml) and different amounts of EFNB2-Fc (A); a fixed optimal amount of EFNB2-Fc (10 µg/ml) and different amounts of anti-CD3 (B); or a suboptimal amount of anti-CD3 (0.8 µg/ml) along with optimal amounts of anti-CD28 or EFNB2-Fc (both at 10 µg/ml) (C). Magnetic bead-purified CD4+ or CD8+ cells were also cultured in wells coated with EFNB2-Fc, NHIgG, or anti-CD28 in the presence of suboptimal solid-phase anti-CD3 (D). The cells were cultured for 48 h, and their [3H]thymidine uptake in the last 16 h was measured. Means ± SD of the cpm from triplicate samples are shown.

 
The T cell response to EFNB2 was also assessed by their expression of activation markers. T cells were stimulated with solid-phase anti-CD3 alone (at a suboptimal concentration) or solid-phase anti-CD3 plus EFNB2-Fc (at an optimal concentration). As shown in Fig. 4, anti-CD3 alone (shaded area) at such a concentration was not able to up-regulate the activation markers examined, i.e., CD25, CD44, CD54, and CD69. After combined stimulation with anti-CD3 and EFNB2-Fc, CD69 but not CD25, CD44, or CD54 was significantly up-regulated. In contrast, the classical costimulation by anti-CD28 drastically enhanced all of the activation markers examined, and CD69-positive T cells reached 61.5%, which was 2-fold higher than that of EFNB2-Fc-costimulated cells. The results indicate that EFNB2R can costimulate T cells, but this is quantitatively and qualitatively different from CD28.



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  		FIGURE 4. Activation marker expression of EFNB2-Fc-costimulated T cells. All experiments were conducted more than three times and were reproducible. Representative results are shown. BALB/c T cells were cultured in wells coated with anti-CD3 mAb (0.8 µg/ml) plus EFNB2-Fc or anti-CD28 (both at 10 µg/ml). Their activation marker expression at 24 h was assessed by two-color flow cytometry, using anti-Thy1.2-PE for T cell gating, and anti-CD25-FITC, anti-CD44-FITC, anti-CD54-FITC, and anti-CD69-FITC for activation markers. Percentages represent activation marker-positive cells among Thy1.2-positive cells (solid lines) after deducting fluorescence in cells stimulated with suboptimal anti-CD3 alone (shaded areas).

 
EFNB2 enhances T cell effector functions

We next examined the effect of EFNB2 on T cell effector functions, such as lymphokine production and CTL activity.

Again, T cells were stimulated with solid-phase anti-CD3 alone (at a suboptimal concentration) or in combination with EFNB2-Fc or anti-CD28 (both at optimal concentrations and on solid phase). NHIgG was used as a negative control for EFNB2-Fc. Anti-CD3 alone did not trigger lymphokine production. Anti-CD28 costimulation drastically induced IL-2, IL-4, and IFN-{gamma}, as expected (Fig. 5). However, EFNB2-Fc costimulation augmented only IFN-{gamma} but not IL-2 or IL-4 release. The IFN-{gamma} level stimulated by EFNB2-Fc was lower than, but of the same order of magnitude of that induced by anti-CD28 costimulation. The lack of IL-2 and IL-4 production was not due to a shift of secretion kinetics during EFNB2 costimulation, because no production of these lymphokines was observed during any time between days 1 and 3 after culture. This again demonstrated the qualitative difference between costimulation mediated by CD28 and EFNB2R.



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  		FIGURE 5. Solid-phase EFNB2-Fc strongly augments IFN-{gamma} but not IL-2 or IL-4 production by anti-CD3-stimulated T cells. Mouse spleen T cells were stimulated with solid-phase EFNB2-Fc (10 µg/ml) in the presence of a suboptimal concentration of anti-CD3 (0.8 µg/ml). Anti-CD28 (10 µg/ml) and NHIgG were used as positive and negative controls, respectively. The culture supernatants were harvested from days 1 to 3, and cytokines in the supernatants were measured by ELISA. The experiment was performed more than twice and was reproducible. Means ± SD of representative results are shown.

