Skip to main content
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1983 Feb 1;157(2):461–472. doi: 10.1084/jem.157.2.461

Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content

PMCID: PMC2186946  PMID: 6296263

Abstract

Cell-surface antigens that are induced to appear on T cells activated by the lectin phytohemagglutinin-P (PHA) can be classified both on the basis of the kinetics of their appearance and on their growth- association properties. Seven distinct T cell activation antigens, defined by monoclonal antibodies, were classified as early, intermediate, or late antigens based on their temporal appearance relative to DNA synthesis. Four antigens, the transferrin receptor, the T cell activation antigen Tac, the 4F2 antigen, and the 49.9 antigen were early antigens, whereas the OKT10 antigen appeared at intermediate times and both HLA-DR and antigen 19.2 appeared late. The use of a dye, Hoechst 33342, which stains DNA stoichiometrically, allowed the simultaneous analysis of immunofluorescence and cell cycle position of individual cells. This analysis unexpectedly revealed that essentially all cells in the proliferative phase of the cell cycle expressed each of the four early-activation antigens. The correlation between expression of the four early-activation antigens and T cell proliferation suggests that these molecules are important for the growth of all T cells. The relationship of two of these activation antigens, known to be the receptors for transferrin and interleukin 2, a T cell growth factor, is discussed with special reference to the roles of their ligands in supporting the growth of T cells.

