Genomic disruption of Fyn has not been associated with an immune-deficient phenotype, notwithstanding the profound impairment in IL-2 production by T cells derived from Fyn-deficient animals observed in vitro . The results presented demonstrate that Fyn deficient animals succumb to influenza infection ahead of the protective expansion of lung infiltrating T cells and viral clearance observed in wild-type hosts. Formal proof that Fyn-dependent IL-2 production mediates T cell expansion in vivo is provided using a model of T cell induced enteropathy. Specifically, Fyn deficient naive T cells do not induce colitis in SCID animals due to their lack of expansion, and Fyn re-expression rescues both IL-2 production and its capacity to support in vivo expansion leading to colitis. These results reconcile the obligatory role of Fyn in T cell activation and autocrine IL-2 supported growth; and underscore the mechanism through which its function is integrated with and regulated by Lck.
KeywordsFynLckTcR SignalingImmunityAutoimmunity
Appleby, M.W., Gross, J.A., Cooke, M.P., Levin, S.D., Qian, X. and Perlmutter, R.M. (1992) Defective T Cell Receptor Signaling in Mice Lacking the Thymic Isoform of p59fyn. Cell, 70, 751-763. http://dx.doi.org/10.1016/0092-8674(92)90309-Z
Stein, P.L., Lee, H., Rich, S. and Soriano, P. (1992) pp59fyn Mutant Mice Display Differential Signaling in Thymocytes and Peripheral T Cells. Cell, 70, 741-750. http://dx.doi.org/10.1016/0092-8674(92)90308-Y
Mamchak, A.A., Sullivan, B.M., Hou, B., Lee, L.M., Gilden, J.K., Krummel, M.F., Locksley, R.M. and DeFranco, A.L. (2008) Normal Development and Activation but Altered Cytokine Production of Fyn-Deficient CD4+ T Cells. The Journal of Immunology, 181, 5374-5385. http://dx.doi.org/10.4049/jimmunol.181.8.5374
Eberl, G., Lowin-Kropf, B. and MacDonald, H.R. (1999) Cutting Edge: NKT cell Development Is Selectively Impaired in Fyn-Deficient Mice. The Journal of Immunology, 163, 4091-4094.
Filipp, D., Leung, B.L., Zhang, J., Veillette, A. and Julius, M. (2004) Enrichment of LCK in Lipid Rafts Regulates Colocalized Fyn Activation and the Initiation of Proximal Signals through TCR Alpha Beta. The Journal of Immunology, 172, 4266-4274. http://dx.doi.org/10.4049/jimmunol.172.7.4266
Filipp, D., Zhang, J., Leung, B.L., Shaw, A., Levin, S.D., Veillette, A. and Julius, M. (2003) Regulation of Fyn through Translocation of Activated LCK into Lipid Rafts. The Journal of Experimental Medicine, 197, 1221-1227. http://dx.doi.org/10.1084/jem.20022112
Filipp, D., Moemeni, B., Ferzoco, A., Kathirkamathamby, K., Zhang, J., Ballek, O., Davidson, D., Veillette, A. and Julius, M. (2008) Lck-Dependent Fyn Activation Requires C-Terminus-Dependent Targeting of Kinase-Active Lck to Lipid Rafts. Journal of Biological Chemistry, 283, 26409-26422. http://dx.doi.org/10.1074/jbc.M710372200
Leach, M.W., Bean, A.G., Mauze, S., Coffman, R.L. and Powrie, F. (1996) Inflammatory Bowel Disease in C.B-17 Scid Mice Reconstituted with the CD45RB High Subset of CD4+ T Cells. American Journal of Pathology, 148, 1503- 1515.