 
In EFNB2-Fc-coated wells, H-2d (BALB/c)-stimulated CTL development of the C57BL/6 T cells (Fig. 6A) or Ld-specific 2C T cells (B) was greatly enhanced, whereas control NHIgG had no such effect. As 2C T cells are predominantly CD8 cells (>85% Thy1.2+ cells were CD8 cells, and <3% Thy1.2+ cells were CD4+ cells), our result suggests that EFNB2 can directly costimulate CD8 cells to achieve CTL differentiation with little dependence on CD4 help. This result is consistent with the fact that CD8 cells in the absence of CD4 cells could be directly costimulated to proliferate by solid-phase EFNB2-Fc (Fig. 3D). The costimulatory effect could be observed only when EFNB2-Fc was on the solid phase in the beginning of the culture. Soluble EFNB2-Fc added to the culture on the last day before the CTL assay or during the CTL assay had no effect (data not shown). This is in agreement with the notion that EFNB2 provides costimulation, which is an early event during T cell activation and differentiation.



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  		FIGURE 6. Effect of solid-phase EFNB2-Fc on CTL development. C57BL/6 mouse (A) or 2C mouse (B) spleen cells were mixed with an equal amount of mitomycin C-treated BALB/c mouse spleen cells and seeded in flat-bottom 24-well plates, which were precoated with EFNB2-Fc or NHIgG (both at 10 µg/ml), or not coated (Medium). After 6 days, CTL activity in the stimulated cells was measured by a standard 4-h 51Cr release assay, using P815 cells as targets. The samples were tested in triplicate, and means ± SD of the percentage of target cell lysis are shown. The experiments were performed twice with similar results, and the data of a representative experiment are presented.

 
The results of this section demonstrate that EFNB2 can selectively enhance certain T cell effector functions, likely by promoting costimulation to T cells during their early stage of activation.

EFNB2 triggers signaling events in T cells

To understand the molecular basis of EFNB2 costimulation, we examined translocation of EFNB2Rs and TCR, and their relationship with lipid rafts on T cell membranes immediately after TCR cross-linking. T cells were preincubated with anti-CD3-biotin, followed by a second Ab on ice. CD3 cross-linking started when the cells were transferred to 37°C. The TCR complex was stained by streptavidin-Alexa Fluor 594 in red; EFNB2Rs were stained by EFNB2-Fc followed by anti-human IgG-Alexa Fluor 488 in green; and the lipid rafts in the T cell membrane were stained by cholera toxin-Alexa Fluor 594 in red. In resting T cells, rafts, TCR, and EFNB2Rs were distributed throughout the cell surface as small speckles. After 10-min cross-linking with anti-CD3, TCR rapidly polarized and formed a cap in one end of the cell. EFNB2Rs also congregated, and they colocalized with TCR (Fig. 7A). Such cocapping lasted >20 min (data not shown). After CD3 cross-linking, rafts underwent congregation and formed caps, but this process was slower than TCR capping and was completed at 20 min (Fig. 7B). EFNB2R congregation preceded raft congregation, but eventually at 20 min, EFNB2Rs translocated into the raft caps, suggesting that EFNB2Rs, probably like the TCR complex, only transiently associated with the rafts during TCR activation. Taken together, these data indicate that TCR and EFNB2Rs first cocap, and then both congregate to a raft cap on the cell surface after TCR-cross-linking. This provides a morphological base for EFNB2 to enhance TCR signaling, because now both TCR and EFNB2Rs are closely associated and located in aggregated rafts, which are scaffolds accommodating many signaling molecules.