Full Text

The Full Text of this article is available as a PDF (868.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arndt-Jovin D. J., Jovin T. M. Analysis and sorting of living cells according to deoxyribonucleic acid content. J Histochem Cytochem. 1977 Jul;25(7):585–589. doi: 10.1177/25.7.70450. [DOI] [PubMed] [Google Scholar]
  2. Barnes D., Sato G. Methods for growth of cultured cells in serum-free medium. Anal Biochem. 1980 Mar 1;102(2):255–270. doi: 10.1016/0003-2697(80)90151-7. [DOI] [PubMed] [Google Scholar]
  3. Cotner T., Hemler M., Strominger J. L. Human T cell proteins recognized by rabbit heteroantisera and monoclonal antibodies. Int J Immunopharmacol. 1981;3(3):255–268. doi: 10.1016/0192-0561(81)90019-9. [DOI] [PubMed] [Google Scholar]
  4. Cotner T., Mashimo H., Kung P. C., Goldstein G., Strominger J. L. Human T cell surface antigens bearing a structural relationship to HLA antigens. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3858–3862. doi: 10.1073/pnas.78.6.3858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeWolf W. C., Schlossman S. F., Yunis E. J. DRw antisera react with activated T cells. J Immunol. 1979 May;122(5):1780–1784. [PubMed] [Google Scholar]
  6. Evans R. L., Faldetta T. J., Humphreys R. E., Pratt D. M., Yunis E. J., Schlossman S. F. Peripheral human T cells sensitized in mixed leukocyte culture synthesize and express Ia-like antigens. J Exp Med. 1978 Nov 1;148(5):1440–1445. doi: 10.1084/jem.148.5.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Gootenberg J. E., Ruscetti F. W., Mier J. W., Gazdar A., Gallo R. C. Human cutaneous T cell lymphoma and leukemia cell lines produce and respond to T cell growth factor. J Exp Med. 1981 Nov 1;154(5):1403–1418. doi: 10.1084/jem.154.5.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Greaves M. F., Verbi W., Festenstein H., Papasteriadis C., Jaraquemada D., Hayward A. "Ia-like" antigens on human T cells. Eur J Immunol. 1979 May;9(5):356–362. doi: 10.1002/eji.1830090504. [DOI] [PubMed] [Google Scholar]
  10. Haynes B. F., Hemler M. E., Mann D. L., Eisenbarth G. S., Shelhamer J., Mostowski H. S., Thomas C. A., Strominger J. L., Fauci A. S. Characterization of a monoclonal antibody (4F2) that binds to human monocytes and to a subset of activated lymphocytes. J Immunol. 1981 Apr;126(4):1409–1414. [PubMed] [Google Scholar]
  11. Haynes B. F., Hemler M., Cotner T., Mann D. L., Eisenbarth G. S., Strominger J. L., Fauci A. S. Characterization of a monoclonal antibody (5E9) that defines a human cell surface antigen of cell activation. J Immunol. 1981 Jul;127(1):347–351. [PubMed] [Google Scholar]
  12. Helderman J. H., Strom T. B. Role of protein and RNA synthesis in the development of insulin binding sites on activated thymus-derived lymphocytes. J Biol Chem. 1979 Aug 10;254(15):7203–7207. [PubMed] [Google Scholar]
  13. Hercend T., Ritz J., Schlossman S. F., Reinherz E. L. Comparative expression of T9, T10, and Ia antigens on activated human T cell subsets. Hum Immunol. 1981 Nov;3(3):247–259. doi: 10.1016/0198-8859(81)90021-5. [DOI] [PubMed] [Google Scholar]
  14. Iscove N. N., Melchers F. Complete replacement of serum by albumin, transferrin, and soybean lipid in cultures of lipopolysaccharide-reactive B lymphocytes. J Exp Med. 1978 Mar 1;147(3):923–933. doi: 10.1084/jem.147.3.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Judd W., Poodry C. A., Strominger J. L. Novel surface antigen expressed on dividing cells but absent from nondividing cells. J Exp Med. 1980 Nov 1;152(5):1430–1435. doi: 10.1084/jem.152.5.1430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ko H. S., Fu S. M., Winchester R. J., Yu D. T., Kunkel H. G. Ia determinants on stimulated human T lymphocytes. Occurrence on mitogen- and antigen-activated T cells. J Exp Med. 1979 Aug 1;150(2):246–255. doi: 10.1084/jem.150.2.246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Larsson E. L., Coutinho A. The role of mitogenic lectins in T-cell triggering. Nature. 1979 Jul 19;280(5719):239–241. doi: 10.1038/280239a0. [DOI] [PubMed] [Google Scholar]
  18. Leonard W. J., Depper J. M., Uchiyama T., Smith K. A., Waldmann T. A., Greene W. C. A monoclonal antibody that appears to recognize the receptor for human T-cell growth factor; partial characterization of the receptor. Nature. 1982 Nov 18;300(5889):267–269. doi: 10.1038/300267a0. [DOI] [PubMed] [Google Scholar]
  19. Loken M. R. Simultaneous quantitation of Hoechst 33342 and immunofluorescence on viable cells using a fluorescence activated cell sorter. Cytometry. 1980 Sep;1(2):136–142. doi: 10.1002/cyto.990010208. [DOI] [PubMed] [Google Scholar]
  20. Morgan D. A., Ruscetti F. W., Gallo R. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science. 1976 Sep 10;193(4257):1007–1008. doi: 10.1126/science.181845. [DOI] [PubMed] [Google Scholar]
  21. Omary M. B., Trowbridge I. S., Minowada J. Human cell-surface glycoprotein with unusual properties. Nature. 1980 Aug 28;286(5776):888–891. doi: 10.1038/286888a0. [DOI] [PubMed] [Google Scholar]
  22. Palacios R. Concanavalin A triggers T lymphocytes by directly interacting with their receptors for activation. J Immunol. 1982 Jan;128(1):337–342. [PubMed] [Google Scholar]
  23. Ruscetti F. W., Gallo R. C. Human T-lymphocyte growth factor: regulation of growth and function of T lymphocytes. Blood. 1981 Mar;57(3):379–394. [PubMed] [Google Scholar]
  24. Shapiro H. M. Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry. 1981 Nov;2(3):143–150. doi: 10.1002/cyto.990020302. [DOI] [PubMed] [Google Scholar]
  25. Smith K. A. T-cell growth factor. Immunol Rev. 1980;51:337–357. doi: 10.1111/j.1600-065x.1980.tb00327.x. [DOI] [PubMed] [Google Scholar]
  26. Snow E. C., Feldbush T. L., Oaks J. A. The effect of growth hormone and insulin upon MLC responses and the generation of cytotoxic lymphocytes. J Immunol. 1981 Jan;126(1):161–164. [PubMed] [Google Scholar]
  27. Stadler B. M., Dougherty S. F., Farrar J. J., Oppenheim J. J. Relationship of cell cycle to recovery of IL 2 activity from human mononuclear cells, human and mouse T cell lines. J Immunol. 1981 Nov;127(5):1936–1940. [PubMed] [Google Scholar]
  28. Strom T. B., Bangs J. D. Human serum-free mixed lymphocytes response: the stereospecific effect of insulin and its potentiation by transferrin. J Immunol. 1982 Apr;128(4):1555–1559. [PubMed] [Google Scholar]
  29. Suciu-Foca N., Susinno E., McKiernan P., Rohowsky C., Weiner J., Rubinstein P. DRw determinants on human T cells primed against allogeneic lymphocytes. Transplant Proc. 1978 Dec;10(4):845–848. [PubMed] [Google Scholar]
  30. Terhorst C., van Agthoven A., LeClair K., Snow P., Reinherz E., Schlossman S. Biochemical studies of the human thymocyte cell-surface antigens T6, T9 and T10. Cell. 1981 Mar;23(3):771–780. doi: 10.1016/0092-8674(81)90441-4. [DOI] [PubMed] [Google Scholar]
  31. Trowbridge I. S., Lopez F. Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits human tumor cell growth in vitro. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1175–1179. doi: 10.1073/pnas.79.4.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Trowbridge I. S., Omary M. B. Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin. Proc Natl Acad Sci U S A. 1981 May;78(5):3039–3043. doi: 10.1073/pnas.78.5.3039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Uchiyama T., Broder S., Waldmann T. A. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J Immunol. 1981 Apr;126(4):1393–1397. [PubMed] [Google Scholar]
  34. Williams J. M., Shapiro H. M., Milford E. L., Strom T. B. Multiparameter flow cytometric analysis of lymphocyte subpopulation activation in lectin-stimulated cultures. J Immunol. 1982 Jun;128(6):2676–2681. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES