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. 1994 Sep 1;180(3):1115–1127. doi: 10.1084/jem.180.3.1115

Protein tyrosine kinase p56-Lck regulates lymphocyte function- associated 1 adhesion molecule expression, granule exocytosis, and cytolytic effector function in a cloned T cell [published erratum appears in J Exp Med 1995 Jan 1;181(1):442]

PMCID: PMC2191636  PMID: 7914906

Abstract

Elevated levels of p56-Lck kinase activity were achieved in an interleukin 2 (IL-2)-dependent cloned cytolytic T cell CTLL-2 through gene transfer approaches. CTLL-2-Lck cells remained dependent on IL-2 for growth and survival in culture but exhibited markedly elevated, IL- 2-independent cytolytic activity against a variety of tumor targets. This immune cell effector function was similar to the non-major histocompatibility complex-restricted cytolytic activity previously described for lymphokine activated killer (LAK) cells, and involved a cytolytic mechanism that was independent of protein synthesis in either the T cells or the tumor targets. Characterization of CTLL-2-Lck cells revealed markedly elevated levels of both the alpha (CD11a) and beta (CD18) chains of the cell adhesion molecule lymphocyte function- associated 1 (LFA-1) and increased binding of these T cells to a recombinant protein representing the extracellular domain of the LFA- ligand, intercellular adhesion molecule 1 (ICAM-1). Antibodies to CD11a partially abrogated cytolytic killing of tumor target cells by CTLL-2- Lck cells, suggesting that the upregulation in LFA protein levels potentially accounts at least in part for the phenotype of these T cells. Gene transfer-mediated elevations in p56-Lck kinase activity in an IL-3-dependent myeloid cell clone 32D.3 also resulted in increased LFA-1 expression, demonstrating that the findings are not unique to CTLL-2 cells. In addition to upregulation of LFA-1 expression, CTLL-Lck cells also exhibited more efficient exocytosis of cytotoxic granules upon activation with Ca(2+)-ionophore and phorbol ester, relative to control transfected and untransfected CTLL-2 cells. The findings functionally link the Lck kinase to a T cell effector pathway involved in cell-mediated cytotoxicity.

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Selected References

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  1. Altomonte M., Gloghini A., Bertola G., Gasparollo A., Carbone A., Ferrone S., Maio M. Differential expression of cell adhesion molecules CD54/CD11a and CD58/CD2 by human melanoma cells and functional role in their interaction with cytotoxic cells. Cancer Res. 1993 Jul 15;53(14):3343–3348. [PubMed] [Google Scholar]
  2. Barnekow A., Jahn R., Schartl M. Synaptophysin: a substrate for the protein tyrosine kinase pp60c-src in intact synaptic vesicles. Oncogene. 1990 Jul;5(7):1019–1024. [PubMed] [Google Scholar]
  3. Bell G. M., Bolen J. B., Imboden J. B. Association of Src-like protein tyrosine kinases with the CD2 cell surface molecule in rat T lymphocytes and natural killer cells. Mol Cell Biol. 1992 Dec;12(12):5548–5554. doi: 10.1128/mcb.12.12.5548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berke G., Rosen D., Ronen D. Mechanism of lymphocyte-mediated cytolysis: functional cytolytic T cells lacking perforin and granzymes. Immunology. 1993 Jan;78(1):105–112. [PMC free article] [PubMed] [Google Scholar]
  5. Bolen J. B. Signal transduction by the SRC family of tyrosine protein kinases in hemopoietic cells. Cell Growth Differ. 1991 Aug;2(8):409–414. [PubMed] [Google Scholar]
