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Keywords:

  • Cell trafficking;
  • Human;
  • Kinases/phosphatases;
  • T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements

We have previously demonstrated that binding of ephrin-A1 to Eph receptors on human CD4+ T cells stimulates migration. Here, we show that a distinct population of CD8+ T lymphocytes, expressing the chemokine receptor CCR7, also binds ephrin-A1 and is stimulated to migrate after binding. The Eph receptor signaling pathway taking part in the migration event was here investigated. Induced tyrosine phosphorylation of several proteins was seen after ephrin-A1 binding. In particular, induced phosphorylation and kinase activity of the Src kinase family member Lck was observed. An Lck inhibitor inhibited ephrin-A1-induced migration, indicating the involvement of Lck in the migration event. In addition, we observed an induced association of the focal adhesion-like kinase proline-rich tyrosine kinase 2 (Pyk2) and the guanidine exchange factor Vav1 with Lck. PI3K inhibitors also inhibited migration, and studies in transfectants indicate an association of PI3K with EphA1. Further, ephrin-A1-induced migration could be related to the activation of Rho GTPases. This was also observed by using an inhibitor of the Rho-associated kinase ROCK, a downstream effector of Rho. Our results suggest that stimulation of Eph receptors on CD8+CCR7+ T cells leads to migration involving activation of Lck, Pyk2, PI3K, Vav1 and Rho GTPase.

Abbreviations:
GEF:

guanine nucleotide exchange factor

PYK2:

proline-rich tyrosine kinase 2

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements

Eph receptors are the largest subfamily of receptor tyrosine kinases, interacting with the ephrin family of ligands. These receptors can be divided into two subgroups, EphA and EphB. The EphA class consists of nine members that in general bind ephrin-A members, linked to the plasma membrane through a GPI anchor. The EphB class consists of six members that in general bind ephrin-B members, which transverse the cell membrane 1. Eph receptors have been shown to be important mediators of cell-cell communication regulating cell attachment, shape and mobility. Functional aspects of Eph receptor signaling have in particular been studied in neuronal and endothelial cells 26.

The identification of Eph receptors and ephrins on cells belonging to the immune system indicates a function for these molecules in immunity. Eph receptors have been identified on different types of DC. Specifically, the EphB1 receptor has been reported in plasmacytoid DC 7, and expression of different Eph receptors has been identified on Langerhans- and interstitial-type DC 8, 9. Signaling through EphA2 on Langerhans DC has been shown to affect adhesion and antigen presentation capacity 8, 9. T lymphocytes also express certain Eph receptors and ephrins of both subfamilies in blood, thymus and in lymphoid organs 1016. Several aspects of T cell function have been suggested for Eph receptors on T cells, like enhancement of TCR signaling strength, regulation of T cell mobility and maintenance of lymphoid organ structural integrity 1218. In view of the highly complex nature of lymphocyte traffic and the role of Eph receptors in other cell systems, it is possible that these receptors are involved in cell migration or cell positioning in lymphatic tissues.

We have previously observed effects on migration after EphA receptor signaling in CD4+ T cells 11. Effects on migration as an outcome of Eph receptor signaling have been shown in different cell types 1924. Molecules identified to be involved in Eph signaling events leading to migration are PI3K, Src kinases, MAPK and different adapter molecules 1924. The small GTPases of the Rho family, like RhoA, Rac1 and Cdc42, have distinct effects on the actin cytoskeleton 2528. They have been shown to be involved in biological effects such as migration and adhesion, after Eph receptor stimulation in neuronal cells, melanoma cells and muscle cells 19, 2932.

In the present study, we describe the expression of receptors binding ephrin-A1 on CD8+CCR7+ T cells, and show that this binding stimulates migration. We present data on the identification of molecules involved in signaling through Eph receptors in CD8+ T cells and relate them to the functional outcome. In particular, Lck, proline-rich tyrosine kinase 2 (Pyk2), PI3K, Vav1 and Rho GTPases are involved in Eph receptor signaling leading to migration of CD8+ T cells.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements

CD8+ T lymphocytes express ephrin-A1-binding receptor(s)

