SEARCH

SEARCH BY CITATION

Keywords:

  • Biphasic modulation · EphB · Ephrin-B · TCR

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Bidirectional signals via Eph receptors/ephrins have been recognized as major forms of contact-dependent cell communications such as cell attraction and repulsion. T cells express EphBs, and their ligands, the ephrin-Bs, have been known as costimulatory molecules for T-cell proliferation. Recently, another remarkable feature of ephrin-As has emerged in the form of a concentration-dependent transition from promotion to inhibition in axon growth. Here we examined whether this modification plays a role in ephrin-B costimulation in murine primary T cells. Low doses of ephrin-B1 and ephrin-B2 costimulated T-cell proliferation induced by anti-CD3, but high concentrations strongly inhibited it. In contrast, ephrin-B3 showed a steadily increasing stimulatory effect. This modulation was virtually preserved in T cells from mice simultaneously lacking four genes, EphB1, EphB2, EphB3, and EphB6. High concentrations of ephrin-B1/B2, but not ephrin-B3, inhibited the anti-CD3-induced phosphorylation of Lck and its downstream signals such as Erk and Akt. Additionally, high doses of any ephrin-Bs could phosphorylate EphB4. However, only ephrin-B1/B2 but not ephrin-B3 recruited SHP1, a phosphatase to suppress the phosphorylation of Lck. These data suggest that EphB4 signaling could engage in negative feedback to TCR signals. T-cell activation may be finely adjusted by the combination and concentration of ephrin-Bs expressed in the immunological microenvironment.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Eph receptors are the largest known family of receptor tyrosine kinases (RTKs). These receptors can be classified into two groups, EphAs and EphBs, based on their sequence homology. Ligands for Eph receptors, so called ephrins, are also divided into two classes. Some are membrane anchored by a glycosylphosphatidylinositol linkage (ephrin-A) and the others through a transmembrane domain (ephrin-B). In mammals, there are nine EphAs that bind to five ephrin-As, and five EphBs (B1, B2, B3, B4, B6) that bind to three ephrin-Bs (B1, B2, B3). The interactions between Ephs and ephrins are promiscuous; one Eph can bind to multiple ephrins and vice versa, including some exceptional interactions between different classes [[1, 2]]. The ephrins can also function as reciprocal receptors for Ephs and this axis works as a bidirectional signal transduction system between two cells upon direct contact [[2, 3]]. The functions of Ephs and ephrins have been extensively demonstrated in the control of accurate spatial patterning and cell positioning in the development and repair after injury of the nervous system [[2, 3]]. Recent studies have also elucidated cross-talk with many other signaling pathways [[4]] and the critical roles in a wide variety of fields, such as angiogenesis, glucose homeostasis, bone maintenance and remodeling, intestinal homeostasis, and cancer development [[2]].

While some members of Ephs/ephrins are also expressed in the lymphoid organs [[2, 5, 6]], their physiological role in immune responses are still not known. Studies have shown that a deficiency of certain Ephs leads to a defect in thymocyte maturation because of abnormal development of the stromal cells [[7-10]]. The effects of Eph receptors expressed on mature T cells have been reported, such as modulation of chemotaxis by certain ephrin-As and ephrin-Bs [[11-14]]. Eph signaling in thymocytes has been reported to blunt the effects of high T-cell receptor (TCR) signaling [[15-17]], suggesting the possible inhibition of negative selection of self-reactive thymocytes. In contrast, Wu and colleagues have proposed promotional TCR costimulatory effects of all ephrin-Bs by using their original ephrin-B-Fc chimeric proteins [[18-20]]. However, the molecular basis for an Eph/ephrin system to inhibit or promote TCR signaling in each cell type remains unknown.

In the central nervous system, it is now clear that Eph receptors have functional versatility, namely, both repulsive and attractive signals [[21-25]]. This bifunctional guidance cue may be regulated by developmental time and location, most likely characterized by the concentration and combination of the ligands. Recently, another remarkable feature of ephrins, a concentration-dependent transition from promotion to inhibition in retinal axon growth, has emerged for ephrin-As [[21]]. Although biphasic effects in cell adhesion and migration in response to different concentration of ephrin-B2 have subsequently been shown to exert by using EphB6-transfected human embryonic kidney (HEK293T) cells [[26]], there is no other direct evidence that this unique concentration-dependent positive and negative effect of Eph/ephrin acts in primary tissues/cells than the aforementioned axon growth.