  6. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  7. Chalupny N. J., Ledbetter J. A., Kavathas P. Association of CD8 with p56lck is required for early T cell signalling events. EMBO J. 1991 May;10(5):1201–1207. doi: 10.1002/j.1460-2075.1991.tb08061.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan I. T., Limmer A., Louie M. C., Bullock E. D., Fung-Leung W. P., Mak T. W., Loh D. Y. Thymic selection of cytotoxic T cells independent of CD8 alpha-Lck association. Science. 1993 Sep 17;261(5128):1581–1584. doi: 10.1126/science.8372352. [DOI] [PubMed] [Google Scholar]
  9. Collins T. L., Uniyal S., Shin J., Strominger J. L., Mittler R. S., Burakoff S. J. p56lck association with CD4 is required for the interaction between CD4 and the TCR/CD3 complex and for optimal antigen stimulation. J Immunol. 1992 Apr 1;148(7):2159–2162. [PubMed] [Google Scholar]
  10. Dennert G., Anderson C. G., Prochazka G. High activity of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase and cytolytic perforin in cloned cell lines is not demonstrable in in-vivo-induced cytotoxic effector cells. Proc Natl Acad Sci U S A. 1987 Jul;84(14):5004–5008. doi: 10.1073/pnas.84.14.5004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Doherty P. C. Cell-mediated cytotoxicity. Cell. 1993 Nov 19;75(4):607–612. doi: 10.1016/0092-8674(93)90480-e. [DOI] [PubMed] [Google Scholar]
  12. Duke R. C., Chervenak R., Cohen J. J. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6361–6365. doi: 10.1073/pnas.80.20.6361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dustin M. L., Springer T. A. Role of lymphocyte adhesion receptors in transient interactions and cell locomotion. Annu Rev Immunol. 1991;9:27–66. doi: 10.1146/annurev.iy.09.040191.000331. [DOI] [PubMed] [Google Scholar]
  14. Ferrini S., Miescher S., Zocchi M. R., von Fliedner V., Moretta A. Phenotypic and functional characterization of recombinant interleukin 2 (rIL 2)-induced activated killer cells: analysis at the population and clonal levels. J Immunol. 1987 Feb 15;138(4):1297–1302. [PubMed] [Google Scholar]
  15. Filippini A., Taffs R. E., Sitkovsky M. V. Extracellular ATP in T-lymphocyte activation: possible role in effector functions. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8267–8271. doi: 10.1073/pnas.87.21.8267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Finke J. H., Zea A. H., Stanley J., Longo D. L., Mizoguchi H., Tubbs R. R., Wiltrout R. H., O'Shea J. J., Kudoh S., Klein E. Loss of T-cell receptor zeta chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res. 1993 Dec 1;53(23):5613–5616. [PubMed] [Google Scholar]
  17. Gershenfeld H. K., Weissman I. L. Cloning of a cDNA for a T cell-specific serine protease from a cytotoxic T lymphocyte. Science. 1986 May 16;232(4752):854–858. doi: 10.1126/science.2422755. [DOI] [PubMed] [Google Scholar]
  18. Gillis S., Smith K. A. Long term culture of tumour-specific cytotoxic T cells. Nature. 1977 Jul 14;268(5616):154–156. doi: 10.1038/268154a0. [DOI] [PubMed] [Google Scholar]
  19. Glaichenhaus N., Shastri N., Littman D. R., Turner J. M. Requirement for association of p56lck with CD4 in antigen-specific signal transduction in T cells. Cell. 1991 Feb 8;64(3):511–520. doi: 10.1016/0092-8674(91)90235-q. [DOI] [PubMed] [Google Scholar]