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  		FIGURE 7. Rapid colocalization of EFNB2Rs with TCR and raft caps after anti-CD3 cross-linking. BALB/c spleen T cells were cross-linked with anti-CD3 for 0, 10, or 20 min, as indicated. The locations of TCR (stained with biotin-anti-CD3 followed by Alexa Fluor 594-streptavidin in red) (A), EFNB2Rs (stained with EFNB2-Fc followed by Alexa 488-anti-human IgG in green) (A and B), and rafts (stained with Alexa Fluor 594-cholera toxin (CT) in red) (B) were revealed by confocal microscopy. All experiments were conducted more than three times and were reproducible. Representative results are shown.

 
Next, in EFNB2-costimulated T cells, we examined MAPK activity, which is modulated in other cell types when some Eph kinases are activated (31, 32, 33, 34). As shown in Fig. 8A, a combination of solid-phase EFNB2-Fc and suboptimal anti-CD3 stimulation for 2 h led to increased p38 MAPK and p44/42 MAPK phosphorylation, a sign of their activation, whereas anti-CD3 at suboptimal concentration had little effect, according to immunoblotting. The membranes were reprobed with anti-p38 MAPK Ab or anti-p44/42 MAKP Ab, respectively, and the total protein levels of these kinases in all of the lanes of their respective membranes were similar. Therefore, these MAPK were activated after EFNB2R-cross-linking, and such activation might be part of the EFNB2 costimulation program. To assess the relevance of the EFNB2-up-regulated MAPK activity in T cell activation, p38 MAPK- and p44/42 MAPK-specific inhibitors, SB203580 and PD98059, respectively, were used. As shown in Fig. 8B, both inhibitors, but not their nonfunctional structural analog SB272474, inhibited EFNB2-enhanced T cell proliferation. This indicates that up-regulation of the MAPK activity is an essential part of EFNB2 costimulation signaling.



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  		FIGURE 8. Activation of p38 MAPK and p44/42 MAPK in T cells by solid-phase EFNB2-Fc. A, Immunoblotting of p38 and p44/42 MAPK. T cells were added to wells coated with EFNB2-Fc or NHIgG (both at 10 µg/ml) in the presence of a suboptimal amount of anti-CD3 (0.8 µg/ml). The cells were harvested after 2 h and analyzed by immunoblotting. Arrows indicate signals of p38 phospho-MAPK and total p38 MAPK of the same membrane, and signals of p44/42 phospho-MAPK and total p44/42 MAPK of the same membrane. B, p38- and p44/42-specific inhibitors inhibit EFNB2-costimulated T cell proliferation. T cells were preincubated for 1 h in complete culture medium containing the p38 MAPK-specific inhibitor SB203580, p44/42-specific inhibitor PD98059, its nonfunctional structural analog SB272474 (all at 10 µM), or vehicle DMSO (0.1%). The cells were then transferred to wells coated with EFNB2-Fc (10 µg/ml), anti-CD3 mAb (0.8 µg/ml), or both, and cultured for 48 h. [3H]Thymidine was added to the culture for the last 8 h, and thymidine uptake by the cells was measured.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the novel functions of EFNB2 on T cell activities were revealed. In the immune system, EFNB2 was expressed on T cells and monocytes/macrophages, and its receptors were mainly expressed on T cells. Solid-phase EFNB2 could costimulate T cells in the presence of suboptimal TCR ligation. Such costimulation led to enhanced T cell proliferation and IFN-{gamma} production. These functional studies were consistent with our previous report (17) in the human system using mAb against EphB6, which is one of the receptors EFNB2 binds. However, the current findings went significantly beyond the said report in several ways: 1) a physiological relevant molecule, i.e., mouse EFNB2, was used in this mouse system, and thus, the results reflected not only the effect of EFNB2 through EphB6, but its overall effect through all the EphBRs it triggered, as would occur in vivo; 2) EFNB2 up-regulated CTL activity, which was a new finding and a logical one; 3) ligation of TCR resulted in rapid congregation of EFNB2Rs to rafts, to which TCR complex also translocated; and 4) EFNB2 costimulation augmented activation of not only p38 MAPK, as with anti-EphB6-stimulated human T cells, but also p44/42 MAPK.