The activation and inhibition of TCR signaling by costimulation with particular molecules for each consequence have been extensively studied in T-cell proliferation [[27, 28]]. Therefore, we postulated that the concentration-dependent functional transition by the same ligand would be suitable for the delicate tuning of immune responses according to the intensity of signals from the immunological microenvironment. In this study, the modulatory effects of ephrin-Bs on TCR-mediated activation of murine primary T cells were carefully evaluated. The results revealed certain ephrin-Bs/EphBs as a novel class of costimulatory molecules with a unique action: concentration-dependent switching from costimulation to inhibition.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Biphasic modulation of T-cell proliferation by ephrin-B1 and ephrin-B2

To elucidate the details of the regulation of primary T-cell function by EphB/ephrin-B system, 3H-thymidine uptake assay was performed. Interestingly, solid phase ephrin-B1 and ephrin-B2 ligands exhibited unique biphasic effects in T-cell proliferation by the suboptimal solid phase anti-CD3 stimulation: stimulatory effect at lower concentration and inversely suppressive effect at higher concentration (Fig. 1A). On the other hand, ephrin-B3 costimulation showed simply promotional effect as previously reported [[18]]. These unique modulation patterns were background independent (C57BL/6: Fig. 1A, Icr mix: Fig. 1B) and conserved even by the more intense TCR signaling with higher anti-CD3 concentration (Supporting Information Fig. 1). The magnitude of response to the anti-CD3 stimulation depended on the genetic background of mice employed in each experiment. The level of peak promotional effects by each ephrin-B (ephrin-B1/B2: at 2.5–5 μg/mL, ephrin-B3: at 20 μg/mL) were comparable with those by optimal anti-CD28 addition (10 μg/mL) (Fig. 1B).

image

Figure 1. Unique modulation of TCR-mediated T-cell proliferation by ephrin-Bs T cells from (A) C57BL/6 and (B) Icr mix background mice were stimulated with immobilized anti-CD3 (0.8 μg/mL) together with each ephrin-B immobilized at indicated concentrations. Solid phase anti-CD28 (10 μg/mL) was used instead of ephrin-B in some experiments in order to show that the peak promotional effect of ephrin-B is at a level similar to that of anti-CD28. Dotted line: NHIgG instead of ephrin-B as a control. Results are expressed as mean counts per minute ± SEM of triplicate cultures and are representative of three independent experiments.

Download figure to PowerPoint

The cytokine production by T cells in this culture system was also assessed. After 48 h incubation, the concentrations of TNF-α, IL-2, and IFN-γ in culture supernatants were similar to the pattern of T-cell proliferation (Fig. 2). On the other hand, secretion of IL-4 was very low and not altered by different ephrin-B-Fc, and IL-5 was under detectable level in all wells. Collectively, the functional consequence of T-cell activation was confirmed to be uniquely modulated by each ephrin-B ligand in cooperation with TCR stimulation.

image

Figure 2. Modulation of cytokine secretion from TCR-stimulated T cells by ephrin-Bs. Supernatants of T-cell proliferation cultures were harvested at 48 h after initiation of culture. Cytokine concentrations were measured by a mouse Th1/Th2 cytokine bead array assay and are expressed as mean + SEM of n = 3 samples/replicates pooled from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student's t-test.

Download figure to PowerPoint

Modulation of T-cell proliferation by ephrin-Bs is independent from EphB1, EphB2, EphB3, and EphB6

According to the binding studies, EphA receptors bind to ephrin-As and EphB receptors bind to ephrin-Bs [[29]], although some exceptions have been found [[30]], such as, (i) EphA4 binds to ephrin-B2 and ephrin-B3, as well as ephrin-A ligands [[31, 32]] and (ii) EphB2 interacts with ephrin-A5 in addition to ephrin-B ligands [[33]]. Thus, we have focused on the all EphBs (EphB1, EphB2, EphB3, EphB4, and EphB6 in mouse) as the candidate receptors responsible for the unique modulation of TCR-mediated proliferation by ephrin-Bs in our culture system. To address this issue, T cells from mice deficient in single and multiple EphB receptors were analyzed.

