Mouse EphrinB3 Augments T-cell Signaling and Responses to T-cell Receptor Ligation

Ephrins (EFN) are cell-surface ligands of Ephs, the largest family of cell-surface receptor tyrosine kinases. The function of EFNs in the immune system has not been well studied, although some EFNs and Ephs are expressed at high levels on certain leukocytes. We report here that EFNB3 and its receptors (collectively called EFNB3Rs, as EFNB3 binds to multiple EphBs) were expressed in peripheral T cells and monocytes/macrophages, with T cells being the dominant EFNB3+ and EFNB3R+ cell type. Solid-phase EFNB3-Fc in the presence of suboptimal anti-CD3 crosslinking enhanced T-cell responses in terms of proliferation, activation marker expression, interferon-γ but not interleukin-2 production, and cytotoxic T-cell activity. EFNB3R costimulation in the presence of phorbol 12-myristate 13- acetate was insensitive to cyclosporin A, similar to CD28 costimulation, suggesting they might share a part of the signaling pathway. After crosslinking, T-cell receptor and EFNB3R congregated into aggregated rafts, and this provided a morphological basis for signaling pathways of T-cell receptor and EFNB3R to interact. Solidphase EFNB3-Fc augmented p38 and p44/42 MAPK activation further downstream of the signaling pathway. These data suggest that EFNB3 is important in T-cell/T-cell and T-cell/antigen-presenting cell collaboration to enhance T-cell activation and function. Ephrins (EFN) are cell-surface ligands of Ephs, the largest family of cell-surface receptor tyrosine kinases. The function of EFNs in the immune system has not been well studied, although some EFNs and Ephs are expressed at high levels on certain leukocytes. We report here that EFNB3 and its receptors (collectively called EFNB3Rs, as EFNB3 binds to multiple EphBs) were expressed in peripheral T cells and monocytes/macrophages, with T cells being the dominant EFNB3+ and EFNB3R+ cell type. Solid-phase EFNB3-Fc in the presence of suboptimal anti-CD3 crosslinking enhanced T-cell responses in terms of proliferation, activation marker expression, interferon-γ but not interleukin-2 production, and cytotoxic T-cell activity. EFNB3R costimulation in the presence of phorbol 12-myristate 13- acetate was insensitive to cyclosporin A, similar to CD28 costimulation, suggesting they might share a part of the signaling pathway. After crosslinking, T-cell receptor and EFNB3R congregated into aggregated rafts, and this provided a morphological basis for signaling pathways of T-cell receptor and EFNB3R to interact. Solidphase EFNB3-Fc augmented p38 and p44/42 MAPK activation further downstream of the signaling pathway. These data suggest that EFNB3 is important in T-cell/T-cell and T-cell/antigen-presenting cell collaboration to enhance T-cell activation and function. Full T-cell activation requires costimulation in addition to T-cell receptor (TCR) 1The abbreviations used are: TCRT-cell receptorEFNephrinPBSphosphate-buffered salineNHIgGnormal human IgGMLRmixed lymphocyte reactionPEphycoerythrinFITCfluorescein isothiocyanateCTLcytotoxic T lymphocyteILinterleukinIFNinterferonmAbmonoclonal antibodyMAPKmitogen-activated protein kinasePMAphorbol 12-myristate 13-acetateCTcholera toxin. ligation. Costimulation can be conferred by cell-surface molecules. For example, the well studied B7.1 and B7.2 are two prototype costimulatory molecules expressed on antigen-presenting cells (1Hathcock K.S. Laszlo G. Pucillo C. Linsley P. Hodes R.J. J. Exp. Med. 1994; 180: 631-640Crossref PubMed Scopus (648) Google Scholar). Additional members of the B7 family with costimulatory function have been documented recently (2Carreno B.M. Collins M. Annu. Rev. Immunol. 2002; 20: 29-53Crossref PubMed Scopus (750) Google Scholar). Certain cell-surface adhesion molecules (3Sims T.N. Dustin M.L. Immunol. Rev. 2002; 186: 100-117Crossref PubMed Scopus (122) Google Scholar) and some membrane-bound tumor necrosis factor family members (4Fotin-Mleczek M. Henkler F. Samel D. Reichwein M. Hausser A. Parmryd I. Scheurich P. Schmid J.A. Wajant H. J. Cell Sci. 2002; 115: 2757-2770Crossref PubMed Google Scholar, 5Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2470) Google Scholar) are capable of costimulation as well. In general, the costimulation increases the affinity and prolongs the interaction between T cells and antigen-presenting cells; it also allows the integration of costimulation-triggered signaling pathways with that of TCR. As a result, the T-cell response threshold to Ag is reduced, and the response to Ag is enhanced. T-cell receptor ephrin