CD8+ T cells isolated from blood were tested for their ability to bind ephrin-A1. Approximately half of the CD8+ T cells bound ephrin-A1-Fc (Fig. 1A). Co-staining with Ab directed to the chemokine receptors CCR5 and CCR7, and L-selectin (CD62L), was performed to investigate the phenotype of the ephrin-A1-binding cells. Both CCR7 and CD62L coexpressed with ephrin-A1-Fc binding (Fig. 1A). The majority of CCR5+ cells did not bind ephrin-A1 (Fig. 1A). These data indicate that recirculating naive and central memory CD8+ T cells 3335 express EphA receptors comparable with the expression pattern of EphA receptors previously reported on CD4+ T cells (CD4+, CD62L+) 11. EphA1 protein expression and EphA4 mRNA expression have previously been described in CD4+ T cells 11. Here, we show EphA1 mRNA expression in CD8+ T cells (Fig. 1B). In addition, EphA1 protein expression was detected by Western blot analysis in CD4+ and CD8+ T cells, but not in CD19+ B cells (Fig. 1B). CD19+ B cells do not bind ephrin-A1 11. EphA4 mRNA expression could not be detected in CD8+ T cells by Northern blot analysis (data not shown). We could not detect expression of functional ephrin-A members on T cells. No binding of soluble EphA2-Fc, which binds to all ephrin-A members 36, 37, could be detected on CD8+ T cells (Fig. 1C). No ephrin-A1 mRNA expression could be detected in freshly isolated blood T cells by Northern blot analysis (data not shown). Freshly isolated blood T cells express low or no ephrin-A4 mRNA that can be induced after activation of the cells 10.

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Figure 1. Ephrin-A1 binds to CD8+CCR7+ lymphocytes. (A) Co-staining of CD8+ T cells with control-Fc or ephrin-A1-Fc and either irrelevant Ig, anti-CCR5, anti-CCR7 or anti-CD62L Ab. Ephrin-A1-binding cells are predominantly CCR5CCR7+CD62L+. (B) Expression of EphA1 mRNA (upper left panel) by Northern blot analysis of CD4+ and CD8+ T cells (0 – freshly isolated), and after CD3/CD28 stimulation of CD8+ T cells (1 – day 1). Lower left panel shows 18S and 28S rRNA bands on the same ethidium bromide-stained agarose gel. Expression of EphA1 protein is shown by Western blot analysis of total cell lysates (upper right panel). CD4+ and CD8+ T cells but not CD19+ B cells express EphA1. Lower right panel shows actin used for loading control. (C) Co-staining of CD8+ T cells with control-Fc, ephrin-A1-Fc or EphA2-Fc and anti-CCR7 Ab.

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To conclude, ephrin-A1-binding receptors (EphA receptors) on CD8+ T cells are mainly found on the CCR7+ naive and central memory subsets.

Ephrin-A1 binding to CD8+ T lymphocytes induces migration

We have previously shown that ephrin-A1 binding to CD4+ T cells stimulates migration and increases CXCL12- and CCL19-induced chemotaxis 11. The effect of ephrin-A1 on CD8+ T cells was also tested in migration assays. Two different chemokines were tested: CCL21 which binds to the CCR7 receptor and CXCL12 which binds to the CXCR4 receptor 38. The expression of CXCR4 corresponds to CCR7 expression on CD8+ naive and central memory subsets 39. Transwell assay plates were used to study the migration of CD8+ T cells. CD8+ T cells were incubated with ephrin-A1-Fc or control-Fc under serum-free conditions, and the cells that passed through the membrane were counted by flow cytometry. The results show that ephrin-A1-Fc induced migration of CD8+ cells when added to the cells in the upper well (Fig. 2A, B). Typically, 20–25% of input cells migrated through the membrane after ephrin-A1 stimulation, meaning that approximately half of the EphA receptor-positive cells migrated within the time limit of the experiment. No significant increase in migration could be observed when ephrin-A1-Fc was added only to the lower chamber. This indicates that ephrin-A1 did not induce a chemotactic-like response (Fig. 2A) but rather induced a random migration effect (chemokinesis). No significant difference in ephrin-A1-induced migration could be observed when cells were exposed to ephrin-A1-Fc both in the lower chamber and in the upper well, compared with ephrin-A1-Fc added only in the upper well (data not shown). Minimal migration was observed with the control-Fc protein only (Fig. 2A). Addition of CCL21 to the lower chamber increased ephrin-A1-Fc-induced migration. Cells exposed to the control-Fc protein were also stimulated to migrate after CCL21 gradient exposure (Fig. 2A). Increasing concentrations of CXCL12 (ranging from 100 to 300 ng/mL) were tested in combination with ephrin-A1-Fc to determine its effect on migration. The data shows increased migration with ephrin-A1 and increasing CXCL12 concentrations (Fig. 2B). The CXCL12-induced migrating effect was diminished when CXCL12 was added to both the upper and lower wells (Fig. 2B).