First, the study tried to reconfirm that EphB6 deficiency compromised T-cell proliferation by anti-CD3 stimulation as previously reported [[34]]. T cells from EphB6–/– mice of Icr mix background showed impaired proliferation compared with wild-type littermates; however, it was not compromised in T cells from EphB6–/– mice on C57BL/6 background (Supporting Information Fig. 2). This finding indicated that the phenotype is genetic background dependent. EphB6–/– mice were then employed on Icr mix background for subsequent studies.

We first speculated that the unique modulations of T-cell proliferation by ephrin-Bs might be, at least partially, mediated by EphB6, because EphB6 transfected in HEK293T cells had been shown to induce biphasic effects in cell adhesion and migration in response to different concentrations of ephrin-B2 [[26]]. Although EphB6 is required to activate T-cell proliferation fully, the unique comodulatory pattern by each ephrin-B was virtually preserved in EphB6–/– T cells (Fig. 3A). Considering the redundancy of Eph function and the expression of all EphBs in T cells (Supporting Information Fig. 3), generation of multiple knockout mice lacking four genes, EphB1, EphB2, EphB3, and EphB6, was further investigated. EphB1, B2, B3, B6 quadruple knockout mice were viable and no apparent abnormality in appearance, however, showed similarly low survival and decreased lymphoid organ cellularity (Supporting Information Fig. 4) as previously reported in EphB2, B3 double mutants [[8]]. Surprisingly, no further alteration was observed in T cells from the quadruple knockout mice (Fig. 3B) compared with the EphB6 single deficiency (Fig. 3A), which suggested that the lack of either EphB6 or the four EphBs (EphB1/B2/B3/B6) negatively affects T-cell stimulation, and other Eph receptors were required for the unique modulation of T-cell proliferation by ephrin-Bs. Taken together, with the fact that EphB5 does not exist in mammals, these results suggest that the unique modification by ephrin-Bs might be regulated by EphB4 and/or EphA4.

image

Figure 3. Preserved unique modulation of TCR-mediated T-cell proliferation by ephrin-Bs in the absence of certain EphB receptors. T cells from mice deficient in (A) EphB6 and (B) EphB1/EphB2/EphB3/EphB6 were stimulated with immobilized anti-CD3 (0.8 μg/mL) together with each ephrin-B immobilized at the indicated concentrations. Results are expressed as mean counts per minute ± SEM of triplicate cultures and are representative of two or three independent experiments. Solid line, wild type; dotted line, EphB knockout mice. The scale of knockout mice data is magnified to show that the modulation pattern was not altered.

Download figure to PowerPoint

Cross-talk between Eph forward and TCR signaling pathways in biphasic modulation

The cross-talk of EphB forward signaling with the TCR pathway was next examined. Costimulatory receptors are needed to activate TCR signaling pathway optimally [[35]]. Wu and colleagues suggested that the EphB receptor and TCR were located closely in aggregated rafts and ephrin-B ligand enhanced TCR signaling, in which p38 and p44/42 MAPK activations were essential parts of ephrin-B1, B2, B3 costimulatory signaling [[18-20]]. To elucidate the importance of p38 and p44/42 MAPKs as ephrin-B-induced costimulatory signaling, inhibitors for these kinases were added in our culture system. The inhibitor of p38 MAPK and p44/42 MAPK significantly reduced the TCR-mediated proliferation, but did conserve the concentration-dependent effects by each ephrin-B (Fig. 4), suggesting that the interference with EphB signaling in TCR signal transduction occurred at the upstream of MAPKs, which is important for cell growth and survival.

image

Figure 4. The unique modulation pattern of TCR-mediated T-cell proliferation by ephrin-Bs is unchanged by inhibition of p38 MAPK and p44/42 MAPK. T cells from C57BL/6 mice were preincubated for 1 h with a p38 MAPK inhibitor (SB203580), p44/42 inhibitor (PD98059), or vehicle (dimethyl sulfoxide (DMSO), 0.1%). The cells were then stimulated with immobilized anti-CD3 (0.8 μg/mL) together with each ephrin-B immobilized at the indicated concentrations. Results are expressed as mean counts per minute ± SEM of triplicate cultures and are representative of two or three independent experiments.