phosphate-buffered saline normal human IgG mixed lymphocyte reaction phycoerythrin fluorescein isothiocyanate cytotoxic T lymphocyte interleukin interferon monoclonal antibody mitogen-activated protein kinase phorbol 12-myristate 13-acetate cholera toxin. Ephrins (EFN) are cell-surface ligands of Eph receptor tyrosine kinases. EFNs are classified into A and B subfamilies; the former consists of six glycosylphosphatidylinositol-anchored membrane proteins (EFNA1–6), and the latter consists of three transmembrane proteins (EFNB1–3) (6Eph Nomenclature Committee Cell. 1997; 90: 403-404Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). EFNBs are capable of reverse transmission of signals into cells, although they are ligands (7Holland S.J. Gale N.W. Mbamalu G. Yancopoulos G.D. Henkemeyer M. Pawson T. Nature. 1996; 383: 722-725Crossref PubMed Scopus (462) Google Scholar). 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In this study, we investigated the expression of EFNB3 and its receptors in immune cells and explored its function in regulating T-cell activity. In Situ Hybridization—A 522-bp cDNA fragment of mouse EFNB3 cDNA from positions 160 to 681 (accession number AF025288) was fetched with PCR from a mouse embryonic tissue cDNA library and cloned into pGEM-4Z (Invitrogen). The resulting construct, pGEM-4Z-mB3, served to transcribe antisense probes with SP6 RNA polymerase or to transcribe sense probes with T7 RNA polymerase using digoxigenin RNA labeling kits (Roche Diagnostics, Laval, Quebec, Canada). In situ hybridization was carried out according to instructions from the kit manufacturer. Generation of Mouse EFNB3-Fc—The coding sequence of the extracellular domains of mouse EFNB3 from positions 34 to 717 was cloned in-frame upstream of the human IgG1-Fc coding sequence in expression vector pCMVhFc. The constructs and pcDNA3 were then transfected into Chinese hamster ovary/dhfr- cells with LipofectAMINE. The cells were cultured in selection medium (α-minimum Eagle's medium without ribonucleosides and deoxyribonucleosides containing 5% dialyzed fetal calf serum, 0.01 mm methotrexate, 0.8 mg/ml G418, and 0.1 mg/ml gentamycin). Stably transfected clones were handpicked after 2 weeks of culture. Fusion proteins were isolated from supernatants by protein A columns and verified by N-terminal peptide sequencing (Sheldon Biotechnology Center, McGill University, Montreal, Quebec, Canada). Lymphocyte Preparation and Culture—Cells were flushed out from the BALB/c mouse spleen, and red blood cells were lysed with 0.84% NH4Cl, as described elsewhere (30Chen H. Luo H. Xu D. Loh D.Y. Daloze P.M. Veillette A. Qi S. Wu J. J. Immunol. 1996; 157: 4297-4308PubMed Google Scholar). The resulting cells were referred to as spleen cells. Spleen T cells were purified by mouse IgG cells from spleen cells with T-cell columns according to the instructions Canada). In some and cells were from the spleen cells by The cells were cultured in medium with fetal calf serum, and Solid-phase EFNB3 and anti-CD3 were by with in phosphate-buffered saline at by EFNB3-Fc as a normal human IgG of at for 2 by on In some and cells were from the spleen cells by For mixed lymphocyte reaction spleen cells from BALB/c mice were with and mixed with spleen cells at a cells at The cells were cultured in the presence of or Ephs and for being were cultured in with proteins, or in The cells were with for the of and was as described previously H. Chen H. Daloze P. G. Wu J. 1992; PubMed Scopus (71) Google Scholar). was for the of EFNB3 expression as well as EFNB3R expression in T-cell BALB/c spleen cells were with by or with EFNB3-Fc by was used for the the expression of activation on T cells, spleen T cells were with in with or of were from T Lymphocyte was as J. Wan X. Ullrich S. B. T. P. Qi S. Chen H. Y. Wu J. J. Clin. Invest. 2001; PubMed Scopus Google Scholar) using mouse spleen cells with of their T cells to as BALB/c mouse spleen cells as and cells as system is very and has a high of the high of T cells (30Chen H. Luo H. Xu D. Loh D.Y. Daloze P.M. Veillette A. Qi S. Wu J. J. Immunol. 1996; 157: 4297-4308PubMed Google Scholar). The of the was as of the of of of supernatants of T cells in were after the of culture. and in the supernatants were by according to the BALB/c T cells were on for with of EFNB3-Fc and of anti-CD3 hamster After the cells were with IgG for on were then with and to to the crosslinking at for this was by with For and EFNB3R the cells were with and on for For and EFNB3R the was similar to that described but cholera was used in of The were a were with anti-CD3 at After the were with EFNB3-Fc or both at and at for to 2 and then at for 2 BALB/c spleen T cells were in the at and the were at for to between the cells and the of the The cells were then cultured at for 2 being The of the is in our (30Chen H. Luo H. Xu D. Loh D.Y. Daloze P.M. Veillette A. Qi S. Wu J. J. Immunol. 