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Figure 2. Ephrin-A1 stimulates migration. (A) CCL21 chemotaxis assay with CD8+ T cells. Fc – control-Fc, A1 – ephrin-A1-Fc. Fc and A1 were added to the upper well, unless described otherwise. FcL – Fc only in the lower chamber, A1L – A1 only in the lower chamber. Where indicated, 250 ng/mL of CCL21 was added to the lower chamber and Fc and A1 added to the upper well. Standard error of the mean (SEM) is shown for four separate experiments. (B) CXCL12 chemotaxis assay with CD8+ T cells. Numbers in the figure (100, 200 and 300) relate to the amount of CXCL12 in ng/mL added to the lower chamber. U/L refers to CXCL12 added both in the upper well (U) and in the lower chamber (L). SEM is shown for four separate experiments.

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To conclude, ephrin-A1 binding to EphA receptors induces migration of CD8+ T cells similar to what was previously described for CD4+ T cells 11.

Activation of the Lck kinase after EphA receptor stimulation

A Src family kinase inhibitor, SU6656, did not inhibit migration, but surprisingly increased ephrin-A1-induced migration of CD4+ T cells 11. Here, we show that SU6656 also increased ephrin-A1-induced migration of CD8+ T cells (Fig. 3A). The effect was most pronounced when the cells were incubated with SU6656 for only a few minutes before assay start, while 30 min of incubation led to a less stimulatory effect (data not shown). SU6656 did not have an effect on control-Fc-treated cells (data not shown). Src family kinases have previously been shown to be activated through chemokine receptors and to be involved in migration events 4042. The observed effect of SU6656, and previous reports indicating Src family kinase members to be involved in EphA receptor signaling 22, 4345, led us to investigate if EphA receptor signaling affected the Lck kinase. First, the effect on migration of an Lck kinase inhibitor, Damnacanthal 38, 46, was tested. Damnacanthal clearly inhibited ephrin-A1-induced migration of CD8+ T cells at both 1 and 0.1 µM concentration (Fig. 3A), indicating that the Lck kinase could be involved in the signaling events leading to migration. Damnacanthal did not have an effect on control-Fc-treated cells (data not shown). Lck was immunoprecipitated from ephrin-A1-stimulated CD8+ T cell lysates to further investigate the effect of EphA receptor signaling on Lck phosphorylation and activity. Tyrosine phosphorylation of Lck was detected using a general anti-phospho-tyrosine Ab (4G10) and an anti phospho-Src Ab. The anti phospho-Src Ab cross-reacts with Lck (and other Src family kinase members) and recognizes a phosphorylated tyrosine residue in the activation loop of the kinase domain of Src family kinases, tyrosine residue 394 (Y394) in Lck. Phosphorylation of this tyrosine leads to up-regulation of the Lck kinase activity 47. Our results show that Lck is indeed phosphorylated after EphA receptor stimulation of CD8+ T cells (Fig. 3B). In particular, induced tyrosine phosphorylation of Y394 was observed, indicating induced activation of the Lck kinase. Some differences in the phosphorylation kinetics of Y394 were observed between different experiments (Fig. 3B, 4B), but repeatedly Y394 induced phosphorylation was observed between 5 and 10 min after addition of ephrin-A1-Fc. Phosphorylation of Lck Y394 was also observed in HEK293T cells co-transfected with EphA receptor and Lck followed by stimulation with ephrin-A1 (see below). Next, an Lck kinase assay was performed to investigate if the observed phosphorylation of Y394 in Lck led to increased kinase activity. A kinase activity assay was performed using Lck immunoprecipitates from cell lysates followed by incubation with ATP and the Src family kinase substrate Sam 68 (see Materials and methods). The results from CD8+ cells show induced kinase activity of Lck after 5 min of ephrin-A1 incubation (Fig. 3C). Correspondingly, the results from HEK293T cells co-transfected with EphA receptor and Lck clearly show induced kinase activity after 5 min of incubation with ephrin-A1 (Fig. 3D). The effect of Damnacanthal on Lck activation was investigated in EphA1 + Lck transfectants. Inhibition of Lck Y394 phosphorylation was observed after ephrin-A1 stimulation of Damnacanthal-treated transfectants, while Damnacanthal did not affect ephrin-A1-induced phosphorylation of EphA1 (data not shown).