  20. Grandori C., Hanafusa H. p60c-src is complexed with a cellular protein in subcellular compartments involved in exocytosis. J Cell Biol. 1988 Dec;107(6 Pt 1):2125–2135. doi: 10.1083/jcb.107.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grimm E. A., Mazumder A., Zhang H. Z., Rosenberg S. A. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med. 1982 Jun 1;155(6):1823–1841. doi: 10.1084/jem.155.6.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hatakeyama M., Kono T., Kobayashi N., Kawahara A., Levin S. D., Perlmutter R. M., Taniguchi T. Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association. Science. 1991 Jun 14;252(5012):1523–1528. doi: 10.1126/science.2047859. [DOI] [PubMed] [Google Scholar]
  23. Horak I. D., Gress R. E., Lucas P. J., Horak E. M., Waldmann T. A., Bolen J. B. T-lymphocyte interleukin 2-dependent tyrosine protein kinase signal transduction involves the activation of p56lck. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1996–2000. doi: 10.1073/pnas.88.5.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hudig D., Ewoldt G. R., Woodard S. L. Proteases and lymphocyte cytotoxic killing mechanisms. Curr Opin Immunol. 1993 Feb;5(1):90–96. doi: 10.1016/0952-7915(93)90086-8. [DOI] [PubMed] [Google Scholar]
  25. Johnston S. C., Dustin M. L., Hibbs M. L., Springer T. A. On the species specificity of the interaction of LFA-1 with intercellular adhesion molecules. J Immunol. 1990 Aug 15;145(4):1181–1187. [PubMed] [Google Scholar]
  26. Jones J., Hallett M. B., Morgan B. P. Reversible cell damage by T-cell perforins. Calcium influx and propidium iodide uptake into K562 cells in the absence of lysis. Biochem J. 1990 Apr 15;267(2):303–307. doi: 10.1042/bj2670303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Karnitz L., Sutor S. L., Torigoe T., Reed J. C., Bell M. P., McKean D. J., Leibson P. J., Abraham R. T. Effects of p56lck deficiency on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line. Mol Cell Biol. 1992 Oct;12(10):4521–4530. doi: 10.1128/mcb.12.10.4521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kato K., Sato N., Tanabe T., Yagita H., Agatsuma T., Hashimoto Y. Establishment of mouse lymphokine-activated killer cell clones and their properties. Jpn J Cancer Res. 1991 Apr;82(4):456–463. doi: 10.1111/j.1349-7006.1991.tb01870.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kawakami A., Tian Q., Duan X., Streuli M., Schlossman S. F., Anderson P. Identification and functional characterization of a TIA-1-related nucleolysin. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8681–8685. doi: 10.1073/pnas.89.18.8681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kuhlman P., Moy V. T., Lollo B. A., Brian A. A. The accessory function of murine intercellular adhesion molecule-1 in T lymphocyte activation. Contributions of adhesion and co-activation. J Immunol. 1991 Mar 15;146(6):1773–1782. [PubMed] [Google Scholar]
  31. Lollo B. A., Chan K. W., Hanson E. M., Moy V. T., Brian A. A. Direct evidence for two affinity states for lymphocyte function-associated antigen 1 on activated T cells. J Biol Chem. 1993 Oct 15;268(29):21693–21700. [PubMed] [Google Scholar]
  32. Lowrey D. M., Aebischer T., Olsen K., Lichtenheld M., Rupp F., Hengartner H., Podack E. R. Cloning, analysis, and expression of murine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9. Proc Natl Acad Sci U S A. 1989 Jan;86(1):247–251. doi: 10.1073/pnas.86.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Luo K. X., Sefton B. M. Cross-linking of T-cell surface molecules CD4 and CD8 stimulates phosphorylation of the lck tyrosine protein kinase at the autophosphorylation site. Mol Cell Biol. 1990 Oct;10(10):5305–5313. doi: 10.1128/mcb.10.10.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Luo K., Sefton B. M. Activated lck tyrosine protein kinase stimulates antigen-independent interleukin-2 production in T cells. Mol Cell Biol. 1992 Oct;12(10):4724–4732. doi: 10.1128/mcb.12.10.4724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Maeda K., Lafreniere R., Jerry L. M. Production and characterization of tumor infiltrating lymphocyte clones derived from B16-F10 murine melanoma. J Invest Dermatol. 1991 Aug;97(2):183–189. doi: 10.1111/1523-1747.ep12479562. [DOI] [PubMed] [Google Scholar]
  36. Marth J. D., Cooper J. A., King C. S., Ziegler S. F., Tinker D. A., Overell R. W., Krebs E. G., Perlmutter R. M. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). Mol Cell Biol. 1988 Feb;8(2):540–550. doi: 10.1128/mcb.8.2.540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mavilio F., Kreider B. L., Valtieri M., Naso G., Shirsat N., Venturelli D., Reddy E. P., Rovera G. Alteration of growth and differentiation factors response by Kirsten and Harvey sarcoma viruses in the IL-3-dependent murine hematopoietic cell line 32D C13(G). Oncogene. 1989 Mar;4(3):301–308. [PubMed] [Google Scholar]
  38. Minami Y., Kono T., Yamada K., Kobayashi N., Kawahara A., Perlmutter R. M., Taniguchi T. Association of p56lck with IL-2 receptor beta chain is critical for the IL-2-induced activation of p56lck. EMBO J. 1993 Feb;12(2):759–768. doi: 10.1002/j.1460-2075.1993.tb05710.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mizoguchi H., O'Shea J. J., Longo D. L., Loeffler C. M., McVicar D. W., Ochoa A. C. Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science. 1992 Dec 11;258(5089):1795–1798. doi: 10.1126/science.1465616. [DOI] [PubMed] [Google Scholar]
  40. Molina T. J., Bachmann M. F., Kündig T. M., Zinkernagel R. M., Mak T. W. Peripheral T cells in mice lacking p56lck do not express significant antiviral effector functions. J Immunol. 1993 Jul 15;151(2):699–706. [PubMed] [Google Scholar]
  41. Molina T. J., Kishihara K., Siderovski D. P., van Ewijk W., Narendran A., Timms E., Wakeham A., Paige C. J., Hartmann K. U., Veillette A. Profound block in thymocyte development in mice lacking p56lck. Nature. 1992 May 14;357(6374):161–164. doi: 10.1038/357161a0. [DOI] [PubMed] [Google Scholar]
  42. Newell M. K., Haughn L. J., Maroun C. R., Julius M. H. Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen. Nature. 1990 Sep 20;347(6290):286–289. doi: 10.1038/347286a0. [DOI] [PubMed] [Google Scholar]
  43. Parsons S. J., Creutz C. E. p60c-src activity detected in the chromaffin granule membrane. Biochem Biophys Res Commun. 1986 Jan 29;134(2):736–742. doi: 10.1016/s0006-291x(86)80482-x. [DOI] [PubMed] [Google Scholar]
  44. Podack E. R., Hengartner H., Lichtenheld M. G. A central role of perforin in cytolysis? Annu Rev Immunol. 1991;9:129–157. doi: 10.1146/annurev.iy.09.040191.001021. [DOI] [PubMed] [Google Scholar]
  45. Reed J. C., Sabath D. E., Hoover R. G., Prystowsky M. B. Recombinant interleukin 2 regulates levels of c-myc mRNA in a cloned murine T lymphocyte. Mol Cell Biol. 1985 Dec;5(12):3361–3368. doi: 10.1128/mcb.5.12.3361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Reed J. C., Tadmori W., Kamoun M., Koretzky G., Nowell P. C. Suppression of interleukin 2 receptor acquisition by monoclonal antibodies recognizing the 50 KD protein associated with the sheep erythrocyte receptor on human T lymphocytes. J Immunol. 1985 Mar;134(3):1631–1639. [PubMed] [Google Scholar]
  47. Reed J. C., Torigoe T., Turner B. C., Merida I., Gaulton G., Saragovi H. U., Rapp U. R., Taichman R. Protooncogene-encoded protein kinases in interleukin-2 signal transduction. Semin Immunol. 1993 Oct;5(5):327–336. doi: 10.1006/smim.1993.1039. [DOI] [PubMed] [Google Scholar]