1996; 157: 4297-4308PubMed Google Scholar). the cells were and lysed in for the were in with and were then The were with MAPK by MAPK or with MAPK by MAPK of the used in were from Canada). were by enhanced EFNB3 and EFNB3R in expression at the in the spleen was by in situ hybridization with the as a control In the spleen, the EFNB3 signal was in the suggesting that The expression of EFNB3 and on and T cells was investigated with by or EFNB3 was in T cells in and activation of T cells with anti-CD3 not the of EFNB3 expression EFNB3R was expressed on T cells; no of expression after T-cell activation was The expression of EFNB3 and EFNB3R in and cells in medium for was by or EFNB3-Fc of and cells The expression of EFNB3 on and cells was similar and although cells expressed cells and EFNB3 and EFNB3R were in B cells and but were expressed in a of and EFNB3 T-cell and are expressed in a of T cells, it is that EFNB3 a in T-cell function. Solid-phase in the presence of was used to this but not a control protein human T-cell in the presence of a suboptimal of anti-CD3 T cells were with of EFNB3-Fc and of they in response to the latter We also the costimulation by EFNB3-Fc with that by mAb Costimulation by was at a that by the costimulatory a suboptimal or EFNB3-Fc or no T-cell proliferation, two columns of The response of and cells to EFNB3 costimulation was similar the costimulation EFNB3R was insensitive to cyclosporin A As in which T-cell activation P. S. D. Blood. 2002; PubMed Scopus Google Scholar, D.M. J. 1999; PubMed Scopus Google Scholar), and T-cell but to for T cells by or that CD28 and EFNB3R costimulation both the pathway. we the EFNB3 can costimulation in the presence of CD28 for EFNB3 no at suboptimal or EFNB3 enhance the that CD28 and EFNB3R not two EFNB3 is to interact with three Eph EphA4, and K. M. Pan L. J. V. S. R. Yancopoulos G.D. Gale N.W. 2001; PubMed Scopus Google Scholar, R. K. D. J.G. R. Cell. Neurosci. 1996; Scopus Google Scholar, P.M. D.M. V. Wilkinson D.G. A.W. 1999; PubMed Google Scholar). We investigated which of EphA4, or or in were to and was As in and but not was to suggesting the of the former two in the The expression of certain activation on T cells after EFNB3 costimulation was then T cells were with anti-CD3 a suboptimal or anti-CD3 EFNB3-Fc As in anti-CD3 was to the activation and After with anti-CD3 and was and was although and were In of were T cells were by These that can enhance T-cell activation to but from that of EFNB3 and the of EFNB3 on T-cell as and activity. T cells were with anti-CD3 a suboptimal or in with EFNB3-Fc or at and on was used as a control for and were from to after with or without not costimulation and as EFNB3-Fc to a with but that by costimulation but not and The of and after EFNB3 costimulation was not by a of no of or was at between and after culture. that EFNB3 and costimulation have mixed lymphocyte reaction was in development was enhanced control normal human IgG no that EFNB3 costimulation important function of T cells, activity. in T by the mechanisms of EFNB3 costimulation, we the of and on the T-cell and their to membrane after was by anti-CD3 by The was by in EFNB3Rs, by EFNB3-Fc by in the in the T-cell by cholera in In T cells, rafts, and were the cell After crosslinking with and a in of the also and they with After crosslinking, the and as well. into the that the by by and the by a morphological for to enhance both and are and in aggregated rafts, and their signaling pathways can interact In T cells, we MAPK which is in other cell some Eph kinases are A. Sharfe N. Roifman C.M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Immunol. 1999; Google Scholar, M.L. J.A. Nat. Immunol. 2000; PubMed Scopus Google Scholar, H. D.M. T. E. B. Nat. Cell. 2001; 3: PubMed Scopus Google Scholar). As in a of EFNB3-Fc and suboptimal anti-CD3 for 2 to p38 MAPK and p44/42 MAPK a of their anti-CD3 at suboptimal according to The were with MAPK and MAPK p38 MAPK and p44/42 MAPK protein levels were similar with were after MAPK activation was and in EFNB3 costimulation, we used p38 and p44/42 and to T cells by but not their EFNB3R costimulation in terms of that p38 and p44/42 MAPK activation is and part of the EFNB3R signaling pathway. We have reported that EFNB3 and were expressed in T cells and T cells by EFNB3-Fc in the presence of suboptimal anti-CD3 enhanced and of but not and expression of certain T-cell activation as into the to which also MAPK was enhanced by EFNB3-Fc These suggest that EFNB3 and its receptors important in T-cell activation and function. The in the receptors and functions of EFNs is that each of binds to multiple Eph family In the of it is to interact with K. M. Pan L. J. V. S. R. Yancopoulos G.D. Gale N.W. 2001; PubMed Scopus Google Scholar), R. K. D. J.G. R. Cell. Neurosci. 