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Figure 3. Ephrin-A1 binding activates the Lck kinase. (A) Effect of the Lck inhibitor Damnacanthal (DA) and the Src kinase inhibitor SU6656 (SU) on ephrin-A1-stimulated CD8+ T cell migration. Fc – control-Fc, A1 – ephrin-A1-Fc. Fc and A1 were added to the upper well. The numbers reflect concentrations of inhibitors in µM. SEM is shown for four separate experiments. (B) Stimulation of CD8+ T cells with ephrin-A1-Fc for the indicated times (min). Lck was immunoprecipitated from cell lysates. The Western blot was incubated with anti-phospho-tyrosine (4G10), anti-phospho-Src (p-Src) and anti-Lck Ab. The Western blot was stripped between the different Ab incubations. (C) In vitro Lck kinase assay. Left panel CD8+ T cells, right panel transfected HEK293T cells. Ephrin-A1 stimulation for the indicated times (min). Lck was immunoprecipitated from cellular lysates and subjected to in vitro kinase assay using Sam 68-GST as substrate. Sam 68 phosphorylation was detected with 4G10. The filters were incubated with anti-Lck Ab as a control for equal loading of immunoprecipitates, and with anti-GST Ab for detection of equal addition of Sam 68-GST into the kinase reaction.

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To conclude, our data from CD8+ T cells and HEK293T transfectants indicate a link between EphA1 receptor stimulation and phosphorylation and activation of the Lck kinase, suggesting that Lck is involved in ephrin-A1-induced migration.

Association of Lck and Pyk2 after ephrin-A1 stimulation

Previously, we described induced tyrosine phosphorylation of Pyk2, a focal adhesion-like kinase, after ephrin-A1 stimulation of CD4+ T cells 11. The tyrosine phosphorylation status of this kinase was therefore also investigated after EphA receptor stimulation of CD8+ T cells. Induced tyrosine phosphorylation of Pyk2 was observed in CD8+ T cells (Fig. 4A). In particular, phosphorylation of the tyrosine residues 402 and 580 was detected. Pyk2 has previously been shown to associate with Src family kinase members including Lck 48, 49. It was therefore investigated if phosphorylated Pyk2 could associate with Lck after ephrin-A1 incubation. Immunoprecipitation of Pyk2 from ephrin-A1-stimulated CD8+ T cells repeatedly co-precipitated Lck (Fig. 4A), shown by the anti-phospho-Src Ab described above and anti-Lck Ab detection. In addition, immunoprecipitation of Lck from ephrin-A1-stimulated CD8+ T cells repeatedly co-precipitated Pyk2 (Fig. 4B). Both a phospho-Y402-specific and a general anti-Pyk2 Ab detected this interaction at the time points corresponding to Y394 phosphorylation of Lck. Phosphorylated Pyk2 Y580 interaction with Lck could be detected at the same time points as Y402 phosphorylation (data not shown). These data indicate that Pyk2 and Lck are associated after EphA receptor activation in T cells.

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Figure 4. Ephrin-A1 induces tyrosine phosphorylation of Pyk2 in CD8+ T lymphocytes. CD8+ T cells were stimulated for the indicated times (in min) with ephrin-A1-Fc. (A) PYK2 immunoprecipitation. The Western blot was incubated with anti-phospho-Pyk2 Ab (Pyk2 Y580 and Pyk2 Y402), anti-Pyk2, anti-phospho-Src (p-Src) and anti-Lck Ab and stripped between incubations. Lck co-precipitated with Pyk2. (B) Lck immunoprecipitation. The Western blot was incubated with the indicated Ab and stripped between incubations. Pyk2 co-precipitated with Lck.

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PI3K is involved in EphA receptor signaling

Ephrins have previously been shown to induce migration in endothelial cells through the PI3K pathway 1921. The effect of a PI3K inhibitor, LY294002, on ephrin-A1-induced migration was therefore tested. A concentration range of LY294002 from 25 to 0.5 µM was used in transwell migration experiments. At 25 µM, LY294002 nearly completely inhibited the migrating effect of ephrin-A1, and effects could be observed even at the lowest LY294002 concentrations (0.5 µM) (Fig. 5A). Treatment with the PI3K inhibitor wortmannin gave a similar inhibition of migration (data not shown). These data indicate an involvement of PI3K in the migratory events after ephrin-A1-Fc stimulation.

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Figure 5. PI3K, Vav1 and Rho GTPase involved in EphA signaling. (A) CD8+ T cells were treated or not with different concentrations of the PI3K inhibitor LY294002 (LY) before the migration assay. Fc – control-Fc, A1 – ephrin-A1-Fc. Fc and A1 were added to the upper well. Numbers show different concentrations of LY294002 (µM). SEM is shown for four separate experiments. (B) Association of Vav1 with Lck. Lck immunoprecipitation was performed on CD8+ T cell lysates followed by Western blot analysis. The blot was incubated with the indicated Ab and stripped between the incubations. Correspondingly, Vav1 was immunopresipitated from ephrin-A1-stimulated cell lysates followed by Western blot analysis. The blot was incubated with the indicated Ab and stripped between the incubations. (C) GTP-bound Rho was precipitated from CD8+ T cells using Rhotekin-GST (upper panel). Time points for ephrin-A1-Fc stimulation (min) are shown. Western blot analysis of total Rho input (lower panel). (D) CD8+ T cells were incubated with different concentrations of the ROCK inhibitor Y-27632 before the start of the migration assay. Numbers indicate Y-27632 concentrations in µM. SEM is shown for four separate experiments.