Download figure to PowerPoint

To ensure the Eph signaling interaction with TCR pathway, the signaling events in T cells stimulated by ephrin-B1, ephirn-B2, and ephrin-B3 together with anti-CD3 were analyzed. Immunoblot analyses revealed that high concentrations of ephrin-B1 and ephrin-B2, but not ephrin-B3, clearly inhibited the anti-CD3-induced phosphorylation of Lck and its downstream signaling molecules, such as ZAP70, c-Raf, MEK1/2, Erk, and Akt (Fig. 5). This was not due to the insufficient contact of T cells with anti-CD3-coated culture bottom because the phosphorylation of Fyn and CD3-ζ was not inhibited by high concentrations of any ephrin-Bs (Fig. 5). In the absence of the anti-CD3 stimulation, these inhibitions of TCR signals were not observed by solely stimulation of ephrin-Bs (Supporting Information Fig. 5).

image

Figure 5. Immunoblotting analysis of TCR signaling pathway. T cells from C57BL/6 mice were stimulated with immobilized anti-CD3 (0.8 μg/mL) together with each ephrin-B immobilized at the indicated concentrations. Cells were harvested after 2 h and extracted proteins were analyzed for the phosphorylation of TCR signaling molecules by Western blot. The expression level of ERK indicates that equivalent amounts of cell lysates were loaded in each lane. Data are representative of two or three independent experiments. Red frame: suppression of phosphorylation by high dose ephrin-B1/B2; Blue frame: no suppression by ephrin-B3.

Download figure to PowerPoint

These data indicate that Eph signaling upon stimulation by high concentrations of ephrin-B1/B2 may engage in negative feedback to TCR signals via Lck.

EphB4 may function as the negative regulator of TCR signaling pathway through SHP1 recruitment

The biphasic modification of T-cell proliferation by ephrin-B1/B2 could be regulated by EphB4 and/or EphA4, as described above. Thus, we next investigated whether EphB4 forward signaling could be involved in this biphasic modulation. First, the phosphorylation of EphB4 receptor in the presence of low or high concentration of ephrin-Bs was examined by immunoprecipitation assay. Tyrosine phosphorylation of EphB4 receptor in WT T cells stimulated in the same culture system as proliferation assay for 2 h was clearly induced by high dose of ephrin-B1/B2 as well as ephrin B3, but not by low concentration (Fig. 6A upper panel). A protein tyrosine phosphatase (PTP), SHP1, is highly expressed in T cells [[36]], and has been known to dephosphorylate Lck specifically at Tyr-394 [[37]]. We speculated that EphB4 could be pivotal in this Eph cross-talk with TCR pathway via suppression of Lck by recruiting SHP1. As expected, the phosphorylated EphB4, which was activated by high concentration of ephrin-B1 and ephrin-B2, strongly recruited SHP1 (Fig. 6A).

image

Figure 6. Co-immunoprecipitation of EphB4 and SHP1 protein in T cells. T cells from C57BL/6 mice were stimulated with immobilized anti-CD3 (0.8 μg/mL) together with each ephrin-B immobilized at the indicated concentrations. Cells were harvested after 2 h and (A) EphB4 (n = 3) or (B) EphA4 (n = 3) was co-immunoprecipitated with SHP1. The quantification of immunoblots is on the right. Data are expressed as mean + SEM of n = 3 samples/replicates and are representative of three experiments. (C) Schematic model of EphB/ephrin-B-mediated suppression of TCR signaling shows the interaction of ephrin-B1/B2 and the EphB4 receptor on primary T lymphocyte when costimulated by low (2.5 μg/mL) or high (20 μg/mL) concentration of ephrin-B1/B2 under TCR stimulation. A high concentration of ephrin-B1/B2 induces the recruitment of SHP1 by EphB4, which inhibits pLck in the TCR signaling cascade.

Download figure to PowerPoint

This SHP1 recruitment was observed only under the TCR stimulation (Supporting Information Fig. 6). On the other hand, ephrin-B3 stimulation did not show SHP1 association with activated EphB4 (Fig. 6A). In addition to EphBs, EphA4 is known to interact with ephrin-B ligands. The previous study has reported EphA4 expression in peripheral T cells [[11]]. Then, we also examined the association of EphA4 with SHP1 after the stimulation by ephrin-Bs. Immunoblotting assay revealed the apparent phosphorylation of EphA4 by high concentration of any ephrin-Bs, however, none of these activation signals resulted in SHP1 recruitment (Fig. 6B). EphB6 seems to be partly involved in T-cell proliferation as described above (Fig. 3A), but ligand-activated phospho-EphB6 as well as phospho-EphA4 did not interact with SHP1 (Supporting Information Fig. 7).