Powrie, F., Leach, M.W., Mauze, S., Caddie, L.B. and Coffman, R.L. (1993) Phenotypically Distinct Subsets of CD4+ T Cells Induce or Protect FROM Chronic Intestinal Inflammation in C. B-17 Scid Mice. International Immunology, 5, 1461-1471. http://dx.doi.org/10.1093/intimm/5.11.1461
Powrie, F., Mauze, S. and Coffman, R.L. (1997) CD4+ T Cells in the Regulation of Inflammatory Responses in the Intestine. Research in Immunology, 148, 576-581. http://dx.doi.org/10.1016/S0923-2494(98)80152-1
Ennis, F.A., Verbonitz, M., Reichelderfer, P. and Daniel, S. (1976) Recombination of Influenza A Virus Strains: Effect on Pathogenicity. Developments in Biological Standardization, 33, 220-225.
Lin, G.H., Sedgmen, B.J., Moraes, T.J., Snell, L.M., Topham, D.J. and Watts, T.H. (2009) Endogenous 4-1BB Ligand Plays a Critical Role in Protection from Influenza-Induced Disease. Journal of immunology, 182, 934-947. http://dx.doi.org/10.4049/jimmunol.182.2.934
Wan, Y.Y., Leon, R.P., Marks, R., Cham, C.M., Schaack, J., Gejewski, T.F. and Degregori, J. (2000) Transgenic Expression of the Coxasackie/Adenovirus Receptor Enables Adenoviral-Mediated Gene Delivery in Naive T Cells. Proceedings of the National Academy of Sciences of the United States of America, 97, 13784-13789. http://dx.doi.org/10.1073/pnas.250356297
Cottey, R., Rowe, C.A. and Bender, B.S. (2001) Influenza Virus. In: Coico, R., Ed., Current Protocols in Immunology, John Wiley and Sons, Hoboken. http://dx.doi.org/10.1002/0471142735.im1911s42
Poussier, P., Ning, T., Chen, J., Banerjee, D. and Julius, M. (2000) Intestinal Inflammation Observed in IL-2R/IL-2 Mutant Mice Is Associated with Impaired Intestinal T Lymphopoiesis. Gastroenterology, 118, 880-891. http://dx.doi.org/10.1016/S0016-5085(00)70174-0
Quah, B.J., Warren, H.S. and Parish, C.R. (2007) Monitoring Lymphocyte Proliferation in Vitro and in Vivo with the Intracellular Fluorescent Dye Carboxyfluorescein Diacetate Succinimidyl Ester. Nature Protocols, 2, 2049-2056. http://dx.doi.org/10.1038/nprot.2007.296
Marmour, M.D. and Julius, M. (2001) Role for Lipid Rafts in Regulating Interleukin-2 Receptor Signaling. Blood, 98, 1489-1497. http://dx.doi.org/10.1182/blood.V98.5.1489
Filby, A., Seddon, B., Kleczkowska, J., Salmond, R., Tomlinson, P., Smida, M., Lindquist, J.A., Schraven, B. and Zamoyska, R. (2007) Fyn Regulates the Duration of TCR Engagement Needed for Commitment to Effector Function. Journal of Immunology, 179, 4635-4644. http://dx.doi.org/10.4049/jimmunol.179.7.4635
Sugie, K., Jeon, M. and Grey, H.M. (2004) Activation of Naive CD4 T Cells by Anti-CD3 Reveals an Important Role for Fyn in Lck-Mediated Signaling. Proceedings of the National Academy of Sciences of the United States of America, 101, 14859-14864. http://dx.doi.org/10.1073/pnas.0406168101
da Silva, A., Rosenfield, J., Mueller, I., Bouton, A., Hirai, H. and Rudd, C. (1997) Biochemical Analysis of p120/130: A Protein-Tyrosine Kinase Substrate Restricted to T and Myeloid Cells. The Journal of Immunology, 158, 2007-2016.
Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T.B., Oukka, M., Weiner, H.L. and Kuchroo, V.K. (2006) Reciprocal Developmental Pathways for the Generation of Pathogenic Effector TH17 and Regulatory T Cells. Nature, 441, 235- 238. http://dx.doi.org/10.1038/nature04753
Veldhoen, M., Hocking, R.J., Atkins, C.J., Locksley, R.M. and Stockinger, B. (2006) TGFβ in the Context of an Inflammatory Cytokine Milieu Supports de Novo Differentiation of IL-17-Producing T Cells. Immunity, 24, 179-189. http://dx.doi.org/10.1016/j.immuni.2006.01.001
Khanolkar, A., Fuller, M.J. and Zajac, A.J. (2004) CD4 T Cell-Dependent CD8 T Cell Maturation. Journal of Immunology, 172, 2834-2844. http://dx.doi.org/10.4049/jimmunol.172.5.2834
Williams, M.A., Tyznik, A.J. and Bevan, M.J. (2006) Interleukin-2 Signals during Priming Are Required for Secondary Expansion of CD8+ Memory T Cells. Nature, 441, 890-893. http://dx.doi.org/10.1038/nature04790
Wilson, E.B. and Livingstone, A.M. (2008) Cutting Edge: CD4+ T Cell-Derived IL-2 Is Essential for Help-Dependent Primary CD8+ T Cell Responses. Journal of Immunology, 181, 7445-7448. http://dx.doi.org/10.4049/jimmunol.181.11.7445
Thomas, P.G., Keating, R., Hulse-Post, D.J. and Doherty, P.C. (2006) Cell-Mediated Protection in Influenza Infection. Emerging Infectious Diseases, 12, 48-54. http://dx.doi.org/10.3201/eid1201.051237
Gillis, S., Ferm, M.M., Ou, W. and Smith, K.A. (1978) T Cell Growth Factor: Parameters of Production and a Quantitative Microassay for Activity. Journal of Immunology, 120, 2027-2032.
Robb, R.J. and Smith, K.A. (1981) Heterogeneity of Human T-Cell Growth Factor(s) Due to Variable Glycosylation. Molecular Immunology, 18, 1087-1094. http://dx.doi.org/10.1016/0161-5890(81)90024-9
Sojka, D.K., Bruniquel, D., Schwartz, R.H. and Singh, N.J. (2004) IL-2 Secretion by CD4+ T Cells in Vivo Is Rapid, Transient, and Influenced by TCR-Specific Competition. Journal of Immunology, 172, 6136-6143. http://dx.doi.org/10.4049/jimmunol.172.10.6136
Sadlack, B., Merz, H., Schorle, H., Schimpl, A., Feller, A.C. and Horak, I. (1993) Ulcerative Colitis-Like Disease in Mice with a Disrupted Interleukin-2 Gene. Cell, 75, 253-261. http://dx.doi.org/10.1016/0092-8674(93)80067-O
Willerford, D.M., Chen, J., Ferry, J.A., Davidson, L., Ma, A. and Alt, F.W. (1995) Interleukin-2 Receptor Alpha Chain Regulates the Size and Content of the Peripheral Lymphoid Compartment. Immunity, 3, 521-530. http://dx.doi.org/10.1016/1074-7613(95)90180-9
Schorle, H., Holtschke, T., Hunig, T., Schimpl, A. and Horak, I. (1991) Development and Function of T Cells in Mice Rendered Interleukin-2 Deficient by Gene Targeting. Nature, 352, 621-624. http://dx.doi.org/10.1038/352621a0
Poussier, P., Ning, T., Banerjee, D. and Julius, M. (2002) A Unique Subset of Self-Specific Intraintestinal T Cells Maintains Gut Integrity. Journal of Experimental Medicine, 195, 1491-1497. http://dx.doi.org/10.1084/jem.20011793