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Vav1 and Rho GTPase are involved in EphA receptor signaling

The effect of ephrin-A1 on T cell migration led us to investigate Vav (Vav1–3) and Rho family members (Rho, Rac, Cdc42) after ephrin-A1 stimulation of CD8+ T cells. Vav1 is restricted to cells of hematopoietic origin and has previously been shown to associate with Lck in T cells 50, 51. Association of Vav1 with immunoprecipitated Lck correlated with the activation of Lck, detected by the anti-phospho-Src Ab (Fig. 5B). Correspondingly, immunoprecipitation of Vav1 also co-precipitated Lck in an ephrin-A1-induced manner (Fig. 5B). Our results here indicate an involvement of Vav1 in EphA1 signaling and a possible association in a complex with Lck.

Rho activation was investigated by precipitating active GTP-bound Rho with the Rho binding domain of Rhotekin 52. Our data show that Rho is activated within 2 min after ephrin-A1 stimulation (Fig. 5C). Which Rho GTPase member is involved was not defined here, because the anti-Rho Ab used in this study detects all members (Rho A, B and C). Further, activation of the Rho pathway was investigated with a Rho-associated kinase (p160 ROCK) inhibitor (Y-27632). p160 ROCK acts downstream of Rho 53. The effect of Y-27632 was tested in the concentration range of 10–0.1 µM in a transwell migration assay. A pronounced effect of Y-27632 was observed on ephrin-A1-induced migration (Fig. 5D). These data indicate that ephrin-A1 stimulation leads to Rho GTPase activation involved in migration of CD8+ T cells. We also observed an effect of this inhibitor on the background migration in wells with control-Fc, which might well also involve the Rho pathway (Fig. 5D). We could not observe induced activation of Rac or Cdc42 after ephrin-A1 stimulation. GTP-bound Rac or Cdc42 was precipitated with Pak 54, which binds GTP-bound Rac and Cdc42, using the same time points as presented for Rho activation (data not shown).

Identification of an EphA1 signaling complex

To further understand the signaling events occurring after ephrin-A1 binding to EphA1, we sought to identify proteins associating with EphA1. Due to difficulties using anti-EphA1 Ab in immunopreciptation studies from CD8+ T cells (low affinity or low expression levels), we used transfectants to further investigate protein interactions. HEK293T cells were transfected with EphA1 and either Lck, Pyk2 or both. Detection of phosphorylated proteins from total cell lysates showed induced phosphorylation on a number of proteins, in particular in the EphA1 + Lck + Pyk2 transfectants (Fig. 6A). EphA1-interacting proteins were investigated after EphA1 immunoprecipitation. Induced EphA1 phosphorylation was observed after ephrin-A1-Fc stimulation. In addition both phosphorylated Lck (Y394) and phosphorylated Pyk2 (Y402, Y580) co-precipitated with EphA1 (Fig. 6B, C). Interaction of EphA1 with Lck and Pyk2 corresponds with induced phosphorylation of EphA1. Immunoprecipitation of either Lck or Pyk2 (Fig. 6D) from transfectants identified the same components of the EphA1 receptor signaling complex as described above. In addition, also the endogenously expressed p85 subunit of PI3K could be detected in both the Lck and Pyk2 immunoprecipitates. As shown above, inhibitors of PI3K inhibited ephrin-A1-induced migration of CD8+ T cells (Fig. 5A). In transfectants, we could observe inducible association of endogenously expressed PI3K (p85 and p110) with EphA1 after ephrin-A1-Fc stimulation (Fig. 6E).

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Figure 6. Detection of an EphA1 receptor signaling complex. HEK293T cells were transfected with EphA1 (E) and either Lck (L), Pyk2 (P) or both, and stimulated with ephrin-A1 for the indicated times (min). (A) Western blot analysis of total cell lysates. Tyrosine-phosphorylated proteins detected with 4G10 and anti-EphA1-Ab for loading control. (B) Western blot analysis of EphA1 immunoprecipitates to investigate Lck interaction. Western blots were incubated with the indicated Ab, and stripped between incubations. (C) Western blot analysis of EphA1 immunoprecipitates to investigate Pyk2 interaction. Western blots were incubated with the indicated Ab, and stripped between incubations. (D) Western blot analysis of Lck or Pyk2 immunoprecipitates from EphA1 + Lck + Pyk2 transfectants. The Western blot was incubated with the indicated Ab, and stripped between incubations. (E) Association of EphA1 with endogenously expressed PI3K in transfectants. HEK293T cells were transfected with EphA1 and either Lck, Pyk2 or both. Western blot analysis of EphA1 immunoprecipitates to investigate PI3K interaction. Western blots were incubated with the indicated Ab, and stripped between incubations.