Collectively, these data suggest that EphB4 may contribute to the unique biphasic modulatory effect by ephrin-B1/B2 through the recruitment of SHP1 (Fig. 6C). In contrast to ephrin-B1/B2, the phospho-EphB4 induced by ephrin-B3 could not couple with SHP1, which has the inhibitory effect of Lck phosphorylation.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

In this study, we elucidated that ephrin-B1 and ephrin-B2 belong to a novel class of costimulatory molecules with unique action, namely, a concentration-dependent switch from costimulation to inhibition; whereas, ephrin-B3 simply exerts a steadily increasing stimulatory effect in TCR-mediated regulation of primary T cells via Eph receptors other than EphB1/B2/B3/B6. The unique inhibitory effects by the high concentrations of ephrin-B1/B2 occur as a consequence of cross-talk of EphB4 signaling on TCR cascade, most likely targeting Lck.

Although Eph receptors/ephrin ligands were initially recognized as mediators of repulsive signals in growing axons, it is now clear that their functions are versatile, including attractive and adhesive property. In vivo, ephrin-Bs have been shown to act as both attractants and repellents for retinal axons during the developmental stage of the visual system [[24]]. However, it remains unclear whether the reciprocal effects in vivo are mediated by the same ephrin ligand in the same cell since these effects are dependent in time and space where the expression of Eph receptors/ephrin ligands would be variable. Definitive demonstration of biphasic action of this system can be done in in vitro system. Recently, Hansen et al. elegantly demonstrated that ephrin-As induced the biphasic retinal axon growth [[21]]. Alfaro et al. [[7]] demonstrated that immobilized Eph-B2-Fc and ephrin-B1-Fc modulated the anti-CD3 antibody-induced apoptosis of CD4+CD8+ thymocytes in a concentration-dependent manner. Our present study has addressed the direct proof of biphasic effect of ephrin-Bs on the proliferation of the primary immune cells. In addition to the function in cell positioning including attraction, adhesion, and repulsion which have been mainly investigated in the nervous system, our study demonstrated for the first time that this biphasic regulation is functional in cell proliferation, as well.

According to the studies for functional determination, Eph receptors can promote adhesion/attraction in a kinase-independent manner; whereas, repulsive function requires tyrosine kinase activity and receptor phosphorylation [[26, 38, 39]]. Eph receptors may possess two distinct functional sites, (i) adhesion by extracellular kinase-independent domain and (ii) repulsive/inhibitory signaling by intracellular kinase-dependent domain. The concentration of ephrin ligands would be one of the factors to determine the balance between these two functions. Ephrin-B1/B2/B3 may promote TCR response via the extracellular domain of EphB receptor as a consequence of stabilization of T cells on anti-CD3 antibody. On the other hand, high dose of ephrin-B1/B2 strongly suppresses T-cell proliferation via inhibitory cross-talk signal with TCR pathway (Fig. 7). Since it has been shown that EphB forms a clustering cap on T cells together with TCR upon stimulation by ephrin-Bs [[18-20]], high density of Eph receptors in lipid raft may be critical for their phosphorylation. Interestingly, as a similar system, ligand concentration-dependent switch of cell behavior has been documented in platelet-derived growth factor (PDGF) signaling. NIH3T3 fibroblasts switch the behavior from migration to a proliferation in response to increasing concentrations of PDGF [[40]]. In oligodendrocyte precursor cells (OPCs), only low concentration of PDGF induces phosphoinositol 3-kinase (PI3K) activation for cell motility, and conversely, only high concentration induces PLC-γ activation for proliferation [[41, 42]]. This provides an elegant model of “rheostat” control mechanism by RTKs to interpret ligand levels to stimulate cell migration in a zone of low ligand level and inhibit migration in high ligand level to recruit them where they should be.

image

Figure 7. Model of the positive and negative effects of ephrin-Bs on TCR-mediated T-cell proliferation. As the ephrin-B concentration increases (green wedge), a simple adhesive interaction leads to the stabilization of anti-CD3-mediated TCR stimulation (orange), which results in an increasing positive effect (red shading, top module). At higher concentrations, a strong negative effect emerges due to the triggered intracellular suppressive cross-talk signaling on Lck phosphorylation (blue shading, top module). Ephrin-B3 lacks this suppressive effect on Lck phosphorylation (right). The overall effect of these positive and negative molecular influences would be a concentration-dependent transition from net positive to net negative effects on T-cell proliferation.