Papiernik, M., de Moraes, M.L., Pontoux, C., Vasseur, F. and Penit, C. (1998) Regulatory CD4 T Cells: Expression of IL-2R Alpha Chain, Resistance to Clonal Deletion and IL-2 Dependency. International Immunology, 10, 371-378. http://dx.doi.org/10.1093/intimm/10.4.371
Kameyama, K., Nemoto, Y., Kanai, T., Shinohara, T., Okamoto, R., Tsuchiya, K., Nakamura, T., Sakamoto, N., Totsuka, T., Hibi, T. and Watanabe, M. (2010) IL-2 Is Positively Involved in the Development of Colitogenic CD4+ IL-7R Alpha High Memory T Cells in Chronic Colitis. European Journal of Immunology, 40, 2423-2436. http://dx.doi.org/10.1002/eji.200939764
Latour, S., Roncagalli, R., Chen, R., Bakinowski, M., Shi, X., Schwartzberg, P.L., Davidson, D. and Veillette, A. (2003) Binding of SAP SH2 Domain to FynT SH3 Domain Reveals a Novel Mechanism of Receptor Signalling in Immune Regulation. Nature Cell Biology, 5, 149-154. http://dx.doi.org/10.1038/ncb919
Qian, D., Lev, S., van Oers, N.S., Dikic, I., Schlessinger, J. and Weiss, A. (1997) Tyrosine Phosphorylation of Pyk2 Is Selectively Regulated by Fyn during TCR Signaling. Journal of Experimental Medicine, 185, 1253-1260. http://dx.doi.org/10.1084/jem.185.7.1253
Davidson, D., Shi, X., Zhang, S., Wang, H., Nemer, M., Ono, N., Ohno, S., Yanagi, Y. and Veillette, A. (2004) Genetic Evidence Linking SAP, the X-Linked Lymphoproliferative Gene Product, to Src-Related Kinase FynT in TH2 Cytokine Regulation. Immunity, 21, 707-717. http://dx.doi.org/10.1016/j.immuni.2004.10.005
Beinke, S.R., Phee, H., Clingan, J.M., Schlessinger, J., Matloubian, M. and Weiss, A. (2010) Proline-Rich Tyrosine Kinase-2 Is Critical for CD8 T-Cell Short-Lived Effector Fate. Proceedings of the National Academy of Sciences of the United States of America, 107, 16234-16239. http://dx.doi.org/10.1073/pnas.1011556107
Veale, M., Raab, M., Li, Z., da Silva, A.J., Kraeft, S.K., Weremowicz, S., Morton, C.C. and Rudd, C.E. (1999) Novel Isoform of Lymphoid Adaptor FYN-T-Binding Protein (FYB-130) Interacts with SLP-76 and Up-Regulates Interleukin 2 Production. Journal of Biological Chemistry, 274, 28427-28435. http://dx.doi.org/10.1074/jbc.274.40.28427
Griffiths, E.K., Krawczyk, C., Kong, Y.-Y., Raab, M., Hyduk, S.J., Bouchard, D., Chan, V.S., Kozieradzki, I., Oliveira-dos-Santos, A.J., Wakeham, A., et al. (2001) Positive Regulation of T Cell Activation and Integrin Adhesion by the Adapter Fyb/Slap. Science, 293, 2260-2263. http://dx.doi.org/10.1126/science.1063397
Yablonski, D., Kadlecek, T. and Almeida, A.R. (2001) Identification of a Phospholipase C-γ1 (PLC-γ1) SH3 Domain-Binding Site in SLP-76 Required for T-Cell Receptor-Mediated Activation of PLC-γ1 and NFAT. Molecular and Cellular Biology, 21, 4208-4218. http://dx.doi.org/10.1128/MCB.21.13.4208-4218.2001
Meldrum, E., Parker, P.J. and Carozzi, A. (1991) The PtdIns-PLC Superfamily and Signal Transduction. Biochimica et Biophysica Acta, 1092, 49-71. http://dx.doi.org/10.1016/0167-4889(91)90177-Y
Hogan, P.G., Chen, L., Nardone, J. and Rao, A. (2003) Transcriptional Regulation by Calcium, Calcineurin, and NFAT. Genes & Development, 17, 2205-2232. http://dx.doi.org/10.1101/gad.1102703