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To conclude from these studies, ephrin-A1-induced EphA1 receptor signaling can generate a signaling complex that includes EphA1, Lck, Pyk2 and PI3K.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements

Previously, we described the expression of ephrin-A1-binding EphA receptors on CD4+ T cells isolated from blood 11. In addition, expression of ephrin-A1 was identified on high endothelial venules, the entrance site for recirculating lymphocytes into lymphoid organs. Ephrin-A1 binding induced migration of CD4+ T cells in vitro. We therefore suggested that the interaction of Eph receptor and ligand might be involved in processes leading to transendothelial migration into secondary lymphoid organs 11. Here, we show that CD8+ T cells, in particular CCR7+ cells that include naive and central memory cells, bind ephrin-A1 and express EphA receptors. In particular, EphA1 expression was identified at both mRNA and protein levels. The chemokine receptor CCR7 is a key factor in the coordinate migration of T cells and DC into, and their localization within, secondary lymphoid organs 55, 56. The CD8+CCR7+EphA1+ cells also express CD62L (L-selectin), a prerequisite for entrance through high endothelial venules 57. Similar to what was seen for CD4+ T cells, ephrin-A1 stimulated migration of CD8+ T cells.

The observation that the Src kinase family inhibitor SU6656 stimulated migration, and previous reports indicating the involvement of Src family kinases in Eph receptor signaling 22, 4446, led us to investigate the involvement of the T cell Src family member Lck. SU6656 has been reported to have a low or no effect on Lck, but particularly inhibits Src and Fyn 58, 59. In line with this, we have not been able to observe stimulation of Fyn after EphA receptor stimulation in CD4+ or CD8+ T cells (data not shown). Ephrin-A1-induced phosphorylation of Lck Y394 and increased enzymatic activity was observed, both in CD8+ T cells and in HEK293T cells transfected with Eph receptor and Lck. This indicates that Lck takes part in the EphA1 receptor signaling cascade in T cells. A few previous reports have linked Lck to migratory events in T cells 40, 60. Our results, based on the inhibiting effect of the Lck inhibitor Damnacanthal on migration and the induced Lck kinase activity, indicate that Lck may be directly involved in the migration events occurring after ephrin-A1 stimulation of T cells. Damnacanthal did not affect EphA1 or Pyk2 phosphorylation in transfectants, and the association of PI3K with EphA1 seemed not to be affected (data not shown). We have also transfected the Lck-deficient Jurkat-derived cell line, JCAM-1, with Lck and investigated the effect of ephrin-A1 on migration in these cells. JCAM-1 cells express EphA receptors 61 and bind ephrin-A1 (data not shown), but we could not draw any conclusion from these experiments regarding the effect of Lck on migration in these cells.

Induced phosphorylation of Lck and Pyk2 is observed after stimulation of the EphA1 receptor. Pyk2 has been shown to be involved in coupling signaling through several receptors, including integrin and chemokine receptors 6264. It has previously been suggested that Src kinases, like Fyn and Lck, can associate with and activate Pyk2 in T cells 65, 66. Our data indicate an association of Lck with Pyk2 in CD8+ T cells. In transfectants, Pyk2 co-precipitated with EphA1 in the absence of transfected Lck, which could indicate a direct association of Pyk2 with EphA1, although we cannot exclude the involvement of endogenously expressed Src kinases in the interaction between EphA1 and Pyk2. A protein similar to Pyk2, focal adhesion kinase, has previously been shown to be physically associated with the EphA2 receptor, and to be involved in morphological changes after ephrin stimulation of the EphA2 receptor 67, 68. Although not proven at the enzymatic level, the association of PI3K with phosphorylated EphA1 in transfectants and the observed migration inhibition of CD8+ T cells by PI3K inhibitors indicate that active PI3K plays an important role in EphA1 signaling and migration of T cells. PI3K has previously been shown to be involved in Eph signaling leading to migration, and might be essential for migration events connected to Eph signaling 1921.