As a general rule, an EFNB subfamily ligand associates with more than one specific EphB receptor. For EFNB2, it can bind to EphB1, -2, -3, -4 (35), and -6 (36). Among these, EphB2 (14), EphB4 (15) and EphB6 (16) are relevant to our study, because they are expressed in the T cell compartment. According to in situ hybridization (data not shown), both EphB4 and EphB6 were expressed in the same region of the lymphoid organs, i.e., thymic cortex and spleen white pulp, like EFNB2. Such colocalization creates a possibility for interactions between EphB4+ and EFNB2+ cells, and between EphB6+ and EFNB2+ cells. We are currently examining whether other EphBs are also expressed in these regions. It is quite possible that other EphBs, in addition to EphB6, are involved in mediating EFNB2-triggered T cell costimulation: soluble EphB4 moderately inhibited solid-phase EFNB2 costimulation on wild-type T cells, but such inhibition was more significant in EphB6 gene knockout T cells (data not shown), although the inhibition was still not complete. This suggests the involvement of both EphB4 and EphB6 in EFNB2-triggered costimulation, and the remainder of the uninhibited costimulation could be mediated by other EphBs.

We noticed that, in this study, EFNB2 costimulation failed to enhance CD25 and CD54 expression on T cells, whereas in our previous study, anti-EphB6 mAb was able to do so (17). If EphB6 is one of the receptors EFNB2 acted on, EFNB2 stimulation, in theory, should achieve effects no less than that achieved by anti-EphB6 mAb. How do we explain the discrepancy? First, our previous study was conducted in the human system, whereas the current one was performed in the murine system. The species difference is always a possible explanation. More likely, this might be caused by a different affinity between the mAb and the natural ligand EFNB2.

A recent crystal structure study has suggested that, before binding to its receptors, EFNB2 might form homodimers on the cell surface (37). Upon binding with EphB2 on another cell, two EFNB2 molecules and two EphB2 molecules on two different cells might form very stable tetramer complexes. It is conceivable that EFNB2 might similarly interact with other EphBs. This might be the molecular basis for EFNB2 to provide strong and lasting costimulation to T cells. Lymphoid organs are densely packed with EFNB2+ T cells as well as other EFNB2+ cells. EFNB2 on these cells might constantly stimulate EFNB2R on the neighboring T cells, and reduce their response threshold to foreign Ags. This is reminiscent of recent findings that, in vivo, T cells constantly receive low level stimulation from self-Ag on neighboring cells, and such stimulation promotes T cell sensitivity to the foreign Ags (38, 39). To maintain active signaling pathway downstream of EFNB2R due to the possible constant interaction with its ligands, continuous expression of new EFNB2Rs with simultaneous shedding of the old ones might be a valid strategy. We found that T cells cultured in untreated serum had low EFNB2R and EFNB2 expression, but the expression drastically augmented when the serum was heat inactivated. This suggests that rapid shedding of these surface molecules is indeed a possibility, although this needs confirmation. With that said, we cannot exclude that this is due to serum inhibitory factors. Further investigation in this regard is warranted.

EFNB2 costimulation seems qualitatively different from anti-CD28 costimulation in that only IFN-{gamma} but not IL-2 or IL-4 was produced in the former. In fact, similar findings were observed using anti-EphB6 mAb (17), or EFNB2 (the current study), or EFNB1 and EFNB3 (data not shown). Thus, this seems to be an intrinsic property of costimulation mediated by EphBs. Consistent with this finding, we found that solid-phase EFNB2 augmented CTL activity, for which increased IFN-{gamma} is likely a contributing factor. Naturally, the enhanced CTL might benefit from other cellular activities that EFNB2 promoted, such as T cell proliferation, which could amplify the number of the CTL precursors.