Download figure to PowerPoint

EphB4 receptor plays important roles in a variety of biologic processes, including cell aggregation and migration, neural development, embryogenesis, and angiogenesis/vascular development [[43-45]]. Among all mice deficient in each EphB receptor, only EphB4 deficiency appears to be lethal during the embryonic period due to the impaired morphogenesis of the capillary vessel network, which requires an extremely precise organization [[46]]. Our data from multiple EphB knockout mice (Fig. 3B) and high-dose ephrin-B1/B2-induced EphB4 phosphorylation in association with SHP1 recruitment (Fig. 6A) strongly suggest that the inhibitory cross-talk signal is most likely mediated by EphB4. EphB4 forward signaling has been shown to inhibit cellular proliferation and decrease MAPK activity in other cells as shown in mouse primary T cells [[47-49]].

In contrast to ephrin-B1/B2, ephrin-B3 stimulated EphB4 phosphorylation without recruitment of SHP1 (Fig. 6A), indicating that the different ligands can induce different signals through a same receptor. Another class of RTKs, ErbB/EGF-family receptors, have been shown to lead to differential phosphorylation by binding of different growth factor ligands, possibly due to differential receptor aggregation and conformation [[50, 51]]. This discrimination results in the different recruitment of signaling molecules and attributes to the diversity of RTK functions.

SHP1 has been known to negatively regulate T-cell signaling [[36]] and to dephosphorylate Lck tyrosine protein kinases at Tyr-394 [[37]]. It seems to be reasonable that SHP1 participates in EphB4-mediated TCR signal suppression for following reasons, (i) suppression of pLck was confirmed by the anti-Y394 (Fig. 5), (ii) another EphB family receptor, EphB6, is shown to form the complex with SHP1 in Jurkat cell [[52]]. The tyrosine phosphatase CD45 has also been known as a regulator of Lck which could dephosphorylate both Tyr-394 and Tyr-505 regulatory sites [[53]]. Because we found no significant change in phosphorylation at Tyr-505 of Lck under the ephrin-Bs costimulation (data not shown), the association between Eph and CD45 may not be involved.

Wu and colleagues [[18-20]] have previously reported that EphB receptors and TCR were located closely in aggregated rafts and ephrin-B ligand simply enhanced TCR signaling, in which p38 and p44/42 MAPK activations were essential parts of ephrin-B1/B2/B3 costimulation. However, in our study, the suppressive phase in primary T-cell proliferation induced by solid-phase ephrin-B ligands with CD3 stimulation has been newly revealed. Cytokine assay also showed the different costimulation effects from Wu and colleagues’ previous data. In their studies, the lymphokinetic pattern induced by ephrin-B1, B2, and B3 ligand costimulation was different from that of CD28 in T-cell proliferation; wherein, it remarkably stimulated production of IFN-γ but not IL-2 possibly due to the absence of Akt activation. In our assay, IL-2 production, as well as IFN-γ and TNF-α, is regulated biphasically by costimulation with ephrin-B1/B2, and was simply promoted by ephin-B3. This implies that IL-2 secretion is evident, as well as IFN-γ and TNF-α, in ephrin-B costimulation. In the promotion phase, EphB receptor functions as one of the costimulatory molecules like CD28. We speculate that the discrepancy between the results may be due to the differences in the origin and concentration of ephrin-B ligands (Wu and colleagues utilized their own ephrin-B-Fc chimeric proteins, while we purchased from R&D systems) and the genetic background of the mouse. One could argue that the unique modification patterns that we observed might be due to the replacement of anti-CD3 antibody by high-dose ephrin-Bs during the coating procedure. But it is very unlikely because of following three reasons, (i) each concentration of normal human IgG instead of ephrin-Bs leads to no inhibition of the anti-CD3 induced T-cell proliferation (Fig. 1A), (ii) high dose of ephrin-B3 did not inhibit (rather promoted) the proliferation in the same culture system, (iii) SHP1 recruitment by EphB4 (Fig. 6A), but not by EphA4 (Fig. 6B) or EphB6 (Supporting Information Fig. 7), reasonably explains the functional inhibition of TCR signaling. We also conducted the culture with wells coated with ephrin-Bs in the presence of soluble anti-CD3 antibody. In this assay, however, the modification patterns by ephrin-Bs were not observed (Supporting Information Fig. 8). Considering the fact that Eph/ephrin system basically works in the cell-cell contact and that other costimulatory or inhibitory molecules in TCR signaling needs to be gathered in the lipid raft where TCR aggregates, it is strongly suggested that both anti-CD3 antibody and ephrin-Bs have to be presented at the same site with a high density to observe the unique modification of TCR signaling by ephrin-Bs.