The small GTPases of the Rho family (Rho, Cdc42, Rac) are key regulators of actin cytoskeleton dynamics in cells, in addition to regulating a variety of other important cellular processes 69. We have previously shown an effect on actin polymerization in CD4+ T cells after ephrin-A1 stimulation 11. Activation of EphA receptors on CD8+ T cells is shown here to involve the activation of Rho GTPase. Further, we show that inhibition of p160 ROCK, acting downstream of Rho-GTPase, leads to inhibition of ephrin-A1-induced migration. Several reports have shown Rho activation through Eph receptors 2931, 70. Ephrin-A5 stimulation can activate RhoA and inhibit Rac1 in cultured retinal ganglion cells, accompanied by chemorepulsive axonal growth cone collapse 30. Ephrin-A5 has been shown to stimulate cell rounding and loss of adhesion in EphA3-expressing HEK293T cells involving the activation of Rho 31. Activation of EphA4 can stimulate RhoA activity in developing neurons and vascular smooth muscle cells, facilitating growth cone collapse and contractility, respectively 29, 70. Several examples of involvement of the Rho GTPase family members Rac and Cdc42 in Eph receptor signaling have been previously described 71. No activation of Cdc42 or Rac could be observed in our study within the time points investigated. Vav proteins are members of the Dbl-homology family of proteins that act as guanine nucleotide exchange factors (GEF) for Rho family proteins. Vav2 has previously been shown to associate with EphA4 in neuronal cells 72 and Vav2 and Vav3 associate with the EphA2 receptor in endothelial cells 73. We show here that Vav1 associates with active Lck kinase in CD8+ T cells. Interaction between Lck and Vav1 has been observed previously 50, 51. Thus, our data indicate a link between Lck and Vav1 that might affect migration through activation of Rho GTPases. One possibility is that Vav1 is recruited to EphA1 and activated by Lck, similar to what has been suggested for Vav2 associating with EphA4 in neuronal cells 72. The products of PI3K activation, phospholipids PIP3, can also activate Vav GEF 50, 74. Thus, it is possible that the coordinate action of Lck and PI3K activated after ephrin-A1 stimulation leads to Vav1 activation. Vav1 has been shown to be a GEF for RhoA, Cdc42 and Rac1 75; thus, the observed Rho activation could be performed by Vav1 in T cells.

So far, no viable EphA1 or ephrin-A1 gene-targeted mice have been reported in the literature. The study of such mice could give indications of the significance of these molecules in immunity and in recirculation of lymphocytes. Thus, the in vivo role of EphA receptor expression on T lymphocytes is still unclear, although our in vitro data indicate functional importance. However, it is intriguing that T cells able to recirculate express EphA1 receptors and bind ephrin-A1 while B cells do not 11. It remains to be investigated in animal models if this difference reflects differences in migration kinetics into lymph nodes.

To conclude, ephrin-A1-binding receptors are found on CD8+ CCR7+ T cells in blood, and in particular EphA1 expression was identified. Stimulation of the EphA1 receptor by ephrin-A1 leads to migration. Signaling through EphA1 involves Lck, Pyk2 and PI3K taking part in the migration events induced by ephrin-A1. We also suggest a role for Vav1 and Rho GTPase activation involved in the cytoskeletal changes associated with migration. These data indicate a function for Eph receptors on CD8+ T cells in the entrance into lymphoid tissue.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements

Ab and reagents

The following Ab were used in this study: anti-EphA1 and FITC-labeled anti-CCR7 (R&D Systems, McKinley Place, MN), anti-Pyk2, anti-Lck, anti-PI3K p110 and anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho Pyk2 sampler kit (Biosource, Camarillo, CA), anti-phospho-Src family Tyr416 (Cell Signaling Technology, Beverly, MA), anti-Rho (A, B, C), anti-PI3K p85, anti-Vav1 and anti-phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY), FITC-labeled anti-CD62L and FITC-labeled anti-CCR5 (BD Biosciences, San Jose, CA), goat anti-mouse Ig-RPE (Southern Biotechnology Associates, Birmingham, AL), HRP-labeled goat anti-rabbit, rabbit anti-mouse and rabbit anti-goat (DakoCytomation), mouse gamma globulin (Jackson ImmunoResearch Laboratories, West Grove, PA). Inhibitors used in this study are: Damnacanthal, SU6656, Y-27632, LY294002 and wortmannin, all from Calbiochem (Darmstadt, Germany). Recombinant human CXCL12 (SDF-1α) and CCL21 were from R&D Systems, and recombinant Sam 68 was purchased from Santa Cruz Biotechnology. The Rho assay reagent (Rhotekin RBD, agarose) was from Upstate Biotechnology.