1996; Scopus Google Scholar), and P.M. D.M. V. Wilkinson D.G. A.W. 1999; PubMed Google Scholar), and its interaction with Eph members is also We to the receptors the of EphA4, and were to and but not were to the T-cell that the former two might the of as Ephs and EFNs are capable of and the and can interact with EFNs in addition to we the that the is to the of and to certain EFNs which signals into T cells. the receptors for the or functions of EFNB3 be to mice each and Eph EphBs) our is the of EFNB3 here functions of receptors by and this is a of EFNB3 can in the EFNB3 signal was not in the spleen by N.W. Flenniken A. N. D.J. Davis S. Wilkinson D.G. Yancopoulos G.D. Oncogene. 1996; 13: Google Scholar), we demonstrated that spleen expressed EFNB3 according to in situ and this was by of EFNB3 expression on T cells and at the protein by The between the report and our is of the of the Because EFNB3 is expressed on T cells and that cells can enhance or the response threshold of EFNB3R+ T cells to might the for T cells to be in lymphoid organs leukocytes are costimulation from antigen-presenting cells to T cells is well studied, we can that the signaling between is important for T-cell A of this can be in T-cell in which cell is for T-cell is as a for a interaction cell-surface receptors and ligands on the T cells is a J. A. Yu P. Chen Y. K. Chen L. J. Clin. Invest. 2001; PubMed Scopus Google Scholar) have demonstrated that which is a cell-surface to the tumor necrosis factor family and is expressed on T cells, can T-cell activation both in and in its receptor on T cells. We have that in T-cell the interaction between EphB6 and its T-cell can enhance T-cell response to (20Luo H. Yu G. Wu Y. Wu J. J. Clin. Invest. 2002; 110: 1141-1150Crossref PubMed Scopus (71) Google Scholar). The expression of both EFNB3 and EFNB3R on T cells and the of EFNB3 on costimulation, as documented in this study, the that two molecules are also in T-cell/T-cell We T-cell by CD28 and The former of and but the latter a T cells can be or the former but not the latter is to P. S. D. Blood. 2002; PubMed Scopus Google Scholar, D.M. J. 1999; PubMed Scopus Google Scholar). CD28 costimulation in the presence of the and is to P. S. D. Blood. 2002; PubMed Scopus Google Scholar, D.M. J. 1999; PubMed Scopus Google Scholar). In the EFNB3R costimulation was also to suggesting that it also the pathway. was used at suboptimal but not EFNB3 was to further enhance the T-cell proliferation, suggesting a certain between CD28 and EFNB3R data that the signaling pathways of CD28 and EFNB3R are but not A that membrane in T-cell activation and the T-cell proteins, as kinases that are for T-cell signaling R. B. Immunol. 1999; 11: PubMed Scopus Google Scholar). the of and the rafts, and this is with signaling and T-cell activation M.L. J.A. Nat. Immunol. 2000; PubMed Scopus Google Scholar). In our EFNB3R into the after crosslinking in by it is that in with crosslinking leads to EFNB3R into the it with and the EFNB3R signaling with that of and the For T-cell and signaling pathways to be activation leads to activation of p44/42 MAPK in in the and activation of the other activation of and proteins p38 MAPK which is for Such is to be for T-cell signaling M.L. J.A. Nat. Immunol. 2000; PubMed Scopus Google Scholar). We have that the of both p44/42 and p38 MAPK are enhanced in the presence of EFNB3 costimulation, and this is with the of MAPK in T-cell it has been reported that activation in the of p44/42 MAPK in cells S. Holland S.J. S. Pawson T. Cell. Biol. 2001; 21: PubMed Scopus Google Scholar), and that activation leads to of this kinase in cell of and H. D.M. T. E. B. Nat. Cell. 2001; 3: PubMed Scopus Google Scholar). with cell that are from in our study, but the is as well. the of EFNB3 in regulating T-cell function. functions were with and not suggesting that molecules have but functions in T-cell These understanding the of and Ephs in the immune We Center, for We for of the EFNB3-Fc expression

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