In this study, the TCR-mediated primary T-cell activation is demonstrated to be highly governed by EphB/ephrin-B axis with a complexity determined by the combination, as well as, the concentration of different ephrin-Bs expressed in immunological microenvironments. EphB4 involved in negative feedback of T-cell activation could be a novel therapeutic target to inhibit the most proximal TCR signaling molecule through the recruitment of SHP1. The generation of strong signaling molecule, which could mimic ephrin-B1/B2, would be an effective strategy to control T cell-mediated immune disorders.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Mice

EphB1, EphB2, EphB3–deficient (EphB1–/–, EphB2–/–, EphB3–/–: Icr background) and EphB6-deficient (EphB6–/–) mice (C57BL/6/129Sv hybrids, which were crossed onto C57BL/6 background for nine generations, and Icr/129Sv hybrid (Icr mix) were generated as previously described [[54-57]]. Multiple EphB-deficient mice were generated by intercrossing EphB1–/–, EphB2–/–, EphB3–/–, and EphB6–/– (Icr mix) mice. C57BL/6J mice were purchased from Japan CLEA (Chiba, Japan). All animal experiments were approved by the Institutional Animal Care and Use Committee and were carried out according to the Kobe University Animal Experimentation Regulations.

T-cell isolation

Splenic T cells from 8–12-wk-old C57BL/6 or Icr mice were isolated by immunomagnetic beads (pan T-cell isolation kit for negative selection) with autoMACS (Miltenyi Biotec, Germany). The purity of CD4/CD8 T cells was more than 90%.

Culture for T-cell proliferation and 3H-thymidine uptake assay

Solid-phase ephrin-Bs and anti-CD3 were prepared by coating 96-well U-bottom Falcon Plates (Falcon 35–3077, Becton Dickinson, Franklin Lakes, NJ, USA), by firstly incubating with anti-CD3 (clone 145–2C11, BD Pharmingen, San Diego, CA, USA) in phosphate buffered saline (PBS) at 37°C for 2 h, and after washing with PBS twice subsequently followed by incubation with different concentrations of ephrin-B1-Fc (473-EB, R&D systems, Minneapolis, MN, USA), ephrin-B2-Fc (496-EB, R&D systems), ephrin-B3-Fc (395-EB, R&D systems), or normal human IgG (NHIgG as a control, I4506, Sigma, St Louis, MO, USA) in PBS at 37°C for 2h. T cells (2 × 105 cells per well) were cultured in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum (FBS), 1 × nonessential amino acid, 50 μM β-mercaptoethanol, 100 μg/mL penicillin-streptomycin at 37°C, and 5% CO2 for 48 h. In some experiments, the liquid phase (for RT-PCR) and solid phase (for other experiments) anti-CD28 (clone 37.51, BD Pharmingen) were used for costimulation, instead of solid-phase ephrin-Bs. In another assay, the soluble anti-CD3 was employed. Antibodies and ephrin-B-Fc chimeric proteins were used at indicated concentrations. Cell proliferation was determined by adding 1 μCi of 3H-thymidine per well 16 h before the end of the incubation. The cultures were harvested with Filter Mate cell harvester and estimated by using Top Count (PerkinElmer, Waltham, MA, USA). In some experiments, the following pharmacological inhibitors were used; p44/42 MAPK (Erk1/2) inhibitor, PD98059 (Promga, Madison, WI, USA), and p38 inhibitor, SB203580 (Promega) at 10 μM.

Cytokine detection

Supernatants of T-cell proliferation cultures were harvested at 48 h after initiation of culture. Concentrations of IFN-γ, TNF-α, IL-2, IL-4, and IL-5 were measured by mouse Th1/Th2 cytokine bead array assay (BD Bioscience) according to manufacturer's recommendation.

Flow cytometry

Thymus was obtained from 6-wk-old C57BL/6 mice, and cell suspensions were stained for 15 min in PBS+0.5% BSA with specific mAbs against CD4-PE and CD8-FITC (BD Bioscience). After staining, cell suspensions were washed and resuspended for analysis. Flow cytometric analysis was performed by a FACScan (BD Bioscience).