The critical role of lipid rafts in TCR signaling has been increasingly appreciated. Some TCR signaling molecules, such as Src kinase, linker for activation of T cells, and Ras are constitutively situated in the rafts (40, 41, 42), whereas others, such as CD3{zeta}, {zeta}-associated protein-70, Vav, phospholipase C-{gamma}1, protein kinase C-{theta}, and I{kappa}B kinase components translocate into rafts after TCR triggering (43, 44, 45, 46). Such translocation enables TCR to use these molecules in the scaffold of rafts to accomplish its signaling. We studied the relationship between EFNB2R and rafts under various conditions. Cross-linking EFNB2Rs using EFNB2 in the absence or presence of suboptimal TCR cross-linking did not alter the EFNB2Rs or raft distribution, and they remained evenly scattered on T cell surface (data not shown). Only rather strong TCR cross-linking (as shown in Fig. 7) resulted in caps containing EFNB2Rs, rafts, and TCR. EFNBR2 capping had different kinetics from raft capping, suggesting that EFNB2Rs are not constitutively located in rafts. This indicates that EFNB2R belongs to the second category of signaling molecules (likely CD3{zeta} et al., as discussed above) that move to the rafts to assist TCR signaling only after TCR have already been triggered.

For proper T cell activation, Ras and Rac signaling pathways need to be mobilized. Ras activation leads to activation of p44/42 MAPK kinases, which, in turn, results in the synthesis and activation of various transcription factors. In contrast, activation of Rac and Cdc42 small G proteins leads to p38 MAPK activation, which is essential for cytoskeleton reorganization. Such reorganization is now known to be pivotal for the T cell signaling (47). We have found that the activities of both p44/42 and p38 MAPK are enhanced in the presence of EFNB2 costimulation, and this is consistent with the roles of these MAPKs in T cell activation. Recently, it has been reported that EphB2 activation results in inhibition of p44/42 MAPK in neuronal cells (34), and that EphA activation leads to inhibition of this kinase in several cell lines of endothelial and epithelial origin (48). Obviously, these reports deal with cell types different from those in our study. The consequence is different as well. In neuronal cells, endothelial cells, and epithelial cells, Eph activation does not induce cell proliferation, while in T cells, it does. Further comparative studies on the EFNB2R signaling in T cells and nonimmune cells will be interesting.

We have demonstrated the costimulatory effects of EFNB2 on mouse T cells in this study. The function of Ephs and their ligands in immune regulation represents a new domain of research in immunobiology and deserves more attention.


   Acknowledgments
 
We acknowledge the editorial assistance of Mr. Ovid Da Silva (Research Support Office, Research Center, Centre Hospitalier de l’Université de Montréal-Hotel-Dieu, Montreal, Canada).


   Footnotes
 
1 This work was supported by grants from Canadian Institutes of Health Research (MT-15673, MOP57697, and PPP57321); Canadian Institutes of Health Research/Canadian Blood Service Partnership Program; Kidney Foundation of Canada; Heart and Stroke Foundation of Quebec; Roche Organ Transplantation Research Foundation, Switzerland (474950960); Juvenile Diabetes Research Foundation (5-2001-540); and J.-Louis Levesque Foundation (to J.W.). J.W. is a National Scholar of the Fonds de la Recherche en Santé du Québec. Back

2 Address correspondence and reprint requests to Dr. Jiangping Wu, Laboratory of Immunology, Research Center, Notre Dame Hospital, Centre Hospitalier de l’Université de Montréal, Pavilion DeSève, Room Y-5616, 1560 Sherbrooke Street East, Montreal, Quebec H2L 4 M1, Canada. E-mail address: jianping.wu{at}umontreal.ca Back

3 Abbreviations used in this paper: RTK, receptor tyrosine kinase; EFN, ephrin; MAPK, mitogen-activated protein kinase; DIG, digoxigenin; NHIgG, normal human IgG. Back

Received for publication December 20, 2002. Accepted for publication April 25, 2003.


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 Materials and Methods
 Results
 Discussion
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