Cell separation procedures

CD8+ T cells were isolated from buffy coats using anti-CD8 Ab-coated beads (Invitrogen, Carlsbad, CA) after which the cells were kept in serum-free medium X-Vivo 15 (Bio-Whittaker, Verviers, Belgium) overnight at 37°C. For Northern blot analysis, CD8+ T cells were stimulated with anti-CD3/CD28 Ab-coated magnetic beads (Invitrogen), with the ratio of one bead per two cells, in RPMI 1640 with 10% fetal bovine serum. All experiments presented in this article, either for functional aspects or for identification of signaling events after ephrin-A1 binding, were performed on total CD8+ T cells isolated from blood, including the Eph receptor-negative cell population (CD8+CCR5+).

Expression analysis

The generation, expression and isolation of the fusion proteins, control-Fc and ephrin-A1-Fc, have been described 10, 11. CD8+ T cells were stained with control-Fc (20 µg/mL) or ephrin-A1-Fc (20 µg/mL), followed by a PE-labeled anti-mouse second layer. The cells were then pre-incubated with mouse IgG followed by incubation with a FITC-labeled Ab. Surface expression was analyzed by FACSCalibur flow cytometer (Becton Dickinson).

Total RNA was extracted from cells or tissues by standard methods, and 10 µg was size-fractionated on a 1% agarose formaldehyde denaturing gel and transferred to nitrocellulose membranes. Hybridization was performed with [32P]dCTP-labeled EphA1- or EphA4-specific cDNA probes as previously described 10.

Migration assay

The CD8+ T cell migration assays were performed in standard transwell plates using 5 µm diameter pores (Costar, Corning, NY) as described 11. In some experiments, cells were pre-incubated with Damnacanthal for 1 h, Y-27632 and LY294002 for 30 min, and SU6656 for 2 min. CD8+ T cell migration assays were performed in RPMI 1640 without serum. Cells (3 × 105) were added to the upper well (inset) in the presence of control-Fc or ephrin-A1-Fc (10 µg/mL). Control-Fc and ephrin-A1-Fc were cross-linked with anti-mouse Ig for 20 min at 37°C before addition to cells. In experiments with chemokines, CXCL12 or CCL21 was added to the lower chamber, or in some cases to both chambers (CXCL12 only). Assays were done in duplicates at 37°C for 1.5 h. A fluorescent bead internal control (Bangs laboratories, Fishers, IN) was added to the cells that had passed through the membrane into the lower chamber. The cells were collected and counted by flow cytometry and normalized to the internal fluorescent bead control.

Cell signaling

HEK293T cells were transfected using Lipofectamine (Invitrogen) according to the manufacturer's instructions. HEK293T cells were transfected with EphA1 (obtained from OriGene Technologies, Rockville, MD), Pyk2 (lymphoid-specific isoform; accession no. BI911409) and Lck expression plasmids 76. The cells were serum-starved overnight prior to signaling experiments. Transfectants or CD8+ T cells were washed once in warm (37°C) PBS before at least 30 min of incubation at 37°C prior to the experiments. Ephrin-A1-Fc was cross-linked with anti-mouse Ig for 20 min at 37°C before being added to the cells at a concentration of 20 µg/mL. At different time points, cold PBS was added to the cells followed by a fast spin (17 s 10 000 × g) to pellet the cells. The cells were then lysed in 50 mM Tris-HCl pH 7.5, 0.150 mM NaCl, 1% NP40 supplemented with phosphatase and protease inhibitors. The following protocol was used for immunopreciptation of cellular proteins: 10 µL protein G Dynabeads (Invitrogen) were coated with Ab (0.5 µg) for 1 h at room temperature. To occupy free protein G sites, the beads were incubated with 5–10 µg control-Fc protein for 30 min. The beads were washed in PBS and added to cell lysates for incubation (2 h or overnight). Beads were then washed twice with lysis buffer and solved in 3× SDS sample buffer. The samples were boiled followed by denaturating SDS-PAGE and transfer to filter by blotting.

A Rho activation assay kit was used to assay for active GTP-bound Rho using the Rho binding domain of Rhotekin (Upstate Biotechnology).

Kinase assay

Cells stimulated or not with ephrin-A1-Fc were lysed in lysis buffer and immunoprecipitated with anti-Lck Ab. Immunoprecipitates were washed twice in 10 mM Tris-HCl pH 7.5, 300 mM NaCl and once in kinase assay buffer (50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 75 mM MgCl2, 15 mM DTT). The immunoprecipitates were solved in kinase assay buffer with 500 µM ATP, and recombinant Sam 68-GST (100 ng/sample) was added at the assay start. The assay was performed at 37°C for 15 min and terminated with the addition of 3× SDS sample buffer. Phosphorylation of Sam 68 was detected by the anti-phospho-tyrosine Ab 4G10.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements

We thank Halvor Holen for his critical comments on the manuscript. This study was supported by the Norwegian Cancer Society and the Norwegian Health Region South.

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