  48. Rudd C. E., Trevillyan J. M., Dasgupta J. D., Wong L. L., Schlossman S. F. The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5190–5194. doi: 10.1073/pnas.85.14.5190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ruegemer J. J., Ho S. N., Augustine J. A., Schlager J. W., Bell M. P., McKean D. J., Abraham R. T. Regulatory effects of transforming growth factor-beta on IL-2- and IL-4-dependent T cell-cycle progression. J Immunol. 1990 Mar 1;144(5):1767–1776. [PubMed] [Google Scholar]
  50. Salcedo T. W., Kurosaki T., Kanakaraj P., Ravetch J. V., Perussia B. Physical and functional association of p56lck with Fc gamma RIIIA (CD16) in natural killer cells. J Exp Med. 1993 May 1;177(5):1475–1480. doi: 10.1084/jem.177.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Straus D. B., Weiss A. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell. 1992 Aug 21;70(4):585–593. doi: 10.1016/0092-8674(92)90428-f. [DOI] [PubMed] [Google Scholar]
  52. Taichman R. S., Torigoe T., Tanaka S., Miyashita T., Reed J. C. Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: implications for mechanisms of IL-2-signal transduction. Cytokine. 1992 Nov;4(6):441–453. doi: 10.1016/1043-4666(92)90004-b. [DOI] [PubMed] [Google Scholar]
  53. Taichman R., Merida I., Torigoe T., Gaulton G. N., Reed J. C. Evidence that protein tyrosine kinase p56-Lck regulates the activity of phosphatidylinositol-3'-kinase in interleukin-2-dependent T-cells. J Biol Chem. 1993 Sep 25;268(27):20031–20036. [PubMed] [Google Scholar]
  54. Takayama H., Trenn G., Sitkovsky M. V. A novel cytotoxic T lymphocyte activation assay. Optimized conditions for antigen receptor triggered granule enzyme secretion. J Immunol Methods. 1987 Nov 23;104(1-2):183–190. doi: 10.1016/0022-1759(87)90502-3. [DOI] [PubMed] [Google Scholar]
  55. Torigoe T., O'Connor R., Fagard R., Fischer S., Santoli D., Reed J. C. Interleukin 4 inhibits IL-2-induced proliferation of a human T-leukemia cell line without interfering with p56-LCK kinase activation. Cytokine. 1992 Sep;4(5):369–376. doi: 10.1016/1043-4666(92)90080-b. [DOI] [PubMed] [Google Scholar]
  56. Torigoe T., O'Connor R., Santoli D., Reed J. C. Interleukin-3 regulates the activity of the LYN protein-tyrosine kinase in myeloid-committed leukemic cell lines. Blood. 1992 Aug 1;80(3):617–624. [PubMed] [Google Scholar]
  57. Veillette A., Bookman M. A., Horak E. M., Bolen J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. doi: 10.1016/0092-8674(88)90053-0. [DOI] [PubMed] [Google Scholar]
  58. Velotti F., Palmieri G., D'Ambrosio D., Piccoli M., Frati L., Santoni A. Differential expression of granzyme A and granzyme B proteases and their secretion by fresh rat natural killer cells (NK) and lymphokine-activated killer cells with NK phenotype (LAK-NK). Eur J Immunol. 1992 Apr;22(4):1049–1053. doi: 10.1002/eji.1830220426. [DOI] [PubMed] [Google Scholar]
  59. Zamoyska R., Derham P., Gorman S. D., von Hoegen P., Bolen J. B., Veillette A., Parnes J. R. Inability of CD8 alpha' polypeptides to associate with p56lck correlates with impaired function in vitro and lack of expression in vivo. Nature. 1989 Nov 16;342(6247):278–281. doi: 10.1038/342278a0. [DOI] [PubMed] [Google Scholar]
  60. Zychlinsky A., Zheng L. M., Liu C. C., Young J. D. Cytolytic lymphocytes induce both apoptosis and necrosis in target cells. J Immunol. 1991 Jan 1;146(1):393–400. [PubMed] [Google Scholar]

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