Immunoblotting and Immunoprecipitation

Splenic T cells from C57BL/6J mice were starved in RPMI1640 + 0.1% FBS at 37°C and 5% CO2 for 4 h at the cell concentration of 1 × 106/mL. After the starvation, cells were resuspended in RPMI1640 + 0.1% bovine serum albumin (BSA), and seeded in the plates precoated with anti-CD3/ephrin-Bs at 2 × 105 cells/well. The plates were centrifuged at 350 rpm for 3 min to achieve rapid contact between the cells and the bottom of the culture wells. The cells were incubated at 37°C and 5% CO2 for 2 h. Then, the cells were harvested and washed with ice-cold PBS. Cell lysis and subsequent Western blotting were performed as previously described [[58]] with minor modifications. Briefly, cells were lysed in cell lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM sodium vanadate, 50 mM sodium fluoride, and protease inhibitor cocktail (Sigma Aldrich). For immunoprecipitation, RIPA lysing buffer (50 mM Tris-HCl, pH 7.5, 137 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM sodium vanadate, 50 mM sodium fluoride, and protease inhibitor cocktail) was used. The lysates were boiled with SDS-loading buffer. Equal amount of sample proteins (35 μg) were separated on 7.5–16% SDS-PAGE and transferred onto PVDF membranes (Immobilon, Millipore, Billerica, MA, USA). The membranes were first incubated with TBST (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween20) containing 5% nonfat dried milk and probed with specific antibodies using primary and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA, USA). Immune complexes were detected by chemiluminescence (Immobillon Western, Millipore). For immunoprecipitation, total cell lysates were incubated with anti-PY antibody (clone 4G10, Millipore) and protein G-sepharose (GE Healthcare Bio-Sciences AB, Sweden) for 18 h at 4°C. The immunoprecipitates were washed with lysis buffer and then with PBS. The blotting membranes were incubated with biotinylated rabbit anti-goat IgG (BA-5000, Vector Laboratories, Burlingame, CA, USA) followed by the amplification with ABC system (Vectastain Elite ABC Reagent, Vector Laboratories). The rabbit anti-phospho-Lck (Tyr394) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit anti-phospho-ZAP70 (Tyr319/Tyr352), anti-phospho-Akt (Ser473), anti-phospho-Erk1/2 (Thr202/Tyr204), anti-Erk1/2, anti-phospho-MEK1/2 (Ser217/221), and anti-phospho-c-Raf (Ser338) antibodies were purchased from Cell Signaling Technologies. The rabbit anti-phospho-CD3 (Tyr142) and rabbit anti-phospho-Fyn (Tyr530) antibodies were purchased from abcam (Cambridge, MA, USA). The goat anti-EphB4 antibody (AF446) was purchased from R&D Systems.

Coimmunoprecipitation

2% CHAPS buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl2 was used in this assay. Total cell lysates containing 130 μg protein was incubated with goat anti-EphB4 antibody (AF446, R&D Systems) or anti-EphA4 antibody (AF641, R&D systems) or anti-EphB6 antibody (AF611, R&D systems), and protein G-sepharose (GE Healthcare Bio-Sciences AB) for 18 h at 4°C. Following procedures were same as the immunoprecipitation assay, except for using biotinylated horse anti-mouse IgG (BA-2000, Vector Laboratories) to detect SHP1. The mouse SHP1 antibody was purchased from Santa Cruz Biotechnology. Image quantification was determined by National Institutes of Health ImageJ software (Bethesda, MD, USA).

Statistical analysis

All values were reported as mean ± SEM. Statistical significance for two unpaired groups was assessed by the Student's t-test. Significance was set at *p< 0.05, **p< 0.01, ***p< 0.001.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

This work was supported by the Grants-in-Aid for the Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) (#20012033), from Japan Society for the Promotion of Science (JSPS) (#21591243), and from the Ministry of Health, Welfare, and Labor in Japan (H22-GANNRINSHO-Ippan032), and a Grant to YK from The Uehara Memorial Foundation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information
Abbreviations
RTK

receptor tyrosine kinase

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2264-sup-0001-Figures1.ppt9040K

Figure 1. Fluorescence-activated cell sorter (FACS) analysis of spleen cells from RA/EG and RA/EG × CD11cCre mice.

Figure 2. Comparison of HIF1αflox, cHIF1αCCL17, and cHIF1αCD11c bone marrow derived dendritic cell (BMDC) for expression of maturation markers.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.